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RYR1 and TTN: The challenge of giant genes implicated in Centronuclear Myopathy (CNM)
Heinz Jungbluth MD PhD & Mathias Gautel MD PhD 1Department of Paediatric Neurology, Neuromuscular Service, Evelina Children’s Hospital, London;
2Randall Division for Cell and Molecular Biophysics, Muscle Signalling Section, and3Clinical Neuroscience Division, IoPPN, King’s College London
ZNM Myotubular Trust European Family Conference, London, July 14th , 2018
Repairing an engine…
Striated muscles are formed
from long chains of sarcomeres.
Sarcomeres are giant, regular protein complexes
Sarcomeres: the most precisely assembled machines in biology
skeletal cardiac
Braun & Gautel, Nat Rev Mol Cell Biol 2011
Sarcomeres: smallest contractile units of skeletal and heart muscle
What is Big?
Titin molecules are over 1 µm long and slender
Single titindimersSingle myosin
Single titinmonomers
Anti-Titin Z-disk
Anti-Titin M-band
1 µm Klaus Weber Dieter Fürst
TTN intron-exon structure and domains
Bang M.-L. et al., Circ Res 2001
364 exons, 169 Ig domains, 132 fibronectin domains, 1 kinase, “other stuff”
TTN splice variants Big, and very big.
Chauveau et al., Human Mutation 2014
Titin/connectin – ruler of the sarcomeredomain layout of cardiac titin isoforms & known interactions
• Telethonin / T-cap (MLP, Myostatin, Calsarcins)
• sAnkyrin (Telethonin, RyR)
• α-Actinin(MLP. Actin)
• Obscurin (sAnk, Novex3)
• Molecular Ruler
I-bandZ-disk A-band M-band
• αB-crystallin • DRAL/FHL-2
(MM-CK, AK, PFK, β-Catenin, AR, ...)
• MARP (Ankrd2, CARP, DARP),• Myopalladin• Calpain-1, calpain-3• Smyd2/HSP90• Protein kinases A, G
• I-band= Spring, Molecular Ruler
• Myosin• MyBP-C• MyBP-H
• Molecular Ruler
• Myomesin,M-Protein
• DRAL/FHL-2 (MM-CK, AK, PFK, β-Catenin, AR, ...)
• p94/calpain-3• Nbr1 (p62, MuRF)
• Obscurin• MURFs• Myospryn
I15
NH2
I1 N2-B I20
I28 I29
I58-I61
I68 - I79
I55
I58
I59
N2-A PEVK
COOH
(cardiac N2B)
(cardiac N2A + N2B)
M1is2 M10
TK
Ig-like
FN3-like
Insertion Sequence
Kinase
MW > 3000 kDa Gene > 300 kb
117
Z-repeatsZ1
21
364 exons 169 Ig domains 132 fibronectin domains
Titin: a highly modular giant protein
multifunctional
modularBigdynamicelastic
Too big to fail…?
Titin mutations: Frequent cause of myopathies
Stop-gained and missense mutations
• LGMD2J, TMD (rec/dom, (Ig169/M10)
• Other distal myopathies
• Salih Myopathy (rec., C-term truncating)
• Hereditary Myopathy with Early Respiratory Failure, HMERF (dom, Fn119)
• Autosomal Recessive Multi-minicore Disease with Heart Disease (AR MmD-HD) (rec. missense/truncating, compound-heterozygous)
• Centronuclear Myopathy, CNM with/without cardiomyopathy (rec. truncating)
• Left ventricular compaction, LVNC (dom?, Ig2/Z2)
• Dilated cardiomyopathy, DCM (rec/dom? Truncating, mostly A-band) Peripartum cardiomyopathy
• Hypertrophic cardiomyopathy, HCM (rec/dom? Missense, mostly A-band)
Disease entities linked to titin
Titin truncating mutations Frequent in dilated cardiomyopathies
Titin variants in 1000 genomes (all)
Status May 2016
1000 genomes….
A glimpse on human evolution
goodbad…ugly…
How can we discriminate them?
0
2
5
7
9
0 1993 3985 5978 7970 9963 11956 13948 15941 17933 19926 21919 23911 25904
Z I I A M
N2B
PEVK
Residues (in 1000)
Incidence
Kinase
HCM EOM HMERF
Titin missense variants cause a wide variety of diseases
TMD/LGMD2J
Chauveau et al., HMG 2014
A recurrent, recessive TK mutation leading to myopathy: W34072R
Compound heterozygous, truncation after Z-disk (maternal), missense in kinase (paternal). No FH Cardiomyopathy, transplantation at age 3, skeletal myopathy.
New case (A. Ferreiro, Paris): Compound heterozygous, truncation in skeletal I-band (paternal), W34072R missense in kinase (maternal). No FH No cardiomyopathy, only skeletal myopathy.
W34072R is a recurrent, recessive pathogenic mutation in TK, a constitutively expressed domain.
With Ana Ferreiro, Carsten Bönnemann
A recessive TK mutation leading to myopathy: W34072R
Soluble, monomeric, no aggregation
Chauveau et al., HMG 2014
Computational models of TTN Fn3 domains are too inaccurate for mutation impact analysis
(iTasser)
Experimental structures and homology-based structural models required for accurate impact prediction
SG Tm: 54°C
WT Tm: 64°C
A63 WT (1.65Å)A63 SG (1.43Å)
A63 WT A63 SG
Ser Gly
Temperature (°C)
Rel
ativ
e Fl
uore
scen
ce
D zone C zoneI / A
1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11A63
serine side chain forms H-bond with valine.
S14141G variant destabilises A63 domain
Common Titin missense variants are not destabilising
D zone C zoneI / A
1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11A81 A110
RC:62°C
WT:62°C
WT:62°C
IV:63°C
A81 A110
Temperature (°C)
Rel
ativ
e Fl
uore
scen
ce
Temperature (°C)
TITINdb, a webtool to assess titinopathy-linked missense variants
Laddach, A., Gautel, M., and Fraternali, F., TITINdb-a computational tool to assess titin's role as a disease gene. Bioinformatics, 2017. 33(21): p. 3482-3485.
-3
-2.25
-1.5
-0.75
0
-40 -30 -20 -10 0 10
Titinopathy-linked missense variants destabilise domains
mCSM
ΔTm
In early-onset myopathies and TMD, TTN mutations strongly destabilise the domain by more than 15 degrees.
> Established semi-automated pipeline for mutation assessment
TMD, Bel Mutation, impact questioned
Neutral, R24947C
Neutral, I27775V
unfolded
Myopathies caused by misfolded toxic proteins
Desminopathies desmin Crystallinopathies αB-crystallin Myofibrillar myopathies BAG3, filamin-C centronuclear myopathies titin Hypertrophic cardiomyopathy many; titin? Dilated cardiomyopathy many; titin?
Neurodegeneration by misfolded toxic proteins
Tipping, K.W., P. van Oosten-Hawle, E.W. Hewitt, and S.E. Radford Trends Biochem Sci, 2015. 40: 719-27.
Conclusions
• TTN is the major scaffold of the sarcomere and a major gene for hereditary myopathies
• Recessive missense variants can become dominant in compound heterozygous settings when expressed with recessive truncating variants
• Discriminate bad from neutral and good?
• Truncating as well as destabilising missense variants frequent in “normal” population
• Accurate assessment of missense variants requires a rational assessment strategy including structural and functional data.
• Destabilising mutations can be recessive or dominant
• TITINdb designed to help with initial assessment of TTN missense variants
King’s College London Alexander AlexandrovichBirgit BrandmeierAtsushi FukuzawaAndrea GhisleniMark HoltKatharina JennichesAy Lin KhoEva MasierioRoksana NikoopourJing QiMartin ReesLuke SmithJessica Stuart
Anna LaddachFranca Fraternali
Mark Pfuhl
Stefano PernigoRoberto Steiner
Sergi Garcia-Manyes
King’s BHF Centre for Research Excellence
Göttingen UniversityJing QiKaomei Guan
Max-Planck-Institute DortmundStefan Raunser
Max-Perutz-Labs ViennaKristina Djinovic-Carugo
EMBL HamburgMatthias Wilmanns
Oxford UniversityKatja GehmlichHugh Watkins
University CologneMarcus Krüger
UKE HamburgLucie Carrier
UCL/Bart’s Luis LopesPetros SyrrisPerry Elliot
UCL ICH/GOSH Francesco Muntoni
Evelina London Children’s Hospital/ Tom Cullup, Heinz JungbluthGSTT Gerald Carr-White
West of Scotland Regional Genetics Glasgow Cheryl Longman
Université Paris Diderot-CNRS Claire ChauveauVirginie CarmignacAna Ferreiro
NIH Bethesda Carsten Bönnemann
University Helsinki Peter HackmanAnna ViholaBjarne Udd
Cologne University Hospital Sebahattin Cirhak
Göttingen University Hospital Elke HobbiebrunkenGabriele DekomienEkkehard Wilichowski
Radboud University Medical Centre Nicol VoermansNijmegen
Hôpital des Enfants Joël FlussGenève
Titin mutations in myopathies: open questions
• Why do truncating mutations mostly appear to be silent?
• Do missense mutations lead to phenotypic penetrance of truncating mutations, or vice versa?
• Do recessive missense mutations cause similar phenotypes in compound heterozygosity or homozygosity?
• What is the cellular fate of truncated titin? • What is the cellular fate of missense titin?
• Is perturbed protein homeostasis a common denominator?
• How does perturbed proteostasis impact on myocyte function?
Sarcomeric M-band links to protein quality control Disease implications
Fukuzawa et al., JCS 2008
Titin
Obsl1
Obscurin
Titin
Myomesin
Nbr1
Cullin-7
MURFs myospryn
SQSTM1
COP9
Calpain-3
TK mutations in HCM, specific myopathies
LGMD2A
LGMD2J, Salih myopathy
• Titin, a molecular ruler – blueprint to determine the length of myosin filaments,
the length of the I-band, and the thickness of the Z-disk
• Titin, a molecular spring – maintains myosin filaments centrally in the sarcomere – Ensures balanced forces between both halves of
sarcomere
• Titin, a “signal transducer” – Titin organises numerous cellular communication
pathways
Titin: a multitasking giant The biggest protein in the human body
myomesin
α-actinin
myosin filaments
M ZZ I IAA
NH2 COOHTitin
actin filaments
NH2
titin
COOH
The sarcomere and regulators of sarcomeric turnover
} }}MyBP-C
ZASP/cypher
calsarcin/myozenin/FATZNbr1/SQSTM1
Obscurin/Obsl1 *
myotilin
MURF1/2/3
calpain-3
MLP
BAG3
γ-filaminCapZ
Fbxl22
tropomodulin myopalladin
MARP
FHL2
telethonin
NH2Nebulin(nebulette)
COOH