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Genetic basis of hypertrophic cardiomyopathy
Bos, J.M.
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Citation for published version (APA):Bos, J. M. (2010). Genetic basis of hypertrophic cardiomyopathy.
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GENETIC BASIS OF HYPERTROPHIC CARDIOMYOPATHY
© 2010 by Johan Martijn Bos
Genetic basis of hypertrophic cardiomyopathy Johan Martijn Bos / University of Amsterdam, 2010. Thesis
Printed by Ipskamp Drukkers B.V.
ISBN: 978-90-9024905-6
No parts of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means without permission of the author
GENETIC BASIS OF HYPERTROPHIC CARDIOMYOPATHY
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
prof. dr. D.C. van den Boom
ten overstaan van een door het college voor promoties ingestelde
commissie, in het openbaar te verdedigen in de Agnietenkapel
op vrijdag 15 januari 2010, te 12.00 uur
doorJohan Martijn Bos geboren te Vorden
Promotiecommissie
Promotor: Prof. dr. A.A.M. Wilde
Co-promotor: Prof. dr. M.J. Ackerman
Leden: Prof. dr. R.J.G Peters Prof. dr. Y.M. Pinto Prof. dr. R.J.A. Wanders Prof. dr. N.A. Blom Prof. dr. P.A.F.M Doevendans Dr. L. Kapusta
Faculteit der Geneeskunde
The research described in this thesis was carried out in the Mayo Clinic Windland Smith Rice Sudden Death Genomics Laboratory in Rochester, MN (USA) in collaboration with the Heart Failure Research Center of the Academic Medical Center, Amsterdam (The Netherlands).
Financial support by the Netherlands Heart Foundation for the publication of this thesis is gratefully acknowledged.
Additional support was generously provided by the Medtronic Bakken Research Center, Maastricht; AstraZeneca, Zoetermeer; St. Jude Medical, Veenendaal and the University of Amsterdam.
Voor mijn ouders
Table of contents Page
Chapter 1 Introduction Genetics of hypertrophic cardiomyopathy: one, two, or more diseases? 9
Curr Opin Cardiol 2007; 22(3): 193 – 199 [Review]
Chapter 2 Genotype-phenotype relationships involving hypertrophic cardiomyopathy - associated mutations in titin, muscle LIM protein, and telethonin. 27
Mol Genet Metab 2006; 88(1): 78 – 85
Chapter 3 Cardiac ankyrin repeat protein gene (ANKRD1) mutations in hypertrophic cardiomyopathy. 49
J Am Coll Cardiol 2009; 54(4): 334 – 42
Chapter 4 Echocardiographic-determined septal morphology in Z-disc hypertrophic cardiomyopathy. 71
Biochem Biophys Res Commun 2006; 351(4): 896 – 902
Chapter 5 Relationship between sex, shape and substrate in hypertrophic cardiomyopathy. 89 Am Heart J 2008; 155: 1128 – 1134
Chapter 6 TGFß-inducible early gene-1 (TIEG1): a novel hypertrophic cardiomyopathy susceptibility gene. 107
Manuscript in preparation
Chapter 7 Diagnostic, prognostic and therapeutic implications of genetic testing for hypertrophic cardiomyopathy. 129
J Am Coll Cardiol 2009; 54(3): 201 – 211 [Review]
The growing field of genetic contributors to the pathogenesis of HCM or,About ‘lumpers’ and ‘splitters’: McKusick revisited. 161
Summary 169
Samenvatting 173
Acknowledgements 177
List of publications 179
Chapter 1
Genetics of Hypertrophic Cardiomyopathy: One, Two, or More Diseases?
J. Martijn Bos, Steve R. Ommen, Michael J. Ackerman
Curr Opin Cardiol 2007; 22(3): 193 – 9 [Review]
Abstract
Purpose of review. Hypertrophic cardiomyopathy (HCM), affecting 1 in 500 persons, is the most common identifiable cause of sudden death in the young. This review details the history of HCM, recent discoveries in its genetic underpinnings and important genotype-phenotype relationships described in recent studies. Recent findings. Since the discovery of the genetic underpinnings of hypertrophic cardiomyopathy in 1989 hundreds of mutations scattered amongst at least 10 sarcomeric genes confer the pathogenetic substrate for this “disease of the sarcomere/myofilament”. More recently, the genetic spectrum of HCM has expanded to encompass mutations in Z-disc associated genes (Z-disc hypertrophic cardiomyopathy) and glycogen storage diseases mimicking HCM (metabolic hypertrophic cardiomyopathy). Recent genotype-phenotype studies have discovered an important relationship between morphology of the left ventricle, its underlying genetic substrate and long-term outcome of this disease. Summary. Genomic medicine has entered the clinical practice and the diagnostic utility of genetic testing for HCM diseases is clearly evident, but with the growing number of hypertrophic cardiomyopathy-associated genes strategic choices have to be made. With recent discoveries in genotype-phenotype relationships, especially pertaining to the echocardiographic septal shape and the underlying pathogenetic mutation, time has come to subdivide the one disease we call HCM.
Keywords
Genetic testing, hypertrophic cardiomyopathy, myofilament, septal morphology
10
Introduction
Hypertrophic cardiomyopathy (HCM) is a disease of enormous phenotypic and genotypic heterogeneity. Affecting 1 in 500 people, it is the most prevalent genetic cardiovascular disease, and more importantly the most common cause of sudden cardiac death in young athletes[1]. Anatomically/physiologically, HCM can manifest with negligible to extreme hypertrophy, minimal to extensive fibrosis and myocyte disarray, absent to severe left ventricular outflow tract obstruction, and distinct septal contours/morphologies such as reverse curve-, sigmoidal-, and apical variant-HCM. The clinical course varies extremely, ranging from an asymptomatic lifelong course to dyspnea/angina refractory to pharmacotherapy to sudden death as the sentinel event.
HCM was fully described for the first time by Teare in 1958 as ‘asymmetrical hypertrophy of the heart in young adults’[2]. It has since been known by a confusing array of names, reflecting its clinical heterogeneity and its uncommon occurrence in daily cardiologic practice. In 1968, the World Health Organization (WHO) defined cardiomyopathies as ‘diseases of different and often unknown etiology in which the dominant feature is cardiomegaly and heart failure’[3]. This statement was updated in 1980 and defined cardiomyopathies as ‘heart muscles diseases of unknown cause’, thereby differentiating it from specific identified heart muscle diseases of known cause, like myocarditis[4].
Throughout the years, names such as idiopathic hypertrophic subaortic stenosis[5], muscular subaortic stenosis[6] and hypertrophic obstructive cardiomyopathy[7] have been widely and interchangeably used to define the same disease. In 1995, a WHO/International Society and Federation of Cardiology Task Force on cardiomyopathies classified the different cardiomyopathies by dominant pathophysiology , or if possible, by etiological/pathogenetic factors[8]. The four most important cardiomyopathies - dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), arrhythmogenic right ventricular cardiomyopathy (ARVC) and HCM - were recognized, next to a number of specific and mostly acquired cardiomyopathies, like ischemic- or inflammatory cardiomyopathy[8].
11
Accordingly, HCM is described as ‘left and/or right ventricular hypertrophy, usually asymmetric and involving the interventricular septum with predominant autosomal dominant inheritance involving sarcomeric contractile proteins’[8]. This nomenclature has been upheld in the most recent ACC/ESC expert consensus document of 2003[9], although with expanding knowledge on the genetic background of these diseases voices have recently been subclassified into primary cardiomyopathies into genetic - , mixed - and acquired cardiomyopathies[10]. Under this approach, the genetic subgroup entails HCM, ARVC and glycogen storage diseases presenting as HCM, but also includes ion channel disorders such as long QT syndrome (LQTS)[10].
Genetic background of HCM
Since the sentinel discovery of the first locus for familial HCM (1989) and the first mutations involving the MYH7-encoded beta myosin heavy chain (1990) as the pathogenic basis for HCM[11, 12], over 300 mutations scattered among at least 24 genes encoding various sarcomeric, calcium handling and mitochondrial proteins have been identified (Table 1). The most common genetically-mediated form of HCM is myofilament-HCM, with hundreds of disease-associated mutations in 8 genes
encoding proteins critical to the sarcomere’s thick myofilament [ -myosin heavy chain (MYH7)[12], regulatory myosin light chain (MYL2) and essential myosin light chain (MYL3)][13], intermediate myofilament [myosin binding protein C (MYBPC3)][14], and thin myofilament [cardiac troponin T (TNNT2), -tropomyosin (TPM1)[15], cardiac troponin I (TNNI3)[16], and actin (ACTC)[17, 18]. Targeted screening of giant sarcomeric TTN-encoded titin, which extends throughout half of the sarcomere, has thus far revealed only one mutation[19]. More recently, mutations have been described in the myofilament protein alpha-myosin heavy chain encoded by MYH6[20]. Although up until 2001 it was thought that specific mutations in these myofilament genes were inherently ‘benign’ or ‘malignant’ [21, 22, 23, 24, 25, 26, 27, 28], genotype-phenotype studies involving a large cohort of unrelated patients have indicated that great caution must be exercised with assigning particular prognostic significance to any particular mutation[29, 30, 31].
12
Furthermore, those studies have demonstrated that the two most common forms of genetically mediated HCM – MYH7-HCM and MYBPC3-HCM – are phenotypically indistinguishable[32]. The prevalence of mutations in the 8 most common myofilament associated genes, currently comprising the commercially available HCM genetic test (www.hpcgg.org) in different international cohorts ranges from 30 to 61%, leaving still a large number of patients with genetically unexplained disease[33].
Over the last few years, the spectrum of HCM-associated genes expanded outside the myofilament to encompass additional subgroups that could be classified as ‘Z-disc-HCM’, ‘calcium-handling HCM’, and ‘metabolic HCM’; all genes currently implicated in the pathogenesis of HCM are shown in Table 1. As a result of its close proximity to the contractile apparatus of the myofilament and its specific structure-function relationship with regards to cyto-architecture, as well as its role in the stretch-sensor mechanism of the sarcomere, recent attention has been focused on the cardiac Z-disc. Initial mutations were described in muscle LIM protein encoded by CSRP3[34] and telethonin encoded by TCAP[35], an observation replicated in our large cohort of unrelated patients with HCM[36]. LDB3-encoded LIM domain binding 3, ACTN2-encoded alpha actinin 2 and VCL-encoded vinculin/metavinculin have been added to that list[37]. Interestingly, although the first HCM-associated mutation in vinculin was found in the cardiac-specific insert of the gene, yielding the protein called metavinculin[38], the follow up study also identified a mutation in the ubiquitously expressed protein vinculin[39].
As the critical ion in the excitation-contraction coupling of the cardiomyocyte, calcium and proteins involved in calcium induced calcium release (CICR) have always been of high interest in the pathogenesis of HCM. Although with very low frequency, mutations have been described in the promoter – and coding region of PLN-encoded phospholamban, an important inhibitor of cardiac muscle sarcoplasmic reticulum Ca(2+)-ATPase (SERCA)[40, 41] as well as in the RyR2-encoded cardiac ryanodine receptor[42]. Recently, our HCM genetic research program discovered three novel mutations in JPH2-encoded junctophilin 2 in three, previously genotype negative, patients with HCM. This is the first time that JPH2, which is thought to play a role in approximating the sarcoplasmic reticulum calcium release channels and plasmalemmal L-type calcium channels, has been implicated in the pathogenesis of HCM[43].
13
Tabl
e 1:
Sum
mar
y of
hyp
ertro
phic
car
diom
yopa
thy
(HC
M)-
susc
eptib
ility
gen
es a
nd t
he e
stim
ated
/ext
rapo
late
d fre
quen
cy (
%)
of s
peci
fic
mut
atio
ns b
y m
orph
olog
ic s
ubgr
oup.
Gen
eLo
cus
Prot
ein
Rev
erse
Cur
ve H
CM
Si
gmoi
dal
HC
MA
pica
l HC
M
Myo
filam
ent H
CM
70 –
85
10 -1
5 30
– 4
0
Gia
ntfil
amen
t T
TN
2q24
.3
Titin
-
- -
Thi
ckfil
amen
t M
YH
714
q11.
2-q1
2 -m
yosi
n he
avy
chai
n 30
- 40
<
5 10
- 15
MY
H6
14q1
1.2-
q12
-myo
sin
heav
y ch
ain
- -
-
MY
L212
q23-
q24.
3 V
entri
cula
r reg
ulat
ory
myo
sin
lig
ht c
hain
<
5 0
2 - 4
MY
L33p
21.2
-p21
.3
Ven
tricu
lar e
ssen
tial m
yosi
n
light
cha
in
- -
-
Inte
rmed
iate
fil
amen
t M
YB
PC
3 11
p11.
2 C
ardi
ac m
yosi
n-bi
ndin
g pr
otei
n C
30
- 40
5
10 -
15
Thi
n fil
amen
t T
NN
T2
1q32
C
ardi
ac tr
opon
in T
5
- 10
<1
< 5
T
NN
I319
p13.
4 C
ardi
ac tr
opon
in I
1-2
<1
0
T
PM
115
q22.
1 -tr
opom
yosi
n 1-
2 0
0
AC
TC
15q1
4 -c
ardi
ac a
ctin
<1
0
0
Z-di
sc H
CM
0
5 - 1
0 <
5
LB
D3
10q2
2.2-
q23.
3 LI
M b
indi
ng d
omai
n 3
(A
lias:
ZA
SP
) 0
3 3
C
SR
P3
11p1
5.1
Mus
cle
LIM
pro
tein
0
<1
0
TC
AP
17q1
2-q2
1.1
Tele
thon
in
0 <1
0
V
CL
10q2
2.1-
q23
Vin
culin
/met
avin
culin
0
<1
<1
A
CT
N2
1q42
-q43
A
lpha
-act
inin
2
0 1
0
Cal
cium
han
dlin
g H
CM
-
--
R
yR2
1q42
.1-q
43
Car
diac
ryan
odin
e re
cept
or
- -
-
JP
H2
20q1
2 Ju
ncto
phili
n-2
<1
<1
0
P
LN6q
22.1
P
hosp
hola
mba
n -
- -
Met
abol
ic H
CM
P
RK
AG
2 7q
35- q
36.3
6 A
MP
-act
ivat
ed p
rote
in k
inas
e -
- -
LA
MP
2Xq
24
Lyso
som
e-as
soci
ated
m
embr
ane
prot
ein
2 -
- -
G
LAXq
22
Alp
ha-g
alac
tosi
dase
A
- -
-
FX
N9q
13
Frat
axin
-
- -
- in
dica
tes
that
no
geno
type
-phe
noty
pe s
tudi
es in
volv
ing
a la
rge
coho
rt of
unr
elat
ed p
atie
nts
have
bee
n pe
rform
ed to
est
imat
e th
e fre
quen
cy o
f tha
t gen
e’s
parti
cula
r inv
olve
men
t in
that
par
ticul
ar m
orph
olog
ical
sub
type
of H
CM
.
The last important genetic subgroup of HCM is that of the metabolic HCM, involving mitochondrial and lysosomal proteins. In 2005, Arad et al. first described mutations in lysosome-associated membrane protein-2 encoded by LAMP2 and protein kinase gamma-2 encoded by PRKAG2 in glycogen storage disease-associated genes mimicking the clinical phenotype of HCM[44, 45, 46, 47]. In 2005, a mutation in FXN-encoded frataxin was described in a patient with HCM. Although this patient also harbored a myofilament mutation in MYBPC3-encoded myosin binding protein C, functional characterization showed significant influence of the FXN-mutant on the phenotype, suggesting that the observed alterations in energetics may act in synergy with the present myofilament mutation[48]. Similar to PRKAG2 and LAMP2, Fabry’s disease can express predominant cardiac features of left ventricular hypertrophy. Over the years, mutations in GLA-encoded alpha-galactosidose A have been found in patients with this multi-system disorder [49, 50, 51].
Although up to 24 HCM-susceptibility genes involving different pathways have been identified, the search for novel mutations in new genes continues. Recently, a genome wide-linkage study identified a new locus for HCM in a large family with left ventricular hypertrophy located to chromosome 7. Subsequent studies of genes located to this region however have thus far not yielded the causative gene[52]. As a result of the increasing genetic heterogeneity of HCM, a classification based on functional genetics might seem very helpful, but in light of the low yield of mutations in a large number of these genes as well as the commercial availability of just a small number of these genes, a phenotypic classification might be a more useful tool in looking at this disease from a clinical practice vantage point.
16
Genotype-phenotype analyses in HCM
Numerous studies have tried to identify phenotypic characteristics most indicative of myofilament/sarcomeric-HCM to facilitate genetic counseling and strategically direct clinical genetic testing[29, 31, 32, 53, 54, 55, 56]. Although several phenotype-genotype relationships have emerged to enrich the yield of genetic testing, these patient profiles have not been particularly clinically informative. An important discovery, linking the echocardiographically determined septal morphology to the underlying genetic substrate, was recently made.
The first link to be drawn between septal morphologies was a result of HCM study by Lever and colleagues in the 1980s, in which septal contour – classified as reverse septal contour, sigmoidal septal contour, apical - and neutral contour - was found to be age-dependent with a predominance of sigmoidal-HCM being present in the elderly[57]. In the early 90’s Seidman et al described an early genotype-phenotype observation involving a small number of patients and family members and discovered that patients with mutations in the beta myosin heavy chain (MYH7-HCM) generally had reversed curvature septal contours (reverse curve-HCM)[58].
Inspired by these two initial observations, we recently finished a large genotype-phenotype analysis correlating the septal morphology with the underlying genotype. After extensive analysis of the echocardiograms of 382 previously genotyped and published patients[32, 53, 56], we observed that sigmoidal-HCM (47% of cohort) and reverse curve-HCM (35% of cohort) were the two most prevalent anatomical subtypes of HCM, and discovered that the septal contour was the strongest predictor for the presence of a myofilament mutation, regardless of age [59]. Multivariate analysis in this cohort demonstrated septal morphology was the only independent predictor of myofilament HCM with an odds ratio of 21 (p<0.001), when reverse curve morphology was present[59]. Apical HCM, in which the hypertrophy is mostly concentrated around the apex of the heart, was found in 10% (n=37) of our cohort. The yield of the commercially available HCM genetic test for myofilament-HCM was 79% in reverse curve-HCM but only 8% in patients with sigmoidal-HCM. Of the smaller subgroup of patients with apical HCM, 32% had a mutation in one of the myofilaments [56].
17
These observations may facilitate echo-guided genetic testing by enabling informed genetic counseling about the a priori probability of a positive genetic test based upon the patient’s expressed anatomical phenotype (Figure 1). In addition, the paucity of myofilament mutations in sigmoidal-HCM opens the door for research to elucidate the molecular/genetic determinants of sigmoidal HCM.
Figure 1: Functional subgroups of genetic hypertrophic cardiomyopathy (HCM) and the yield of genetic testing for the two most common septal morphologies with their respective subgroup. Shown are the most important functional subgroups of genetically mediated HCM and the yield of mutations over various cohorts. Blue arrows indicate the functional relationship between the different elements. The black arrows show the yield of genetic testing for the subgroups of myofilament HCM and Z-disc HCM and their morphologic subgroups. LAMP2, lysosome-associated membrane protein 2; PLN, phospholamban; PRKAG2, AMP-activated protein kinase; SR, sarcoplasmic reticulum;RyR2, cardiac ryanodine receptor.
18
With the majority of known myofilament proteins studied, except for a complete analysis of the giant protein TTN-encoded titin, recent research has been focused proteins beyond the cardiac myofilaments, especially proteins involved in the cyto-architecture and cardiac stretch sensor mechanism of the cardiomyocyte localized to the cardiac Z-disc (Figure 1). The Z-disc is an intricate assembly of proteins at the Z-line of the cardiomyocyte sarcomere. Extensively reviewed, proteins of the Z-disc are important in the structural and mechanical stability of the sarcomere as they appear to serve as a docking station for transcription factors, calcium signaling proteins, kinases and phosphatases [60, 61]. In addition, this assembly of proteins seems to serve as a way station for proteins that regulate transcription by aiding in their controlled translocation between the nucleus and the Z-disc[60, 61].
With all of these roles, a main implication for the Z-disc is its involvement in the cardiomyocyte stretch sensing and response systems[62]. Mutations in three such proteins localized to the cardiac Z-disc, CSRP3-encoded muscle LIM protein (MLP), TCAP-encoded telethonin and VCL-encoded vinculin, including its cardiac specific insert of exon 19 that yields metavinculin, have previously been established as both HCM[34, 35, 36, 38, 39] and dilated cardiomyopathy (DCM)-susceptibility genes[34, 35, 36, 38, 63, 64]. Additionally, it is now fully appreciated that these divergent cardiomyopathic phenotypes of HCM and DCM are partially allelic disorders with ACTC, MYH7, TNNT2, TPM1, MYBPC3, TTN, MLP, TCAP, and VCL established as both HCM- and DCM-susceptibility genes[34, 35, 38, 63, 65, 66, 67, 68, 69].
Mutations in ACTN2-encoded alpha-actinin-2 (ACTN2) and LDB3-encoded LIM domain binding 3 (LDB3) as novel HCM-susceptibility genes[37] were described. Building on our discovery linking reverse-curve HCM to the presence of myofilament mutation, and recognizing that the Z-disc may transduce multiple signaling pathways during stress, translating into hypertrophic responses, cell growth and remodeling [70], we have observed that Z-disc HCM, in contrast to myofilament HCM, is preferentially sigmoidal. Eleven out of 13 patients with Z-disc HCM had a sigmoidal septal contour and no reverse septal curvatures were seen [37]. We speculate that Z-disc HCM leads to a hypertrophic response that is expressed in the areas of highest stress (i.e. LVOT) and therefore predisposes to a sigmoidal septal contour.
19
Intriguing conclusions can be drawn from these observations. Whereas in initial morphologic studies, sigmoidal-HCM seemed to be associated with older age [57], the underlying genotype rather than age appears to be the predominant determinant of septal morphology[59]. Furthermore, Z-disc HCM seems to have a predilection for sigmoidal contour status. Given that the vast majority of our patients with sigmoidal HCM still lack a putative disease-causing mutation, the molecular underpinnings responsible for a sigmoidal morphology remain to be elucidated. Alternatively, it seems plausible that a HCM-predisposing mutation might not be the principle determinant for many patients with sigmoidal-HCM. Instead, a multi-factorial model may be responsible for this subtype of clinically diagnosed HCM.
In this model, the sum of all contributors – the presence or absence of a mutation or LVH promoting polymorphisms[71], an unidentified genetic substrate, environmental factors and hypertension, culminates in what is clinically labeled as HCM. This multi-factorial model for sigmoidal-HCM is supported by the significantly older age at diagnosis of patients with sigmoidal-HCM (49 years) compared to those with reverse curve-HCM (32 years)[59] and the fact that nearly 20% of patients classified with sigmoidal-HCM were noted to have mild hypertension[59]. Diagnosed with HCM by experienced physicians, a subset of this group may have a basal septum more sensitive to the pro-hypertrophy trigger of increased afterload, precipitating basal septal hypertrophy (sigmoidal disease).
20
Conclusions
Genomic medicine has entered the clinical practice as it pertains to the evaluation and management of HCM. The diagnostic utility of genetic testing for HCM diseases is clearly evident, but strategic choices have to be made with the growing number of genes implicated in this disease. With recent discoveries in genotype-phenotype relationships, especially pertaining the echocardiographic septal shape and the underlying pathogenetic mutation, time has come to further subdivide the one disease we call HCM.
Clinical HCM specialists are accustomed already to prefacing the HCM label with physiological descriptors of obstructive- and non-obstructive-HCM and anatomical/morphological descriptors: reverse curve- , sigmoidal- , and apical-HCM. Accordingly, a pathogenetic subdivision seems warranted. Just as there is no prerequisite for clinically diagnosed HCM to necessarily be obstructive or reverse curve in nature, it should not be mandated that clinically diagnosed HCM requires a genetic perturbation in one of the sarcomeric myofilaments. Instead, what is emerging is a clear picture that the two most common anatomical/morphological subtypes of HCM (reverse curve- and sigmoidal-HCM) largely emanate from fundamentally distinct pathogenetic mechanisms. Herein, most (but not all) of reverse curve-HCM is indeed a “disease of the sarcomere” and most (but not all) sigmoidal-HCM is in search of its etiology.
21
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24
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53. Van Driest SL, Jaeger MA, Ommen SR, Will ML, et al. Comprehensive analysis of the beta-myosin heavy chain gene in 389 unrelated patients with hypertrophic cardiomyopathy. JAm Coll Cardiol 2004; 44(3): 602-610.
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55. Richard P, Charron P, Carrier L, Ledeuil C, et al. Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation 2003; 107(17): 2227-2232.
56. Van Driest SL, Ellsworth EG, Ommen SR, Tajik AJ, et al. Prevalence and spectrum of thin filament mutations in an outpatient referral population with hypertrophic cardiomyopathy. Circulation 2003; 108: 445-451.
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58. Solomon SD, Wolff S, Watkins H, Ridker PM, et al. Left ventricular hypertrophy and morphology in familial hypertrophic cardiomyopathy associated with mutations of the beta-myosin heavy chain gene. J Am Coll Cardiol 1993; 22(2): 498-505.
59. Binder J, Ommen SR, Gersh BJ, Van Driest SL, et al. Echocardiography-guided genetic testing in hypertrophic cardiomyopathy: septal morphological features predict the presence of myofilament mutations. Mayo Clin Proc 2006; 81(4): 459-467.
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25
62. Knoll R, Hoshijima M, Hoffman HM, Person V, et al. The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy. Cell 2002; 111(7): 943-955.
63. Mohapatra B, Jimenez S, Lin JH, Bowles KR, et al. Mutations in the muscle LIM protein and alpha-actinin-2 genes in dilated cardiomyopathy and endocardial fibroelastosis. Mol Genet Metab 2003; 80(1-2): 207-215.
64. Olson TM, Illenberger S, Kishimoto NY, Huttelmaier S, et al. Metavinculin mutations alter actin interaction in dilated cardiomyopathy. Circulation 2002; 105(4): 431-437.
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71. Perkins MJ, Van Driest SL, Ellsworth EG, Will ML, et al. Gene-specific modifying effects of pro-LVH polymorphisms involving the renin-angiotensin-aldosterone system among 389 unrelated patients with hypertrophic cardiomyopathy. Eur Heart J 2005; 26(22): 2457-2462.
26
Chapter 2
Genotype-Phenotype Relationships Involving Hypertrophic Cardiomyopathy-Associated Mutations in
Titin, Muscle LIM Protein and Telethonin
J. Martijn Bos, Rainer N. Poley, Melissa Ny, David J. Tester, Xiaolei Xu, Matteo Vatta, Jeffrey A. Towbin, Bernard J. Gersh, Steve R. Ommen, Michael J. Ackerman
Mol Genet Metab 2006; 88(1): 78 – 85
Letter to editor:
Mol Genet Metab 2006; 88(2): 199 – 200
Mol Genet Metab 2006; 89(3): 286 – 287
Abstract Background: TTN-encoded titin, CSRP3-encoded muscle LIM protein, and TCAP-
encoded telethonin are Z-disc proteins essential for the structural organization of the
cardiac sarcomere and the cardiomyocyte’s stretch sensor. All 3 genes have been
established as cardiomyopathy-associated genes for both dilated cardiomyopathy
(DCM) and hypertrophic cardiomyopathy (HCM). Here, we sought to characterize the
frequency, spectrum, and phenotype associated with HCM-associated mutations in
these 3 genes in a large cohort of unrelated patients evaluated at a single tertiary
outpatient center.
Methods: DNA was obtained from 389 patients with HCM (215 male, left ventricular
wall thickness of 21.6 ± 6 mm) and analyzed for mutations involving all translated
exons of CSRP3 and TCAP and targeted HCM-associated exons (2, 3, 4 and 14) of
TTN using polymerase chain reaction (PCR), denaturing high performance liquid
chromatography (DHPLC), and direct DNA sequencing. Clinical data was extracted
from patient records and maintained independent of the genotype.
Results: Overall, 16 patients (4.1%) harbored a Z-disc mutation: 12 had a MLP
mutation and 4 patients a TCAP mutation. No TTN mutations were detected. Seven
patients were also found to have a concomitant myofilament mutation. Seven patients
with a MLP-mutation were found to harbor the DCM-associated, functionally
characterized W4R mutation. W4R-MLP was also noted in a single white control
subject. Patients with MLP/TCAP-associated HCM clinically mimicked myofilament-
HCM.
Conclusions: Approximately 4.1% of unrelated patients had HCM-associated MLP or
TCAP mutations. MLP/TCAP-HCM phenotypically mirrors myofilament-HCM and is
more severe than the subset of patients who still remain without a disease-causing
mutation. The precise role of W4R-MLP in the pathogenesis of either DCM or HCM
warrants further investigation.
Keywords
Genetics, genes, hypertrophy, cardiomyopathy, Z-disc, muscle LIM protein, telethonin,
TCAP, titin
28
Introduction Affecting one in 500 persons, hypertrophic cardiomyopathy (HCM) is a disease
associated with remarkable genotypic and phenotypic heterogeneity[1, 2]. Clinical
outcomes range from an entirely asymptomatic course with normal longevity to chronic
progressive heart failure or sudden cardiac death (SCD). Indeed, HCM is one of the
leading causes of SCD in young persons [1].
The most common genetically mediated form of HCM is myofilament-HCM with
hundreds of disease-associated mutations in 8 genes encoding proteins critical to the
sarcomere’s thick - [ -myosin heavy chain (MYH7)[3], regulatory myosin light chain
(MYL2) and essential myosin light chain (MYL3)][4], intermediate - [myosin binding
protein C (MYBPC3)][5], and thin myofilament [cardiac troponin T (TNNT2), -
tropomyosin (TPM1)[6], cardiac troponin I (TNNI3)[7], and actin (ACTC)[8, 9]].
Myofilament-HCM accounts for approximately 40-65% of HCM among cohorts of
unrelated patients[10]. In general, patients with myofilament-HCM have greater
hypertrophy and present at a younger age than those who remain without an
established disease-causing mutation[11]. The 2 most common genotypes of
myofilament-HCM, MYBPC3- and MYH7-HCM, are phenotypically indistinguishable
from each other[12, 13, 14, 15, 16, 17, 18, 19, 20].
Besides perturbations involving the sarcomere’s myofilaments, the Z-disc,
which comprises a cadre of proteins involved in cardiomyocyte cytoarchitecture and
mechano-sensor- signaling, has emerged recently as host to several HCM-associated
mutations extending the spectrum of “sarcomeric”-HCM. To date, 3 genes encoding
critical Z-disc proteins: TTN-encoded titin, CSRP3-encoded muscle LIM protein (MLP),
and the TCAP-encoded telethonin, have been implicated in the pathogenesis of both
dilated cardiomyopathy (DCM) and HCM[21, 22, 23, 24].
29
As part of the cardiomyocyte stretch response machinery, TTN-encoded titin,
which extends throughout half of the sarcomere from the M-line to the Z-disc is the
largest of the three proteins; mapped on chromosome 2q31, TTN encodes for a giant
26,926 amino-acid protein with a molecular weight of 2,993 kD[25]. CSRP3-encoded
MLP and TCAP-encoded telethonin are mapped to 11p15.1 and 17q12 respectively
and contain 194 and 167 amino acids respectively [26, 27]. Prior to this study, 1 HCM-
associated mutation in TTN (R740L-TTN)[28], 3 HCM-associated mutations in MLP
(L44P-MLP, C58G-MLP and S54R/E55G-MLP)[22], and 2 HCM-associated mutations
in TCAP (T137I-TCAP and R153H-TCAP) have been reported[21].
Having completed a comprehensive mutational analysis involving all translated
exons of the 8 genes responsible for myofilament-HCM[14, 15, 29, 30], we sought to
determine the frequency, spectrum, and phenotype associated with these 3 genes that
encode essential Z-disc proteins among a large cohort of unrelated patients diagnosed
clinically with HCM.
30
Methods Study population
Following a written informed consent for this IRB-approved research protocol, blood
samples were obtained from 389 unrelated patients with HCM (215 male, left
ventricular wall thickness of 21.6 6 mm) evaluated at the Mayo Clinic’s HCM clinic
between April 1997 and December 2001. Subsequently DNA was extracted from the
blood samples using Purgene DNA extraction kits (Gentra, Minneapolis, Minnesota).
HCM-associated mutational analysis of TTN, CSRP3, and TCAP
Using polymerase chain reaction (PCR) and denaturing high performance liquid
chromatography (DHPLC) (WAVE, Transgenomic, Omaha, Nebraska), the 3 genes
implicated in Z-disc-HCM: TTN-encoded titin, CSRP3-encoded muscle LIM protein,
and TCAP-encoded telethonin, were analyzed. Abnormal elution profiles were further
characterized by direct DNA sequencing (ABI Prism 377; Applied Biosystem, Foster
City, California).
For TTN, only a targeted analysis of the exons (2, 3, 4 and 14) hosting
cardiomyopathy-associated mutations was performed while a comprehensive open
reading frame/splice-site analysis was conducted for all translated exons of CSRP3 (5
exons) and TCAP (2 exons). A topological schematic of both MLP and telethonin
including key functional domains is depicted in Figure 1. Primers, annealing
temperatures and optimized WAVE conditions are available upon request. Four
hundred reference alleles, derived from 100 white and 100 black healthy controls
(Coriell Cell Repositories), were also examined to determine whether an identified
amino acid variant was a common polymorphism. The non-synonymous mutations
were annotated using the single letter convention as in L44P whereby the wild type
leucine (L) at residue 44 has been replaced by proline (P).
Statistical analysis
Analysis of variance tests were used to assess differences between continuous
variables; contingency tables or z-tests were used as appropriate to analyze nominal
variables independency of the different variables. Student’s T-tests were performed to
elucidate differences between the different subgroups. A p-value less than 0.05 was
considered statistically significant.
31
Figure 1: Topological schematic of muscle LIM protein and telethonin. Shown are the important domains of the protein. For MLP, the TCAP-binding domain, both LIM-domains and its nuclear localization signal (NLS) are shown. For telethonin, the MLP -, titin- and minK-binding domains are shown. Amino-acid localization of the specific domains between parentheses
32
Results
Table 1 summarizes the phenotype of the entire HCM cohort including those with
perturbations involving either MLP or telethonin. The mean age at diagnosis for our
total cohort was approximately 41 ± 19 years with 216 patients (55%) having cardiac
symptoms at presentation and 60 (15%) having received an implantable cardioverter-
defibrillator (ICD). The mean maximum left ventricular wall thickness (LVWT) was 21.6
± 6 mm. Of the 389, 161 (41%) were treated in part by a surgical myectomy, reflecting
the surgical referral bias and subsequent over-representation of obstructive HCM in
this cohort. Approximately one-third had a family history of HCM whereas one-seventh
was found to have a family history of sudden cardiac death. Myofilament-HCM was
demonstrated previously for 147 of the 389 subjects (38%)[14, 15, 30].
Overall, 16 (4.1%) individuals with HCM hosted possible mutations in the genes
underlying Z-disc-HCM: TTN (0), CSRP3 (12), and TCAP (4). The clinical phenotypes
of these patients are described in Table 2. The average at diagnosis for MLP (CSRP3)
- and TCAP-associated HCM was 48.5 ± 17 and 38.8 ± 9 years, respectively, while the
mean maximal left ventricular wall thickness (MLVWT) was 20.1 ± 3 mm and 29.5 ± 12
mm, respectively. Three patients (25%) with a MLP-mutation and 1 patient (25%) with
a TCAP-mutation reported a family history of HCM, while 2 and 1 patient (17 and 25%)
respectively had a family history of SCD. A total of 8 patients underwent a surgical
myectomy due to refractory symptoms despite optimal medical treatment.
33
Table 1: Clinical characteristics of HCM cohort
HCM-cohort
Genotype negative
Singlemyofilament
mutationMLP TCAP
No. of
individuals 389 233 140 12 4
Sex,
male/female 215/174 127/106 79/61 7/5 2/2
Age at Dx 41.2 ±19 45.1 ± 19 34.5 ± 17 48.5 ± 17 38.8 ± 9
Cardiac
symptoms 216 (56%) 128 (55%) 74 (53%) 9 (75%) 4 (100%)
Max LVWT
(mm) 21.6 ± 6 20.6 ± 6 23.0 ± 7 20.1 ± 3 29.5 ± 12
LVWT 25 mm 78 (20%) 38 (16%) 38 (28%) 0 2 (50%)
Resting LVOTO
(mmHg) 46.6 ± 42 46.6 ± 42 42.8 ± 42 80 ± 43 75 ± 38
Pos. FH for
HCM 121 (31%) 54 (23%) 61 (44%) 3 (25%) 1 (25%)
Pos. FH for
SCD 54 (14%) 26 (11%) 27 (19%) 2 (17%) 1 (25%)
Myectomy 160 (41%) 92 (39%) 62 (44%) 5 (42%) 3 (75%)
Pacemaker 67 (17%) 35 (15%) 26 (19%) 5 (42%) 2 (50%)
ICD 60 (15%) 23 (10%) 36 (26%) 1 (8%) 0
Multiple or
concomitant
myofilament
mutation
147 - 10/140 6/12 1/4
Values are mean ± SD or % (n). Dx indicates diagnosis; FH, family history; HCM, Hypertrophic cardiomyopathy; ICD, implantable cardioverter-defibrillator; LVOTO, left ventricular outflow tract obstruction; LVWT, left ventricular wall thickness; SCD, sudden cardiac death
34
HCM-associated MLP mutations
Figure 2 depicts the mutations found in the CSRP3-encoded MLP; novel mutations
are indicated by an asterisk. Five CSRP3 variants were identified in 12 patients,
including 4 missense mutations and 1 frame-shift mutation, involving residues highly
conserved across species (data not shown) and not seen in 400 reference alleles.
Figure 2: Schematic representation of mutations in muscle LIM protein and telethonin. Representation of mutations found in our cohort of 389 patients with HCM. The L44P-MLP has been previously published as a HCM-associated mutation. The W4R-MLP mutation has been previously published and functionally characterized in patients with DCM. Novel mutations are indicated with an asterisk.
35
Clinical phenotypes are described in Table 2. K42fs/165 and Q91L were
detected in patients having no HCM-associated myofilament mutations (cases 8 and 12). The previously published HCM-causing mutation (L44P, case 9) localized to the
LIM1 -actinin binding domain while the R64C and Y66C mutations (cases 10 and 11)
localized to the 6 amino acid nuclear localization signal (NLS). These 3 mutations
(cases 9-11) were detected in patients also hosting HCM-associated myofilament
mutations.
The missense mutation, W4R-MLP, which localizes to telethonin’s binding domain,
was noted in 7 patients (cases 1-7). Three of these patients (cases 5-7) also had a
mutation involving either the beta myosin heavy chain or myosin binding protein C.
W4R was also observed in one of the 400 reference alleles examined (a healthy
Caucasian control).
36
HCM-associated TCAP mutations
Three different, novel TCAP mutations were identified in 4 patients with HCM (Table 2,
cases 13 – 16). Two patients (cases 13 and 14) had an in-frame deletion involving
glutamic acid at position 13 (E13del). The R70W mutation was located in the reciprocal
MLP-binding domain of telethonin in a patient (case 15) with a MLVWT of 46 mm and
a positive family history for HCM. The titin-binding domain of telethonin was host to a
missense mutation, P90L, for one patient (case 16) who also had a missense mutation
involving myosin binding protein C.
Genotype-phenotype relationships in MLP/TCAP-HCM
Compared to patients still lacking a mutation (genotype negative) and patients with
myofilament-HCM, patients with mutations involving either MLP or TCAP more closely
resembled the subset with myofilament-HCM (Figure 3 a-c). The subset with MLP-
HCM were, however, more obstructive (80 ± 43 mmHg) than both myofilament-HCM
(42.8 ± 42 mmHg; p = 0.01) and genotype negative-HCM (46.6 ± 42 mmHg; p =
0.007). Despite the small sample size, patients with TCAP-HCM had significantly
greater MLVWT (29.5 ± 12 mm) compared with either genotype negative- (20.6 ± 6
mm; p = 0.006), myofilament- (23.0 ± 7.0 mm; p = 0.04), or MLP-HCM (20.1 ± 3 mm; p
= 0.01) and a similar age at diagnosis as myofilament positive-HCM (38.8 ± 9 vs. 34.5
± 17 years old; p = 0.6). When a subset analysis of patients with either Z-disc only
(n=9) mutations or Z-disc mutation plus a concomitant myofilament (n=7) mutation was
performed, the phenotypes of these two subgroups did not differ from each other on
MLVWT (23.9 ± 9 mm vs. 20.6 ± 3 mm; p = 0.3), MLVOTO (67.4 ± 49 mmHg vs. 93.5
± 20 mmHg; p = 0.2) or age at diagnosis (46.3 ± 6 yrs vs. 45.8 ± 6.8 yrs; p = 0.9),
supporting the role of MLP/TCAP mutations in pathogenesis of HCM.
37
Tabl
e 2.
Clin
ical
pro
files
of P
atie
nts
with
a H
CM
-ass
ocia
ted
CS
PR
3 (M
LP) o
r TC
AP
Mut
atio
n
C a s eG
ene
Mut
atio
n(e
xon)
M
yofil
amen
t M
utat
ion
Age/
Se
x
Age
at DxRa
ce*
Sym
ptom
s a
tPr
esen
tatio
n Su
bseq
uent
sy
mpt
oms
A F
Max
.LV
WT
(mm
)
Res
ting
LVO
TO
(mm
Hg)
FH of HC
M
FH o
f SC
D(A
ge a
t SC
D) †
Tr
eatm
ent
1C
SR
P3
W4R
(1)
80/F
69
1An
gina
, dys
pnea
An
gina
, dy
spne
a Y
1521
Yes
No
PM
2C
SR
P3
W4R
(1)
29/M
161
Asym
ptom
atic
D
yspn
ea
N25
0N
oN
o…
3C
SR
P3
W4R
(1)
56/M
411
Asym
ptom
atic
An
gina
, dy
spne
a,
(pre
)syn
cope
N17
32No
NoPM
4C
SR
P3
W4R
(1)
78/F
68
1n/
aD
yspn
ea
N20
0N
oN
oM
yect
omy
5C
SR
P3
W4R
(1)
F111
3I-
MYB
PC3
59/M
501
Asym
ptom
atic
D
yspn
ea,
(pre
)syn
cope
N
2311
7No
NoM
yect
omy,
PM
, IC
D
6C
SR
P3
W4R
(1)
T137
7M-
MYH
750
/F
431
n/a
Angi
na,
dysp
nea,
(p
re)s
ynco
pe
N18
86Ye
sN
oM
yect
omy
7C
SR
P3
W4R
(1)
I511
T-M
YH7
60/F
53
1D
yspn
ea
Dys
pnea
, (p
re)s
ynco
pe
N16
0Ye
sN
o…
8C
SR
P3
K42
fs/1
65
(2)
53/M
462
Angi
na, d
yspn
ea
Angi
na,
dysp
nea,
(p
re)s
ynco
pe
N18
112
NoNo
…
9C
SR
P3
L44P
(2)
G10
41 fs
/5-
MYB
PC3
71/F
62
n/a
Pres
ynco
pe
Angi
na,
dysp
nea,
(p
re)s
ynco
pe
N25
100
Yes
Yes
(40,
32,
39)
Mye
ctom
y,
PM
10C
SR
P3
R64
C (2
) I1
131T
-M
YBPC
372
/M65
n/a
Dys
pnea
, (p
re)s
ynco
pe
Angi
na,
dysp
nea
Y23
58No
No…
11C
SR
P3
Y66C
(2)
R16
2Q-
TNN
I3
36/M
281
n/a
Asym
ptom
atic
N
1910
0N
oYe
s(4
2)M
yect
omy
12C
SR
P3
Q91
L (2
) 57
/M44
1An
gina
An
gina
, dy
spne
a,
(pre
)syn
cope
Y22
18No
NoPM
13T
CA
PE1
3del
(1)
53/M
471
Dys
pnea
, (p
re)s
ynco
pe
Dys
pnea
, (p
re)s
ynco
pe
N22
100
NoNo
….
14T
CA
PE1
3del
(1)
42/M
371
Angi
na, d
yspn
ea
Angi
na,
dysp
nea
N30
81Ye
sYe
s(5
4)M
yect
omy,
IC
D
15T
CA
PR
70W
(2)
65/F
44
1As
ympt
omat
ic
Dys
pnea
Y
4619
Yes
No
Mye
ctom
y,
PM
16T
CA
PP9
0L (
2)
Q99
8R-
MYB
PC3
45/F
26
1D
yspn
ea
Angi
na,
dysp
nea,
pr
esyn
cope
Y20
100
NoNo
Mye
ctom
y,
PM
AF, a
trial
fibr
illatio
n; D
x, d
iagn
osis;
FH
, fam
ily h
isto
ry; H
CM
, Hyp
ertro
phic
card
iom
yopa
thy;
ICD
, im
plan
tabl
e ca
rdio
verte
r def
ibril
lato
r; LV
OTO
, lef
t ven
tricu
lar
outfl
ow tr
act o
bstru
ctio
n; L
VWT,
left
vent
ricul
ar w
all t
hick
ness
; n/a
, not
ava
ilabl
e; P
M, p
acem
aker
;SC
D, s
udde
n ca
rdia
c de
ath;
* 1 =
Cau
casia
n, 2
= H
ispan
ic; †
, in
a fir
st d
egre
e re
lativ
e
Figure 3a
Figure 3b
40
Figure 3c
Figure 3a – c: Degree of hypertrophy (a), degree of left ventricular outflow tract obstruction (b) and age at diagnosis (c) for genotyped subjects. Genotyped patients with hypertrophic cardiomyopathy are grouped on the X-axis as hosting as hosting no putative mutation (genotype negative), hosting a myofilament mutation (myofilament-HCM), a MLP-mutation or a TCAP-mutation. Unless otherwise noted, all pair wise comparisons are not statistically significant. *, p<0.05 compared to all other groups
41
Discussion
As critical components of the dynamic protein scaffolding between the sarcomere and
cytoskeleton at the Z-line, the titin-muscle LIM protein-telethonin complex is involved in
both cyto-architecture and mechano-signaling, thus serving as a potential link between
myofilament-HCM and Z-disc-HCM. 1 Prior to this study, 1 HCM-associated mutation
in titin[28], 4 HCM-associated mutations in MLP[22, 31] and 2 HCM-associated
mutations in telethonin have been reported[21]. In addition, consistent with the notion
that HCM and DCM are often allelic disorders, several DCM-associated mutation in
these 3 Z-disc proteins have been discovered as well[23, 24, 32, 33, 34]. Based upon
our observations in this study, the genes encoding Z-disc proteins currently implicated
so far as only DCM-susceptibility genes constitute rational candidate genes to explore
in HCM.
This study represents the largest series of patients examined for the 3 known
subtypes of Z-disc-HCM whereby approximately 4% of unrelated patients harbored a
mutation in either MLP (CSRP3) or TCAP. We did not observe any mutations in the
giant protein, titin, which extends across half of the entire sarcomere. However, only
those regions implicated previously in either HCM or DCM were examined. Among the
12 patients with a non-synonymous, amino-acid altering variant in the CSRP3-encoded
MLP, a compelling case for disease-association exists at the present time for 5
patients (cases 8-12). Besides the L44P-MLP, R64C-MLP, and Y66C-MLP missense
mutations, 3 patients (cases 9-11) also possessed a concomitant myofilament
mutation: G1041fs/5-MYBPC3, I1131T-MYBPC3 and R162Q-TNNI3 respectively. The
L44P-MLP variant along with the K42fs/165-MLP frameshift mutation localize to the
LIM1-domain which is responsible for binding to -actinin. In a yeast 2-hybrid assay,
Geier et al. recently showed a significantly impaired binding affinity for -actinin due to
C58G-MLP[22].
42
The pathogenic mechanism for HCM in these patients hosting both MLP
variants and myofilament mutations may be due to synergistic heterozygosity (two-hit
hypothesis) as we have previously demonstrated in a patient hosting a known myosin
binding protein C missense mutation and a functionally-compromised frataxin
mutation[35]. Previously, we demonstrated that among the 140 patients in our cohort
previously established to have solely myofilament-HCM, 10 patients (7%) hosted 2
myofilament mutations with one of the variants usually involving myosin binding protein
C[14]. Supporting the notion that both variants contributed to the expressed
phenotype, these patients with multiple myofilament-HCM were younger at diagnosis
and had greater hypertrophy than those having a single myofilament mutation. Herein,
proportionately more patients with putative Z-disc-HCM also had a myofilament
mutation raising the possibility that some of these variants may represent false
positives. Future studies of the families represented by these HCM cases may shed
light on the relative contributions of both the myofilament and the Z-disc mutation in the
expressed phenotype.
The precise contribution of W4R-MLP (seen in 7 patients, cases 1-7) in the
pathogenesis of HCM remains an enigma. Four of the 7 patients with W4R-MLP in the
present study also have a published HCM-associated myofilament mutation. Initially,
W4R was discovered as a DCM-associated mutation and was reportedly absent in 640
normal reference alleles[34]. Localizing to the telethonin-binding domain of MLP, it was
not surprising to see in vitro assays demonstrating markedly reduced
interaction/localization with telethonin[34]. Transgenic mouse models of W4R-MLP
yield mice with a rather pronounced cardiomyopathy characterized by significant
ventricular dilation and systolic dysfunction[36].
Recently, W4R-MLP was observed in 1 of 137 unrelated patients with
HCM[31]. This variant was found in a patient with predominant apical HCM in which no
myofilament mutations were identified. However, these investigators also observed
W4R in 3 of 500 reference alleles (0.6% allelic frequency). We have now observed
W4R in 1/400 reference alleles. While clearly a phenotype producing mutation in an
overexpression transgenic mouse model, further studies are necessary to elucidate the
precise role of W4R-MLP in the pathogenesis of cardiomyopathies in humans.
43
Finally, 4 patients hosted mutations in telethonin with 1 patient also having a
myofilament Q998R-MYBPC3 genotype. These patients had severe hypertrophy
(mean MLVWT = 29.5 mm) whereas previously published TCAP probands had a mean
MLVWT of 20 mm. In particular, the patient in our study with R70W-TCAP had
massive hypertrophy with a septal wall thickness of 46 mm. No other mutations in
known HCM genes have been found in this individual. R70W-TCAP localizes to the
functional domain essential for binding MLP.
Most of the HCM- and DCM-associated mutations reported in these 3 Z-disc
proteins have not been characterized functionally. It remains to be determined whether
or not the various mutations selectively perturb force generating (HCM-predisposing)
or force transmitting (DCM-predisposing) functions.
44
Conclusions
In this study, HCM-susceptibility mutations in CSRP3 and TCAP represent uncommon
causes of HCM, with a prevalence similar to troponin I- and actin-HCM. The combined
clinical phenotype of MLP/TCAP-HCM resembles that of myofilament-HCM. Co-
segregation and functional studies are now needed to dissect the relative contributions
of the various Z-disc mutations to the pathogenesis and phenotypic expression of
HCM.
Acknowledgements
We are grateful to the patients seen at the HCM Clinic for their participation in this
study and to Mr. Doug Kocer, the nurse coordinator of the HCM Clinic.
45
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16. Erdmann J, Raible J, Maki-Abadi J, Hummel M, et al. Spectrum of clinical phenotypes and gene variants in cardiac myosin-binding protein C mutation carriers with hypertrophic cardiomyopathy. J Am Coll Cardiol 2001; 38(2): 322-330. 17. Morner S, Richard P, Kazzam E, Hellman U, et al. Identification of the genotypes causing hypertrophic cardiomyopathy in northern Sweden. J Mol Cell Cardiol 2003; 35(7): 841-849. 18. Garcia-Castro M, Reguero JR, Batalla A, Diaz-Molina B, et al. Hypertrophic cardiomyopathy: low frequency of mutations in the beta-myosin heavy chain (MYH7) and cardiac troponin T (TNNT2) genes among Spanish patients. Clinical Chemistry 2003; 49(8): 1279-1285. 19. Jaaskelainen P, Soranta M, Miettinen R, Saarinen L, et al. The cardiac beta-myosin heavy chain gene is not the predominant gene for hypertrophic cardiomyopathy in the Finnish population. J Am Coll Cardiol 1998; 32(6): 1709-1716. 20. Jaaskelainen P, Kuusisto J, Miettinen R, Karkkainen P, et al. Mutations in the cardiac myosin-binding protein C gene are the predominant cause of familial hypertrophic cardiomyopathy in eastern Finland. J Mol Med 2002; 80: 412-422. 21. Hayashi T, Arimura T, Itoh-Satoh M, Ueda K, et al. Tcap gene mutations in hypertrophic cardiomyopathy and dilated cardiomyopathy. J Am Coll Cardiol 2004; 44(11): 2192-2201. 22. Geier C, Perrot A, Ozcelik C, Binner P, et al. Mutations in the human muscle LIM protein gene in families with hypertrophic cardiomyopathy. Circulation 2003; 107(10): 1390-1395. 23. Gerull B, Gramlich M, Atherton J, McNabb M, et al. Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy. Nat Genet 2002; 30(2): 201-204. 24. Itoh-Satoh M, Hayashi T, Nishi H, Koga Y, et al. Titin mutations as the molecular basis for dilated cardiomyopathy. Biochem Biophys Res Commun 2002; 291(2): 385-393. 25. Labeit S, Kolmerer B. Titins: giant proteins in charge of muscle ultrastructure and elasticity. Science 1995; 270(5234): 293-296. 26. Valle G, Faulkner G, De Antoni A, Pacchioni B, et al. Telethonin, a novel sarcomeric protein of heart and skeletal muscle. FEBS Lett 1997; 415(2): 163-168. 27. Fung YW, Wang RX, Heng HH, Liew CC. Mapping of a human LIM protein (CLP) to human chromosome 11p15.1 by fluorescence in situ hybridization. Genomics 1995; 28(3): 602-603. 28. Satoh M, Takahashi M, Sakamoto T, Hiroe M, et al. Structural analysis of the titin gene in hypertrophic cardiomyopathy: Identification of a novel disease gene. Biochem Biophys Res Commun 1999; 262: 411-417. 29. Ackerman MJ, Van Driest SV, Ommen SR, Will ML, et al. Prevalence and age-dependence of malignant mutations in the beta-myosin heavy chain and troponin T gene in hypertrophic cardiomyopathy: a comprehensive outpatient perspective. J Am Coll Cardiol 2002; 39(12): 2042-2048. 30. Van Driest SL, Ellsworth EG, Ommen SR, Tajik AJ, et al. Prevalence and spectrum of thin filament mutations in an outpatient referral population with hypertrophic cardiomyopathy. Circulation 2003; 108: 445-451.
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31. Newman B, Cescon D, Woo A, Rakowski H, et al. W4R variant in CSRP3 encoding muscle LIM protein in a patient with hypertrophic cardiomyopathy. Mol Genet Metab 2005; 84(4): 374-375. 32. Hayashi T, Arimura T, Ueda K, Shibata H, et al. Identification and functional analysis of a caveolin-3 mutation associated with familial hypertrophic cardiomyopathy. Biochem Biophys Res Commun 2004; 313(1): 178-184. 33. Mohapatra B, Jimenez S, Lin JH, Bowles KR, et al. Mutations in the muscle LIM protein and alpha-actinin-2 genes in dilated cardiomyopathy and endocardial fibroelastosis. Mol Genet Metab 2003; 80(1-2): 207-215. 34. Knoll R, Hoshijima M, Hoffman HM, Person V, et al. The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy. Cell 2002; 111(7): 943-955. 35. Van Driest SL, Gakh O, Ommen SR, Isaya G, et al. Molecular and functional characterization of a human frataxin mutation found in hypertrophic cardiomyopathy. Mol Genet Metab 2005; 85(4): 280-5 36. Arber S, Hunter JJ, Ross J, Jr., Hongo M, et al. MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell 1997; 88(3): 393-403.
48
Chapter 3
Cardiac Ankyrin Repeat Protein Gene (ANKRD1)
Mutations in Hypertrophic Cardiomyopathy
Takuro Arimura*, J. Martijn Bos*, Akinori Sato, Toru Kubo, Hiroshi Okamoto, Hirofumi Nishi, Haruhito Harada, Yoshinori Koga, Mousumi Moulik, Yoshinori L. Doi, Jeffrey A. Towbin, Michael J. Ackerman, Akinori Kimura* These authors equally contributed to the work
J Am Coll Cardiol 2009; 54(4): 334 – 42
Editorial comment by:
L. Mestroni. Phenotypic Heterogeneity of Sarcomeric
Gene Mutations: a Matter of Gain and Loss?
J Am Coll Cardiol 2009; 54(4): 343 – 5
Abstract
Objectives: The purpose of this study was to explore a novel disease gene for
hypertrophic cardiomyopathy (HCM) and evaluate functional alteration(s) caused by
mutations.
Background: Mutations in genes encoding myofilaments or Z-disc proteins of the
cardiac sarcomere cause HCM, but the disease-causing mutations can be found in half
of the patients, indicating that novel HCM-susceptibility genes await discovery. We
studied a candidate gene ANKRD1 encoding for cardiac ankyrin repeat protein (CARP)
that is a Z-disc component interacting with N2A domain of titin/connectin and N-terminal
domain of myopalladin.
Methods: We analyzed 384 HCM patients for mutations in ANKRD1 and in the N2A
domain of titin/connectin gene (TTN). Interaction of CARP with titin/connectin or
myopalladin was investigated using co-immunoprecipitation assay to demonstrate the
functional alteration caused by ANKRD1 or TTN mutations. Functional abnormalities
caused by the ANKRD1 mutations were also examined at the cellular level in neonatal
rat cardiomyocytes.
Results: Three ANKRD1 missense mutations, Pro52Ala, Thr123Met and Ile280Val,
were found in 3 patients. All mutations increased binding of CARP to both titin/connectin
and myopalladin. In addition, TTN mutations, Arg8500His and Arg8604Gln, in the N2A
domain were found in 2 patients and these mutations increased binding of
titin/connectin to CARP. Myc-tagged CARP showed that the mutations resulted in
abnormal localization of CARP in cardiomyocytes.
Conclusion: CARP abnormalities may be involved in the pathogenesis of HCM.
Keywords
Hypertrophic cardiomyopathy, mutation, Z-disc, CARP, titin/connectin
50
Abbreviations Ab Antibody
ANKRD1 Ankyrin repeat domain 1
CARP Cardiac ankyrin repeat protein
cDNA Complementary deoxyribonucleic acid
Co-IP Co-immunoprecipitation
DAPI 4’6-diamidino-2-phenylindole
DCM Dilated cardiomyopathy
HCM Hypertrophic cardiomyopathy
PCR Polymerase chain reaction
WT Wild-type
51
Introduction
Cardiomyopathy is a primary heart muscle disorder caused by functional abnormalities
of cardiomyocytes. There are several clinical subtypes of cardiomyopathy and the most
prevalent subtype is hypertrophic cardiomyopathy (HCM)[1,2]. HCM is characterized by
hypertrophy and diastolic dysfunction of cardiac ventricles accompanied by
cardiomyocyte hypertrophy, fibrosis and myofibrillar disarray[1]. Although the etiology of
HCM has not been fully elucidated, 50-70% of the patients with HCM have apparent
family histories consistent with autosomal dominant genetic trait[2], and recent genetic
analyses have revealed that a significant percentage of HCM is caused by mutations in
the genes encoding for myofilaments and Z-disc proteins of the cardiac sarcomere with
the majority of mutations identified in MYH7-encoded beta myosin heavy chain and
MYBPC3-encoded myosin binding protein C[2].
ANKRD1 (ankyrin repeat domain 1)-encoded “cardiac adriamycin responsive
protein” [3] or “cardiac ankyrin repeat protein”(CARP)[4], is a transcription co-factor and
an early differentiation marker of cardiac myogenesis, expressed in the heart during
embryonic and fetal development. CARP expression is up-regulated in the adult hearts
at end-stage heart failure [5]. In addition, increased CARP expression was found in
hypertrophied hearts from experimental murine models [6, 7]. These observations
suggest a pivotal role of CARP in cardiac muscle function in both physiological and
pathological conditions. Although CARP is known to be involved in the regulation of
gene expression in the heart, Bang et al. demonstrated that CARP located to both the
sarcoplasm and nucleus, suggesting a shuttling of CARP in cellular components [8].
Within the I-band region of sarcomere, CARP bound to both N2A domain of
titin/connectin encoded by TTN and the N-terminal domain of myopalladin encoded by
MYPN. Hence, titin/connectin and myopalladin function in part as anchoring proteins of
“sarcomeric CARP” [8, 9].
52
Titin/connectin is the most giant protein expressed in the striated muscles,
which is involved in sarcomere assembly, force transmission at the Z-disc, and
maintenance of resting tension in the I-band region[10, 11]. In cardiac muscle, there are
two titin isoforms, N2B and N2BA. The N2B isoform contains a cardiac specific N2B
domain, and the N2BA isoform contains both N2B and N2A domains. Both N2A and
N2B domains, within the extensible I-band region, function as a molecular spring that
develops passive tension; the expression of N2B isoform results in a higher passive
stiffness than that of N2AB isoform. We previously reported an HCM-associated
mutation localizing to the N2B domain[12], and Gerull et al.[13] reported other TTN
mutations in the Z/I transition domain. These observations suggest that the I-band
region of titin/connectin contains elastic components extending with stretch to generate
passive force, which plays an important role in the maintenance of cardiac function.
Another protein that anchors CARP at the Z/I band is myopalladin, a
cytoskeletal protein containing 3 proline-rich motifs and 5 Ig domains. The proline-rich
motifs in the central part is required for binding to nebulin/nebulette, and the Ig domains
at the N-terminus and C-terminus are involved in the binding to CARP and sarcomeric
-actinin, respectively[8]. It was suggested that myopalladin played key roles in
sarcomere/Z-disc assembly, myofibrillogenesis, recruitment of the other Z/I-band
elements, and signaling in the Z/I-band[8].
In this study, we analyzed unrelated patients with heretofore
genotype-negative HCM for mutations in ANKRD1 and found 3 mutations that showed
abnormal binding to myopalladin and titin/connectin. In addition, we searched for
mutations in the recipcrocal CARP-binding N2A domain of titin/connectin and identified
2 HCM-associated mutations in TTN causing abnormal binding to CARP. We report
here that abnormal CARP assembly in the cardiac muscles may be involved in the
pathogenesis of HCM.
53
Methods Subjects
A total of 384 unrelated patients with HCM were included in this study. The patients
were diagnosed based on medical history, physical examination, 12-lead
electrocardiogram, echocardiography, and other special tests if necessary. The
diagnostic criteria for HCM included LV wall thickness >13mm on echocardiography, in
the absence of coronary artery disease, myocarditis, and hypertension. The patients
had been analyzed previously for mutations in previously published myofilament- and
Z-disc associated genes and no mutation was found in any of the known
HCM-susceptibility genes (15-18). Ethnically-matched healthy individuals (400 and 300
from Japan and USA, respectively) were used as controls. Blood samples were
obtained from the subjects after given informed consent. The protocol for research was
approved by the Ethics Reviewing Committee of Medical Research Institute, Tokyo
Medical and Dental University (Japan) and by the Mayo Foundation Institutional Review
Board (US).
Mutational analysis
Using intronic primers, each translated ANKRD1 exon was amplified by polymerase
chain reaction (PCR) from genomic DNA samples. TTN exons 99 to 104 corresponding
to the N2A domain including binding domains to CARP and p94/calpain were amplified
by PCR in exon-by-exon manner. Sequence of primers and PCR conditions used in this
study are available upon request. PCR products were analyzed by direct sequencing or
by denaturing high performance liquid chromatography (DHPLC) followed by
sequencing analysis. Sequencing was performed using Big Dye Terminator chemistry
(version 3.1) and ABI3100 DNA Analyzer (Applied Biosystems, CA, USA).
54
Co-immunoprecipitation (co-IP) assay
We obtained cDNA fragments of human ANKRD1 and TTN by RT-PCR from adult heart
mRNA. A wild-type (WT) full-length CARP cDNA fragment spanned from bp249 to
bp1208 of GenBank Accession No. NM_014391 (corresponding to aa1-aa319). Three
equivalent mutant cDNA fragments containing C to G (Pro52Ala mutation), C to T
(Thr123Met mutation) or A to G (Ile280Val mutation) substitutions were obtained by
primer-directed mutagenesis method. A WT TTN cDNA fragment encoding N2A
domains (from bp25535 to bp26465 of NM_133378 corresponding aa8437-aa8747)
was obtained and 3 TTN mutants carrying T to C (non disease-associated Ile8474Thr
polymorphism), G to A (HCM-associated Arg8500His mutation) or G to A
(HCM-associated Arg8604Gln mutation) substitutions were created by the
primer-mediated mutagenesis method. The cDNA fragments of ANKRD1 were cloned
into myc-tagged pCMV-Tag3 (Stratagene, CA, USA), while TTN and MYPN cDNA
fragments were cloned into pEGFP-C1 (Clontech, CA, USA). These constructs were
sequenced to ensure that no errors were introduced.
Cellular transfection and protein extractions were performed as described
previously [14], and co-IP assays were performed using the Catch and Release v2.0
Reversible Immunoprecipitation System according to the manufacturer‘s instructions
(Millipore, Billerica, MA). Immunoprecipitates were separated on SDS-PAGE gels and
transferred to a nitrocellulose membrane. After a pre-incubation with 3% skim milk in
PBS, the membrane was incubated with primary rabbit anti-myc polyclonal or mouse
anti-GFP monoclonal Ab (1:100, Santa Cruz Biotechnology, CA, USA), and with
secondary goat anti-rabbit (for polyclonal Ab) or rabbit anti-mouse (for monoclonal Ab)
IgG HRP-conjugated Ab (1:2000, Dako A/S, Grostrup, Denmark). Signals were
visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA,
USA) and Luminescent Image Analyzer LAS-3000mini (Fujifilm, Tokyo, Japan), and
their densities were quantified by using Multi Gauge ver3.0 (Fujifilm, Tokyo, Japan).
Numerical data were expressed as means ± SEM. Statistical differences were
analyzed using one-way ANOVA and Student’s t test for paired values. Means were
compared by independent samples t-tests without correction for multiple comparisons.
A p-value<0.05 was considered to be statistically significant.
55
Indirect immunofluorescence microscopy
All care and treatment of animals were in accordance with “Guidelines for the Care and
Use of Laboratory Animals” published by the National Institute of Health (NIH
Publication 85-23, revised 1985) and subjected to prior approval by the local animal
protection authority. Neonatal rat cardiomyocytes were prepared as described
previously[14]. Eighteen and 48 hours after the transfection, cardiomyocytes were
washed with PBS, fixed for 15 min in 100% ethanol at -20°C. Transfected cells were
incubated in blocking solution, and stained by primary rabbit anti-myc polyclonal Ab
(1:100, Santa Cruz Biotechnology) and mouse anti- -actinin monoclonal Ab (1:800,
Sigma-Aldrich), followed by secondary sheep anti-rabbit IgG FITC-conjugated Ab
(1:500, Chemicon, Victoria, Australia) and Alexa fluor 568 goat anti-mouse IgG (1:500,
Molecular Probes, OR, USA). All cells were mounted on cover-glass using Mowiol 4-88
Reagent (Calbiochem, Darmstadt, Germany) with 4’6-diamidino-2-phenylindole (DAPI,
Sigma-Aldrich) and images from at least 200 transfected cells were analyzed with an
LSM510 laser-scanning microscope (Carl Zeiss Microscopy, Jena, Germany).
56
Results Identification of ANKRD1 (CARP) and TTN mutations in HCM
Eleven distinct sequence variations in ANKRD1 were identified among the 384 patients
with HCM (Figure 1A). Four intronic variants, 2 non-synonymous substitutions and 1
synonymous variation were polymorphisms, because they were also found in the
controls. A nonsense mutation (c.423C>T in exon 2 yielding Gln59ter) was found in
2patients with familial HCM and was absent in the controls, but was not co-segregated
with the disease in both families, suggesting that they were not associated with HCM. In
contrast, 3 missense mutations, Pro52Ala (c.402C>G in exon 2), Thr123Met (c.616C>T
in exon 4) and Ile280Val (c.1086A>G in exon 8), identified in three unrelated HCM
patients, were not found in the controls.
Sequence variations in TTN at the N2A domain containing binding region to
CARP and p94/calpain were searched for in the patients and 8 variations were
identified (Figure 1B). An intronic variation and 3 synonymous variations were
polymorphisms observed in the controls. Two non-synonymous variations, Ile8474Thr
(c.25645T>C in exon 99) and Asp8672Val (c.26239A>T in exon 102), were not
associated with HCM, because Ile8474Thr was found in the controls and Asp8672Val
did not co-segregate with the disease in a multiplex family. On the other hand, 2
missense mutations, Arg8500His (c.25723G>A in exon 99) and Arg8604Gln
(c.26035G>A in exon 100), identified in familial HCM patients, were not found in the
controls.
Clinical phenotypes
Clinical findings of the patients carrying the ANKRD1 or TTN mutations are summarized
in Table 1. All patients manifested with HCM except CM1288 II-2 who had mild cardiac
hypertrophy. Her father had died suddenly of unknown etiology at the age of 30. Two
unaffected brothers of the patient did not harbor the mutation (Figure 1C). The proband
patient with the TTN Arg8606Gln mutation (CM1480, Table 1) showed asymmetric
septum hypertrophy. A family study revealed that his father had unexplained sudden
cardiac death. His son (CM1481, Table 1) was affected and carried the same mutation
(Figure 1D).
57
58
Figure 1: Mutational analyses of ANKRD1 and TTN in HCM. (A) Sequence variations found in ANKRD1. Single letter code was used to indicate the amino acid residue. Solid boxes represent protein coding region corresponding to exons 1-9. Dotted boxes indicate ankyrin repeat domains encoded by exons 5-8. (B) Sequence variations found in TTN. Solid boxes represent Ig domains corresponding to exons 98, 99 and 102-104. Dotted boxes indicate tyrosine- rich motif encoded by exons 99-101. (C and D) Pedigrees of HCM families with the ANKRD1 T123M (C, CM 1288 family) and the TTN R8604Q (D, CM 1480 family). Filled square and filled circle indicate affected male and female, respectively. Open square and open circle represent unaffected or unexamined male and female with HCM, respectively. An arrow indicates the proband patient. Presence (+) or absence (-) of the mutations is noted.
Tabl
e 1:
Clin
ical
cha
ract
eris
tics
of in
divi
dual
s ca
rryin
g A
NK
RD
1 or
TT
N m
utat
ions
IDM
utat
ion
Age
,ge
nder
Age at
onse
tC
linic
alD
xA
ge a
t cl
inic
alex
amFH
of
HC
MNY
HALV
Dd
(mm
) LV
Ds
(mm
) IV
S(m
m)
PW (mm
)%
FS%
EFO
ther
rem
arks
May
o I
AN
KR
D1
P52A
44
,
mal
e 30
H
CM
32
N
o II
- -
22
- -
70
LVH
on
ECG
; pro
voca
ble
grad
ient
100
mm
HG
, but
as
ympt
omat
ic
May
o II
AN
KR
D1
P52A
65
,
mal
e 41
H
CM
54
N
o III
38
16
14
14
-
84
Mid
-ven
tricu
lar-
apic
al
hype
rtrop
hy w
ith m
idve
ntric
ular
w
all t
hick
ness
up
to 3
5mm
CM
1288
II-
2 A
NK
RD
1 T1
23M
62
, fe
mal
e 40
H
CM
40
N
o I
41
22
13
13
46
78
Late
ral L
V h
yper
troph
y (1
5mm
), LA
D=3
7 m
m, E
CG
; abn
orm
al
Q-w
ave
in II
, III
,aV
f, V
4-6
May
o III
A
NK
RD
1 I2
80V
82
, fe
mal
e 61
H
CM
73
N
o III
52
30
20
14
-
70
Sep
tal a
blat
ion
(relie
ved
obst
ruct
ion
73m
mH
g ->
22
mm
Hg)
CM
89
TT
N
R85
00H
59
,
mal
e 53
H
CM
59
N
o I
42
25
28
8 40
79
LV
H (A
SH)
CM
1480
II-
4 T
TN
R
8604
Q
52,
m
ale
43
HC
M
43
Yes
I 41
24
18
10
41
80
LV
H (A
SH
), A
trial
fibr
illatio
n E
CG
; Inv
erte
d T-
wav
e in
V4-
V6
CM
1481
III
-1
TT
N
R86
04Q
25
,
mal
e 16
H
CM
16
Ye
s I
45
27
22
9 40
66
LV
H (A
SH
),
EC
G; I
nver
ted
T-w
ave
in V
1-V
3
Dx,
dia
gnos
is; E
F, e
ject
ion
fract
ion;
FH
, fam
ily h
isto
ry; L
AD
, lef
t atri
al d
imen
sion
; LVH
, lef
t ven
tricu
lar
hype
rtrop
hy; L
VDd,
left
vent
ricul
ar d
imen
sion
dia
stol
e,
LVD
s, le
ft ve
ntric
ular
dim
ensi
on s
ysto
le, I
VS
, int
rave
ntric
ular
sep
tum
; FS
, fra
ctio
nal s
horte
ning
; NY
HA
, New
Yor
k H
eart
Ass
ciat
ion
Altered interaction between titin/connectin and CARP caused by the TTN
or CARP mutations
To investigate the functional alterations caused by the CARP mutations in the binding to
titin/connectin N2A domain, WT-, Pro52Ala-, Thr123Met-, or Ile280Val-CARP construct
was co-transfected with the WT TTN-N2A construct into COS-7 cells. Western blot
analyses of immunoprecipitates from the transfected cells demonstrated that
HCM-associated CARP mutations significantly increased binding to TTN-N2A
(2.22±0.76 AU, p<0.05, 1.98±0.52 AU, p<0.01 or 2.16±0.64 AU, p<0.05, respectively)
(Figure 2A and B). Reciprocally, the effect of titin/connectin mutations in binding to
CARP was assessed. TTN-N2A constructs, WT-, HCM-associated mutants
(Arg8500His- and Arg8604Gln-TTN), or non-disease-related variant (Ile8474Thr)
TTN-N2A were co-transfected with WT CARP. Western blot analyses showed that
Arg8500His and Arg8604Gln significantly increased the binding to CARP (2.78±0.40 or
3.16±0.40 AU, respectively, p<0.001 in each case) (Figure 2A and B), while the
non-disease related variant (Ile8474Thr) did not alter the binding (1.18±0.11 AU),
despite equal expression of proteins.
Altered interaction between myopalladin and CARP caused by the CARP
mutations
Because CARP bound also to myopalladin, we investigated the effects of CARP
mutations in binding to myopalladin. WT or mutant CARP construct was co-transfected
with a MYPN construct. Western blot analysis revealed that binding of mutant CARPs,
Pro52Ala, Thr123Met or Ile280Val, to myopalladin was significantly increased
(3.60+/-0.67 AU, p<0.001, 1.87+/-0.47 AU, p<0.01 or 2.48+/-0.45 AU, p<0.001,
respectively) (Fig. 2C and D).
60
Figure 2: Binding of CARP to titin/connectin and myopalladin. Binding of CARP to titin/connectin (TTN) or myopalladin (MYPN) was analyzed by co-IP assays. (A) Myc-tagged CARPs co-precipitated with GFP-tagged TTN-N2A domain were shown (top panel). Expressions of GFP-tagged TTN- N2A (middle panel) and myc-tagged CARP (lower panel) were confirmed by immunobloting of whole cell supernatants. Binding pairs were WT CARP in combination with WT, I8474T, R8500H or R8604Q mutant TTN-N2A , or WT TTN-N2A with WT, P52A, T123M or I280V mutant CARP. Dashes indicate no GFP- or myc-tagged proteins (transfected only with pEGFP-C1 or pCMV-Tag3 vectors, respectively). (B) Densitometric data obtained in the co-IP assay. Data for WT CARP with WT TTN-N2A were arbitrarily defined as 1.00 arbitrary unit (AU). Data are represented as mean ± SEM. (n= 6 for each case). *** p<0.001 vs WT; ** p<0.01 vs WT; * p<0.05 vs WT.
61
Figure 2 cont’d. (C): Myc-tagged CARP co-precipitated with GFP-tagged full-length MYPN was detected by immunobloting using anti-myc antibody (top panel). Expressed amounts of GFP-tagged MYPN (middle panel) and myc-tagged CARP (lower panel) were confirmed as in(A). Binding pairs were full-length WT-MYPN with WT, P52A, T123M or I280V mutant CARP. (D) Densitometric analysis of myc-blotting data in (C). Data were arbitrarily represented as intensities and that for WT CARP with full length or N-terminal half WT MYPN was defined as 1.00 AU. Data are expressed as mean± SEM. (n = 9 for each case). *** p<0.001 vs WT; ** p<0.01 vs WT.
62
Altered localization of CARP caused by the mutations
To further investigate the functional consequence of the CARP mutations, we examined
cellular distribution of the mutant CARP proteins expressed in neonatal rat primary
cardiomyocytes. Cells were transfected with myc-tagged WT or mutant CARP
constructs, co-immunostained for myc (a marker for CARP) and -actinin (a marker for
Z-disc). WT and mutant myc-CARP proteins were expressed at a similar level in the
transfected cells as assessed by Western-blot analyses, suggesting that the mutations
did not affect the expression level and stability of CARP proteins (data not shown).
Control cells expressing myc-tag alone showed negative staining for myc-tag with
striated staining pattern of sarcomeric -actinin at the Z-disc (data not shown). In
premature cardiomyocytes containing Z-bodies (Z-disc precursors), myc-tagged WT
CARP was mainly targeted to nucleus and colocalization of CARP with -actinin, which
formed patchy dense bodies in the cytoplasm, was observed (Figure 3A-C). No
apparent changes in localization of mutant CARP proteins were observed in the
nascent and immature cardiomyocytes (Figure 3D-F, G-I and J-L).
In the mature cardiomyocytes where Z-discs were well organized, myc-tagged
WT CARP was assembled in the striated pattern at the Z-I bands and co-localized with
-actinin (Figure 4A-C). It was found that most ( 90%) of mature cardiomyocytes did
not contain nuclear CARP (Figure 4A-C). On the other hand, higher intensity of
CARP-related fluorescence at the Z-I bands and diffused localization in the cytoplasm
was observed in the most ( 80%) of mature cardiomyocytes expressing myc-tagged
mutant CARPs, albeit that the Z-disc assembly was not impaired (Figure 4D-F, G-I and J-L). Quite interestingly, myc-tagged mutant CARP proteins displayed localization
within the nuclear and/or at nuclear membrane in 60% of mature cardiomyocytes
(Figure 4D-F, G-I and J-L).
63
Discussion
CARP encoded by ANKRD1 is a nuclear transcription co-factor expressing in the
embryonic hearts. Its expression progressively decreases in adult hearts [3, 4] and
reappears in the hypertrophied or failing adult heart [5, 15], suggesting that CARP may
be involved in the regulation of muscle gene expression. CARP also localizes in cardiac
sarcomere although the roles of “sarcomeric CARP” are not fully elucidated. Several
reports have demonstrated that CARP binds titin/connectin [9] , myopalladin [8] and
desmin [16] at the Z/I-region of sarcomere. In this study, we found that the
HCM-associated ANKRD1 mutations increased the binding of CARP to titin/connectin
and myopalladin, and HCM-associated TTN mutations in its reciprocal CARP
N2A-binding domain increased the binding of titin/connectin to CARP. These
observations in association with HCM suggested that the assembly or binding of
sarcomeric CARP with titin/connectin and/or myopalladin would be required for the
maintenance of cardiac function.
In the nascent myofibrils, myc-tagged CARP proteins were detected within the
nucleus irrespective of mutations. Because CARP is an early differentiation marker
during heart development, recruitment of CARP into nuclei may be important in the
embryonic gene expression. Interestingly, abnormal intra-nuclear accumulation of
myc-tagged mutant CARP proteins was observed in mature myofibrils. It is well known
that the embryonic and fetal gene program of cardiac cytoskeletal proteins is initiated
during the cardiac remodeling [17, 18]. Hence, one could hypothesize that nuclear
CARP may cause embryonic/fetal gene expression in mature myofibrils and this
abnormal gene expression is a possible mechanism leading to the pathogenesis of
HCM. It was reported that CARP negatively regulated expression of cardiac genes
including MYL2, TNNC1 and ANP [3, 4].
64
Conversely, another report suggested that different expression level of CARP
did not correlate with the altered expression of cardiac genes such as MYL2, MYH7,
ACTC, CACTN, TPM1, ACTN2 and DES [19]. Thus, the role of CARP as a regulator of
cardiac gene expression remains to be resolved. During the preparation of this paper,
Cinquetti et al. [20] reported other CARP mutations, rearrangements or Thr116Met, in
association with the cyanotic congenital heart anomaly known as total anomalous
pulmonary venous return (TAPVR). These mutations were demonstrated to be
associated with increased expression or stability of CARP. It is not clear whether the
mutations associated with HCM altered expression or stability of CARP, though our data
suggested that HCM-associated CARP mutations did not alter the stability. The
molecular mechanisms underlying the CARP-related pathogenesis should be different
between TAPVR and HCM.
65
Figure 3: Distribution myc-tagged CARP in immature rat cardiomyocytes. Neonatal rat cardiomyocytes transfected with myc-tagged WT (A-C) or mutant (P52A, T123M or I280V) (D-F, G-I or J-L, respectively) CARP constructs were fixed 18 h after the transfection, and stained with DAPI and anti- -actinin antibody followed by secondary antibody (B, E, H, and K). Merged images were shown in C, F, I, and L. In the immature cardiomyocytes showing nascent myofibrils with Z bodies (Z-disc precursors), myc-tagged CARPs were preferentially localized to the nucleus and mutant CARP showed relatively low expression in the cytoplasm. Scale bars=10 m.
66
Figure 4: Distribution of myc-tagged CARP in mature rat cardiomyocytes. Neonatal rat cardiomyocytes transfected with myc-tagged WT (A-C) or mutant (P52A, T123M or I280V) (D-F, G-I or J-L, respectively) CARP constructs were fixed 48 h after the transfection, and stained with DAPI and anti- -actinin antibody followed by secondary antibody (B, E, H, and K). Merged images were shown in C, F, I, and L. In the mature cardiomyocytes showing myofibrils with Z-discs, normal localization of myc-tagged WT CARP at the Z-discs was observed (A-C). In contrast, myc-tagged mutant CARP proteins showed intense localization at the I-discs (colocalization with -actinin) and diffused localization in the cytoplasm (D-F, G-I and J-L). In addition, myc-tagged mutant CARPs expressed at high levels around the nuclear membrane (white arrow) and/or in the nucleus (white arrowhead).
67
Conclusions
We identified 3 missense CARP mutations in < 1% of unrelated patients with HCM,
which not only increased the binding of sarcomeric CARP to I-band components but
also resulted in the mis-localization of CARP to the nucleus. Although the molecular
mechanisms of HCM due to the CARP mutations remain to be elucidated, our findings
imply that HCM may be associated with the abnormal recruitment of CARP in
cardiomyocytes leading to pathological hypertrophy.
Acknowledgements
We thank Drs. H. Toshima, C. Kawai, K. Kawamura, M. Nagano, T. Sugimoto, S.
Ogawa, A. Matsumori, S. Sasayama, R. Nagai, and Y. Yazaki for their contributions in
clinical evaluation and blood sampling from patients with cardiomyopathy, and Ms. M.
Yanokura, M. Emura and A. Nishimura for their technical assistance.
68
References
1. Richardson P, McKenna W, Bristow M, Maisch B, et al. Report of the 1995 World Health Organization/International Society and Federation of Cardiology task force on the definition and classification of cardiomyopathies. Circulation 1996; 93(5): 841-842. 2. Bos JM, Ommen SR, Ackerman MJ. Genetics of hypertrophic cardiomyopathy: One, two, or more diseases? Curr Opin Cardiol 2007; 22(3): 193-199. 3. Jeyaseelan R, Poizat C, Baker RK, Abdishoo S, et al. A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes. J Biol Chem 1997; 272(36): 22800-22808. 4. Zou Y, Evans S, Chen J, Kuo HC, et al. CARP, a cardiac ankyrin repeat protein, is downstream in the nkx2-5 homeobox gene pathway. Development 1997; 124(4): 793-804. 5. Zolk O, Frohme M, Maurer A, Kluxen FW, et al. Cardiac ankyrin repeat protein, a negative regulator of cardiac gene expression, is augmented in human heart failure. Biochem Biophys Res Commun 2002; 293(5): 1377-1382. 6. Ihara Y, Suzuki YJ, Kitta K, Jones LR, et al. Modulation of gene expression in transgenic mouse hearts overexpressing calsequestrin. Cell Calcium 2002; 32(1): 21-29. 7. Baudet S. Another activity for the cardiac biologist: CARP fishing. Cardiovasc Res 2003; 59(3): 529-531. 8. Bang ML, Mudry RE, McElhinny AS, Trombitas K, et al. Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies. JCell Biol 2001; 153(2): 413-427. 9. Miller MK, Bang ML, Witt CC, Labeit D, et al. The muscle ankyrin repeat proteins: CARP, Ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules. JMol Biol 2003; 333(5): 951-964. 10. Granzier HL, Labeit S. The giant protein titin: A major player in myocardial mechanics, signaling, and disease. Circ Res 2004; 94(3): 284-295. 11. LeWinter MM, Wu Y, Labeit S, Granzier H. Cardiac Titin: Structure, functions and role in disease. Clin Chim Acta 2007; 375(1-2): 1-9. 12. Itoh-Satoh M, Hayashi T, Nishi H, Koga Y, et al. Titin mutations as the molecular basis for dilated cardiomyopathy. Biochem Biophys Res Commun 2002; 291(2): 385-393. 13. Gerull B, Gramlich M, Atherton J, McNabb M, et al. Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy. Nat Genet 2002; 30(2): 201-204. 14. Arimura T, Matsumoto Y, Okazaki O, Hayashi T, et al. Structural analysis of Obscurin gene in hypertrophic cardiomyopathy. Biochem Biophys Res Commun 2007; 362(2): 281-287. 15. Aihara Y, Kurabayashi M, Saito Y, Ohyama Y, et al. Cardiac ankyrin repeat protein is a novel marker of cardiac hypertrophy: Role of m-cat element within the promoter. Hypertension 2000; 36(1): 48-53. 16. Witt SH, Labeit D, Granzier H, Labeit S, et al. Dimerization of the cardiac ankyrin protein CARP: Implications for MARP titin-based signaling. J Muscle Res Cell Motil 2006; 26(6-8): 401-408.
69
17. Swynghedauw B. Molecular mechanisms of myocardial remodeling. Physiol Rev 1999; 79(1): 215-262. 18. Swynghedauw B, Baillard C. Biology of hypertensive cardiopathy. Curr Opin Cardiol 2000; 15(4): 247-253. 19. Torrado M, Lopez E, Centeno A, Castro-Beiras A, et al. Left-right asymmetric ventricular expression of CARP in the piglet heart: Regional response to experimental heart failure. Eur J Heart Fail 2004; 6(2): 161-172. 20. Cinquetti R, Badi I, Campione M, Bortoletto E, et al. Transcriptional deregulation and a missense mutation define ANKRD1 as a candidate gene for total anomalous pulmonary venous return. Hum Mutat 2008; 29(4): 468-474.
70
Chapter 4
Echocardiographic-Determined Septal Morphology in Z-Disc Hypertrophic Cardiomyopathy
Jeanne L. Theis*, J. Martijn Bos *, Virginia B. Bartleson, Melissa L. Will, Josepha Binder, Matteo Vatta, Jeffrey A. Towbin, Bernard J. Gersh, Steve R. Ommen, Michael J. Ackerman * These authors contributed equally to this study
Biochem Biophys Res Commun 2006; 351(4): 896 – 902
Abstract Hypertrophic cardiomyopathy (HCM) can be classified into at least 4 major anatomic
subsets based upon the septal contour, and the location and extent of hypertrophy:
reverse curvature-, sigmoidal-, apical-, and neutral contour-HCM. Here, we sought to
identify genetic determinants for sigmoidal-HCM and hypothesized that Z-disc HCM
may be associated preferentially with a sigmoidal phenotype. Utilizing PCR, DHPLC,
and direct DNA sequencing, we performed mutational analysis of five genes encoding
cardiomyopathy associated Z-disc proteins. The study cohort consisted of 239
unrelated patients with HCM previously determined to be negative for mutations in the
8 genes associated with myofilament-HCM. Blinded to the Z-disc genotype status, the
septal contour was graded qualitatively using standard transthoracic
echocardiography. Thirteen of the 239 patients (5.4%) had one of 13 distinct HCM-
associated Z-disc mutations involving residues highly conserved across species and
absent in 600 reference alleles: LDB3 (6), ACTN2 (3), TCAP (1), CSRP3 (1) and VCL
(2). For this subset with Z-disc-associated HCM, the septal contour was sigmoidal in
11 (85%) and apical in 2 (15%). While Z-disc-HCM is uncommon, it is equal in
prevalence to thin filament-HCM. In contrast to myofilament HCM, Z-disc HCM is
associated preferentially with sigmoidal morphology.
Keywords Hypertrophy, cardiomyopathy, septum, echocardiography, genes, Z-disc
72
Introduction Affecting 1 in 500 persons, hypertrophic cardiomyopathy (HCM) is the most common
identifiable cause of sudden death in young athletes and is the most common heritable
cardiovascular disease[1]. Characterized by unexplained myocardial hypertrophy in the
absence of precipitating factors such as hypertension or aortic stenosis, HCM is
underscored by profound genetic and phenotypic heterogeneity. Since the sentinel
discovery of mutations involving the MYH7-encoded -myosin heavy chain as the
pathogenetic basis for HCM in 1990[2], more than 300 mutations scattered among at
least 12 HCM-susceptibility genes encoding sarcomeric proteins have been identified.
The first link to be drawn between septal morphologies was a result of a pre-
genomics era HCM study by Lever and colleagues where septal contour was found to
be age-dependent with a predominance of the sigmoid septum with normal curvature
being present in the elderly[3]. This was followed up by an early genotype-phenotype
observation by Seidman and colleagues involving a small number of patients and
ultimately revealed that patients with mutations in the beta myosin heavy chain (MYH7-
HCM) generally had reversed curvature septal contours[4]. Most recently, we
discovered that myofilament-HCM may have a predilection for a reverse curvature
septal phenotype regardless of age[5]. After analyzing all echocardiograms of 382
previously genotyped and published patients[6, 7, 8], multivariate analysis
demonstrated that reverse septal curvature was the only, independent predictor of
myofilament HCM with an odds ratio of 21[5]. Moreover, the yield of the commercially
available HCM genetic test (panel A and panel B) which examines 8 genes responsible
for myofilament-HCM was 79% in reverse curve-HCM but only 8% in sigmoidal-HCM.
These observations provide the rationale for seeking novel genetic
determinants that confer susceptibility for sigmoidal-HCM. Recent attention has been
focused on proteins outside the cardiac myofilament, involved in the cyto-architecture
and cardiac stretch sensor mechanism of the cardiomyocyte. Mutations in three such
proteins localized to the cardiac Z-disc, CSRP3-encoded muscle LIM protein (MLP),
TCAP-encoded telethonin and VCL-encoded vinculin, including its cardiac specific
insert of exon 19 that yields metavinculin, have previously been established as both
HCM[9, 10, 11, 12, 13] and dilated cardiomyopathy (DCM)-susceptibility genes[9, 10,
11, 12, 14, 15].
73
Additionally, it has been recognized that these divergent cardiomyopathic
phenotypes of HCM and DCM are partially allelic disorders with ACTC, MYH7, TNNT2,
TPM1, MYBPC3, TTN, MLP, TCAP, and VCL established as both HCM- and DCM-
susceptibility genes[10, 11, 12, 14, 16, 17, 18, 19, 20].
These observations prompted us to consider perturbations in the cardiac Z-disc
as another pathway for hypertrophic or dilated cardiomyopathy. Besides the three
aforementioned Z-disc proteins implicated in HCM, we considered two additional
genes: ACTN2-encoded alpha-actinin 2 and LDB3-encoded LIM domain binding 3
(official HUGO nomenclature; also known as ZASP-encoded Z-band associated
alternatively spliced PDZ-motif protein), as candidates for HCM. Both genes have been
implicated in the pathogenesis of DCM and encode proteins that are key binding
partners of the previously mentioned HCM-associated, Z-disc proteins[14, 21]. The
published Q9R-ACTN2 missense mutation inhibited cellular function and was
associated with extra-nuclear localization in cultured cells with co-immunoprecipitation
studies showing its failure to bind to MLP[14]. LDB3-associated animal models reveal
that null mice completely devoid of this protein lose their ability to maintain structural
integrity of the Z-disc, leading to impaired contraction and perinatal death[22].
Because of the specific structure-function relationship of the proteins in the
cardiac Z-disc[23, 24] and the specific cardiomyocyte stretch response mechanism of
these proteins[25], we hypothesized that Z-disc HCM might be preferentially sigmoidal.
We speculate that in the presence of Z-disc mutations, the compensatory hypertrophic
response may be greatest in areas of highest stress (i.e. LVOT), thereby resulting in
the basal septal bulge and sigmoidal shaped contour.
74
Methods
Between April 1997 and December 2001, a total of 382 unrelated patients (210 male,
mean maximum left ventricular wall thickness (MLVWT) 21.5 ± 6mm) had both
comprehensive echocardiographic examination and evaluation in Mayo Clinic’s HCM
clinic and genetic testing. HCM was diagnosed according to WHO criteria as
unexplained cardiac hypertrophy (>13 mm) in the absence of hypertrophy inciting
factors such as aortic stenosis. Following written informed consent for this IRB-
approved research protocol, DNA was extracted from the blood samples using
Purgene DNA extraction kits (Gentra, Inc., Minneapolis, Minnesota). After a
comprehensive analysis of the eight most common myofilament HCM-associated
genes, 239 patients (131 male, mean MLVWT 20.7 ± 6mm) remained without a
pathogenetic explanation and are in this study referred to as “myofilament genotype
negative” [6, 7, 8, 26].
This subset was analyzed for mutations in all translated exons of all published,
cardiomyopathy-associated Z-disc genes: CSRP3-encoded muscle LIM protein (MLP),
TCAP-encoded telethonin (TCAP), VCL-encoded vinculin, ACTN2-encoded alpha-
actinin 2 (ACTN2) and LDB3-encoded LIM domain binding protein 3 (LDB3), using
polymerase chain reaction (PCR), denaturing high performance liquid chromatography
(DHPLC) (Transgenomic, Omaha NE) and direct DNA sequencing(ABI Prism 377;
Applied Biosystem, Foster City, California). Primer sequences and DHPLC-methods
are available upon request. To exclude common non-synonymous polymorphisms, we
examined 600 ethnically matched reference alleles.
Echocardiography
Septal curvature and cavity contour were evaluated in the long axis view at end-
diastole. Sigmoid septal morphology was defined as a generally ovoid left ventricular
(LV) cavity with the septum being concave toward the LV with a pronounced basal
septal bulge. Reverse curve septal morphology was defined as a predominant mid-
septal convexity toward the left ventricular cavity with the cavity itself having an overall
crescent shape. Apical variant HCM was defined as a predominant apical distribution
of hypertrophy. Neutral septal contour was defined by an overall straight or variable
convexity that was neither predominantly convex nor concave toward the LV cavity.
Septal contours were assessed by two independent reviewers (JB and SRO) and
genotypic data was kept in a database blinded to all clinical and echocardiographic
data.
75
Results
The demographics of the myofilament genotype negative cohort are shown in Table 1.
As indicated in the study design exclusion criteria, no mutations in the eight genes
underlying myofilament-HCM (beta myosin heavy chain, myosin binding protein C,
etc.) were present in this cohort. This cohort consisted of 239 patients (131 male) with
an average age at diagnosis of 45.1 years old and a mean MLVWT of 20.7 mm. Fifty-
six percent of patients presented with cardiac symptoms, 24% had a family history of
HCM in a first degree relative and 16% had a family history of sudden cardiac death.
Forty percent of patients underwent surgical myectomy because of refractory
symptoms. In comparison, this subset of patients with myofilament genotype negative-
HCM are older, have less hypertrophy, and are less likely to have a reverse curvature
shaped septum compared to the 143 patients with myofilament-HCM (Table 1) [5, 6, 7,
8, 26].
Table 1: Demographics of Myofilament Genotype Negative HCM Cohort
Myofilament Negative(N = 239)
Myofilament Positive(N = 143)
p-value
Male/female 131/108 79/64 NS
Age at diagnosis (years) 45.1 ± 19 35.7 ± 17 < 0.001
MLVWT (mm) 20.7 ± 6 22.8 ± 7 0.002
Mean peak LVOT gradient (mmHG) 48.3 ± 42 45.6 ± 42 NS
Sigmoidal-shaped septal contour 166 (69%) 15 (10%) <0.0001
Presenting w/ cardiac symptoms (%) 55.7% 55.8% NS
Positive family history of HCM* 24% 47% <0.001
Positive family history of SCD* 16% 25% NS
Surgical myectomy 95 (40%) 64 (45%) NS
Pacemaker 40 (17%) 28 (19%) NS
ICD 23 (10%) 37 (25%) <0.001 HCM, hypertrophic cardiomyopathy; LVOT, left ventricular outflow tract; MLVWT, maximum left ventricular wall thickness; SCD, sudden cardiac death; ICD, implantable cardioverter-defibrillator * In first degree relative
76
After analysis of all translated exons of LDB3, CSRP3, TCAP, ACTN2 and VCL, 14
mutations in 13 patients (5%) were discovered. Most mutations were missense
mutations conserved over species and absent in 600 ethnically matched reference
alleles. S196L-LDB3 was identified in two patients. Mutations and their location in the
topology of their respective protein are shown in Figure 1. One patient with the Y468S-
LDB3 missense mutation also harbored a second Z-disc mutation, a frame-shift
mutation (K42 fs/165) in the CSRP3-encoded muscle LIM protein.
Figure 1: Schematic topologies of analyzed genes and the mutations found. The legend behind the gene name directs to the binding domain shown in its partner-protein. For vinculin, the cardiac specific insert that yields metavinculin (exon 19) is shown.
77
78
The clinical phenotype of the 13 patients is shown in Table 2. Overall, the average age
at diagnosis was 42.9 ± 18 years with seven of the twelve patients being male. The
MLVWT is 20.9 ± 9 mm and the LVOT gradient averaged 56.8 ± 49 mmHg. Eight
patients (case 1-3, 5, 6, 11-13) underwent surgical septal myectomy because of
refractory symptoms. Pathological reports of the surgical specimens show at least two
of the three characteristics (cardiomyocyte hypertrophy, endocardial fibrosis and
myofibrillar disarray) of HCM in all cases; half of the specimens showed myofibrillar
disarray.
Ta
ble
2: C
linic
al p
heno
type
of p
atie
nts
with
Z-d
isc
HC
M
Cas
e G
ene
Mut
atio
n Se
xA
geat
Dx
(yrs
) M
LVW
T(m
m)
LVO
T(m
mH
g)
Sept
alsh
ape
Fam
Hx
of H
CM
Fa
m H
x of
SC
D
Trea
tmen
t Pa
thol
ogy
repo
rt
1A
CT
N2
G11
1V
M
31.4
20
10
0 S
igm
oid
No
No
Mye
ctom
y M
arke
d m
yocy
te h
yper
troph
y,
foca
l m
yocy
te d
isar
ray,
en
doca
rdia
l fib
rosi
s
2A
CT
N2
T495
M
M
32.5
16
0
Sig
moi
d N
o N
o M
yect
omy
Mar
ked
endo
card
ial f
ibro
sis,
m
yocy
te h
yper
troph
y, i
nter
stiti
al
fibro
sis
3A
CT
N2
R75
9T
M
17.9
16
12
0 S
igm
oid
No
No
Mye
ctom
y N
o re
port
4C
SR
P3
Q91
L M
44
.5
22
18
Sig
moi
d N
o N
o P
acem
aker
N
A
5T
CA
PR
70W
F
44.2
46
19
S
igm
oid
Yes
N
o M
yect
omy,
P
acem
aker
S
ever
e m
yocy
te h
yper
troph
y,
mod
erat
e in
ters
titia
l fib
rosi
s
6LD
B3
S19
6L
F 73
.0
19
64
Sig
moi
d N
o N
o M
yect
omy
Mar
ked
myo
cyte
hyp
ertro
phy,
m
oder
ate
endo
card
ial f
ibro
sis,
fo
cal m
yocy
te d
issa
ray
7LD
B3
S19
6L
F 63
.8
13
0 A
pica
l N
o N
o R
x N
A
8LD
B3
D36
6N
M
68.5
18
16
S
igm
oid
No
No
Rx
NA
9LD
B3
CS
RP
3Y
468S
, K
42 fs
/165
M
46
.8
18
112
Sig
moi
d N
o N
o R
x N
A
10LD
B3
Q51
9P
F 21
.2
15
55
Sig
moi
d Y
es
No
Rx
NA
11LD
B3
P61
5L
M
28.3
27
12
0 S
igm
oid
No
No
Mye
ctom
y M
oder
ate
myo
cyte
, mild
to
mod
erat
e fo
cal e
ndoc
ardi
al fi
bros
is
12V
CL
L277
M
F 76
20
0
Sig
moi
d N
o N
o M
yect
omy
Myo
cyte
hyp
ertro
phy,
ca
rdio
myo
cyte
dis
arra
y, in
ters
titia
l fib
rosi
s
13V
CL
R97
5W
F 42
.8
22
0 A
pica
l N
o N
o M
yect
omy
Mar
ked
myo
cyte
hyp
ertro
phy,
mild
in
ters
titia
l fib
rosi
s, fo
cal m
yofib
rilla
r di
ssar
ray
In contrast to the patients with myofilament-HCM from our previous study, none of the
patients with Z-disc HCM exhibited reverse septal curvature echocardiographically
(104/143 vs. 0/13, p-value < 0.0001, Figure 2). Instead, 11 of the 13 patients (85%)
had a sigmoidal shaped septum and the other 2 patients had apical-HCM (case 7 and 13). For the entire original cohort of 382 unrelated patients, a putative pathogenic
explanation for sigmoidal-HCM has increased from 8% (myofilament genotype
positive) to now 14% with inclusion of Z-disc mediated disease (Figure 2). The
majority of sigmoidal-HCM remains genotypically unexplained.
Figure 2: Overview of the genotype-phenotype relationships between the two most common septal morphologies (bottom) and the presence of mutations in the cardiac Z-disc (top-middle) or the myofilament (top-sides). Arrows pointing towards the morphologies, represent the frequency of that morphology for a particular genotype. Arrows pointing towards the myofilament or Z-disc represent the number of mutations present when showing a particular septal morphology.
80
Discussion
Due to the hundreds of mutations scattered throughout the genes which encode
proteins of the myofilament, HCM has long been considered a disease of the
sarcomere, more specifically, a disease of the myofilament. With the recent discovery
of HCM associated mutations in genes encoding for proteins of the Z-disc[9, 10, 11,
12, 14] and the distinction whereby HCM associated mutations in PRKAG2 and
LAMP2 have categorized certain cases of glycogen storage disease[27, 28], the
spectrum of genetically-mediated disease pathways continues to expand.
Although specific mutations in particular genes may be rare, the question arises
as to whether there may be significant genotype-phenotype correlations associated
with distinct HCM-yielding pathways such as myofilament-, Z-disc, or metabolic-HCM.
To this end, we further explored our recent discovery that linked reverse curvature
HCM with mutations in genes encoding proteins of the myofilament (i.e. myofilament-
HCM) [5]. Here, we demonstrated that reverse septal curvature was the strongest,
independent predictor of the presence of a myofilament mutation (OR 21, p<0.001)
over age and MLVWT[5].
The cardiac Z-disc as a novel target in the pathogenesis of HCM
Focusing on the myofilament negative subgroup, we extended our investigation to
encompass five cardiomyopathy-susceptibility genes that encode important and
interacting proteins that are key constituents of the cardiac Z-disc architecture. The Z-
disc is an intricate assembly of proteins at the Z-line of the cardiomyocyte sarcomere.
Extensively reviewed, proteins of the Z-disc are important in the structural and
mechanical stability of the sarcomere as they appear to serve as a docking station for
transcription factors, Ca2+-signaling proteins, kinases and phosphatases[23, 24]. In
addition, this assembly of proteins seems to serve as a way station for proteins that
regulate transcription by aiding in their controlled translocation between the nucleus
and the Z-disc[23, 24].
81
With all of these roles, a main implication for the Z-disc is its involvement in the
cardiomyocyte stretch sensing and response systems [25]. While this is a critical task
which is an integral component of Z-disc function in the long term, there is the potential
that the Z-disc may transduce multiple signaling pathways during stress, translating
into hypertrophic responses, cell growth and remodeling [29]. Based on this potentially
important structure-function relationship and its role in the cardiomyocyte stretch
response system, we hypothesized that perturbations in the cardiac Z-disc may confer
susceptibility for the development of sigmoidal-HCM.
Z-disc-HCM is preferentially sigmoidal
Indeed, after extensive analysis of the genes encoding these 5 key Z-disc proteins, we
observed a very strong predilection for sigmoidal disease in the presence of a rare
mutation that disrupts a Z-disc protein. In fact, in contrast to the 79% likelihood for
myofilament-HCM in the setting of reverse curvature-HCM, none of the patients within
this subgroup of Z-disc-HCM displayed reverse septal curvature. Although the vast
majority of sigmoidal HCM in our cohort still is genetically unexplained, the yield of
genetic testing for sigmoidal curvature has nearly doubled by extending the genetic
testing from the 8 myofilament-HCM genes that are tested for commercially to include
these 5 genes associated with Z-disc-HCM. We speculate that Z-disc HCM leads to a
hypertrophic response that is expressed in the areas of highest stress (i.e. LVOT) and
therefore predisposes to a sigmoidal septal contour.
These observations generate several intriguing questions regarding HCM in
association with a sigmoidal septal contour. Whereas in previous morphologic studies,
Lever and colleagues associated sigmoidal-HCM with older age [3], the underlying
genotype rather than age appears to be the predominant determinant of septal
morphology [5]. Given that the vast majority of our patients with sigmoidal HCM still
lack a putative disease-causing mutation, it remains to be determined whether such
patients possess, in fact, congenital HCM (i.e. a primary HCM-predisposing genetic
mutation). It can be speculated that, especially in the sigmoidal septal subgroup, the
sum of all contributors – the presence or absence of a mutation or LVH promoting
polymorphisms [30], an unidentified genetic substrate, environmental factors and
hypertension – culminates in what is clinically labeled as HCM.
82
This multi-factorial model for sigmoidal HCM is supported by the significantly
older age at diagnosis of patients with sigmoidal HCM (49 years) compared to those
with reverse curvature-HCM (32 years)[5]. Furthermore, nearly 20% of patients
classified with sigmoidal-HCM were noted to have mild hypertension [5]. Although
diagnosed with HCM and presently showing co-existent hypertension, a subset of this
group may have a basal septum more sensitive to the pro-hypertrophy trigger of
increased afterload, precipitating basal septal hypertrophy (sigmoidal disease), but
nonetheless culminating in a clinical diagnosis of HCM. In this scenario, a Mendelian
genetic mechanism will not be found.
On the other hand, this novel genotype-phenotype association characterized by
predilection for sigmoidal, basal septal hypertrophy in the setting of perturbations in the
cardiac Z-disc raises the possibility that other constituents of the Z-disc (> 20 proteins)
may host additional HCM-susceptibility mutations in general and sigmoidal-HCM
susceptibility mutations in particular. For example, as one of the central proteins of the
Z-disc, ACTN2 binds to a large number of proteins, including ALP-encoded actinin-
associated LIM-protein [31], CapZ-encoded actin capping protein [32] or S100, of
which the S100B-isoform seems to function as an inhibitor of the hypertrophic
response [33]. ALP, CAPZ and S100 may represent the next tier of HCM candidate
genes to further test our hypothesis that sigmoidal septal shaped HCM is associated
with perturbations in the cardiac Z-disc.
83
Conclusions
Thus far, examination of the five established cardiomyopathy susceptibility genes,
encoding key components of the Z-disc, demonstrate that perturbations in the Z-disc is
a much less common cause for HCM compared to the two most common HCM-
associated genotypes of myosin binding protein C- and beta myosin heavy chain-
HCM. Nevertheless, Z-disc HCM is as common as thin filament-HCM (i.e., troponin T-,
troponin I-, tropomyosin-, or actin-HCM). However, unlike myofilament HCM, Z-disc
HCM is preferentially sigmoidal. Whether a significant proportion of sigmoidal disease
will be explained by perturbations in other components of the cardiac Z-disc awaits
further investigation.
84
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85
16. Kamisago M, Sharma SD, DePalma SR, Solomon S, et al. Mutations in sarcomere protein genes as a cause of dilated cardiomyopathy. N Engl J Med 2000; 343(23): 1688-1696. 17. Olson TM, Doan TP, Kishimoto NY, Whitby FG, et al. Inherited and de novo mutations in the cardiac actin gene cause hypertrophic cardiomyopathy. J Mol Cell Cardiol 2000; 32(9): 1687-1694. 18. Olson TM, Kishimoto NY, Whitby FG, Michels VV. Mutations that alter the surface charge of alpha-tropomyosin are associated with dilated cardiomyopathy. J Mol Cell Cardiol 2001; 33(4): 723-732. 19. Gerull B, Gramlich M, Atherton J, McNabb M, et al. Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy. Nat Genet 2002; 30(2): 201-204. 20. Daehmlow S, Erdmann J, Knueppel T, Gille C, et al. Novel mutations in sarcomeric protein genes in dilated cardiomyopathy. Biochem Biophys Res Commun 2002; 298(1): 116-120. 21. Vatta M, Mohapatra B, Jimenez S, Sanchez X, et al. Mutations in Cypher/ZASP in patients with dilated cardiomyopathy and left ventricular non-compaction. J Am Coll Cardiol 2003; 42(11): 2014-2027. 22. Zhou Q, Chu PH, Huang C, Cheng CF, et al. Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy. J Cell Biol 2001;155(4):605-612. 23. Frank D, Kuhn C, Katus HA, Frey N. The sarcomeric Z-disc: a nodal point in signaling and disease. J Mol Med 2006; 84(6): 446-68. 24. Pyle WG, Solaro RJ. At the crossroads of myocardial signaling: the role of Z-discs in intracellular signaling and cardiac function. Circ Res 2004; 94(3): 296-305. 25. Knoll R, Hoshijima M, Hoffman HM, Person V, et al. The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy. Cell 2002; 111(7): 943-955. 26. Van Driest SL, Ommen SR, Tajik AJ, Gersh BJ, et al. Yield of Genetic Testing in Hypertrophic Cardiomyopathy. Mayo Clin Proc 2005; 80(6): 739-744. 27. Blair E, Redwood C, Ashrafian H, Oliveira M, et al. Mutations in the gamma(2) subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis. Hum Mol Genet 2001; 10(11): 1215-1220. 28. Arad M, Maron BJ, Gorham JM, Johnson WH, Jr., et al. Glycogen storage diseases presenting as hypertrophic cardiomyopathy. N Engl J Med 2005; 352(4):362-372. 29. Frey N, Katus HA, Olson EN, Hill JA. Hypertrophy of the heart: a new therapeutic target? Circulation 2004; 109(13): 1580-1589. 30. Perkins MJ, Van Driest SL, Ellsworth EG, Will ML, et al. Gene-specific modifying effects of pro-LVH polymorphisms involving the renin-angiotensin-aldosterone system among 389 unrelated patients with hypertrophic cardiomyopathy. Eur Heart J 2005; 26(22): 2457-2462. 31. Xia H, Winokur ST, Kuo WL, Altherr MR, et al. Actinin-associated LIM protein: identification of a domain interaction between PDZ and spectrin-like repeat motifs. J Cell Biol 1997; 139(2) :507-515. 32. Papa I, Astier C, Kwiatek O, Raynaud F, et al. Alpha actinin-CapZ, an anchoring complex for thin filaments in Z-line. J Muscle Res Cell Motil 1999; 20(2): 187-197.
86
33. Tsoporis JN, Marks A, Kahn HJ, Butany JW, et al. Inhibition of norepinephrine-induced cardiac hypertrophy in s100beta transgenic mice. J Clin Invest 1998; 102(8): 1609-1616.
87
88
Chapter 5
Relationship Between Sex, Shape, and Substrate in Hypertrophic Cardiomyopathy
J. Martijn Bos, Jeanne L. Theis, A. Jamil Tajik, Bernard J. Gersh, Steve R. Ommen, Michael J. Ackerman
Am Heart J 2008; 155(6): 1128 – 34
Abstract
Background: Hypertrophic cardiomyopathy (HCM) is a disease characterized by
substantial genetic, morphologic and prognostic heterogeneity. Recently, sex-related
differences in HCM were reported with females being older at diagnosis and exhibiting
greater left ventricular outflow tract obstruction than men. We sought to evaluate the
influence of sex on the HCM phenotype in a large cohort of unrelated patients with
genetically and morphologically classified HCM.
Methods: Comprehensive genotyping of 13 HCM-susceptibility genes encoding
myofilament and Z-disc proteins of the cardiac sarcomere was performed previously
on 382 unrelated patients with HCM. Blinded to the genotype, the septal morphology
was graded as reverse curvature-, sigmoidal-, apical-, or neutral contour-HCM by
echocardiography.
Results: Overall, females were a) significantly older at diagnosis (45.1 ± 20 vs. 35.8 ±
17 years; p<0.001), b) had greater left ventricular outflow tract obstruction (53.5 ± 45
vs. 41.7 ± 42 mmHg; p = 0.009), c) were more likely to have concomitant hypertension
(19% vs. 11%, p = 0.02), and d) had a higher rate of surgical myectomy (49% vs. 36%,
p = 0.01) than men. Interestingly, these sex-based differences were apparent only
among patients with sigmoidal-HCM (p < 0.001).
Conclusions: In this largest cohort of comprehensively genotyped and
morphologically classified patients with clinically diagnosed HCM, we observed that the
striking sex-related differences in the clinical phenotype are confined largely to the
subset of mutation negative, sigmoidal-HCM. Whereas mutations within the sarcomere
appear to dominate the disease process, in their absence, sex has a significant
modifying effect, specifically noted in cases of sigmoidal-HCM.
Keywords Sex, hypertrophy, hypertrophic cardiomyopathy, septum, echocardiography
90
Abbreviations FH Family history
HCM Hypertrophic cardiomyopathy
LVEDD Left ventricular end-diastolic dimension
LVH Left ventricular hypertrophy
LVOT(O) Left ventricular outflow tract (obstruction)
MLVWT Maximum left ventricular wall thickness
SCA Sudden cardiac arrest
91
Background
Affecting 1 in 500 persons, hypertrophic cardiomyopathy (HCM) is a disease or
diseases characterized by marked genetic and prognostic heterogeneity[1].
Characterized by unexplained myocardial hypertrophy in the absence of precipitating
factors, HCM is the most common cause of sudden death in young athletes[1, 2].
Since the sentinel discovery of the first locus linked to familial HCM[3] and the first
HCM-associated mutations identified in the MYH7-encoded -myosin heavy chain[4],
hundreds of mutations scattered among 16 HCM-associated genes encoding
sarcomeric proteins have been identified.
In a pre-genomics study by Lever et al, a striking correlation between the
echocardiographically classified reverse- and sigmoidal septal contour, and age of
onset was described[5]. This observation was followed by an early shape-genetic
substrate analysis by Seidman et al. showing a correlation between reverse septal
curvature and the presence of an HCM-associated MYH7 mutation[6]. Recently, a
strong relationship between the genetic substrate comprised by all 8 myofilament
genes underlying HCM and the morphological subtype was elucidated in a large cohort
of genotyped patients with HCM[7]. The morphology of the left ventricle and septum
were much more closely related to the presence or absence of an underlying
myofilament mutation than to the age of the patient. In fact, multivariate analysis
revealed reverse septal contour to be the strongest independent predictor of a
myofilament mutation, with an odds ratio of 21[7].
Over the past several years, several studies have described sex differences in
HCM[8, 9, 10, 11]. Most recently, significant sex related differences were reported in a
large cohort of American and Italian patients with HCM. This study, in which women
were underrepresented, showed that women were older and more symptomatic at the
time of initial diagnosis[11]. Furthermore, the aforementioned study noted that women,
usually with left ventricular outflow tract obstruction (LVOTO), were more likely to
progress to advanced heart failure and stroke[11]. The relative contributions between
sex, genetic substrate, and anatomical shape could not be ascertained because this
analysis was performed on a cohort of genetically undefined and morphologically
unclassified patients with HCM. Due to the heterogeneous nature both at the level of
the genotype as well as the specific anatomical morphology, we sought to further
evaluate the influence of sex on the HCM phenotype in a large cohort of unrelated
patients with genetically and morphologically classified HCM.
92
Methods
Between April 1997 and December 2001, a total of 382 unrelated patients (210 male,
mean maximum left ventricular wall thickness (MLVWT, 21.5 ± 6mm) underwent
clinical evaluation including echocardiography in Mayo Clinic’s HCM Clinic, a tertiary
referral center for HCM and surgical septal myectomies. Furthermore, comprehensive
genetic testing for 8 myofilament- and 5 Z-disc-associated, HCM-susceptibility genes
was completed for all patients in Mayo Clinic’s Windland Smith Rice Sudden Death
Genomics Laboratory[12, 13, 14, 15, 16, 17, 18]. Informed consent for this IRB-
approved study was obtained from all patients or parents, if underage.
Evaluation of septal curvature and cavity contour was previously performed and
blinded to genotype, patients were classified morphologically into sigmoidal-, reverse
curve-, apical-, and neutral contour-HCM[7]. The diagnosis of HCM was based on the
echocardiographic demonstration of increased left ventricular wall thickness in the
absence of clear etiology. Data on symptomatic status at initial visit (angina, dyspnea)
was collected and scored in severity using the New York Heart Association (NYHA) -
class, while the overall NYHA-class was assessed as well.
As hypertension is a common disease in the US population, some patients in
this cohort also had mild concomitant hypertension. In these cases, the diagnosis of
HCM was felt to be the appropriate diagnosis, by experienced clinicians dedicated to
the care of patients with HCM, as the severity of hypertrophy was out of proportion to
the concomitant hypertension. As a reference, 317 patients were referred to the Mayo
HCM clinic during this time period and were felt to have either significant hypertension
or aortic valve stenosis rather than HCM, and were therefore not included in this
cohort.
93
Statistical analysis
Student’s t tests and Fisher’s exact tests were applied to calculate overall differences
between the males and females, as well as sex differences for the four difference
morphological subgroups using JMP Statistical Software (JMP 6.0, SAS Institute Inc.
2005). For characteristics with multiple levels, multivariate analyses ( 2) were
performed to assess the distribution of the given character between sexes, and
therefore a single p-value was reported. Multiple logistic and linear regression
analyses which included the sex-by-shape interaction effect were used to assess
whether the difference between sexes were, in fact, dependent on morphology. A p-
value <0.05 was considered statistically significant.
94
Results
The demographics of the entire cohort as well as the independent analysis of males
and females are shown in Table 1. Overall, there were 382 patients (210 male)
diagnosed at an average age of 41.5 ± 19 years with females being significantly older
at diagnosis than males (45.1 ± 20 vs. 35.8 ± 17 years; p < 0.001). As inquired during
the interview, one third of patients had a family history of HCM and 20% of patients
had a family history of sudden cardiac arrest (SCA). Fifty-two patients (14%) were
found to have concomitant hypertension at their evaluation at Mayo Clinic (mean
systolic blood pressure, SBP, 123 ± 17 mmHg), which was more common in women.
Nineteen percent of women (31/172) had concomitant hypertension compared to 11%
of males (21; p = 0.02). Clinically, women were more symptomatic at diagnosis with
respect to dyspnea (p = 0.002) and overall NYHA-class (p = 0.0006). During mean
follow-up of 24 months (range 0.1 – 88 months), 25 patients died of HCM-associated
causes, but no sex-differences were observed in these small numbers.
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Table 1: Sex differences among patients with clinically diagnosed HCM
Total Male Female p-valueN 382 210 172 Age at Dx (years) 41.5 ± 19 35.8 ± 17 45.1 ± 20 < 0.001 Age > 50 (%) 125 (33) 55 (26) 70 (41) 0.003 Angina* n(%) 151 (40) 80 (38) 71 (45) 0.2 Dyspnea* n(%) 250 (65) 126 (60) 134 (78) 0.002 NYHA-class n(%) Class I 116 (30) 82 (39) 34 (20) Class II 74 (19) 48 (23) 26 (45) Class III 164 (43) 76 (36) 88 (51) Class IV 8 (2) 4 (2) 4 (2)
0.0006
FH HCM (%)§ 117 (31) 62 (30) 55 (32) 0.7 FH SCA (%)§ 53 (14) 34 (17) 19 (11) 0.2 Hypertension (%) 52 (14) 21 (11) 31 (19) 0.02 SBP (mmHg) 123 ± 17 122 ± 16 124 ± 19 0.4 DBP (mmHg) 72 ± 11 73 ± 11 71 ± 12 0.1 Septal myectomy (%) 159 (42) 75 (36) 84 (49) 0.01 Septal ablation (%) 15 (4) 5 (2) 10 (6) 0.1 Echocardiography MLVWT (mm) 21.5 ± 6 21.7 ± 6 21.4 ± 7 0.7 Patients w/ obstruction (%) 294/382 (77) 153 (73) 141 (82) 0.04 Resting gradient (mm Hg) 47.3 ± 42 41.7 ± 40 53.5 ± 45 0.009 EF (%) 72.7 ± 8 72.5 ± 8 73.1 ± 8 0.4 Morphology Sigmoid 181 (47) 102 (49) 79 (46) Reverse 131 (35) 69 (33) 62 (36) Apical 37 (10) 22 (10) 15 (9) Neutral 33 (8) 17 (8) 16 (9)
0.83
Genotype positive 157 (41) 86 (41) 71(41) 1.0 Mutation location Thick filament (%) 57 (15) 23 (11) 34 (20) Intermediate filament (%) 57 (15) 37 (18) 20 (11) Thin filament (%) 12 (3) 9 (5) 3 (2) Z-disc (%) 12 (3) 7 (3) 5 (3) Multiple (%)# 19 (5) 10 (4) 9 (5)
0.06
Dx, diagnosis; DBP, diastolic blood pressure; DT, deceleration time; EF, ejection fraction; HCM, hypertrophic cardiomyopathy; LA, LVEDD, left ventricular end-diastolic dimension; MLVWT, maximum left ventricular wall thickness; SBP, systolic blood pressure SCA, sudden cardiac arrest defined as unexpected death, nocturnal or within one hour of witnessed collapse * Symptomatic status as classified by NYHA-class, data shown are class II, III and IV combined; §In a first-degree relative; #Patients harboring more than one HCM mutation, (double/compound heterozygotes)
96
Although there was no difference in mean MLVWT between men and women
(21.7 ± 6 vs. 21.4 ± 7 mm; p = 0.7), a slightly greater portion of women than men
(141/172 (82%) vs. 153/210 (73%); p = 0.04) had obstructive HCM with a significantly
higher LVOT gradient (53.5 ± 45 vs. 41.7 ± 40 mm Hg; p = 0.009). Overall, sigmoidal-
HCM (181 patients, 47%) and reverse curve-HCM (131 patients, 35%) represented the
two major morphological subtypes (Figure 1); only 37 patients (10%) had apical-HCM
and 33 patients (8%) had neutral contour-HCM. As shown previously, only 14% of
patients with sigmoidal-HCM had a probable disease causing mutation following
comprehensive open reading frame/splice site genetic testing of the 13 HCM-
susceptibility genes compared to 79% of the patients with reverse curve-HCM[7, 17].
Overall, there was no statistical difference in the distribution of each morphological
subtype of HCM or distribution of mutations between men and women.
Figure 1: Two most common morphologic subtypes of HCM. Echocardiographic picture and graphic depiction of the 2 most common morphologic subtypes of HCM: sigmoidal-HCM (47%) and reverse curve-HCM (35%). Gene + = presence of HCM-associated mutation.
97
To investigate the influence of septal contour, we subdivided the cohort into the
four septal contour subgroups and further analyzed the sex-related based differences
of the two major sub-groups of sigmoidal- and reverse curve-HCM. Strikingly, the
effect of sex on clinical phenotype that was first observed for the cohort at large was
present only among patients with sigmoidal-HCM (Table 2). Akin to the initial
observations gleaned from the entire cohort, women with sigmoidal-HCM were older at
diagnosis (56.0 ± 15 vs. 42.6 ± 16 years old; p < 0.001), were more likely to show
obstructive HCM (75/79, 95% vs. 83/102, 84%; p = 0.007), had higher LVOT gradient
(63.9 ± 40 vs. 49.7 ± 42 mm Hg; p = 0.02), and were more likely to have concomitant
hypertension (p = 0.05) compared to men with sigmoidal-HCM. Although not
statistically significant, more women (52%) than men (40%) underwent surgical septal
myectomy (p = 0.1).
In contrast to the overall observation, no statistical differences were seen in
symptomatic status (angina, dyspnea and overall NYHA-class) between sexes and the
two morphological subgroups. Specifically, the clinical presentation in women was
similar between obstructive sigmoidal HCM and obstructive reverse curve-HCM
suggesting that symptoms stem from the degree of obstruction regardless of the
morphological substrate for that obstruction. No statistical differences were observed
between men and women in MLVWT (p = 0.6), EF (0.08) or the presence or location of
a HCM-associated mutation (p = 0.1). Sex had no demonstrable effect for patients with
reverse curve-HCM.
To assess whether the observed differences between sexes were dependent
specifically on the morphology, multiple linear and logistic regression analyses were
performed. For women, age at diagnosis (p = 0.01), systolic blood pressure (p =
0.008), and presence of LVOTO (p = 0.04) were in fact directly dependent on the
sigmoidal morphology whereas prevalence of myectomy no longer achieved statistical
significance.
98
Table 2: Sex differences in the two most common morphological subgroups
Sigmoidal-HCM (n = 181)
Reverse Curve-HCM (n = 131)
Male Female p Male Female p
Multiregr.
model(p)
N 102 79 69 62 Age at Dx (years) 42.6 ±16 56.0 ± 15 <0.001 29.2 ± 16 33.3 ± 18 0.2 0.01 Age > 50yrs (%) 36 (35) 47 (59) 0.002 5 (7) 10 (16) 0.2 0.9 Angina* n(%) 49 (48) 35 (44) 0.8 20 (29) 27 (44) 0.4 0.2 Dyspnea* n(%) 73 (72) 67 (85) 0.1 35 (51) 43 (69) 0.03 0.4 NYHA-class n (%) Class I 29 (28) 10 (13) 33 (48) 17 (27) Class II 23 (23) 21 (26) 18 (26) 19 (31) Class III 49 (48) 47 (59 16 (23) 25 (40) Class IV 1 (1) 2 (3)
0.07
2 (3) 1 (2)
0.07 0.7
FH HCM (%)* 25 (25) 14 (18) 0.4 29 (42) 30 (48) 0.5 0.2 FH SCA (%)* 13 (13) 5 (6) 0.2 15 (22) 10 (16) 0.5 0.5 Hypertension (%) 13 (13) 20 (26) 0.05 2 (3) 4 (7) 0.4 0.1 SBP (mmHg) 124 ± 16 131 ± 30 0.01 118 ± 17 115 ± 13 0.2 0.008 DBP (mmHg) 73 ± 10 73 ± 11 0.6 71 ± 11 69 ± 11 0.3 0.7 Septal myectomy 41 (40) 41 (52) 0.1 27 (39) 26 (42) 0.9 0.4 Echocardiography MLVWT (mm) 19.8 ± 5 19.4 ± 5 0.6 24.5 ± 7 24.6 ± 7 0.9 0.9
Patients w/ obstruction (%) 83 (84) 75 (95) 0.007 51 (74) 47 (76) 0.8 0.04
Resting gradient (mmHg) 49.7 ± 42 63.9 ± 40 0.02 38.1 ± 37 50.3 ± 49 0.1 0.9
EF (%) 73.1 ± 6 74.6 ± 5 0.08 71.7 ± 9 72.6 ± 9 0.6 0.8 Genotype positive 15 (15) 10 (13) 0.8 58 (84) 47 (76) 0.3 0.6 Mutation location Thick filament (%) 0 (0) 4 (5) 0.04 17 (25) 23 (37) 0.1
Intermediate filament (%) 6 (6) 2 (3) 0.5 26 (38) 14 (23) 0.09
Thin filament (%) 1 (1) 1 (1) 1.0 7 (10) 2 (3) 0.2 Z-disc (%) 7 (7) 3 (4) 0.5 0 (0) 0 (0) - Multiple (%)# 1 (1) 0 (0) 1.0 8 (12) 8 (13) 1.0
0.3
Dx, diagnosis; DBP, diastolic blood pressure; DT, deceleration time; EF, ejection fraction; HCM, hypertrophic cardiomyopathy; LA, left atrial LVEDD, left ventricular end-diastolic dimension; MLVWT, maximum left ventricular wall thickness; SBP, systolic blood pressure SCA, sudden cardiac arrest, defined as unexpected death, nocturnal or within one hour of witnessed collapse. * Symptomatic status as classified by NYHA-class, data shown are class II, III and IV combined §In a first-degree relative; #Patients harboring more than one HCM mutation, (double/compound heterozygotes)
99
Our prior demonstration that reverse curve-HCM is predominantly genotype
positive whereas sigmoidal-HCM is mostly genotype negative, prompted us to further
homogenize the two most common subsets of morphological/genetic HCM by
comparing patients with mutation positive/reverse curve-HCM (N = 105) to patients
with mutation negative/sigmoidal-HCM (N = 156). Herein, sex-based differences in age
at diagnosis, LVOT gradient and presence of concomitant hypertension were
significantly higher among women than men for the largest subtype of HCM, i.e.
mutation negative/sigmoidal-HCM (Figure 2).
Figure 2: Male-female comparisons among patients with genotype positive, reverse curve-HCM and patients with genotype negative, sigmoidal-HCM. Bar diagrams showing the sex differences between males and females with HCM in the specific subgoups of reverse-curve, genotype positive (gene +), reverse curve-HCM and genotype negative (gene-), sigmoidal-HCM on age at diagnosis (top left panel), resting left ventricular outflow tract gradient (top right panel), percentage with surgical myectomy (bottom right panel), and percentage with mild hypertension (bottom left panel).
100
To investigate the potential confounding influence of concomitant hypertension,
the analysis of sex-differences per septal subgroup was repeated excluding the
patients diagnosed with concomitant hypertension. As shown in Table 3, all previously
observed, statistically significant differences that were confined to the sigmoidal-HCM
subgroup – age at diagnosis, number of patients with obstruction, degree of LVOT
obstruction and rate of surgical myectomies – persisted, and no new statistically
significant differences were seen (data not shown). Again, logistic regression models
showed a clear female sex-sigmoidal shape dependence with respect to age at
diagnosis (p = 0.006) and presence of LVOTO (p = 0.02). Overall, patients with
sigmoidal-HCM and concomitant hypertension were less likely to undergo surgical
myectomy than patient without hypertension (21% vs. 52%; p < 0.001), explaining the
increase of significance in surgical myectomies when hypertension was excluded from
the analysis.
Table 3: Sex differences in the two most common morphological subgroups after exclusion of patients with mild hypertension
Sigmoidal-HCM(n = 148)
Reverse Curve-HCM (n = 125)
Male Female p Male Female p
Multiple regr.
models (p)
N 89 59 - 67 58 - -
Age at Dx 40.9 ± 16 53.8 ± 15 0.001 29.4 ± 16 31.6 ± 16 0.5 0.006
Patients w/ obstruction n(%) 72 (81) 57 (97) 0.005 50 (75) 44 (75) 1.0 0.02
Resting gradient (mm Hg) 50.0 ± 42 66.1 ± 39 0.02 38.4 ± 37 49.0 ± 49 0.2 0.06
Septal myectomy n(%) 37 (42) 38 (64) 0.008 26 (39) 24 (41) 0.9 0.1
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Discussion
Long considered a disease of the sarcomere or, more specifically, a disease of the
myofilament, the discovery of mutations in multiple proteins outside the myofilament
has caused an expansion of the spectrum of genetically-mediated pathways
culminating in the disease phenotype that clinicians diagnose as HCM. Currently, over
18 HCM-susceptibility genes have now been published, with 8 of these genes
encoding the essential cardiac myofilaments for which HCM genetic testing is now
commercially available. With this large number of putative pathogenetic genes, a
variety of genes yielding rare mutations, it is intriguing that 30 to 50% of adults with
clinically diagnosed HCM remain genetically unexplained[19]. Binder et al. recently
described an important genotype-phenotype relationship linking the genotypic
substrate to the morphological shape. The analysis of a large cohort of genotyped –
and echocardiographically characterized patients reveal that nearly 80% of patients
with reverse curve-HCM have a positive genetic test for myofilament-HCM whereas
the same genetic test is positive in fewer than 10% of patients clinically diagnosed with
HCM but having a sigmoidal contour (i.e. sigmoidal-HCM) [7]. More recently, the yield
for sigmoidal-HCM increased from 8% to 14% with the inclusion of 5 Z-disc associated
genes[17]. Conversely, myofilament-HCM preferentially yields reverse curve-HCM
whereas Z-disc-HCM predisposes to the development of sigmoidal-HCM[20].
Now, this present study examines the influence of sex in the interplay between
genetic substrate and anatomical shape. Overall, this cohort mirrors previously
published studies that examined the effect of sex on a presumably heterogeneous
cohort of HCM lacking both morphological and genetic sub-classification[9, 10, 11].
Moreover, the data presented herein suggest that the differences are largely confined
to sigmoidal-HCM which constitutes the anatomical phenotype of nearly half of the
patients with clinically diagnosed HCM in our institution. Furthermore, it shows that
specifically for age at diagnosis, systolic blood pressure and presence of LVOTO,
there is a direct sex-by-morphology interaction for women with sigmoidal-HCM.
102
These observations among distinct subtypes of HCM generate several
questions regarding the influence of sex in the phenotypic expression of disease. More
specifically, it suggests that gender does not seem to be a significant modifier in
reverse curve-HCM. This is supported by our original morphological data that the
underlying genotype rather than sex appears to be the predominant determinant of
septal morphology[7]. Thus, in reverse curve-HCM, the presence of a structural
myofilament mutation is the driver of the phenotype, whereas in sigmoidal-HCM, a
multi-factorial process culminates in a clinical expression of HCM, with significant male
– female differences.
The presence of mild, concomitant hypertension may be a contributing factor in
the pathogenesis of sigmoidal-HCM in women as 1 of 4 females within this
morphological classification of HCM were mildly hypertensive. Several studies have
shown that in response to pressure-overload, sex differences in the hypertrophic
response patterns can be seen. Krumholz et al. showed that in isolated hypertension,
significant sex differences can be observed in cardiac adaptation. In contrast to our
findings, they show females predominantly develop a concentric hypertrophy, whereas
a more eccentric pattern was observed in men[21]. Similar patterns of sex-dependent
hypertrophy were observed in aortic stenosis[22, 23] and as a response to
hemodynamic overload after myocardial infarction[24]. Furthermore, the presence of
concomitant hypertension could mean there has always been a presence of low-grade
hypertension and therefore a higher afterload in these patients. These factors
combined with a (undefined) genetic susceptibility for HCM by means of a
pathogenetic mutation or a LVH-promoting polymorphism [25], or endocrine factors
[26] could all converge in the phenotype of clinically diagnosed, sigmoidal-HCM.
Although recent studies have shown that the prevalence of LVOTO is far more
prevalent than previously believed [27, 28], our study may be biased with its higher
prevalence of patients with obstructive HCM at rest due to our role as tertiary referral
center for the surgical treatment of HCM. This is reflected in higher prevalence of
patients with resting LVOTO (75% vs. ~35-40% in other published HCM cohorts)[27,
28] as well as a higher rate of surgical myectomies (42% vs. ~5-10% in other
published HCM cohorts)[29]. Our observations might therefore be less applicable to a
broader spectrum of patients with HCM, particularly non-obstructive HCM. On the
other hand, the conclusions regarding these important sex-substrate differences
appear robust for the subset of patients with obstructive disease.
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Conclusions
In this large cohort of comprehensively genotyped and morphologically classified,
unrelated patients with clinically diagnosed HCM, we observed that the striking and
previously noted sex-related differences in HCM are confined largely to the subset of
patients with mutation negative, sigmoidal-HCM. Sex does not appear to be a
significant genetic modifier in myofilament-HCM.
Acknowledgements
We are indebted to the statisticians from Mayo Clinic’s NIH funded Center for
Translational Science Activities (CTSA) for their help with the proper study design and
statistical analyses.
104
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17. Theis JL, Bos JM, Bartleson VB, Will ML, et al. Echocardiographic-determined septal morphology in Z-disc hypertrophic cardiomyopathy. Biochem Biophys Res Commun 2006; 351(4): 896-902. 18. Bos JM, Poley RN, Ny M, Tester DJ, et al. Genotype-phenotype relationships involving hypertrophic cardiomyopathy-associated mutations in titin, muscle LIM protein, and telethonin. Mol Genet Metab 2006; 88(1): 78-85. 19. Van Driest SL, Ommen SR, Tajik AJ, Gersh BJ, et al. Sarcomeric genotyping in hypertrophic cardiomyopathy. Mayo Clin Proc 2005; 80(4): 463-469. 20. Bos JM, Ommen SR, Ackerman MJ. Genetics of hypertrophic cardiomyopathy: one, two, or more diseases? Curr Opin Cardiol 2007; 22(3): 193-199. 21. Krumholz HM, Larson M, Levy D. Sex differences in cardiac adaptation to isolated systolic hypertension. Am J Cardiol 1993; 72(3): 310-313. 22. Carroll JD, Carroll EP, Feldman T, Ward DM, et al. Sex-associated differences in left ventricular function in aortic stenosis of the elderly. Circulation 1992; 86(4): 1099-1107. 23. Kostkiewicz M, Tracz W, Olszowska M, Podolec P, et al. Left ventricular geometry and function in patients with aortic stenosis: gender differences. Int J Cardiol 1999; 71(1): 57-61. 24. Jain M, Liao R, Podesser BK, Ngoy S, et al. Influence of gender on the response to hemodynamic overload after myocardial infarction. Am J Physiol Heart Circ Physiol 2002; 283(6): H2544-2550. 25. Perkins MJ, Van Driest SL, Ellsworth EG, Will ML, et al. Gene-specific modifying effects of pro-LVH polymorphisms involving the renin-angiotensin-aldosterone system among 389 unrelated patients with hypertrophic cardiomyopathy. Eur Heart J 2005; 26(22): 2457-2462. 26. Malhotra A, Buttrick P, Scheuer J. Effects of sex hormones on development of physiological and pathological cardiac hypertrophy in male and female rats. Am J Physiol 1990; 259(3 Pt 2): H866-871. 27. Maron MS, Olivotto I, Zenovich AG, Link MS, et al. Hypertrophic cardiomyopathy is predominantly a disease of left ventricular outflow tract obstruction. Circulation 2006; 114(21): 2232-2239. 28. Shah JS, Tome Esteban MT, Thaman R, Sharma R, et al. Prevalence of exercise induced left ventricular outflow tract obstruction in symptomatic patients with non-obstructive hypertrophic cardiomyopathy. Heart 2008; 94(10): 1288-94. 29. Maron BJ. Controversies in cardiovascular medicine. Surgical myectomy remains the primary treatment option for severely symptomatic patients with obstructive hypertrophic cardiomyopathy. Circulation 2007; 116(2): 196-206; discussion 206.
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Chapter 6
TGFß-Inducible Early Gene-1 (TIEG1):A Novel Hypertrophic Cardiomyopathy-
Susceptibility Gene
J. Martijn Bos, Malayannan Subramaniam, John R. Hawse, I. Christiaans, Steve R. Ommen, Arthur A.M. Wilde, Thomas C. Spelsberg, Michael J. Ackerman
Manuscript in preparation
Abstract
Background: Hypertrophic cardiomyopathy (HCM) is the most common heritable
cardiovascular disease and the most common cause of sudden cardiac death in the
young. Over 24 genes have been implicated in the pathogenesis of HCM. However, for
about half of patients with HCM, the genotypic substrate remains elusive. A recent
study showed that male TGF Inducible Early Gene-1 (TIEG1) knock-out (TIEG1-/-)
mice develop HCM after 16 months. Microarray analysis on the mice hearts showed a
13-fold up-regulation of PTTG1-encoded pituitary tumor-transforming gene 1. We
therefore speculated that TIEG1 could be a novel candidate gene in the pathogenesis
of genotype negative HCM, possibly through a loss of its repression on PTTG1
expression.
Methods: For this study, we analyzed a cohort of 923 unrelated patients from two
independent cohorts of patients with HCM (664 male, age at diagnosis 47.6 ± 18,
mean left ventricular wall thickness (MLVWT) 20.0 ± 7mm). All patients were genotype
negative with respect to the 9 genes responsible for myofilament/sarcomeric-HCM.
Open reading frame/splice site mutational analysis of TIEG’s 4 translated exons was
performed using DHPLC and direct DNA-sequencing. Site directed mutagenesis was
performed to clone novel variants. The effect of wild type and mutant TIEG1 on the
PTTG1 and SMAD7 promoters was studied using transient transfection and luciferase-
assays. Cardiac HCM tissue was studied by immunohistochemistry to determine levels
of PTTG1 protein expression.
Results: Six novel missense mutations (A12T, M27T, T216A, E137K, A204T and
S225N) in TIEG1 were discovered in 6/923 patients (2 males/4 females, mean age at
diagnosis 56.2 ± 23 years, MLVWT 20.8 ± 4 mm). Each missense mutation was
absent in 800 ethnically-matched reference alleles and involved residues that were
conserved across species. Compared to the 50% repression of PTTG1 promoter
function by wild type TIEG1, 5 TIEG1 mutants had this repression significantly
attenuated resulting in marked accentuation of PTTG1 promoter function similar to the
TIEG1-/- KO-mice. One TIEG1 mutant significantly altered TIEG1-function on SMAD7-
expression. By immunohistochemistry, PTTG1-protein expression was increased in
myectomy specimens from all patients with HCM, irrespective of TIEG1 mutation
status, compared to normal hearts.
108
Conclusions: This is the first paper to associate mutations in TIEG1 to human
disease with the discovery of 6 novel, HCM associated variants. Functional assays
suggest a role for PTTG1 and SMAD7 in the pathogenesis of TIEG1-mediated HCM.
Up-regulation of PTTG1 could be a final common pathway response in HCM. Future
studies are needed to elucidate the precise role of PTTG1 in the pathogenesis of
TIEG1-HCM as well as HCM in general.
Keywords
Hypertrophic cardiomyopathy, TIEG1, KLF10, genes, PTTG1
109
Background
In the last two decades, over 24 disease-susceptibility genes have been elucidated for
hypertrophic cardiomyopathy (HCM), a disease characterized by unexplained cardiac
hypertrophy that affects approximately 1 in 500 individuals [1, 2]. Currently over 80% of
reverse-curve HCM and 10% of sigmoidal-HCM is explained by mutations in genes
encoding the myofilaments of the cardiac sarcomere[3], making a large portion of
sigmoidal HCM genetically elusive. More recently, rare mutations in genes encoding Z-
disc proteins and calcium handling proteins have been linked to the pathogenesis of
HCM[1], but the search for novel HCM-causing genes continues.
TIEG1-encoded TGF -inducible early gene-1 (TIEG1)(also referred to a
KLF10-encoded Kr ppel-Like Factor 10) was discovered originally as an early
response gene following TGF treatment of human osteoblast and is expressed in
many tissues, including cardiac myocardium[4, 5]. It is a member of the Krüppel-like
family of transcription factors, which are known to be involved in anti-proliferative and
apoptotic inducing functions following TGF -induction[6]. Subsequent studies in
engineered TIEG1-knock out (TIEG-/- ) mice[7] showed that male mice develop HCM
with significant, but relatively late-onset cardiac hypertrophy at 16 months of age[8].
Microscopic examination of TIEG-/--male mice hearts showed characteristic hallmarks
of HCM: cardiomyocyte hypertrophy, fibroblast hyperplasia and myocyte disarray[8].
Furthermore, microarray analysis revealed a significant up-regulation of PTTG1-
encoded pituitary tumor transforming gene-1, demonstrating that TIEG1 plays an
important role in the repression of proliferative and hypertrophic pathways, possibly
through the actions of PTTG1. Based on these findings, we hypothesized that TIEG1
could be a novel HCM-susceptibility gene.
110
Methods
Study cohort
Our study cohort consisted of 923 unrelated patients with HCM from two large cardiac
referral centers – Mayo Clinic (Rochester, MN USA) and the Academic Medical Center
(AMC, Amsterdam, The Netherlands). All patients were genotype negative for
mutations in the 9 HCM-associated genes, currently included in commercially available
genetic tests (MYBPC3, MYH7, TNNT2, TNNI3, TNNC1, TPM1, MYL2, MYL3, and
ACTC). Clinical data were collected on all patients, including pertinent personal and
family history (especially with regard to HCM or sudden cardiac arrest (SCA), and an
echocardiogram to determine maximum left ventricular wall thickness (MLVWT) and
resting left ventricular outflow tract gradient (LVOT). Clinical diagnosis of HCM was
made when subjects had a MLVWT over 13mm in the absence of hypertrophy
inducing conditions such as aortic stenosis or hypertension.
Genetic analysis
DNA of all patients was extracted from peripheral blood lymphocytes (Gentra Inc,
Minneapolis, MN). After design of intronic primers, for each patient all 4 translated
exons of TIEG1 (Nm_005646) were amplified by polymerase chain reaction (PCR) and
subsequently analyzed for genetic variations by denaturing high performance liquid
chromatography (DHPLC)(WAVE®, Transgenomic, Omaha, NE). Abnormal DHPLC-
elution profiles were subjected to direct DNA sequencing (ABI Prism 377, Applied
Biosystems, Foster City, CA) to determine the nature of nucleotide substitution. All
translated exons were analyzed for 800 Caucasian reference alleles from ostensibly
healthy, ethnically-matched controls to distinguish novel HCM-associated mutations
from rare or common polymorphisms.
Site-directed mutagenesis and luciferase assays
After design of sequence specific primers, identified mutations and control variants
were created using site-directed mutagenesis, cloned into the pcDNA4.0 expression
vector (Invitrogen, Carlsbad, CA) and transformed into XL-10 ultracompetent cells
(Quickchange® II, Stratagene, La Jolla, CA). DNA was purified from bacteria
(Miniprep®, Qiagen, Valencia, CA) and all constructs were confirmed by direct DNA
sequencing.
111
Effects of the various TIEG1 mutations on PTTG1-promoter activity (PTTG1-
promoter including the 5’-flanking region (-1,321 to -3) cloned in front of a luciferase
reporter as previously described[8]) were studied using luciferase assays. Blinded to
the observer, the PTTG1-promoter construct (1 g) was transfected into AKR2B mouse
embryo fibroblasts along with 1 g of empty expression vector, wild-type (WT) TIEG1-
expression vector or the various mutant TIEG1 expression vectors. Following 24h of
transfection, cell lysates were prepared and analyzed for luciferase activity. Luciferase
assays were repeated at least 3 times and values were normalized to total protein
concentrations and expressed as fold-change relative to empty-vector promoter
activity.
To assess the effect of mutations on the cardiac expressed SMAD7-promoter,
1 g of empty expression vector, and either WT-TIEG1 or TIEG1 mutants were
transfected into H9c2 rat cardiocytes along with a SMAD7-promoter construct (1ug).
Eight hours after transfection, culture media (DMEM + 10% horse serum (HS)) was
replaced with DMEM with 1% HS to induce cardiocyte differentiation. Thirty-six to forty-
eight hours after transfection, cells were lysed, and luciferase assays were performed
and analyzed as described above.
Immunohistochemistry
To determine expression of PTTG1-protein, cardiac tissue obtained following surgical
myectomy in patients with HCM and from non-failing left ventricular hearts at autopsy
(controls) was stained with monoclonal PTTG1-antibody (Epitomics, Burlingame, CA).
Formalin fixed, paraffin embedded tissue was retrieved from 2 TIEG1-genotype
positive patients, 2 genotype negative HCM patients, and 2 autopsy-negative, non-
cardiac death subjects. Paraffin-blocks were sectioned at 5 m for
immunohistochemical staining. Deparaffinization with xylene and subsequent
rehydration with graded ethanol preceded heat induced epitope retrieval with EDTA
buffer (pH 8) in a Lab Vision PT Module (Fremont, CA). The staining procedure was
carried out by an automated immunohistochemistry-staining machine (DAKO
Techmate 500, DAKO, Denmark) using the Envision program. Titration for correct
dilution of antibody was performed and after review, a dilution of 1:75 was selected for
all assays. We compared expression of PTTG1-protein in tissue of TIEG1-genotype
positive patients to genotype negative HCM patients as well as cardiac tissue of two
autopsy negative, non-cardiac death subjects.
112
Statistical analyses
Statistical analyses were performed using JMP 7.0 statistical software (JMP, Cary, NC)
using analysis of variance (ANOVA) and Student’s t-test. A p-value <0.05 was
considered statistically significant.
113
Results
Demographics of the study cohort are summarized in Table 1. Overall 923 patients
with HCM (664 male) were enrolled in this study – 739 from Mayo Clinic (USA) and
184 from Academic Medical Center (AMC) (NL). Patients had an average age at
diagnosis of 47.6 ± 18 years and mean MLVWT of 20.0 ± 7 mm. Twenty-six percent of
patients reported a family history of HCM and 15% of patients had a family history of
SCA. Thirty percent of patients had undergone surgical myectomy and 14% of patients
had received an ICD. The specific demographics of each cohort (Mayo and AMC) are
detailed in Table 1. Overall, patients from the AMC cohort had a lower MLVWT (17.5
± 5.4 vs. 17.5 ± 5.4 (p =0.03) and were more likely a family history of HCM (35% vs.
25%, p = 0.005) or sudden cardiac arrest (SCA) (47% vs. 15%, p < 0.001). Patients at
Mayo were likely to have undergone surgical septal myectomy (38% vs, 6%, p<0.001),
reflecting the referral bias for Mayo Clinic as a surgical center for treatment of
obstructive HCM.
Table 1: Demographics of study cohort
Total Mayo AMC
N 923 739 184
Sex (male/female) 664/359 441/298 123/61
Age at Dx, years 47.6 ± 18 47.4 ± 18 48.3 ± 19
Septal thickness, mm 20.0 ± 7 20.6 ± 8 17.5 ± 5.4*
LVOT gradient, mm Hg 45.1 ± 40 45.2 ± 44 44.7 ± 29
Family history of HCM, n (%) 249 (26) 184 (25) 65 (35)*
Family history of SCA, n (%) 199 (22) 113 (15) 86 (47)*
Myectomy, n (%) 289 (31) 281 (38) 8 (6)*
ICD, n (%) 126 (14) 103 (14) 23 (14)
Dx, diagnosis; HCM, hypertrophic cardiomyopathy; ICD, implantable cardioverter defibrillator; LVOT, left ventricular outflow tract; SCA, sudden cardiac arrest. *, p<0.05
114
Genetic results
Genetic analysis of TIEG1’s open reading frame (exome) revealed 6 novel missense
mutations (A12T, M27T, E137K, A204T, T216A and S225N) in 6 patients with HCM
that were absent in 800 reference alleles (Figure 1). One novel variant (Q10H) was
discovered in HCM patients as well as healthy controls at similar frequencies (allelic
frequency 0.4%). One previously reported rare polymorphism (S249F, rs4734653) was
seen in 3 HCM-patients, but not in our cohort of 400 ethnically matched controls. This
variant, however, was previously described in 0.8% of healthy Europeans.
Figure 1: Topology of TIEG1-protein. Schematic representation of 480 amino acid containing TIEG1 protein with HCM-associated (black circles) and control variants/polymorphisms (white circles) identified in two cohorts of HCM patients.
115
All variants and surrounding residues found in patients with HCM (Figure 2A) as well as control variants (Figure 2B) were conserved across species and the mutant
residues were not seen in other species. In certain species, general sequence
homology was poor or even absent for the first 90 residues of TIEG1.
Figure 2: Sequence conservation. Shown is the conservation across species of (a) novel, HCM-associated TIEG1-mutations (top panel) and (b) novel and previously described control variants (bottom panel).
116
117
Patient characteristics
Patient characteristics for TIEG1-positive patients are summarized in Table 2. Each
mutation was found once in 2 male and 4 female patients. All patients were of
Caucasian ethnicity. Overall, there did not seem to be a specific phenotype associated
with TIEG1-mediated HCM, although in most cases – except for case 2 - HCM was
late onset (mean age at diagnosis 56.2 ± 23 years). The mean MLVWT of TIEG1-
positive patients was 20.8 ± 4 mm and family history of HCM was found in 2/6 (cases 3 and 6) and SCA in 1 case (case 6). Three patients had undergone surgical septal
myectomy for relief of symptoms (cases 2-4), a number relatively high compared to
the annual average rate of myectomy in HCM (~5-10%). The most severely affected
patient (case 2) was a man with M27T-TIEG1. He was diagnosed at 15 years of age,
with extreme hypertrophy (MLVWT, 26mm) and obstruction (117 mmHg gradient) for
which a surgical myectomy was performed. To date, both parents and most siblings do
not meet the diagnostic criteria of HCM following frequent echocardiographic screening
suggesting variable penetrance of the disease or a de novo mutation in this patient.
His family, as well as the others’, have been contacted but have declined or have not
yet enrolled for co-segregation studies.
Tabl
e 2:
Pat
ient
cha
ract
eris
tics
CO
PD
, ch
roni
c ob
stru
ctiv
e pu
lmon
ary
dise
ase;
Dx,
dia
gnos
is;
HC
M,
hype
rtrop
hic
card
iom
yopa
thy;
HF,
hea
rt fa
ilure
; IC
D,
impl
anta
ble
card
iove
rter
defib
rilla
tor;
LV
OT,
left
vent
ricul
ar o
utflo
w tr
act;
SC
A, s
udde
n ca
rdia
c ar
rest
.
Cas
eC
ohor
tN
ucle
otid
ech
ange
Mut
atio
nSe
xA
ge a
t D
x(y
rs)
MLV
WT
(mm
)LV
OT
grad
ient
(mm
Hg)
FH HC
MFH SC
ATr
eatm
ent
Oth
er
1A
MC
c.
GC
G>A
CG
p.
A12
T M
65
17
13
N
o N
o -
-
2M
ayo
c.A
TG>A
CG
p.
M27
T M
15
26
11
7 N
o N
o M
yect
omy
Par
ents
+ m
ost
sibl
ings
ech
o ne
gativ
e
3M
ayo
c.G
AA
>AA
A
p.E
137K
F
48
20
55
Yes
N
o M
yect
omy,
IC
D
Two
sons
echo
neg
4A
MC
c.
AC
A>G
CA
p.
T216
A
F 58
16
20
N
o N
o M
yect
omy
Fath
er d
ied
sudd
enly
at a
ge
63 u
nkno
wn
caus
e 5
May
o c.
GC
T>A
CT
p.A
204T
F
80
20
49
No
N
o -
6M
ayo
c.A
GT>
AA
T p.
S22
5N
F 71
26
16
Y
es
Yes
-
Pat
ient
dec
ease
d at
age
76
of H
F an
d C
OP
D
SMAD7 promoter activity in H9c2-cardiocytes
To study the effect of TIEG1 on SMAD7-promoter expression in a more native
environment, we performed a luciferase assay in H9c2 rat cardiocytes. Thirty-six to
forty-eight hours after transfection, cardiocytes were lysed and analyzed for luciferase
activity as described above. As expected, WT TIEG1 repressed SMAD7-promoter
activity by ~70%. Four of the 6 HCM-associated variants as well as the two control
variants exhibited normal TIEG1-like function relative to SMAD7 (Figure 3). Two of the
putative TIEG1-HCM mutations – A12T and S225N – altered SMAD7-promoter
expression with S225N-TIEG1 showing significantly increased expression of SMAD7
promoter activity as compared to wild-type (Figure 3).
Figure 3: SMAD7-promoter activity in H9c2-cardiocytes. Bar diagram showing SMAD7-promoter activity in H9c2-cardiocytes. Wild-type TIEG1 (WT) repressed SMAD7-expression. Four of six mutations as well as control variants act like WT, whereas S225N-TIEG1 significantly alters TIEG1-function on SMAD7-expression.
119
PTTG1 promoter activity in AKR2B-fibroblasts
Twenty-four hours after transfection with the PTTG1 promoter and either WT-TIEG1,
mutant TIEG1, or control variant expression constructs, AKR2B-cells were lysed and
analyzed for luciferase activity. As expected, WT TIEG1 repressed PTTG1-promoter
activity by ~55% (Figure 4). Akin to data from TIEG1-/- -mice, 5 of the 6 putative
TIEG1-HCM mutations resulted in luciferase activity that was significantly higher than
that of WT (p < 0.05 compared to WT), and in the case of T216A-TIEG1, up to 2 fold
higher than vector control (Figure 4). In contrast, PTTG1-promoter activity seen in
Q10H-TIEG1 control variant was identical to WT TIEG1 effect, while on the other hand,
S249F-TIEG1 showed increased PTTG1 activity.
Figure 4: PTTG1-promoter activity in AKR2B-fibroblasts. Bar diagram showing PTTG1-promoter activity in AKR2B-fibroblasts. Wild-type TIEG1 (WT) repressed PTTG1-expression, where 5 of 6 mutations altered PTTG1-expression significantly.
120
PTTG1 protein expression in cardiac tissue
In order to determine the expression of PTTG1 protein in HCM patients, we performed
immunohistochemical analysis on paraffin embedded tissue sections derived from
patients with TIEG1-HCM (cases 2 and 3), genotype negative-HCM and autopsy
normal hearts (Figure 5). Characteristic hallmarks of HCM, such as myofibrillar
disarray as well as fibrosis could be seen in all myocardial specimens from all 4
patients with HCM (Figure 5C-F). Only mild to no PTTG1-protein expression was seen
in normal heart tissue (Figure 5A+B) as previously described[9]. In contrast, PTTG1
protein expression was dramatically increased in tissue of both HCM genotype
negative patients (Figure 5C + D) as well as the two patients (Cases 2 and 3) with
TIEG1-HCM (Figure 5E + F). PTTG1 localized mostly to cytoplasm and myofibers of
cardiomyocytes and strongest expression was seen in TIEG1-mediated disease. This
data suggests PTTG1 could be a biomarker for HCM in general, although more data is
needed to determine whether there is a different expression of PTTG1 between
genotype negative and TIEG1-mediated HCM.
121
Figure 5: Immunohistochemistry staining of PTTG1 in cardiac tissue. Immunohistochemical staining for PTTG1 protein expression demonstrates little PTTG1-protein expression in autopsy normal hearts (A and B), but severely increased levels of PTTG1 protein in myocardial specimens derived from patients with genotype negative-HCM (C and D) and TIEG1-HCM. Characteristic hallmarks of HCM – myofibrillar disarray and fibrosis – can also be seen in HCM tissues (C-F).
122
Discussion
Over the past two decades, multiple genes encoding proteins involved in various
processes in the cell have been implicated in the pathogenesis of HCM. Since the
discovery of the first gene, MYH7-encoded -myosin heavy chain for HCM in 1989,
over 24 HCM-susceptibility genes have been reported and commercial genetic testing
is now available for a large subset of these genes[1, 10]. However, for many patients,
the underlying genetic cause remains elusive and research continues to discover novel
HCM-associated genes. Recently, male TIEG1-/- mice exhibited features of late-onset
HCM, including asymmetric cardiac hypertrophy, increased ventricular size at 16
months of age, increased heart weight to body weight ratio, increased fibrosis and
increased wall thickness compared to WT mice[8]. Furthermore, Masson’s Trichrome-
staining demonstrated evidence of myocyte disarray and fibrosis which led us to
hypothesize that TIEG1 could be a candidate gene in the pathogenesis of HCM.
Herein, we analyzed 923 unrelated patients with HCM from the USA and the
Netherlands. After comprehensive analysis of all translated regions of TIEG1, we
discovered 6 novel, HCM-associated missense mutations in patients which were
absent in 800 ethnically-matched Caucasian healthy control subjects. Furthermore, we
discovered a novel control variant. The Q10H-variant was seen in our patients as well
as in our 800 reference alleles (allelic frequency 0.4%). Notably, S249F-TIEG1
(rs4734653) was seen in our patients (Allelic frequency 0.5%), was absent in our
reference alleles; but was seen in 0.8% of European controls.
Overall, no TIEG1-specific phenotype seemed to be associated with human
TIEG1-HCM, although similar to the TIEG -/- mice, the patient’s cardiac hypertrophy
was of late-onset and obstructive in most cases. While HCM-phenotype was seen
exclusively in male- TIEG -/- mice, no gender predilection could be demonstrated
among the small cohort of patients with putative TIEG1-HCM. Female TIEG1-/- mice
are known to have skeletal defects which have been characterized as osteopenia[11].
Careful examination of TIEG1-mutation positive patients’ charts – especially women -
revealed no specific skeletal problems in our patients, although it must be
acknowledged that most patients were specifically referred to a cardiologist for HCM
evaluation.
123
Transforming growth factor- (TGF ) is a key mediator of cardiac adaptations
to hemodynamic overload and plays a critical role in induction of cardiac hypertrophy,
heart failure and fibrosis[12]. This is caused by TGF -induced expression of collagen
mRNA and subsequent collagen deposition in fibroblasts, induced expression of
factors of the fetal gene program (MYH7 and ACTC) in cardiomyocytes, and through
activation of the MAPK signaling pathway and p38-induced transcription factors[13,
14]. TIEG1 plays a critical role in the regulation of TGF in multiple cell types. First, it
was demonstrated that expression of TIEG1 is increased within 30 minutes following
TGF treatment in osteoblast cells[4]. Normal TIEG1 function then subsequently
attenuates TGF -signaling through either activation of SMAD2 or repression of the
inhibitory co-factor SMAD7[15, 16, 17].
SMAD7 is a member of the SMAD-family of proteins involved in TGF -
signaling, and with SMAD6, comprises the subgroup of inhibitory SMADS that
antagonize TGF -family members [18, 19]. While most mice devoid of the
indispensible MH2-domain of SMAD7 die in utero, surviving mice have impaired
cardiac functions (such as ejection – and shortening fraction) and cardiac
arrhythmias[20]. Because of its known role in TIEG1-TGF -signaling, as well as the
mutant mouse phenotype, we sought to examine the effects of our novel HCM-
associated TIEG1-variants on the activity of the SMAD7-promoter. We found that two
of the 6 mutants and none of the control variants alter TIEG1-function on SMAD7, with
one variant showing significantly altered function compared to WT TIEG1.
Further studies of TIEG1-/- male mice demonstrated the mice develop
characteristic features of HCM during aging with a marked upregulation of PTTG1[8].
PTTG1 is typically overexpressed in a variety of endocrine-related tumors, especially
pituitary, thyroid, breast, ovarian, and uterine tumors as well as non-endocrine tumors.
PTTG1 functions in cell replication, proliferation, DNA damage/repair, organ
development, and metabolism (reviewed in [21]). In vitro luciferase assays studies
demonstrated that TIEG1 acts directly on the promoter of PTTG1 causing a 60-70%
drop in PTTG1’s promoter activity suggesting that the observed myocyte hypertrophy
and fibrosis in male TIEG-/- mice may be mediated by loss of TIEG1’s normal inhibition
over PTTG1 and consequential accentuation in PTTG1 gene expression [8]. Akin to
these observations, our current study showed that 5 of our 6 TIEG1 mutations resulted
in a significant increase in PTTG1 promoter activity relative to WT TIEG1.
124
In addition, protein levels of PTTG1 in surgically resected, hypertrophic
myocardium of patients with TIEG1-HCM, was markedly increased. Further,
accentuation in PTTG1 might be a final common pathway hypertrophic response as
two patients with genotype negative HCM also displayed this finding. These data
suggest that these putative HCM-associated TIEG1 mutations dysregulate TIEG1’s
normal repressive control over either SMAD7 or PTTG1 and that PTTG1 protein
expression might be a ‘final common pathway’ biomarker in HCM[22, 23]. Further
studies are therefore needed to dissect the biological role of PTTG1. Conceivably,
gain-of-function PTTG1 mutations in humans or transgenic overexpression of PTTG1
in mice could precipitate HCM.
125
Conclusions
This is the first report to associate mutations in the TIEG1 gene with human disease.
We have identified 6 novel, HCM associated TIEG1 missense mutations and have
demonstrated that a number of these variants have abnormal function with regard to
mutant TIEG1’s ability to regulate either the PTTG1 or SMAD7 promoters, two genes
known to be associated with hypertrophic pathways. Furthermore, tissue expression of
PTTG1 seemed to be associated with HCM in general with highest expression seen in
TIEG1-mediated HCM suggesting PTTG1 might be a biomarker for HCM. While these
studies have implicated TIEG1 in human HCM, additional in vitro and in vivo functional
studies are needed to further elucidate the exact pathway(s) leading to HCM in TIEG1-
genotype positive patients. Furthermore, studies are needed to examine the potential
role of PTTG1 as a biomarker in the pathogenesis of HCM.
126
References 1. Bos JM, Towbin JA, Ackerman MJ. Diagnostic, prognostic, and therapeutic implications of genetic testing for hypertrophic cardiomyopathy. J Am Coll Cardiol 2009; 54(3): 201-211. 2. Maron BJ. Hypertrophic cardiomyopathy: A systematic review. JAMA 2002; 287(10): 1308-1320. 3. Binder J, Ommen SR, Gersh BJ, Van Driest SL, et al. Echocardiography-guided genetic testing in hypertrophic cardiomyopathy: Septal morphological features predict the presence of myofilament mutations. Mayo Clin Proc 2006; 81(4): 459-467. 4. Subramaniam M, Harris SA, Oursler MJ, Rasmussen K, et al. Identification of a novel tgf-beta-regulated gene encoding a putative zinc finger protein in human osteoblasts. Nucleic Acids Res 1995; 23(23): 4907-4912. 5. Subramaniam M, Hefferan TE, Tau K, Peus D, et al. Tissue, cell type, and breast cancer stage-specific expression of a TGF-beta inducible early transcription factor gene. J Cell Biochem 1998; 68(2): 226-236. 6. Dang DT, Pevsner J, Yang VW. The biology of the mammalian kruppel-like family of transcription factors. Int J Biochem Cell Biol 2000; 32(11-12): 1103-1121. 7. Subramaniam M, Gorny G, Johnsen SA, Monroe DG, et al. TIEG1 null mouse-derived osteoblasts are defective in mineralization and in support of osteoclast differentiation in vitro. Mol Cell Biol 2005; 25(3): 1191-1199. 8. Rajamannan NM, Subramaniam M, Abraham TP, Vasile VC, et al. TGFbeta inducible early gene-1 (tieg1) and cardiac hypertrophy: Discovery and characterization of a novel signaling pathway. J Cell Biochem 2007; 100(2): 315-325. 9. Chiriva-Internati M, Ferraro R, Prabhakar M, Yu Y, et al. The pituitary tumor transforming gene 1 (PTTG-1): An immunological target for multiple myeloma. J Transl Med 2008; 6: 15. 10. Geisterfer-Lowrance AA, Kass S, Tanigawa G, Vosberg H, et al. A molecular basis for familial hypertrophic cardiomyopathy: A beta cardiac myosin heavy chain gene missense mutation. Cell 1990; 62: 999-1006. 11. Hawse JR, Iwaniec UT, Bensamoun SF, Monroe DG, et al. TIEG-null mice display an osteopenic gender-specific phenotype. Bone 2008; 42(6): 1025-1031. 12. Brand T, Schneider MD. The TGF beta superfamily in myocardium: Ligands, receptors, transduction, and function. J Mol Cell Cardiol 1995; 27(1): 5-18. 13. Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intracellular signaling pathways. Nat Rev Mol Cell Biol 2006; 7(8): 589-600. 14. Parker TG, Packer SE, Schneider MD. Peptide growth factors can provoke "Fetal" Contractile protein gene expression in rat cardiac myocytes. J Clin Invest 1990; 85(2): 507-514. 15. Johnsen SA, Subramaniam M, Janknecht R, Spelsberg TC. TGFbeta inducible early gene enhances tgfbeta/smad-dependent transcriptional responses. Oncogene 2002; 21(37): 5783-5790. 16. Johnsen SA, Subramaniam M, Katagiri T, Janknecht R, et al. Transcriptional regulation of SMAD2 is required for enhancement of TGFbeta/SMAD signaling by TGFbeta inducible early gene. J Cell Biochem 2002; 87(2): 233-241.
127
17. Johnsen SA, Subramaniam M, Monroe DG, Janknecht R, et al. Modulation of transforming growth factor beta (TGFbeta)/SMAD transcriptional responses through targeted degradation of TGFbeta-inducible early gene-1 by human seven in absentia homologue. J Biol Chem 2002; 277(34): 30754-30759. 18. Heldin CH, Miyazono K, ten Dijke P. TGFbeta signalling from cell membrane to nucleus through smad proteins. Nature 1997; 390(6659): 465-471. 19. Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 2003; 113(6): 685-700. 20. Chen Q, Chen H, Zheng D, Kuang C, et al. SMAD7 is required for the development and function of the heart. J Biol Chem 2009; 284(1): 292-300. 21. Vlotides G, Eigler T, Melmed S. Pituitary tumor-transforming gene: Physiology and implications for tumorigenesis. Endocr Rev 2007; 28(2): 165-186. 22. Bowles NE, Bowles KR, Towbin JA. The "Final common pathway" Hypothesis and inherited cardiovascular disease. The role of cytoskeletal proteins in dilated cardiomyopathy. Herz 2000; 25(3): 168-175. 23. Olivotto I, Cecchi F, Poggesi C, Yacoub MH. Developmental origins of hypertrophic cardiomyopathy phenotypes: A unifying hypothesis. Nat Rev Cardiol 2009; 6(4): 317-321.
128
Chapter 7
Diagnostic, Prognostic and Therapeutic Implications of Genetic Testing for Hypertrophic Cardiomyopathy
J. Martijn Bos, Jeffrey A. Towbin, Michael J. Ackerman
J Am Coll Cardiol 2009; 54(3): 201 – 211 [Review]
Purpose of Review
Over the last two decades the pathogenic basis for the most common heritable
cardiovascular disease, hypertrophic cardiomyopathy (HCM), has been investigated
extensively. Affecting approximately 1 in 500 individuals, HCM is the most common
cause of sudden death in young athletes. In recent years, genomic medicine has been
moving from the bench to the bedside throughout all medical disciplines including
cardiology. Now, genomic medicine has entered clinical practice as it pertains to the
evaluation and management of patients with HCM. The continuous research and
discoveries of new HCM-susceptibility genes, the growing amount of data from
genotype-phenotype correlation studies, and the introduction of commercially available
genetic tests for HCM make it essential that the modern-day cardiologist understand
the diagnostic, prognostic, and therapeutic implications of HCM genetic testing.
130
Affecting 1 in 500 people, hypertrophic cardiomyopathy (HCM) is a disease marked by
phenotypic and genotypic heterogeneity and is the most prevalent, heritable
cardiovascular disease. HCM is the most common cause of sudden cardiac death in
young athletes[1]. HCM can manifest negligible to extreme hypertrophy, minimal to
extensive fibrosis and myocyte disarray on microscopy, absent to severe left
ventricular outflow tract (LVOT) obstruction, and distinct septal morphologies such as
reverse curve-, sigmoidal-, and apical-HCM. The clinical course varies extremely as
well, ranging from an asymptomatic lifelong course to dyspnea/angina refractory to
pharmacotherapy to sudden death as the sentinel event. Fully described for the first
time by Teare in 1958, HCM was regarded as ‘asymmetrical hypertrophy of the heart
in young adults’[2]. It has since been referred to by an array of names – idiopathic
hypertrophic subaortic stenosis[3], muscular subaortic stenosis[4] and hypertrophic
obstructive cardiomyopathy[5] - reflecting its clinical heterogeneity and its relatively
uncommon occurrence in daily cardiologic practice.
131
Diagnostic Implications of HCM Genetic Testing
Identification of HCM-Susceptibility Genes
Nearly 20 years ago, the first chromosome locus for familial HCM and subsequently
mutations involving the MYH7-encoded -myosin heavy chain were elucidated as the
pathogenic basis for HCM[6, 7]. Since then several hundreds of mutations scattered
among at least 27 putative HCM-susceptibility genes encoding various sarcomeric,
calcium-handling and mitochondrial proteins have been identified (Table 1, 2). The
most common genetically-mediated form of HCM is myofilament (sarcomeric)-HCM
with hundreds of disease-associated mutations in 9 genes encoding proteins
(myofilaments) critical to the cardiac sarcomere. This includes -myosin heavy chain
(MYH7)[7], regulatory - (MYL2) and essential myosin light chains (MYL3)[8], myosin
binding protein C (MYBPC3)[9], cardiac troponin T (TNNT2), -tropomyosin (TPM1)
[10], cardiac troponin I (TNNI3)[11], cardiac troponin C (TNNC1)[12] and actin
(ACTC)[13, 14]. Complete screening through a large cohort of patients has not been
performed, yet targeted screening of giant sarcomeric TTN-encoded titin, which
extends throughout half of the sarcomere, has thus far revealed only 1 mutation[15].
132
Table 1: Summary of HCM-Susceptibility Genes
Gene Locus Protein Frequency (%)
Myofilament-HCM
TTN 2q24.3 Titin < 1
MYH7 14q11.2-q12 -myosin heavy chain 15 – 25
MYH6 14q11.2-q12 -myosin heavy chain < 1
MYL2 12q23-q24.3 Ventricular regulatory myosin light < 2
MYL3 3p21.2-p21.3 Ventricular essential myosin light < 1
MYBPC3 11p11.2 Cardiac myosin-binding protein C 15 – 25
TNNT2 1q32 Cardiac troponin T < 5
TNNI3 19p13.4 Cardiac troponin I < 5
TPM1 15q22.1 -Tropomyosin < 5
ACTC 15q14 -Cardiac actin < 1
TNNC1 3p21.3-p14.3 Cardiac troponin C < 1
Z-disc HCM
LBD310q22.2-
q23.3
LIM binding domain 3
(Alias: ZASP) 1 – 5
CSRP3 11p15.1 Muscle LIM protein < 1
TCAP 17q12-q21.1 Telethonin < 1
VCL 10q22.1-q23 Vinculin/metavinculin < 1
ACTN2 1q42-q43 Alpha-actinin 2 < 1
MYOZ2 4q26-q27 Myozenin 2 < 1
Calcium-handling HCM
JPH2 20q12 Junctophilin-2 < 1
PLN 6q22.1 Phospholamban < 1
Bolded genes are available as commercial genetic test
133
Expanding the scope of proteins involved in the pathogenesis of HCM, the
spectrum of HCM-associated genes has moved outside the myofilaments of the
sarcomere to encompass additional subgroups that could be classified as ‘Z-disc-
HCM’ and ‘calcium-handling HCM’ (Table 1). Due to its close proximity to the
contractile apparatus of the myofilament, its specific structure-function relationship with
regards to cyto-architecture, as well as its role in the stretch-sensor mechanism of the
sarcomere, attention subsequently focused on the cardiac Z-disc. This focus has been
fueled by the fact that HCM and DCM are partially allelic disorders, in which mutations
in the same genes – especially the Z-disc – can be responsible for both
cardiomyopathic phenotypes[16, 17, 18, 19, 20, 21, 22, 23, 24]. The first Z-disc
mutations associated with HCM were described in muscle LIM protein encoded by
CSRP3[21] and telethonin encoded by TCAP[23]. Recently, LDB3-encoded LIM
domain binding 3, ACTN2-encoded alpha actinin 2, VCL-encoded
vinculin/metavinculin[24, 25, 26] and MYOZ2-encoded myozenin-2[27] have been
added to that list. In another demonstration of mutations in one gene causing multiple
diseases, MYPN-encoded myopalladin (MYPN) mutations were implicated in the
pathogenesis of DCM, HCM and restrictive cardiomyopathy (RCM) - via disturbed
myofibrillogenesis, abnormal gene expression, and/or abnormality in assembly of Z-
disc and intercalated disc (Purevjav et al. unpublished data).
In yet another signal transduction pathway, proteins involved in calcium
induced calcium release and the hypothesis that errors in this process may lead to
compensatory hypertrophy have always been of high interest in the pathogenesis of
HCM. Mutations have been described in the promoter and coding region of PLN-
encoded phospholamban, an important inhibitor of cardiac muscle sarcoplasmic
reticulum Ca(2+)-ATPase (SERCA)[28, 29]. Recently, mutations in JPH2-encoded
junctophilin 2, which helps approximate the sarcoplasmic reticulum calcium release
channels and plasmalemmal L-type calcium channels, may cause HCM [30].
134
Table 2: HCM phenocopies
Gene Locus Protein Syndrome
TAZ Xq28 Tafazzin (G4.5) Barth syndrome/LVNC
DTNA 18q12 -dystrobrevin Barth syndrome/LVNC
PRKAG2 7q35- q36.36 AMP-activated protein kinase WPW/HCM
LAMP2 Xq24 Lysosome-associated membrane
protein 2 Danon’s syndrome/WPW
GAA 17q25.2-q25.3 -1,4-glucosidase deficiency Pompe’s disease
GLA Xq22 -galactosidase A Fabry’s disease
AGL 1p21 Amylo-1,6-glucosidase Forbes disease
FXN 9q13 Frataxin Friedrich’s ataxia
PTPN11 12q24.1 Protein tyrosine phosphatase,
non-receptor type 11, SHP-2
Noonan’s syndrome,
LEOPARD syndrome
RAF1 3p25 V-RAF-1 murine leukemia viral
oncogene homolog 1
Noonan’s syndrome,
LEOPARD syndrome
KRAS 12p12.1 v-Ki-ras2 Kirsten rat sarcoma viral
oncogene homolog Noonan’s syndrome
SOS1 2p22-p21 Son of sevenless homolog 1 Noonan’s syndrome
LVNC, left ventricular non-compaction; WPW, Wolff-Parkinson-White syndrome. Bolded genes are available as commercial genetic test
135
New insights and approaches to genomics of cardiac hypertrophy and
HCM
Not only in molecular genetics but also in other fields of “-omics”, novel pathways
underlying the pathophysiology of cardiac hypertrophy and HCM have been identified
using several new techniques to study large-scale transcriptional changes[31, 32, 33].
A transcriptomic approach can be performed by using microarray, a technique that
enables to give a snapshot view of gene expression that, combined with complex
analytic tools, can identify genes that are differentially regulated or seem to be co-
regulated and thereby form a transcriptional network of genes and pathways.
Microarray-chips can hold over tens of thousands of genes and can be utilized to
compare expression levels in certain disease states with healthy controls.
In 2002, Hwang et al. studied RNA from heart failure patients with either HCM
or DCM, and found almost 200 genes to be up-regulated and 51 genes down-
regulated in both conditions, as well as several genes differentially expressed between
the two diseases providing information on different pathways and genes involved in the
pathogenesis[34]. Rajan et al. performed microarray analysis on ventricular tissue of
two previously developed transgenic, 2.5 months old HCM-mice ( -TM175 and -
TM180) carrying mutations in alpha-tropomyosin (TPM1). Studying 22,600 genes, 754
differentially expressed genes between transgenic and non-transgenic mice were
detected, of which 266 were differentially regulated between the 2 different mutant
hearts showing most significant changes in genes belonging to the
‘secreted/extracellular matrix’ (up-regulation) and ‘metabolic enzymes’ (down-
regulation) [35].
136
Another emerging field is that of microRNA’s (miR’s) and their role in cardiac
development and (hypertrophic) heart disease. These fundamental cellular regulators
were first described by Lee et al in 1993[36] and consist of approximately 22 non-
coding RNA molecules that silence genes through posttranscriptional regulation.
MicroRNA’s play an important role in cardiac development as well as in orchestrating
organogenesis and early embryonic patterning processes[37, 38]. Furthermore, these
non-coding RNA molecules seem to play an important role in cardiac remodeling and
the development of hypertrophy as initially reported by Van Rooij et al in 2006 (42).
Utilizing 2 mouse models of pathological hypertrophy – transverse aortic constriction
(TAC) and calcineurin transgenic mice, 6 miR’s were up-regulated, which in vitro,
were sufficient to induce hypertrophic growth of cardiomyocytes[39]. Furthermore, a
transgenic mouse model over-expressing one of these miR’s (miR-195) showed that a
single miRNA could induce pathological hypertrophy and heart failure[39]. Over the
last year, multiple studies have been published with miRNA expression profiles in
different settings, in vivo and in vitro, of cardiac hypertrophy[37, 39, 40, 41, 42, 43].
Lastly, PMAGE (polony multiplex analysis of gene expression) is a technique
that detects messenger RNAs (mRNAs) as rare as one transcript per three cells [44].
Using this new technique, early transcriptional changes preceding pathological
manifestations were identified in mice with HCM-causing mutations, including low-
abundance mRNA encoding signaling molecules and transcription factors that
participate in the disease pathogenesis[44].
The development and implementation of these new techniques as well as their
applications in research and clinical models of cardiac hypertrophy and HCM will over
the years teach us more about the pathophysiology of normal and pathologic
hypertrophy as well as HCM. This in turn might lead to discovery of novel disease
causing genes, involved pathways and possible novel therapeutic targets.
137
HCM Genetic Testing in Clinical Practice
Recently, HCM genetic testing has matured from its two-decade long residence in
research laboratories into the realm of clinically available, diagnostic testing for
physicians evaluating and treating patients with this disease [(Harvard Partners,
Correlagen, PGxHealth, and GeneDx. These companies now offer testing for the 8
most common myofilament associated genes; additional genes offered by some are
the genes involved in the glycogen storage diseases or the recently discovered HCM-
associated gene troponin C encoded by TNNC1. The HCM-susceptibility genes
available for commercial genetic testing are highlighted in bold in Table 1 and 2.
Although some of the new HCM-susceptibility genes may surpass the
prevalence of mutations found in some of the myofilament proteins, MYBPC3 and
MYH7 remain by far the most common HCM-associated genes, with an estimated
prevalence of 15 to 25% for both genes. Among the 9 HCM-associated, myofilament
encoding genes, the prevalence of myofilament-HCM has ranged from 35 to 65% in
several different, international cohorts of unrelated patients who met the clinically
accepted definition of HCM[45, 46].
138
Echo-guided genetic testing
While several phenotype-genotype relationships have emerged to enrich the yield of
genetic testing, most of these patient profiles have not been particularly clinically
informative. Recently, the possibility of echo-guided genetic testing has been
explored[47]. Noting a predominance of sigmoidal-HCM among the elderly, Lever et al
suggested over 2 decades ago that there was a strong age-dependence with the
various septal morphologies of HCM, where septal contour was classified as reverse
curve-, sigmoidal-, apical-, and neutral contour-HCM (Figure 1)[48]. In the early 1990s,
Solomon et al observed that patients with mutations in the beta myosin heavy chain
(MYH7-HCM) generally had reversed curvature septal contours (reverse curve-HCM)
[49].
Figure 1: Septal morphologies in HCM. Shown are the most common septal morphologies in HCM. The distribution of septal morphologies among a large cohort of patients with HCM is shown along the top while the yield of genetic testing for each morphological subgroup is shown along the bottom of the figure.
139
Subsequently, a large genotype-phenotype analysis correlating the septal
morphology with the underlying genotype was conducted. After extensive analysis of
the echocardiograms of nearly 400 unrelated patients, sigmoidal HCM (47% of cohort)
and reverse curve-HCM (35% of cohort) represented the two most prevalent
anatomical subtypes of HCM (Figure 1). In this study, the yield of genetic testing for
myofilament-HCM (8 genes) was 80% in reverse curve-HCM but only 10% in patients
with sigmoidal-HCM and septal contour was the strongest predictor of a positive HCM
genetic test, regardless of age (odds ratio 21, p <0.0001) [47]. These observations
may facilitate echo-guided genetic testing by enabling informed genetic counseling
about the pre-test probability of a positive genetic test based upon the patient’s
expressed anatomical phenotype (Figure 2).
Role of HCM genetic testing for both index cases and relatives
Although there may be some prognostic relevance presently and therapeutic relevance
futuristically to the HCM genetic test in the index case who already clinically manifests
the disease, the principal role for index case genetic testing is diagnostic. It can
however, as we will show later on, be of significant importance to the approach and
screening of relatives.
Figure 2 provides a flow chart for clinicians, in which the 2 pathways are
described when an index case (HCM proband) is identified. It must be recognized that,
before a diagnosis of HCM in the proband is made, a full history, examination,
including extensive family history should be performed. This way clues can be picked
up to expose other causes of unexplained LVH –aortic stenosis, hypertension or the
presence of a phenocopy – as being responsible for the patient’s symptoms. For
example, signs of ventricular pre-excitation might point to a PRKAG2-mediated
glycogen storage disease or an inheritance pattern that strictly affects males might
suggest LAMP2-mediated disease. If the phenotype is HCM, echocardiography may
inform genetic counseling by providing an a priori probability for a positive genetic test
and advice on how to proceed with further evaluation and family screening (left arm of
algorithm). If genetic testing of the major genes remains negative, the presence of a
phenocopy with pure cardiac involvement should be considered.
140
Figure 2: Genetic- and echocardiographic-based screening in HCM. Flow-chart showing a possible decision tree to follow in genetic- and echocardiography-based screening in HCM. Noted is the a priori probability for a positive genetic test result based on the echocardiographic scored septal contour, as well as the steps to follow if a patient chooses not to pursue genetic testing.
141
As it stands now, genetic testing of the index case for the index case has the
potential of providing the diagnostic gold standard for his/her offspring, siblings, and
parents and more distant relatives. A positive genetic test would then enable
systematic scrutiny of the index case’s relatives to separate the “haves” from the “have
nots” (positive versus negative test). In other words, the genetic testing of the index
case risk stratifies the family enabling 2 very different courses to be charted: 1) close
surveillance of the genotype-positive, pre-clinical individual and 2) casual observation
or dismissal of the genotype-negative/phenotype-negative relative and his/her future
progeny.
In general and irrespective of genetic testing, once a diagnosis of HCM has
been rendered, all first degree relatives and probably “athletic” second degree relatives
to the index case should be screened by an ECG and echocardiogram. Annual
screenings are recommended for young adults (age 12 to 25 yrs) and athletes and
thereafter every 3 to 5 years. As intimated previously, if an HCM-causing mutation is
established for the index case, first degree relatives should then have confirmatory
genetic testing for that particular HCM-causing mutation. Depending on the established
familial versus sporadic pattern, confirmatory genetic testing should proceed in
concentric circles of relatedness.
For example, if the index case’s mutation is present in his/her father, then the
paternal grandparents and paternal aunts/uncles should be tested. The index case’s
first degree cousins may or may not need genetic testing depending on the results of
the testing among the aunts and uncles and so forth. If a phenotype negative family-
member tests negative for the index case’s mutation, then future cardiologic
evaluations for that relative and his/her progeny may not be necessary. However, a
decision to cease surveillance for HCM in a relative hinges critically on the certainty of
the identified gene/mutation and its causative link as well as the complete absence of
any traditional evidence used to clinically diagnose HCM (i.e. asymptomatic and
normal echocardiogram).
142
Aside from the role of genetic testing described above, there is still concern
among patients and practitioners on social and economic aspects of knowing ones
genetic make-up. For example, genetic testing may or may not be covered by an
individual’s health insurance plan. Some payors view HCM genetic testing as a bona
fide clinical test while others view it as an “investigational” one. Secondly, if genetic
testing is performed, a patient might feel anxiety about the potential clinical dangers of
hosting an HCM-predisposing mutation. Also, there might be fears that the presence of
this information in one’s medical records might influence health insurance premiums
and employment opportunities, although this last issue has been addressed recently
by former President Bush’s signing of the Genetic Information Nondiscrimination Act
(GINA) into law. This is an important step to provide protection against genetic
discrimination. Besides discussing the differential impact in terms of follow-up for a
genotype positive relative compared to a genotype negative relative as described
earlier, these important issues should also comprise the genetic counseling
Novel imaging techniques for early detection of HCM disease gene
expression
With growing knowledge on genetics and other pathogenetic pathways in HCM, it
would be very helpful if parameters could be found that suggest pre-clinical, pre-
hypertrophic expression of the genetic substrate. Tissue Doppler (TD)
echocardiography studies in transgenic rabbit models of HCM[50, 51, 52] and in
humans have shown reduced myocardial Doppler velocities in genotype positive
subjects without LVH[50, 53, 54, 55]. Nagueh et al. provided the first evidence in 2001
demonstrating that myocardial contraction and relaxation velocities as detected by TD
are reduced in familial, mutation positive HCM. In 2002, Ho et al. showed that
abnormalities of diastolic function as assessed by Doppler tissue imaging precede
development of LVH in patients with MYH7-mutations where a combination of Ea
velocity and ejection fraction (EF) was highly predictive of affected phenotype in
patients without hypertrophy[55]. Cardiac MRI (CMR) is also showing potential to
become an important tool in the diagnosis of HCM as it has capacity to acquire images
with tissue contrast and border definition that is often superior to echocardiography[56,
57, 58, 59]. In 2006, Germans et al. performed CMR on 16 mutation positive HCM
patients and detected pre-hypertrophic crypts in the inferoseptal LV wall, possibly
representing early pathological alterations stemming from the pathogenic
substrate[60].
143
Prognostic Implications of HCM Genetic Testing
From the early-beginnings of the genomic-era and since the description of the first
HCM-causing mutation, investigators have attempted to correlate genotypes to
particular clinical phenotypic expressions. Stemming from earlier pedigree studies,
specific missense mutations were associated with a markedly unfavorable prognosis
whereas others had an uneventful natural history. These observations resulted in
specific mutations being designated as either “malignant” mutations or “benign”
mutations [61, 62, 63, 64, 65, 66, 67, 68]. The first study of its kind was published by
Watkins et al. in 1992 in which they described mutations in MYH7 found in 12 out of 25
families with HCM[61]. They concluded that the MYH7-R403C mutation was
associated with a significantly shorter life expectancy, and could therefore be
considered a ‘malignant’ mutation. In contrast, a non-charge change mutation (V606M)
was associated with nearly normal survival and therefore was considered ‘benign’[61].
In 2003, Woo et al. analyzed mutations in functional domains in 15 (out of 70) MYH7-
positive probands and concluded that their may be prognostically informative
domains[69].
Initial reports on the clinical expression from the most common subtype of HCM
– MYBPC-mediated HCM – seems to show a slower, but progressive clinical disease
course, with later onset, milder disease characteristics[68, 70, 71]. Investigators in the
Netherlands and South-Africa have discovered founder mutations in MYBPC3 with
mild phenotypic expression that are present in at least 30% of their cases[72].
Similarly, certain genotype-phenotype correlations were attributed to TNNT2 (troponin
T)-HCM. Far less common than MYBPC-HCM or MYH7-HCM, TNNT2-HCM (affecting
< 5% of patients) was associated with less severe left ventricular wall thickness, but a
higher incidence of premature sudden cardiac death[64, 73, 74]. Overall, these
TNNT2-HCM patients who suddenly died had less hypertrophy and less fibrosis, but
more myocyte disarray, which may have provided the substrate for malignant
arrhythmias[74].
144
Overall, these observations have been gleaned from small cohorts involving
larger families with penetrant disease expression whereas genotype-phenotype
studies involving large cohorts of unrelated patients have indicated that great caution
must be exercised with assigning particular prognostic significance to any particular
mutation[75, 76, 77]. In one such cohort, only 2% hosted one of those formally
annotated ‘benign’ mutations and moreover, these particular hosts displayed a severe
clinical phenotype with all 5 patients requiring surgical myectomy, 3 of the 5 having a
family history of sudden cardiac death, and 1 adolescent requiring an orthotopic heart
transplant[77]. In contrast, 3 patients hosting a so-called ‘malignant’ mutation displayed
a heretofore mild phenotype[75]. Furthermore, these studies have demonstrated that
the two most common forms of genetically mediated HCM – MYH7-HCM and
MYBPC3-HCM – are phenotypically indistinguishable[78].
More recently, in one of the first studies of its kind for HCM, a longitudinal study
in a large cohort of unrelated Italian patients with HCM have shown an increased risk
of cardiovascular death, non-fatal stroke or progression to New York Heart Association
functional class III/IV among patients with a positive HCM genetic test involving any of
the myofilament genes compared to those patients with a negative genetic test (25%
vs. 7%, respectively; p = 0.002) (Figure 3A); multivariate analysis showed myofilament
positive HCM (i.e. a positive genetic test) to be the strongest predictor of an adverse
outcome (hazard ratio 4.27 (CI 1.43 – 12.48), p = 0.008)[79]. Furthermore, patients
with myofilament genotype-positive-HCM had greater probability of developing severe
LV systolic dysfunction (p = 0.021; Figure 3B) and restrictive LV filling (p = 0.018;
Figure 3C).
Lastly, it has been observed that patients with multiple mutations (i.e.
compound or double heterozygotes), detected in about 3-5% of genotype positive
patients, have a more severe phenotype and increased incidence of sudden death[78,
80, 81], suggesting a gene-dosage effect might contribute to disease severity.
Interestingly, in the majority of cases of compound heterozygosity, one of the
mutations usually involves MYBPC3[78]. In their longitudinal study, Olivotto et al.
observed a similar trend showing that patients with double mutations (of which 1 was
usually MYBPC3) had greater disease severity than myofilament negative patients or
patients with a single MYBPC3 -, thick filament – or thin filament mutation combined (p
< 0.05; Figure 3D).
145
Figure 3: Relation of genetic test status to outcome in patients with hypertrophic cardiomyopathy. Follow-up data shows that patients harboring a myofilament mutation (i.e. a positive genetic test) progress to CV Death, ischemic stroke or NYHA-Class III-IV more rapidly than patients with a negative genetic test (A). Furthermore, patients with a myofilament mutation are more likely to develop systolic dysfunction (B) or a restrictive filling pattern (C), independent of the genotype involved (D).
In summary, although clinical prognostication must be rendered with great
caution for specific gene domains or specific genetic mutations, a positive HCM
genetic test in general portends a greater likelihood for disease progression,
particularly as it pertains to systolic and diastolic dysfunction and propensity to develop
symptoms. As such, clinical genetic testing may thereby aid in the prognostication of a
patient’s disease outcome.
146
Interpretation of rare variants and phenocopies
One group of patients that pose an intriguing challenge for clinicians is that of patients
with seemingly unexplained LVH that mimics the HCM-phenotype. These diseases are
usually referred to as phenocopies and the most important ones are listed in Table 2. Phenocopies or rare variants pose a tough dilemma for the clinician. If the phenotype
does not look like typical HCM, other symptoms like ventricular pre-excitation or
muscle weakness are present, the presence of an underlying multi-system disease
should be considered and additional testing should be performed. On the other hand,
if myofilament genetic testing does not reveal an HCM-associated mutation, testing for
mutations in the metabolic genes can reveal that the LVH is the primary presentation
of a multi-system disease process.
A different cardiomyopathy and phenocopy that can present itself with
seemingly unexplained hypertrophy is that of left ventricular non-compaction (LVNC) –
a primary cardiomyopathy characterized by a severely thickened 2-layered
myocardium, numerous prominent trabeculations, and deep intertrabecular
recesses[82, 83]. Although genetically still largely unexplained, mutations in the TAZ-
encoded tafazzin (G4.5) – also associated with Barth syndrome –, DTNA-encoded -
dystrobrevin and LDB3 have been associated in the pathogenesis of LVNC[84, 85, 86].
Recently, Klaassen et al. systematically analyzed a cohort of 63 unrelated patients with
LVNC for mutations in 6 myofilament encoding genes, identifying nine distinct
heterozygous mutations in 11 patients in MYH7, ACTC and TNNT2[87] suggesting that
there might be a shared etiology for the myofilament forms of the common allelic
cardiomyopathies of HCM, DCM and LVNC.
Some diseases presenting chiefly with cardiac hypertrophy turn out to have
clearly distinct underlying pathophysiologies. In 2001, 2 independent groups
discovered PRKAG2 mutations being involved in families with cardiac hypertrophy and
ventricular pre-excitation, conduction abnormalities and signs of Wolff-Parkinson-White
(WPW) syndrome[88, 89]. In 2005, Arad et al. also described mutations in lysosome-
associated membrane protein-2 encoded by LAMP2 and PRKAG2 and found that
underlying glycogen storage diseases mimicked the clinical phenotype of HCM [88, 89,
90, 91]. Yang et al. showed that LAMP2 mutations may account for a significant
portion of patients diagnosed with pediatric- or juvenile onset HCM, especially when
skeletal myopathy and/or WPW are present[92].
147
Role of modifiers in HCM
The role of modifiers of the HCM phenotype, either by the presence of common
polymorphisms or founder-mutations, has become the subject of recent investigations.
The most important subgroup of polymorphisms, studied to date, involve the major
components of the renin- angiotensin-aldosterone system (RAAS). Polymorphisms in
the RAAS-pathway [angiotensionogen-I converting enzyme (ACE), angiotensin
receptor 1 (AGTR1), chymase 1 (CMA), angiotensin I (AGT) and cytochrome P450,
polypeptide 2 (CYP11B2)): DD-ACE, CC-AGTR1, AA-CMA, T174M- and M235T-AGT,
and CC-CYP11B2] appear to influence the HCM phenotype, in particular the severity
of LVH[93, 94]. Among patients with the DD-ACE genotype, there was greater LVH
than among those with an ID or II genotype[95]. Furthermore, a combined ‘pro-LVH’
profile of five RAAS-genes was associated with higher degree of LVH in one particular,
founder MYPBC3-HCM pedigree [93] and in a large cohort of myofilament positive
patients[94].
In 2008, sex hormone polymorphisms were shown to modify the HCM
phenotype(104). Fewer CAG repeats in AR-encoded androgen receptor were
associated with thicker myocardial walls in male subjects (p = 0.008) and male carriers
of the A-allele in the promoter of ESR1-encoded estrogen receptor 1 (SNP rs6915267)
exhibited a 11% decrease in LV wall thickness (p = 0.047) compared to GG-
homozygote male subjects[96]. HCM modifier polymorphisms like these could
contribute to the clinical differences observed between men and women with HCM[97,
98]. The release of the complete human genome sequence and the enormity of
variation in individuals show a growing role for modifier genes and the search for effect
by genome-wide studies. In 2007, Daw et al. performed the first study of this kind for
HCM and they identified multiple loci with suggestive linkage. Effect sizes on left
ventricular mass on this cohort of 100 patients ranged range from ~8g shift from one
locus for the common allele to 90g shift for another locus’ uncommon allele[99].
148
Therapeutic Implications of HCM Genetic Testing
Pharmacogenomics
Currently, there is no available therapy specifically designed to target specific HCM-
causing gene mutations or particular HCM genotypes. Further, no therapies have
been shown to reverse the hypertrophic process in humans. One of the first studies of
its kind was performed in transgenic MYH7-R403Q mice models (designated
MHC403/+). In a randomized trial MHC403/+ -mice treated with diltiazem, a L-type
calcium inhibitor, showed significant improvement as compared to mice treated with
placebo in terms of cardiac systolic function as measured by increased end-diastolic
and end systolic volumes, decreased dP/dTmax values and end-systolic
elastance[100]. Furthermore, diltiazem-treated mice showed significantly less
hypertrophy at 30 and 39 weeks than age-matched MHC403/+-untreated mice as
well as less fibrosis and myocyte disarray on microscopy[100]. Recently, Westermann
et al. showed in a different transgenic mouse model (TNNT2–I79N) – that diltiazem
improved diastolic function and prevented diastolic heart failure and sudden cardiac
death compared to untreated mice[101].
As previously discussed, RAAS polymorphisms modify the phenotype of HCM,
particularly MYBPC3-HCM[93, 94] and there is now growing evidence that ACE-
inhibitors especially combined with low doses of aldosterone receptor blockers may
attenuate the progression of hypertrophy and fibrosis [102, 103, 104, 105, 106]. In
early mouse-models of transgenic cardiac troponin T (cTnT-Q92) that exhibit myocyte
disarray and fibrosis, a randomized, blinded trial comparing losartan (an angiotensin-II
blocker) or placebo demonstrated that losartan significantly reversed fibrosis and
expression of collagen 1 (I) and TGF -1 in the transgenic mice[107]. In a similar
study involving the same transgenic mice, losartan produced a 50% reduction in
myocyte disarray compared to mice treated with placebo as well as complete
normalization of the collagen volume fraction[108].
149
Lastly, another group of drugs, statins, may favorably modify the phenotype of
hypertrophic cardiomyopathy. A study involving 24 transgenic mice harboring the
MYH7 R403Q-mutation showed a regression of hypertrophy and fibrosis, improved
cardiac function and reduced ERK1/2 activity after treatment with simvastatin
compared to 12 non-transgenic mice[109]. Similar results were observed in transgenic
rabbits with this mutation who were treated with atorvastatin[110]. However, a small,
randomized control pilot study failed to show an effect on humans with HCM[111].
Therefore, one can envision that, with increasing knowledge of the patient’s
pathogenic substrate and polymorphism profile, specific therapies may someday
emerge. In other cases for example, the proper and prompt recognition of an HCM
phenocopy such as cardiac Fabry’s disease can facilitate gene-specific
pharmacotherapy such as enzyme-replacement therapy. Albeit rare, such clinical
sleuthing can enable early treatment and prevent the progression of the disease.
Recently, human trials such as the “DELIGHT” (DiltiazEm Long-term In Genotype-
positive Hypertrophic cardiomyopathy as preclinical Treatment) trial have begun and
are examining whether calcium channel inhibitors like diltiazem can prevent the
development of hypertrophy among patients with genotype positive/LVH negative-
HCM.
150
Conclusions
Genomic medicine, as it pertains to HCM, has moved from the bench to the bedside,
but caution is needed to interpret and manage the genetic portfolio of a patient.
Although some prognostic forecasts may be gleaned from the HCM genetic test,
therapeutic decisions regarding use of a defibrillator should not be dictated by the
genetic test result. Instead, knowledge of the genetic background in subjects with HCM
has significant diagnostic implications and echocardiography may help guide genetic
testing by providing anticipatory guidance and a pre-test probability of a positive
genetic test result. Clearly, knowledge of disease-causing mutations in an index case
enables rapid genetic testing and diagnosis of potentially at-risk relatives thereby
providing improved and informed follow-up and treatment decisions for such family
members. The information gained in these subjects can define risk status and, in those
subjects with negative genetic screening, less close follow-up and testing over time
and psychological freedom.
Increasingly, clinical care in HCM and other genetic-based disorders includes
the wise use and wiser interpretation of genetic tests. Therefore, understanding the
genetic underpinnings of disease and the risk placed on these subjects will be
imperative for all patients and their families. The 21st century clinician must be
cognizant of the state-of-the-art of translational genetics in order to best care for their
patients and families, as well as to help to define new clinical guidelines over the next
decade.
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The Growing Field of Genetic Contributors to the Pathogenesis of Hypertrophic Cardiomyopathy
or
About ‘lumpers’ and ‘splitters’: McKusick revisited
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In 1969, Victor McKusick, a medical geneticist, wrote a paper entitled “On lumpers and splitters, or the nosology of genetic disease”[1]. In this paper, McKusick disclosed the two leading principles in genetic nosology (study of classification of disease): ‘pleiotropism’ (multiple effects of a single etiologic factor) and ‘genetic heterogeneity’ (existence of two or more fundamentally distinct entities with essentially one and the same phenotype). And, already in 1969, McKusick articulated that against man’s naturally tendency to ‘lumping’ (recognize similarities and lump a disease under one umbrella), geneticist are forced to be ‘splitters’ instead[1]. This 40-year old paper seems prophetic for the current state of genetics of hypertrophic cardiomyopathy (HCM). Characterized by not only profound genetic heterogeneity, but also vast phenotypic and pathological diversity, for years the field of HCM tried to fit this disease in one, two or more diagnostic corners and continuing genetic discoveries still raise the question whether we should still be ‘lumpers’ or ‘splitters’.
Clinically, since Teare and Brock first described HCM as ‘asymmetrical hypertrophy of the heart in young adults’ [2, 3], this disease has been known by many names, such as idiopathic hypertrophic subaortic stenosis[4], muscular subaortic stenosis[5] and hypertrophic obstructive cardiomyopathy[6]. It wasn’t until 1995 that it was agreed upon to ‘lump’ all these syndromes under one name[7]. Accordingly, HCM is now described as left and/or right ventricular hypertrophy, usually asymmetric and involving the interventricular septum with predominant autosomal dominant inheritance involving sarcomeric contractile proteins[7]. Pathologically, HCM has been characterized by microscopic features of cardiomyocyte hypertrophy, myofibrillar disarray and interstitial fibrosis, although recent research has shown an emerging role of myocardial ischemia, coronary microvascular dysfunction and myocardial bridging[8, 9]. And genetically, over 24 HCM-susceptibility genes have been elucidated with mutations found in genes encoding proteins of the cardiac myofilament, Z-disc and calcium-handling pathways as well as multiple genes involving syndromes mimicking the HCM phenotype [10]. Also, numerous HCM modifier genes have been identified [11, 12, 13].
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And lastly, based on previous studies and observations described in this thesis, we have seen a strong genotype-morphology correlation between reverse-curve HCM and the presence of a myofilament mutation[14] and subsequently observed that Z-disc HCM is predominantly sigmoidal[15], suggesting a strong link between ventricular septal morphology and HCM-substrate. With all these different observations and possible clinical, pathological, genetic or morphological groupings, we can ask ourselves whether we should start ‘splitting’ HCM into for example morphologic and genetic subgroups of HCM, or keep ‘lumping’ and focus our research back onto understanding a proposed ‘final common pathway’ for HCM[16, 17].
This thesis has uncovered some new insights in this disease with the discovery of novel HCM-associated genes located to the cardiac Z-disc (Chapter 2 and 3) [18, 19], a morphologic predilection for sigmoidal morphology and relationships between sex, shape, and genetic substrate (Chapters 4 and 5) [15, 20] as well as the discovery of a novel HCM-susceptibility gene (TIEG1) and possible biomarker (PTTG1) for HCM (Chapter 6). However, multiple challenges and research questions remain, whose answers will help us further understand the pathogenesis of this disease, the role of modifiers, epigenetic factors and environmental influences, and novel therapeutic strategies.
First of all, with 30-50% of HCM genetically unexplained, the number of HCM-associated genes is expected to rise either by candidate gene selection, linkage analysis of large families with unexplained HCM, or large genome wide association studies. It remains to be seen whether a gene explaining a large portion of HCM (like MYH7-HCM and MYBPC3-HCM) will be found, but it is a certainty that novel disease-associated genes will be discovered. However, with the mutations expected to be found at a low frequency, it will raise the question whether the variants are really pathogenic or just rare, innocuous amino-acid alterations. Before such variants are published as disease causing/contributing, it will call for increasingly stringent clinical, familial, functional and bioinformatics studies.
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With the 1000-genome project in its final stages cataloguing the full genetic code and its interpersonal variations of 1000 ostensibly healthy volunteers, a lot more knowledge on normal genetic variation will enter the public databases to help answer an important part of this question. But, especially in a fairly common disease like HCM, one still has to wonder: what is normal? Expressing sometimes at a later age, it can be envisioned that some people participating in the 1000-genome project were considered healthy upon entry in the study, but still develop hypertrophy and HCM later on in life. More than ever, especially in the current age of clinical genetic testing for HCM, it is important to separate the real from the rare. Research programs should be performing appropriate functional assays into possible pathogenic variants and genetic testing companies should be performing their due diligence determining the possible disease associated role of a finding (pathogenic or innocuous?) before reporting back to the clinician and patient.
Secondly, since most research thus far has been focused on translated portions of the genome, a huge part of our genetic code remains poorly understood from the standpoint of HCM pathogenesis. Promoter regions, splice sites, enhancers and other intronic functional and non-functional domains might end up playing an important role in the pathogenesis of many diseases including HCM. Next-generation sequencing techniques will make it easier to read much larger portions of the genome with the possibility to identify novel, disease associated variants located to potentially functional domains of the intronic genome (“introme’). Although putatively pathogenic, novel assays to prove its pathogenicity will have to be developed to add the satisfactory level of clinical significance to these variants. Also on the level of translation, post-translational processes and epigenetics (DNA methylation, chromatin remodeling, RNA transcription etc), a lot remains to be discovered. Not only just amino-acid altering variations or insertions/deletions might contribute to disease development, but also translational and posttranslational modifications that take place in the human cell.
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Steps in this direction have for example already been taken by the discovery and description of the role of microRNA’s (miR’s) in hypertrophic heart disease and their role as negative regulators of gene expression. MiR’s are small, non-coding RNA-molecules that inhibit translation or promote degradation of target mRNAs. Previous studies on two mouse models of pathological hypertrophy – transverse aortic constriction (TAC) and calcineurin transgenic mice – demonstrated 6 miR’s were up-regulated, which in vitro, were sufficient to induce hypertrophic growth of cardiomyocytes[21]. A transgenic mouse model over-expressing miR-195 showed that even a single miR could induce pathological hypertrophy and heart failure[21]. Multiple studies in vivo and in vitro, have since been published with miR expression profiles in different settings of cardiac hypertrophy demonstrating the potential role of miR’s and possible therapeutic target in (hypertrophic) heart disease and HCM [21, 22, 23, 24, 25, 26]. Comprehensive genetic analyses of the genes encoding these miRs or their respective target sites have yet to be performed in a large collection of patients with HCM. Also, no studies have yet demonstrated expression profiles of miRs in human HCM samples.
Thus far, no therapeutic option exists to treat, prevent or reverse cardiac hypertrophy in patients with genotype positive HCM or HCM in general, although animal studies do suggest that disease prevention/regression is a possibility. In a randomized trial, diltiazem (a L-type calcium channel blocker) treated MYH7-HCM transgenic mice showed significant improvement of cardiac systolic function, significantly less hypertrophy at 30 and 39 weeks as well as less fibrosis and myocyte disarray compared to untreated, transgenic mice [27]. These findings were replicated in a troponin T-HCM transgenic mouse model also [28]. Currently, human clinical trials are near completion of their first stages evaluating treatment with diltiazem and the progression of hypertrophy in genotype positive patients without signs of hypertrophy. Similar animal studies have been published showing that other drugs like ace-inhibitors, aldosterone receptor blockers, angiotensin-II blockers or statins might attenuate progression of hypertrophy, fibrosis and myofibrillar disarray in transgenic mouse models, but small human trials have been inconclusive or have yet to be finalized[29, 30, 31, 32, 33, 34, 35]. Prevention of hypertrophy or at least slowing its progression should change its natural history although it remains to be seen whether these or other drugs could also prevent the incidence of sudden cardiac death.
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Even with novel techniques, identification of novel putative pathogenic variants in unexplored regions or discovery of novel pathways, it’s possible that a subset of patients will remain without a genetic explanation of their disease. Furthermore, as HCM is characterized by profound heterogeneity, even within patients of families with the same genetic background, the genetic, epigenetic, environmental basis for incomplete penetrance and variable expressivity remains poorly understood. Already demonstrated by previous modifier and gender studies, one can envision that certain intrinsic or environmental factors such as gender, (low-grade) hypertension, race or smoking amongst others, trigger one of the pro-hypertrophic pathways leading to a disease diagnosed by clinicians as HCM, but studies have yet to study and identify specific environmental factors that influence the disease.
Lastly, research can be focused on understanding the pathogenic pathways of cardiomyopathies as allelic diseases. Particularly when one looks at the HCM-associated genes encoding Z-disc proteins, it is striking that most are also associated in the pathogenesis of DCM with both cardiomyopathic phenotypes sometimes seen in the same family [36, 37, 38, 39, 40, 41, 42, 43, 44]. This poses the question how mutations in one gene can lead to two divergent compensatory responses and whether these pathways are induced by the underlying genotype or that exogenous factors determine whether one develops HCM or DCM. Looking at the role of Z-disc genes in the heart and functional capacities in the stretch-sensor and mechanoreceptor function of the heart, one can see where pathways of possible stretch-induced hypertrophy and dilatation of these diseases overlap, but studies are still needed to dissect the exact pathways whereby these divergent diseases develop. Not only are similar genotypic background seen between HCM and DCM; recently for example sarcomeric genes have been associated in the pathogenesis of left ventricular non-compaction (LVNC) [45] and restrictive cardiomyopathy (RCM)[45, 46]. Presently, the McKusick ‘pleiotropism’ encompasses 4 distinct cardiomyopathies capable of emanating from sarcomeric perturbations.
Thus, in a time where we have gained more and more knowledge on the extent and breadth of clinical, pathological, genetic and morphological heterogeneity of HCM and have witnessed the expansion of cardiomyopathic pleiotropism, perhaps we should heed McKusick’s 40-year old admonition to resist the urge to lump and be a ‘splitter’ instead.
167
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Summary
172
Hypertrophic cardiomyopathy (HCM) is a disease characterized phenotypically by
unexplained left ventricular hypertrophy in the absence of an underlying cause. With a
prevalence of 1 in 500 individuals, HCM is the most common heritable cardiac disease
and the most common cause of sudden cardiac death in young adults, especially
athletes. Pathologically, HCM is characterized by microscopic cardiomyocyte
hypertrophy, myofibrillar disarray and focal, interstitial fibrosis. Genetically, HCM was
considered a disease of the sarcomere, or more specifically the cardiac myofilaments,
with most mutations thus far found in genes encoding cardiac myofilaments. However,
among different international cohorts, the yield of genetic testing for these genes, now
equivalent to commercial genetic tests, ranged between 30 and 70%. More recently, it
was demonstrated that there was a strong correlation between ventricular septal
morphology and the underlying genotype with 80% of reverse-curve HCM being
genotype positive and only 8% of sigmoidal-HCM harboring a pathogenic mutation.
Based on these findings, we set out to study the genetic basis of genotype negative
HCM and study the genotype-phenotype correlations of newly discovered HCM-
susceptibility genes.
Chapter 1 of this thesis is a general introduction on HCM which briefly reviews
the clinical and genetic history of HCM. Furthermore, the state of genetics of HCM in
2007 and recent observation of correlation between septal contour and underlying
genetic substrate are discussed. It also sets the stage for most chapters in this thesis
and the discussion whether HCM is one disease or maybe a disease that can be
categorized in various clinical, genetic or morphological subgroups.
Chapter 2 describes the discovery and subsequent genotype-phenotype
analyses of mutations in three HCM-susceptibility genes that encode key
cytoskeletal/scaffolding proteins of the Z-disc. As a large percentage of patients
remained without genetic explanation for their disease, the research field moved from
the myofilament proteins of the sarcomere to the sarcomere’s Z-disc and its vast array
of proteins as mutations had previously been published in dilated cardiomyopathy
(DCM) or animal models supported a role for certain genes in the pathogenesis in
HCM. We discovered that 4% of unrelated patients with HCM harbored mutations in
CSRP3-encoded muscle LIM protein (MLP) and TCAP-encoded telethonin (TCAP).
Phenotypically these patients mirrored the phenotype seen in myofilament-HCM and
patients were more affected than patients who continued to be genetically elusive.
173
Chapter 3 provides the first description of mutations in ANKRD1-encoded
ankyrin repeat domain 1 (ANKRD1) (also known as cardiac ankyrin repeat protein
(CARP)) associated with HCM. In this collaborative effort with our colleagues in Japan
led by Dr. Akinori Kimura, we describe the discovery of 3 novel mutations in ANKRD1
in two independent cohorts of unrelated patients with HCM. Furthermore, two
mutations were found in the N2A-domain of the large sarcomere-stretching protein titin
encoded by TTN. Functional analyses of these mutations demonstrated increased
binding of titin to ANKRD1 as well as altered localization of ANKRD1-mutants in
cardiomyocytes. Compared to wild type ANKRD1, which localizes to the striated
pattern at the Z-I-bands, mutant ANKRD1 showed increased localization within or at
nuclear membrane in approximately 60% of mature cardiomyocytes.
Chapters 4 and 5 describe genotype-phenotype correlations between the
previously discovered, novel Z-disc associated HCM mutations and their relationship to
sex (Chapter 5) and/or their underlying genotypic substrate. Akin to the strong
correlations between reverse curve-HCM and the presence of a myofilament mutation,
we found that Z-disc HCM is preferentially sigmoidal. Described in Chapter 4, we found
that among the 13 patients with Z-disc HCM (including mutations in the novel HCM-
associated gene alpha-actinin 2 encoded by ACTN2), 85% had a sigmoidal septal
contour. None of the patients demonstrated the myofilament-associated reverse septal
morphology. These findings led us to speculate that the pathogenic mechanism behind
Z-disc HCM might predispose patient to the more sigmoidal septal bulge at the left
ventricular outflow tract.
Chapter 5 focuses on this same subset of patients, but specifically focuses on
the sex-related differences within this cohort. In a previous study involving genetically
uncharacterized patients with HCM from the USA and Italy, women i) were older and
more symptomatic at diagnosis, ii) had more left ventricular outflow tract obstruction,
and iii) were more likely to progress to advanced heart failure and stroke. In our study,
we performed a similar analysis on patients that were genotyped for mutations in the
myofilament genes and scored for septal morphology on echocardiography. Similar to
previous studies, we found striking differences between men and women, but these
differences were confined largely to the subgroup of women with mutation-negative,
sigmoidal HCM.
174
Multiple and linear regression models demonstrated that, for women, age at
diagnosis, systolic blood pressure and presence of left ventricular outflow tract
obstruction were directly dependent on sigmoidal morphology. These observations
demonstrated that whereas mutations within the sarcomere appear to dominate the
disease process, in their absence, sex has a significant modifying effect among
patients with genotype negative, sigmoidal HCM.
In an effort to further explain genotype negative HCM, we subsequently moved
away from the cardiac Z-disc to analyze a novel candidate gene, which in knock-out
mice showed late onset HCM and a distinct gender predilection. Described in Chapter
6 is the discovery of 6 novel HCM-associated mutations in TIEG1-encoded TGF -
inducible early gene-1 in two independent cohorts of unrelated patients with HCM from
the Academic Medical Center (Amsterdam, NL) and the Mayo Clinic (Rochester, MN
USA). Subsequent in vitro studies showed that, akin to data from the TIEG1 knock-out
mouse, 5 out of 6 TIEG1-mutations significantly altered TIEG1 function on the PTTG1-
promoter resulting in a significant upregulation of PTTG1-promoter activity.
Immunohistochemistry analyses showed increased PTTG1-protein staining in HCM
patients in general and even more in patients with TIEG1-HCM suggesting that up-
regulation of PTTG1 might be a final common pathway in HCM and a potential disease
biomarker.
Chapter 7 is a recent review article describing the diagnostic, prognostic and
therapeutic implications of genetic testing for HCM. Besides the past and present state
of HCM genetics, it describes how genetic testing has now moved from the laboratory
bench into the physician’s hands at the patient’s bedside. It provides physicians an
algorithm on when to consider genetic testing and discusses guidelines for screening
family members of patients with HCM. It also discussed the particular situation of
screening children or athletes related to patients with HCM. Lastly, it provides insights
in the current and future options for patients with genotyped HCM and examines where
research is making strides to delineate the underpinnings of this disease.
175
In conclusion, the understanding of HCM has matured from its cornerstone as a
disease of the sarcomere to a compendium of diseases with various clinical, genetic
and morphologic substrates. Research has provided us more insights into i) the
pathogenetic development of HCM, ii) the possible pro-hypertrophic pathways that are
triggered, and iii) the modifiers that influence the disease phenotypes. These findings
have opened the door for individualized medicine and the development of therapeutic
trials aimed at disease prevention or slowing of disease progression in patients with
genotype positive, hypertrophy negative HCM. However, far more research is needed
to understand its exact pathogenic pathways, discover novel drug targets or
understand and prevent its devastating feature of sudden cardiac death.
176
Samenvatting
178
Hypertrofische cardiomyopathie (HCM) is een aandoening gekarakteriseerd door
hypertrofie van de linker ventrikel in de afwezigheid van een aanwijsbare,
onderliggende oorzaak. Met een prevalentie van 1 op 500 is het de meest
voorkomende, erfelijke hartaandoening en de belangrijkste oorzaak voor plotselinge
hartdood in jong volwassenen, voornamelijk atleten. Karakteristieke pathologische
veranderingen zijn hypertrofie van de cardiomyocyt, disorganizatie van de myofibrillen
en focale, interstitiele fibrose. Op genetisch vlak werd HCM gezien als een ziekte van
de sarcomeer, waarin de meeste mutaties tot nu toe zijn gevonden in genen die
coderen voor eiwitten in het contractiele apparaat van de hartspiercel. Echter, meta-
analyses lieten zien dat de frequentie van mutaties in verschillende cohorten lag
tussen de 30 en 70%. Recent onderzoek toonde een sterke correlatie tussen de vorm
van het interventriculare septum van het hart (het “fenotype”) en het onderliggende
genotype; 80% van de patiënten met septum hypertrofie, waarbij hypertrofie de linker
hartkamer inbuigt (reverse curve HCM) had een mutatie, in tegenstelling tot slechts 8%
van de patiënten met hypertrofie rond het uitstroom traject van de linker ventrikel
(sigmoïdal-HCM). Gebaseerd op deze bevindingen, besloten we het onderzoek te
richten op de genetische basis van HCM in patienten waarbij geen afwijking in
sarcomeer genen werd gevonden en genotype-fenotype correlaties van nieuw
ontdekte genen.
Hoofdstuk 1 is een algemene introductie in HCM, waarin de klinische en
genetische geschiedenis van HCM worden besproken. Verder wordt de status van de
genetica van HCM in 2007 besproken alswel de recente observatie van een sterke
correlatie tussen de vorm (de morfologie) van het septum en het onderliggende
genetische substraat. Daarbij is het een opzet voor de meeste hoofdstukken van dit
proefschrift en de discussie of HCM één ziekte is dan wel een ziekte die onderverdeeld
zou moeten worden in verschillende klinische, genetische en/of morfologische
subgroepen.
179
In hoofdstuk 2 worden de ontdekkking en genotype-fenotype relaties
besproken van mutaties in 3 Z-disc geassocieerde genen. Aangezien na analyse van
de sarcomeer genen in een groot gedeelte van HCM patiënten geen mutatie werd
gevonden werd de aandacht gericht op de aanliggende elementen van de Z-disc;
reeds eerder waren mutaties gevonden in dilaterende cardiomyopathie (DCM) en
verscheidende diermodellen onderschreven een potentiele rol van de Z-disc genen in
de pathogenese van HCM. In ons onderzoek beschrijven we de ontdekking dat 4%
van patiënten met HCM een mutatie heeft in de Z-lijn eiwitten muscle LIM-protein
(MLP, gecodeerd door CSRP3) en telethonin (gecodeerd door TCAP). Fenotypisch
gezien leken patiënten met een Z-disc mutatie op patiënten met een mutatie in een
van de myofilament genen en waren ze meer aangedaan dan patiënten zonder HCM
mutatie.
Hoofsdtuk 3 beschrijft de ontdekking van mutaties in Ankyrin repeat domain 1
(gecodeerd door ANKRD1) in patiënten met HCM. In samenwerking met collega’s in
Japan geleid door Dr. Akinora Kimura beschrijven we de ontdekking van 3 nieuwe
mutaties in ANKRD1 in twee onafhankelijke cohorten van HCM-patiënten. Daarnaast
werden twee mutaties gevonden in het N2A-domein van het zeer grote, sarcomeer
eiwit titin (gecodeerd foor het gen TTN). Functionele studies van deze mutaties
vertoonden toegenomen binding van titin en ANKRD1 alswel dislocalisatie van
ANKRD1-mutanten in cardiomyocyten. In tegenstelling tot normaal ANKRD1,
normaalgesproken gelocaliseerd in de Z-I band van de sarcomeer, bleek gemuteerd
ANKRD1 gelocaliseerd te zijn in of rond het membraan van de nucleus in ongeveer
60% van volwassen cardiomyocyten.
In hoofdstuk 4 en 5 worden de genotype-fenotype relaties beschreven tussen
eerder ontdekte, myofilament mutaties en de relatie tot sexe (hoofdstuk 5) en/of de
onderliggende morfologische vorm van het interventriculaire septum. Gelijk aan de
correlatie gezien tussen reverse-curve HCM en de aanwezigheid van een myofilament
mutatie, bleek na analyse Z-disc HCM voornamelijk sigmoïd van vorm te zijn. Van de
13 patiënten met een mutatie in een van de Z-disc genen, bleek 85% een sigmoïdale
contour op echo te hebben; geen van de patiënten had tekenen van reverse-curve
HCM. Deze bevindingen leidden ons tot de speculatie dat het pathogenetische
mechanisme van Z-disc patiënten preferentieel sigmoïdale hypertrofie veroorzaakt in
het uitstroom traject van de linker ventrikel.
180
Hoofdstuk 5 richt zich op dezelfde subgroep, met een speciaal focus op sexe-
verschillen tussen de patiënten. Een eerder gepubliceerde studie op patiënten zonder
bekend genotype met HCM uit de VS en Italie liet zien dat vrouwen over het algemeen
ouder waren bij hun diagnose met HCM, meer obstructie hadden van het linker
ventriculaire uitstroom traject en vaker hartfalen ontwikkelden. Voor onze studie
ondernamen we een soortgelijke analyse op patiënten met HCM, gegenotypeerd voor
mutaties in de sarcomeer genen en gescoord voor de morfologsiche vorm van het
septum. Gelijk aan de voorgaande resulaten zagen wij significante verschillen tussen
mannen en vrouwen, maar verschillen werden alleen gezien in de subgoup van
vrouwen met mutatie-negatief, sigmoïd HCM. Multi en lineair statistische regressie
modellen wezen uit dat, voor vrouwen, diagnose leeftijd, systolische bloeddruk en
aanwezigheid van linker ventrikel uitstroom obstructie direct afhankelijk waren van
sigmoïde hypertrofie. Deze bevindingen demonstreerden dat in HCM patiënten met
een myofilament mutatie de mutatie het ziekteproces lijkt te domineren, en dat in
afwezigheid van een mutatie, sexe een grote invloed lijkt te hebben op het fenotype
van HCM.
In een poging om genotype negatief HCM verder te verklaren, ondernamen we
vervolgens een stap weg van de Z-disc om een nieuw kandidaat gen te bestuderen,
dat in knock-out muizen een beeld liet zien van HCM met een specifieke voorkeur
voor mannetjes muizen. In hoofdstuk 6 beschrijven we de ontdekking van 6 nieuwe
HCM-mutaties in TIEG1 (TGF -inducible early gene-1 gecodeerd door het gen TIEG1)
in twee onafhankelijke cohorten van HCM patiënten van het Academisch Medisch
Centrum in Amsterdam (AMC) en de Mayo Clinic in Rochester, MN in de Verenigde
Staten. In vitro analyses van de gevonden mutaties, liet, gelijk aan studies in de knock-
out muizen, een modificatie van TIEG1 zien op de promoter van PTTG1 uitschrijven
svp voor 5 van de 6 gekloonde mutaties resulterend in een significante overexpressie
van de PTTG1-promoter activiteit. Immunohistochemische analyse van hartweefsel liet
een toename zien van PTTG1-eiwit expressie zien in HCM-patiënten en zelfs meer in
patiënten met een TIEG1-mutatie, aanwijzingen dat PTTG1 wellicht een biomarker is
voor HCM.
181
Hoofdstuk 7 is een recent review artikel waarin de diagnostische,
prognostische en therapeutische implicaties voor genetisch testen in HCM worden
besproken. Naast de geschiedenis en huidige stand van zaken in genetica van HCM,
wordt besproken hoe genetisch testen voor HCM zich heeft verplaatst van het
laboratorium naar de handen van de arts aan het bed van de patiënt. Het geeft artsen
een algoritme dat tot hulp is in de keuze voor genetisch testen en bespreekt de huidige
richtlijnen voor screenen van familieleden van patiënten met HCM; screening van
kinderen en athleten worden tevens besproken. Het biedt inzichten in de huidige en
toekomstige therapeutische opties voor patiënten met een HCM-mutatie en beschrijft
de meest recente ontwikkelingen in het onderzoek omtrent HCM.
Concluderend, de genetische basis van HCM heeft zich de laatste jaren
uitgebreid van een ziekte van de sarcomeer naar een ziekte met verscheidene
klinische, genetische en morfologische verschijningsvormen. Onderzoek heeft ons
nieuwe inzichten gegeven in de pathogenetische ontwikkeling van de ziekte, mogelijke
pathways die aangezet worden en identificatie van modificerende factoren die de
verschillen fenotypes zouden kunnen beinvloeden. Deze bevindingen hebben de deur
geopend naar patiënt-gerichte geneeskunde en de ontwikkeling van trials gericht op
preventie of remming van ziekte progressie in genotype positieve patiënten zonder
hypertrofie. Meer onderzoek is echter nodig om te exacte pathogenetische aspecten te
ontcijferen, nieuwe targets te vinden voor medicijnen en naar het voorkomen van
plotselinge hartdood.
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List of publications
188
Peer reviewed publications
1. Bos JM, Poley RN, Ny M, Tester DJ, Xu X, Vatta M, Towbin JA, Gersh BJ, Ommen SR, Ackerman MJ. Genotype-phenotype relationships involving hypertrophic cardiomyopathy-associated mutations in titin, muscle LIM protein, and telethonin. Molecular Genetics and Metabolism 2006; 88(1): 78 – 85.
2. Bos JM, Hagler DJ, Silvilairat S, Cabalka A, O’Leary P, Daniels O, Miller FA, Abraham TP. Right ventricular function in asymptomatic individuals with a systemic right ventricle. Journal of the American Society of Echocardiography 2006; 19(8): 1033 – 1037.
3. Theis JL*, Bos JM*, Bartleson VB, Will ML, Binder J, Vatta M, Towbin JA, Gersh BJ, Ommen SR, Ackerman MJ. Echocardiographic-determined septal morphology in Z-disc hypertrophic cardiomyopathy. Biochemical and Biophysical
Research Communications 2006; 351(4): 896 – 902 (*co-equal first author).
4. Landstrom AP, Weisleder N, Batalden KB, Bos JM, Tester DJ, Ommen SR, Wehrens XH, Claycomb WC, Ko JK, Hwang M, Pan Z, Ma J, Ackerman MJ. Mutations in JPH2-encoded junctophilin-2 associated with hypertrophic cardiomyopathy. Journal
of Molecular and Cellular Cardiololgy 2007; 42(6): 1026 – 1035
5. Pandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, Martinelli S, Pogna EA, Schackwitz W, Ustaszewska A, Landstrom AP, Bos JM, Ommen SR, Esposito G, Lepri F, Faul C, Mundel P, Siguero JPL, Tenconi R, Selicorni A, Rossi C, Mazzanti L, Torrente I, Marino B, Digilio MC, Zampino G, Ackerman MJ, Dallapiccola B, Tartaglia M, Gelb BD. Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nature Genetics 2007; 39(8): 1007 – 1012.
6. Olivotto I, Girolami F, Ackerman MJ, Nistri S, Bos JM, Zachara E, Ommen SR, Theis JL, Vaubel RA, Re F, Armantano C, Poggessi C, Torricelli F, Cecchi F. Myofilament protein gene mutation screening and outcome in hypertrophic cardiomyopathy. Mayo Clinic Proceedings 2008; 83(6): 630 – 638.
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7. Bos JM, Theis JL, Tajik AJ, Gersh BJ, Ommen SR, Ackerman MJ. Relationship between sex, shape and substrate in hypertrophic cardiomyopathy. American Heart Journal 2008; 155: 1128 – 1134.
8. Landstrom AP, Parvatiyar MS, Pinto JR, Marquardt ML, Bos JM, Ommen SR, Potter JD, Ackerman MJ. Defining TNNC1-Encoded Cardiac Troponin C as Novel Target Gene for Hypertrophic Cardiomyopathy. Journal of Molecular and Cellular
Cardiology. 2008; 45(2):281 – 288.
9. Maron MS, Finley JJ, Bos JM, Hauser TH, Manning WJ, Haas TS, Lesser JR, Udelson JE, Ackerman MJ, Maron BJ. Prevalence, clinical significance, and natural history of left ventricular apical aneurysms in hypertrophic cardiomyopathy. Circulation.
2008; 118(15): 1541 – 1549.
10. Arimura T*, Bos JM*, Sato A, Kubo T, Okamoto H, Nishi H, Harada H, Koga Y, Moulik M, Doi Y, Towbin JA, Ackerman MJ, Kimura A. Cardiac ankyrin repeat protein gene (ANKRD1) mutations in hypertrophic cardiomyopathy. Journal of the American
College of Cardiology 2009; 54(4): 334 – 342 (*co-equal first author).
11. Theis JL, Bos JM, Theis JD, Miller DV, Dearani JA, Schaff HV, Gersh BJ, Ommen SR, Moss RL, Ackerman MJ. Expression Patterns of Cardiac Myofilament Proteins – Genomic and Protein Analysis of Surgical Myectomy Tissue from Patients with Obstructive Hypertrophic Cardiomyopathy. Circulation: Heart Failure 2009; 2: 325 – 333.
12. Rubinshtein R, Glockner JF, Ommen SR, Araoz PA, Ackerman MJ, Sorajja P, Bos JM, Tajik AJ, Valeti US, Nishimura RA, Gersh BJ. Characteristics and Clinical Significance of Late Gadolinium Enhancement by Contrast-Enhanced Magnetic Resonance Imaging in Patients with Hypertrophic Cardiomyopathy. Circulation: Heart
Failure. 2009 Oct 22. [Epub ahead of print]
13. McLeod CJ, Bos JM, Theis JL, Edwards WD, Gersh BJ, Ommen SR, Ackerman MJ. Histologic characterization of hypertrophic cardiomyopathy with and without myofilament mutations. American Heart Journal. 2009; 158(5): 799 – 805.
190
Editorial comments, review articles
1. Ackerman MJ, Van Driest SL, Bos M. Are longitudinal, natural history studies the next step in genotype-phenotype translational genomics in hypertrophic cardiomyopathy? Journal of the American College of Cardiology 2005; 46(9): 1744 – 1746 [Editorial comment].
2. Bos JM, Ommen SR, Ackerman MJ. Genetics of hypertrophic cardiomyopathy: one, two, or more diseases? Current Opinion in Cardiology 2007; 22(3): 193 – 199 [Review].
3. Bos JM, Towbin JA, Ackerman MJ. Diagnostic, Prognostic and Therapeutic Implications of Genetic Testing for Hypertrophic Cardiomyopathy. Journal of the
American College of Cardiology 2009; 54(3): 201 – 211 [Review].
Book Chapters
1. Menon SC, Bos JM, Ommen SR, Ackerman MJ. Arrhythmogenic Malignancies in Hypertrophic Cardiomyopathy. In: Electrical Diseases of the Heart: Genetics, Mechanisms, Treatment, Prevention (Chapter 40). Springer 2007. ISBN: 978-1846288531.
2. Bos JM, Ommen SR, Ackerman MJ. Hypertrophic Cardiomyopathy in the Era of Genomic Medicine. In: Genomic and Personalized Medicine (Chapter 61).Academic Press 2008. ISBN: 978-0123694201.
3. Bos JM, Ommen SR, Ackerman MJ. Hypertrophic Cardiomyopathy in the Era of Genomic Medicine. In: Essential Genomic and Personalized Medicine (Chapter 28).Academic Press 2009. ISBN: 978-0123749345.
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