Whole exome sequencing of patients with diffuse idiopathic ...
Transcript of Whole exome sequencing of patients with diffuse idiopathic ...
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ORIgInAL PAPERS
1. Serviço Especializado de Epidemiologia e Biologia Molecular(SEEBMO) / Hospital de Santo Espírito da Ilha Terceira (HSEIT)2. Comprehensive Health Research Center, CHRC, Lisbon, Portugal3. Centre of Marine Science (CCMAR) / University of Algarve
could be involved in this phenotype in an as yet un-known way.
Keywords: Rheumatic and musculoskeletal diseases ;Genetic association; Rheumatology.
INTRODUCTION
Previous studies undertaken by our group, identifiedand characterized twelve families affected with DiffuseIdiopathic Skeletal Hyperostosis (DISH, MIM 106400)and/or Calcium Pyrophosphate Dihydrate (CPPD)Chondrocalcinosis (CC), hereafter designated,DISH/CC. DISH/CC is a poorly understood phenotypecharacterised by peripheral and axial enthesopathic cal-cifications, frequently fulfilling the radiological criteriafor DISH, and in some cases associated with CPPDChondrocalcinosis. A common pathogenic mechanism,shared by the two conditions, has been suggested1.DISH is a common skeletal disorder characterized byprogressive calcification and ossification of ligamentsand entheses2-3. The exact prevalence and incidence ofDISH is unknown, however it is well known that it ismore frequent in males and its prevalence rapidly in-creases with age, mainly affecting subjects over the ageof 404. The prevalence of DISH in patients over 50 yearsof age is 25% in males and 15% in females,5 and thisdisease is now becoming a serious problem in aging so-cieties. Several lines of evidence suggest that geneticfactors might play a part in the etiology of DISH, suchas the existence of familial cases with early onset (in thethird decade of life)6 and the higher frequency of DISHin the boxer dogs relative to other dog breeds7-8. So far,however, no single gene has been conclusively associa -ted with the disease. Chondrocalcinosis is characteri -zed by deposition of crystals of calcium pyrophosphatedihydrate (CPPD) in articular hyaline and fibro-carti-
Whole exome sequencing of patients with diffuse idiopathic skeletal hyperostosis and calcium
pyrophosphate crystal chondrocalcinosis
Parreira B1,2, Couto AR1,2, Rocha F1,2, Sousa M1,2, Faustino V1, Power DM3, Bruges-Armas J1,2
ACTA REUMATOL PORT. 2020;45:116-126
ABSTRACT
Objectives: DISH/CC is a poorly understood pheno-type characterised by peripheral and axial entheso-pathic calcifications, frequently fulfilling the radiolog-ical criteria for Diffuse Idiopathic Skeletal Hyperostosis(DISH, MIM 106400), and in some cases associatedwith Calcium Pyrophosphate Dihydrate (CPPD) Chon-drocalcinosis (CC). The concurrence of DISH and CCsuggests a shared pathogenic mechanism. In order toidentify genetic variants for susceptibility we performedwhole exome sequencing in four patients showing thisphenotype.Materials and methods: Exome data were filtered inorder to find a variant or a group of variants that couldbe associated with the DISH/CC phenotype. Variantsof interest were subsequently confirmed by Sanger se-quencing. Selected variants were screened in a cohortof 65 DISH/CC patients vs 118 controls from Azores.The statistical analysis was performed using PLINKV1.07.Results:We identified 21 genetic variants in 17 genesthat were directly or indirectly related to mineraliza-tion, several are predicted to have a strong effect at aprotein level. Phylogenetic analysis of altered aminoacids indicates that these are either highly conservedin vertebrates or conserved in mammals. In case-con-trol analyses, variant rs34473884 in PPP2R2D was sig-nificantly associated with the DISH/CC phenotype(p=0.028; OR=1.789, 95% CI= 1.060 - 3.021)).Conclusion: The results of the present and precedingstudies with the DISH/CC families suggests that thephenotype has a polygenic basis. The PPP2R2D gene
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lage9. For the moment two genes, ANKH (CCAL2) andTNFRSF11B, are known to be involved in the devel-opment of Chondrocalcinosis10-11. Previously, we per-formed genetic studies of DISH/CC using a “Wholegenome wide linkage study” and an “Identity byState/Identity by Descent” association study and twogenes were identified as good candidate genes forDISH/CC; RSPO4 on chromosome 20, and LEMD3 onchromosome 12. Several RSPO4 gene variants wereidentified and nucleotide modifications located in theregulatory region were more frequent in control indi-viduals than in the DISH/CC group12. Even though thegenetic basis of DISH/CC is unknown, it is consideredto be a bone forming disease and genes related to cal-cification and ossification are considered good candi-dates. In the present study, we took advantage of wholeexome sequencing (WES) so that even in the absenceof sufficient pedigrees and samples for traditional link-age approaches we could identify rare protein-codingvariants that are the cause of the majority of mono-genic diseases13-14. We performed targeted exome se-quencing on four DISH/CC patients, with an appar-ently autosomal dominant DISH/CC phenotype, inorder to capture rare and pathogenic variants expect-ed to have potentially damaging effects on proteinfunction that lead to modified calcification and/or os-sification. To our knowledge this is the first report ofWES analysis in patients affected with DISH.
MATERIAlS AND METHODS
This study was approved by the “Comissão de Éticado Hospital de Santo Espírito da Ilha Terceira”. Allmethods were performed in accordance with the ap-proved guidelines, obtaining informed consent fromeach subject before conducting the experiments.
ExOME CApTURE
The selection of patients was made after ruling out mu-tations in ANKH and secondary causes for Chondro-calcinosis. DNA was extracted from peripheral bloodsamples using a standard salting out procedure. Sam-ples were resequenced, using an ABI-SOLiD platformand Agilent’s SureSelect Target Enrichment System for38 Mb, by “Sistemas Genómicos, S.L.” in Valencia,Spain. After standard DNA quality control, SOLiDFragment libraries were prepared and enriched withSureSelect All Human Enrichment Target Exon. Thequality and quantity of the libraries were assessed by
analysis with an Agilent 2100 Bioanalyzer and Qubit.Each library went through a process of emulsion PCRfor clonal amplification of the fragments, followed byan enrichment process and chemical modification toallow loading into the reaction chamber. The qualityand quantity of the beads obtained for each librarywere estimated taking into account the parameters giv-en by Work Flow Analysis. Then, ligation sequencingwas done to obtain sequences of 50nt +35nt (Paired-end) reads using a SOLiD4 sequencer. The data qual-ity was estimated using the parameters provided bythe software SETS (SOLiD Experimental Tracking Sys-tem). Single Nucleotide Variants (SNVs) were classifiedusing Ensembl’s nomenclature and grouped using thefollowing scheme: Known and Novel (Coding, Splic-ing, Others). The coding variants were divided intononsynonymous and synonymous. The “Others” in-cluded intronic, UTR, regulatory region, intergenic,downstream and upstream variants.
WES fIlTERINg
The segregation analysis of these families indicated thatthe most likely model for this disease is an autosomaldominant model1. However, given that a previouslinkage study did not identify a major dominant lo-cus, the data were analyzed assuming both a recessiveand a dominant model.Genetic variants were identified in 815, 917, 872
and 593 genes under a dominant model, for indivi -duals AZ1 to AZ4. Assuming that the genetic cause isthe same for the individuals AZ1 to AZ4, identifiedgenes were then filtered to select common, sharedgenes (supplementary Table I, available in supple-mentary file). A group of 52 genes were common to thefour patients; candidate genes for testing were select-ed taking into account their involvement in calcifica-tion and/or ossification or related conditions that couldbe associated with the DISH/CC disease. Gene vari-ants shared between the genes common to the four pa-tients were identified and nonsynonymous, splice site,stop lost/gain and frameshift variants were targeted, inanticipation that synonymous and intronic variantswould be far less likely to be relevant in thepathogenicity.
CANDIDATE gENES – SElECTION By fUNCTION
A group of 20 candidate genes was selected and in-cluded genes that are reported to be involved in bonemetabolism and/or related conditions. These geneswere Alkaline Phosphatase (ALPL/TNAP), the Calcium
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EcToPIc cALcIfIcATIon
TA
BlE
I. R
AD
IOlO
gy
RE
SU
lTS
fR
OM
fO
UR
SE
lE
CT
ED
pA
TIE
NT
S f
OR
WE
S.
RADIOLOGY
Age at
Patient
Sex
C-Spine
T-Spine
L-Spine
Knees
Elbow
onset
Other diseases
AZ1
MNor
mal
Nor
mal
Synde
smop
hytos
is D
ISH
N/A
Periarticular
<40
Obe
sity
calcifications
AZ2
FDISH
DISH
DISH
Arthrosis,
Periarticular
30Lithiasis,
enthesop
athy
calcifications
Diabe
tes mellitus
AZ3
FN/A
DISH
DISH
Enthesop
athy,
Periarticular
N/A
Obe
sity
osteop
hytos
is,
calcifications
arthrosis
AZ4
MNor
mal
Synde
smop
hytos
isSy
nde
smop
hytos
is,
Osteo
phytos
is,
Periarticular
<40
Lithiasis,
anterior
osteo
phytos
iscalcifications in
calcifications
cardiac
caps
ule, a
rthrosis
arrythmia
Definition of Rad
iological terminolog
y 1)
DISH: C
ontinuou
s os
sification
along the an
terolateral a
spect of three or
fou
r co
ntigu
ous ve
rteb
ral b
odies; 2) Sy
nde
smop
hytos
is: V
ertical a
nd
symmetrical c
alcification
of the lateral m
argins of the intervertebr
al disc sp
ace; 3) Osteo
phytos
is: p
resence of sp
urs, w
hich are outgrows of bon
e tissue; 4) Enthesop
athy:
calcification/ossification process at the site of the insertion of liga
men
ts, ten
dons, fascia or
articular caps
ule in
to bon
e; 5) Arthrosis: presence of joint sp
ace narrowing, scleros
is and
osteop
hytos
is.
Abb
reviations: N
/A: n
ot ava
ilab
le, C
-Spine: C
ervical S
pine, T-Spine: Thor
acic spine an
d L-Spine: Lumba
r sp
ine.
Sensing Receptor (CASR), Bone MorphogeneticProtein Receptor Type 1B (BMPR1B), Osteopon-tin (OPN/SPP1), Integrin Binding Sialoprotein(IBSP), Fibroblast Growth Factor 2 (FGF2), Inor-ganic Pyrophosphate Transport Regulator(ANKH), Collagen Type XI Alpha 2 (COL11A2),Nucleotide Pyrophosphatase 1 (ENPP1), Runt-related Transcription Factor (RUNX2), DickkopfWNT Signaling Pathway (DKK-1), Insulin LikeGrowth Factor 1 (IGF1), Matrix Gla Protein(MGP); Vitamin D (VDR), Bone MorphogeneticProtein 4 (BMP-4), Collagen Type 1 Alpha 1(COL1A1), Transforming Growth Factor Beta 1(TGF�1), Solute Carrier Family 29 Member 1(SLC29A1), Bone Morphogenetic Protein 2 (BMP--2) and Collagen Type VI Alpha 1 (COL6A1).
gENETIC vARIANTS – SElECTION By
pATHOgENICITy
In the selection by variant pathogenicity filter-ing strategy we used two levels; a “variant level”and a “knowledge level” (Figure 1). In the “vari-ant level” we first filtered according to varianttype and focused on functional significance(splice sites, frameshift coding, nonsynonymouscoding and loss or a gain of a stop). Only theSIFT prediction with Deleterious and Unknownand GERP values equal or higher than 3 were in-cluded. We excluded variants with MAF valueshigher than 0.05 by filtering the SNP_IDs (rsnumber or chromosome position) against theEnsemble Variant Effect Predictor tool(http://www.ensembl.org/ info/docs/tools/vep/index.html). Then in the selection by “knowl-edge” we focused only on genes associated withcalcification and/or ossification or related con-ditions. Lastly, we evaluated the functional sig-nificance and pathogenic potential of each vari-ant found in the selected genes.
EvAlUATION Of gENES AND vAlIDATION Of
vARIANTS
Information about candidate genes was obtainedfrom several databases, including Ensembl(http://www.ensembl.org/index), National Centerfor Biotechnology Information (NCBI) (http://www.ncbi.nih.gov), Pubmed (https:// www.ncbi.nlm.nih.gov/pubmedwe), GeneCards (http://www.genecards.org/), Online Mendelian Inheri-tance in Man (OMIM) (http://www. omim.org/)
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and MalaCards (http://www.malacards.org/).PCR primers were designed using the software
Primer3 (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) to amplify and validatevariants detected by exome sequencing. Sanger se-quencing using standard protocols was performed us-ing an automated DNA sequencer ABI 3130xl (AppliedBiosystemsÒ). Genetic variants were screened using Se-quencing Analysis and SeqScape (Applied Biosystem-sÒ) and using a reference NCBI sequence.The functional significance and the potential deleteri-
ous effect of each variant was explored using the follow-ing databases: Ensembl (http://www.ensembl. org/index),Human Gene Mutation Database (HGMD) (http://www.hgmd.cf.ac.uk/ac/index.php) and dbSNP (https://www.ncbi.nlm.nih.gov/projects/SNP/), ma king use ofPolyPhen-2 (Polymorphism Phenotyping v2) (http://ge-netics.bwh.harvard.edu/pph2/), SIFT and GERP.PolyPhen is classified as, benign [0-0.2], possibly dam-aging [0.2-0.85], and probably damaging [0.85-1], SIFTas deleterious if less than 0.05 and GERP, ranges from -12.3 to 6.17, with 6.17 being the most conserved15. The MAF values of each variant were also analyzed.
Protein conservation analysis was performed usingClustalW (http://www.genome.jp/tools/clustalw/) tocompare homologous amino acid sequences amongmultiple vertebrates at the sites where the variations oc-cur (accession numbers of transcripts available in sup-plementary Table II, available in supplementary file).
fIgURE 1. The two-level filter approach used to analyze the WES results from 4 patients with DISH/CC disease. SIFT: Sorting Intolerant From Tolerant, GERP: Genomic Evolutionary Rate Profiling score and MAF: Minor Allele Frequency
ASSOCIATION STUDIES AND STATISTICAl
ANAlySIS
In order to verify a possible association between iden-tified genes from WES and the DISH/CC phenotype,selected variants were screened in family members (af-fected with DISH/CC and unaffected), and the mostconserved variants and those that were present in threeor four WES patients, were selected for screening in agroup of 65 DISH/CC patients and a group of 118 con-trols. Association testing was performed by chi-squaredanalysis using PLINK software (V1.07)16: p-values<0.05 were considered significant. ORs with a 95% CIwere calculated for the minor alleles of each variant.OR >1 indicates a susceptibility allele and OR<1 indi-cates a protective allele for the disease.
RESUlTS
The WES was performed using DNA from four patientsfrom unrelated DISH/CC families (AZ1-AZ4) fromAzores. A detailed description of these families is pro-vided in our previous study1. The patients who gaveinformed consent, a blood sample was obtained andstandard X-rays were taken from: the knees, axial skele-ton, wrists, hands, elbows, and pelvis. The 4 patientswere selected because they shared a very evident DISHphenotype. The radiological characterization of pa-tients selected for WES are indicated in Table I.
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TA
BlE
II. l
IST
Of
fIl
TE
RE
D C
AN
DID
AT
E g
EN
ET
IC v
AR
IAN
TS
CO
Nf
IRM
ED
By
SA
Ng
ER
SE
qU
EN
CIN
g.
Mutational
Patient
Gene
Chr
SNP_ID
spectrum
Variant
AA
MAF
SIFT
PolyPhen
AZ1
AZ2
AZ3
AZ4
PPP2R
2D10
rs34
4738
84HC
c.10
72G>A
G35
8S0.18
DB
hm
ht
ht
ht
COL11
A2
6rs92
7793
4RR
c.82
6G>A
E27
6K0.32
TPs D
hm
ht
hm
ht
VDR
12rs22
2857
0CM
c.2T
>CM1T
0.33
DB
hm
hm
ht
ht
CASR
3rs18
0172
6RR
c.30
31G>C
E10
21Q
0.08
TB
hm
hm
hm
FGF2
4rs10
4820
1NA
c.*7
57C>T
R20
9Q0.23
NA
NA
ht
ht
ht
BMP2
20rs23
5768
HC
c.57
0A>T
R19
0S0.24
DPb D
ht
ht
ht
ENPP1
6rs10
4449
8RR
c.51
7A>C
K17
3Q0.34
TB
ht
ht
ht
BMP4
14rs17
563
CM
c.45
5T>C
V15
2A0.37
TB
hm
hm
ht
MGP
12rs42
36RR
c.30
4A>G
T10
2A0.39
TB
ht
ht
ht
PLCG2
16rs13
8158
454
RR
c.20
54+7
G>A
NA
0.01
NA
NA
ht
ht
ALPL
1rs32
0025
4RR
c.78
7T>C
Y26
3H0.27
TB
ht
ht
CASR
3rs18
0172
5HC
c.29
56G>T
A98
6S0.09
TB
ht
ht
COL1A
117
rs37
2029
024
CM
c.32
47G>T
A10
83T
<0.01*
TB
hm
PLCG2
16rs37
4430
619
RR
c.37
86G>C
K12
62N
<0.01*
DB
ht
ALPL
1rs14
9344
982
RR
c.45
5G>A
R15
2H0.01
TB
ht
ABCC6
16rs41
2781
74CM
c.31
90C>T
R10
64W
0.01
DPb D
ht
COL11
A2
6rs22
2979
2RR
c.51
65C>T
P17
22L
0.01
DB
ht
TGFβ1
19rs55
6590
02RR
c.71
3-8d
elC
NA
0.01
NA
NA
ht
AMER3
2rs72
8549
96RR
c.13
00C>G
L43
4V0.05
TB
ht
FLNC
7rs22
9156
9HC
c.47
00G>A
R15
67Q
0.06
DPb D
ht
COL6A
121
rs10
5331
2RR
c.25
49G>A
R85
0H0.27
TB
hm
The va
rian
ts are listed
in the table acco
rding to occurren
ce in
two or
more of the pa
tien
ts A
Z1 to A
Z4 in the study.
Abb
reviations: C
hr: chromos
ome, H
C: h
ighly con
served
, CM: c
onserved
in m
ammals, RR: relax
ed reg
ion, N
A: N
ot A
pplicable, A
A: A
minoa
cid, M
AF: M
inor
Allele Frequ
ency, h
m:
hom
ozyg
ous, ht: heteroz
ygou
s, Blank cells represen
t wild type
, T: T
olerated
, D: D
eleterious, Pb D for
“Proba
bly Dam
aging”
, Ps D for
“Pos
sibly Dam
aging”
and B for
Ben
ign. *
Allele
freq
uen
cies assumed
from N
HLBI GO Exo
me Se
quen
cing Project.
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fIlTERINg RESUlTS
Approximately 38 Mb of sequence per patient was gen-erated and the capture specificity and sensitivity in allsamples was about 55% and 94%, respectively. The re-sults for each sample after SNV calling and indel identi-fication steps are summarized in supplementary Table III.After filtering, 21 missense, deletion and splice site
variants in 17 genes were obtained: PLCG2, ALPL,
CASR, FGF2, COL11A2, ENPP1, MGP, VDR, BMP-4,COL1A1, TGF�1, BMP-2, COL6A1, FLNC, AMER3,PPP2R2D and ABCC6. Ten of the identified variantswere present in the HGMD mutation database, linkedto phenotypes, other than DISH/CC. The identifiedvariants and available functional information are pre-sented in Table II. Sequence conservation of the missense variants were
fIgURE 2. Sequence conservation in six vertebrates of the variants identified in candidate genes possibly associated with the DISH/CCphenotype in humans (some genes are not available in all species). The variant for the FGF2 gene is not presented since it is located inthe 3’ untranslated region.Abbreviations: PLCG2- Phospholipase C Gamma 2, ALPL- Alkaline Phosphatase, Liver/Bone/Kidney, CASR- calcium-sensing receptor,COL11A2- Collagen Type XI Alpha 2 Chain, ENPP1– ectonucleotide pyrophosphatase/phosphodiesterase 1, MGP- Matrix Gla Protein,VDR- Vitamin D Receptor, BMP4- Bone morphogenetic protein 4, COL1A1- Collagen Type I Alpha 1 Chain, BMP2- Bone morphogeneticprotein 2, COL6A1- Collagen Type VI Alpha 1 Chain, FLNC- Filamin C, AMER3- APC Membrane Recruitment Protein 3, PPP2R2D-Protein Phosphatase 2 Regulatory Subunit B delta, ABCC6 – ATP-binding cassete subfamily C, member 6.
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then evaluated in 6 different vertebrates. It is evidentin Figure 2 that the variants in the genes PPP2R2D,BMP2, FLNC, and CASR are highly conserved in all thevertebrates analyzed. Variants of the genes VDR, BMP4,COL1A1 and ABCC6 are only conserved in mammals.The degree of conservation is not directly linked to low-er allele frequency, since several of these variants havehigh Minor Allele Frequencies (MAF).
ASSOCIATION BETWEEN vARIANTS AND THE
DISH/CC pHENOTypE
Two cohorts, one of 65 unrelated DISH/CC patients(45 males, 20 females; age of onset around 40 years)and another of 118 unrelated controls without anysigns of DISH/CC, with a similar ethnic background,were selected after radiological characterization (47males, 71 females; mean current age, 68 years; range,60-104) and used for association studies. In cases weselected younger patients around the age of 40, wheregenetic factors may be associated, however in the con-trols we selected older individuals to ensure that theyhave not developed the disease.All patients had at least 2 highly conserved genetic
variants in combination with other variants that arenormally conserved in mammals (Table II). Variantswith a high degree of conservation, present in 4 or 3WES patients, irrespective of the MAF, were selectedfor the association study. The variants rs34473884,rs9277934 and rs2228570 in PPP2R2D, COL11A2 andVDR genes, respectively, were present in all four WESpatients. The variants rs1048201, rs235768,rs1044498 and rs17563 in FGF2, BMP2, ENPP1 and
BMP4 genes, respectively, were present in three of thefour patients selected for WES. Seven variants werescreened in a group of 65 DISH/CC patients and 118controls and the results are indicated in Table III. Thevariant rs34473884 in the PPP2R2D gene was the onlythat gave significant results. It was found to be muchmore frequent in the DISH/CC group than in the con-trols (p=0.028; OR=1.789, 95% CI= 1.060 - 3.021).
DISCUSSION
In this study, we used WES as a method to identify can-didate genes for DISH/CC aetiology and associationstudies to investigate some of the identified variants.As expected, thousands of protein coding variants perpatient were identified across each exome. Conse-quently, a number of filtering strategies were used to se-lect potential high risk variants of genes potentially as-sociated with DISH. The filtering strategies employedwere based on previous knowledge about gene func-tion. The major limitations of the strategy used to iden-tify candidate genes is that unannotated genes andthose with unknown functions were not investigated inthe study.Very few genetic studies have been published about
DISH so far. DISH susceptibility genes have previous-ly been investigated, including Human Leukocyte Anti-gens (HLA),17-24 Collagen 6A1 gene (COL6A1),25 Fi-broblast Growth factor 2 (FGF2),26 Vitamin D (1,25-Dihydroxyvitamin D3) Receptor (VDR) and CollagenType I�1 (COL1A1),27 but only two genes are known to
TABlE III. ASSOCIATION STUDy BETWEEN SEvEN vARIANTS fROM SEvEN gENES AND THE DISH/CC pHENOTypE
MAFSNP Allele DISH/CC Controls
Chr Gene M/m MAF (N=65) (N=118) OR (95% CI) p-value10 PPP2R2D rs34473884 G/A 0.262 0.165 1.789 (1.060 - 3.021) 0.02814 BMP4 rs17563 T/C 0.454 0.390 1.301 (0.843 - 2.006) 0.2344 FGF2 rs1048201 C/T 0.139 0.131 1.063 (0.569 - 1.985) 0.8496 ENPP1 rs1044498 A/C 0.200 0.203 0.979 (0.574 - 1.670) 0.9386 COL11A2 rs9277934 G/A 0.385 0.360 1.110 (0.713 - 1.728) 0.64320 BMP2 rs235768 A/T 0.292 0.284 1.042 (0.650 - 1.671) 0.86512 VDR rs2228570 T/C 0.400 0.373 1.121 (0.723 - 1.739) 0.609
The minor alleles are indicated in bold.Abbreviations: Chr: Chromossome, SNP: Single nucleotide polymorphism, M/m: major allele/minor allele, MAF: Minor allele frequency andOR (95% CI): Odds Ratio (95% Confidence Interval).
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have a positive association with DISH susceptibility;COL6A128-29 and FGF2.26 However, the COL6A1 andFGF2 variants associated with the disease, are locatedin non-coding regions and are common variants with-in the general population, suggesting they have a smallgenetic effect. In our study we found 3 genetic variants that were
present in all 4 DISH/CC patients and 6 that were pre-sent in 3 DISH/CC patients. Minor allele frequencies ofthese variants were high (mean 0.25), meaning thatthese were common variants in the general population.Rare nonsynonymous SNPs are two times more likelyto be predicted as protein-affecting, when compared tocommon SNPs. However, in our study three commonSNPs, shared by the 4 patients (G358S, E276K andM1T in PPP2R2D, COL11A2 and VDR genes, respecti -vely), according to SIFT and/or PolyPhen algorithmswere predicted to have a strong effect on the protein.Eight of these genetic variants, in heterozygous and/orhomozygous states, were in conserved positions of pro-teins associated with mineralization; four of which werein regions that were highly conserved across the verte-brates. The four WES selected patients had at least 2highly conserved genetic variants in combination withother variants that were conserved in mammals. The as-sociation study indicated that the SNP rs34473884 inthe PPP2R2D gene was significantly associated with theDISH/CC phenotype. In humans, the full PPP2R2D (Protein Phosphatase
2 Regulatory Subunit � delta) gene structure is com-posed of 9 exons and spans 79.19 kb in chromosome10. The gene encodes a crucial serine/threonine proteinphosphatase that regulates basal cellular activities bydephosphorylating substrates. Protein Ser/Thr phos-phatases are a group of enzymes that catalyze the re-moval of phosphate groups from serine and/or threo-nine residues by hydrolysis of phosphoric acidmonoesters. They oppose the action of kinases andphosphorylases and are involved in signal transduc-tion. Protein phosphatases have long been postulatedto influence TGF-� superfamily signaling, which regu-lates numerous cellular responses30. Despite the long-standing suspected influence of protein phosphatasesin TGF-� signaling, concrete data confirming the inter-action only started to emerge recently. In human celllines, PPP2R2D negatively modulates TGF-�/Activin//Nodal signaling by inhibiting the type I receptorsALK4 and ALK531. The variant c.1072G>A (G327S) found in PPP2R2D
gene is a missense variant which causes substitution of
glycine for serine at amino acid 327 in the protein.These two amino acids are hydrophilic but glycine isnon-polar and serine is polar. The glycine is normallytotally conserved in all vertebrates studied, and thevariant has a low, deleterious, SIFT score (0.03) but thePolyphen score (0.33) does not corroborate its harm-ful effect. The frequency of this variant is high in Eu-ropean populations with a MAF of 0.18 and this vari-ant was identified in all four DISH/CC patients usedfor WES; AZ1 was homozygous and AZ2, 3 and 4 wereheterozygous. As far as we known no phenotype hasbeen associated with this gene. Rare variants, with a deleterious effect on proteins in
the human genome, are the basis for the developmentof Mendelian diseases. However, common polymor-phisms that individually exert small effects might, as agroup, also play a substantial genetic role. Despite thelack of association of most of the studied gene variantsin the present study, this may be in part linked to theheterogeneous features of the phenotype, and so im-proved characterization of the disorder under study isessential for the planning of future studies. The resultsof our study lead us to suggest that DISH/CC is poly-genic, and is influenced by the interaction of multiple,small effect gene variants and possibly by unknown en-vironmental factors.
CONClUSION
Our results underline the polygenic nature of DISH anda number of conserved and sometimes deleterious vari-ants were identified in genes with a role in mineraliza-tion. The variant rs34473884 in the PPP2R2D gene wassignificantly associated with the DISH/CC phenotypeand we propose it may contribute to the developmentof this disorder. Further studies will be needed to con-firm the association of PPP2R2D with the phenotypeunder study.
ACkNOWlEDgEMENTS
The authors thank all the patients who participated in this researchand made it possible. We also thank Isa Dutra (MSc), João PauloPinheiro (BsC) and Raquel Meneses (BsC) from Hospital Santo Es-pírito da Ilha Terceira, EPE, Epidemiology and Molecular BiologyService (SEEBMO), Angra do Heroísmo, Portugal, who performedthe DNA extractions and Vânia Machado (MSc), who participatedin the elaboration of the pedigrees. BP was supported by “Fundo Re-gional para a Ciência e Tecnologia (FRCT)” (M3.1.2/F/023/2011).
COMpETINg fINANCIAl INTERESTS
The authors declare no competing interests.
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EcToPIc cALcIfIcATIon
CORRESpONDENCE TO
Jácome Bruges ArmasServiço Especializado de Epidemiologia e Biologia Molecular (SEEBMO) Hospital de Santo Espírito da Ilha Terceira (HSEIT)Canada do Briado9700-049, Angra do HeroísmoE-mail: [email protected]
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PARREIRA B ET AL
SUpplEMENTARy fIlES
SUpplEMENTARy TABlE I. NUMBER Of
CANDIDATE gENES pER SAMplE AND NUMBER
Of CANDIDATE gENES SHARED By DISH/CC
pATIENTS
Number ofcandidate genes
Dominant Recessive Samples model ModelAZ1 815 48AZ2 917 58AZ3 872 47AZ4 593 25AZ1+AZ2 220 6AZ1+AZ3 212 4AZ1+AZ4 139 4AZ2+AZ3 249 6AZ2+AZ4 149 4AZ3+AZ4 162 3AZ1+AZ2+AZ3 118 2AZ1+AZ2+AZ4 65 1AZ2+AZ3+AZ4 78 2AZ1+AZ3+AZ4 77 1AZ1+AZ2+AZ3+AZ4 52 1
SU
pp
lE
ME
NTA
Ry
TA
BlE
II. l
IST
Of
gE
NE
S A
ND
TH
EIR
AC
CE
SS
ION
TR
AN
SC
RIp
T N
UM
BE
RS
US
ED
fO
R C
ON
SE
Rv
AT
ION
AN
Aly
SIS
.
SPECIES
GENES
Human
Chimpanzee
Dog
Mouse
Chicken
Zebrafish
PLCG2
ENST
0000
0564
138.5
ENSP
TRT00
0000
1547
9.3
ENSC
AFT00
0000
3181
5.3
ENSM
UST
0000
0081
232.8
ENSG
ALT
0000
0050
238.1
ENSD
ART00
0000
2139
9.7
ALPL
ENST
0000
0374
840.7
ENSP
TRT00
0000
0059
2.2
ENSC
AFT00
0000
2357
8.3
ENSM
UST
0000
0030
551.10
ENSG
ALT
0000
0068
960.1
ENSD
ART00
0001
3110
1.2
CASR
ENST
0000
0498
619.3
ENSP
TRT00
0000
4399
6.4
ENSC
AFT00
0000
1876
0.3
ENSM
UST
0000
0063
597.13
XM_4
1649
1.5.1
ENSD
ART00
0000
1093
4.8
COL11
A2
ENST
0000
0341
947.6
ENSP
TRT00
0000
4856
4.4
ENSC
AFT00
0000
0140
9.4
ENSM
UST
0000
0087
497.10
Ni
ENSD
ART00
0001
0575
4.4
ENPP1
ENST
0000
0360
971.6
ENSP
TRT00
0000
3435
6.3
ENSC
AFT00
0000
0000
1.3
ENSM
UST
0000
0105
520.7
ENSG
ALT
0000
0066
498.1
ENSD
ART00
0001
2735
0.3
MGP
ENST
0000
0539
261.5
ENSP
TRT00
0000
0873
2.4
ENSF
CAT
0000
0031
714.1 (C
at)
ENSM
UST
0000
0032
342.2
ENSG
ALT
0000
0019
173.4
ENSD
ART00
0001
4962
2.2
VDR
ENST
0000
0229
022.7
Ni
ENSC
AFT00
0000
4347
3.2
ENSM
UST
0000
0023
119.14
ENSG
ALT
0000
0071
682.1
ENSD
ART00
0001
6189
2.1
BMP4
ENST
0000
0245
451.8
ENSP
TRT00
0000
1164
3.4
ENSC
AFT00
0000
2362
4.3
ENSM
UST
0000
0074
077.11
ENSG
ALT
0000
0020
316.5
ENSD
ART00
0000
7515
0.4
COL1A
1ENST
0000
0225
964.9
ENSP
TRT00
0000
1723
1.4
ENSC
AFT00
0000
2695
3.3
ENSM
UST
0000
0001
547.7
XM_0
1527
3228
.1.1
ENSD
ART00
0000
0939
3.7
BMP2
ENST
0000
0378
827.4
ENSP
TRT00
0000
2460
6.3
ENSC
AFT00
0000
4969
0.1
ENSM
UST
0000
0028
836.6
ENSG
ALT
0000
0065
435.1
ENSD
ART00
0001
6665
7.1
COL6A
1ENST
0000
0361
866.7
ENSP
TRT00
0000
2615
6.3
ENSC
AFT00
0000
1891
8.3
ENSM
UST
0000
0001
147.4
ENSG
ALT
0000
0039
669.3
ENSD
ART00
0001
1060
8.3
FLNC
ENST
0000
0325
888.12
ENSP
TRT00
0000
3644
4.5
ENSC
AFT00
0000
0250
4.3
ENSM
UST
0000
0065
090.7
NM_2
0457
3.1.1
Ni
AMER3
ENST
0000
0423
981.1
ENSP
TRT00
0000
2312
4.3
ENSC
AFT00
0000
0674
7.2
ENSM
UST
0000
0052
670.10
ENSG
ALT
0000
0055
960.1
ENSD
ART00
0001
4999
2.2
PPP2R
2DENST
0000
0455
566.5
ENSP
TRT00
0000
0582
9.4
ENSC
AFT00
0000
4441
6.1
ENSM
UST
0000
0041
097.12
ENSG
ALT
0000
0081
063.1
ENSD
ART00
0001
7217
5.1
ABCC6
ENST
0000
0205
557.11
ENSP
TRT00
0000
1439
8.4
ENSC
AFT00
0000
2890
8.3
ENSM
UST
0000
0002
850.7
ENSG
ALT
0000
0048
627.1
ENSD
ART00
0001
7294
3.1
Data was retriev
ed from the Ensembl gen
ome da
taba
se; a
ccessed on
Nov
embe
r 20
16.
Ni: not id
entified
.
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126
EcToPIc cALcIfIcATIon
SUpplEMENTARy TABlE III. kNOWN, NOvEl AND TOTAl NUMBER Of SNvS AND INDElS fOR THE fOUR
SAMplES TESTED (AZ1-4)
SNVs Indels TOTAL/Sample Known Novel Total Known Novel Complex Total sampleAZ1 17281 1619 18900 457 405 51 913 19813AZ2 18086 1759 19845 440 496 66 1002 20847AZ3 18327 1650 19977 524 505 73 1102 21079AZ4 17575 1223 18798 475 424 72 971 19769
SNVs: Single nucleotide variants.