A genome-wide association study identifies two loci
associated with heart failure due to dilated
cardiomyopathy
Villard et al.
Supplementary information, tables and figures
Cardiogenics Consortium:
Tony Attwood1, Stephanie Belz2, Jessy Brocheton3, François Cambien3, Jason Cooper4, Panos
Deloukas5, Abi Crisp-Hihn1, Jeanette Erdmann2, Nicola Foad1, Tiphaine Godefroy3, Alison
Goodall6, Jay Gracey6, Emma Gray5, Stefanie Gulde2, Rhian Gwilliams5, Martina Grassi7,
Susanne Heimerl7, Christian Hengstenberg 7, Jennifer Jolley1, Unni Krishnan6, Patrick LinselNitschke2, Heather Lloyd-Jones1, Ingrid Lugauer7, Per Lundmark8, Seraya Maouche2, Gilles
Montalescot3, Jasbir S Moore6,David Muir1, Elizabeth Murray1, Chris P Nelson, David Niblett5,
Karen O'Leary1, Willem H. Ouwehand1,5, Helen Pollard6, Carole Proust3, Angela Rankin1,
Augusto Rendon9, Catherine M Rice5, Hendrick Sager2, Nilesh J. Samani6, Jennifer Sambrook1,
Gerd Schmitz10, Michael Scholz11, Laura Schroeder, Heribert Schunkert2, Ann-Christine
Syvannen8, Laurence Tiret3, David-Alexandre Tregouet3, Chris Wallace4, on behalf of the
Cardiogenics Consortium
1Department of Haematology, University of Cambridge, Long Road, Cambridge, CB2 2PT, UK
and National Health Service Blood and Transplant, Cambridge Centre, Long Road,
Cambridge, CB2 2PT, UK; 2Medizinische Klinik 2, Universität zu Lübeck, Lübeck Germany;
3INSERM UMRS 937, Pierre and Marie Curie University (UPMC, Paris 6) and Medical School,
91 Bd de l’Hôpital 75013, Paris, France; 4Juvenile Diabetes Research Foundation/Wellcome
Trust Diabetes and Inflammation Laboratory, Department of Medical Genetics, Cambridge
Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building,
Cambridge, CB2 0XY, UK; 5The Wellcome Trust Sanger Institute, Wellcome Trust Genome
Campus, Hinxton, Cambridge CB10 1SA, UK; 6Department of Cardiovascular Sciences,
University of Leicester, Glenfield Hospital, Groby Road, Leicester, LE3 9QP, UK; 12Leicester
NIHR Biomedical Research Unit in Cardiovascular Disease, Glenfield Hospital, Leicester, LE3
9QP, UK; 7 Klinik und Poliklinik für Innere Medizin II, Universitätsklinikum Regensburg,
Germany, 8 Molecular Medicine, Department of Medical Sciences, Uppsala University,
Uppsala, Sweden; 9European Bioinformatics Institute, Wellcome Trust Genome Campus,
Hinxton, Cambridge, CB10 1SD, UK; 10Institut für Klinische Chemie und
Laboratoriumsmedizin, Universität, Regensburg, D-93053 Regensburg, German;, 11 Trium,
Analysis Online GmbH, Hohenlindenerstr. 1, 81677, München, Germany
Patients and controls
CARDIGENE study. Cases1 were French patients with a diagnosis of idiopathic DCM (enlarged
left ventricle end-diastolic volume/diameter >140 ml/m2 on ventriculography or >34 mm/m2
on echocardiography and low ejection fraction (≤40%) confirmed over a six-month period, in
1
the absence of causal factors such as coronary artery disease (coronary angiography was
mandatory if DCM occurred after 35 years of age) or sustained arterial hypertension,
intrinsic valvular disease, documented myocarditis, congenital malformation, insulindependent diabetes. Only apparently sporadic DCM cases without additional (first degree)
relative with DCM were included (but, after careful cardiac examination in relatives, 8% were
retrospectively with familial form). All were of European origin (born in France, from parents
born in France or neighbouring countries). Recruitment was performed in ten hospitals from
six French regions (Lille, Lyon, Nancy, Nantes, Paris-Ile de France, and Strasbourg) from
September 1994 to February 1996. A total of 424 DCM cases were included (338 males and
86 women, among them 224 had undergone a cardiac transplantation). Mean age of
patients at diagnosis was 45.2 ± 10.6 years, mean ejection fraction was 23 ± 7%, mean enddiastolic volume was 195 ± 67 ml/m2. The study was supported by grants from Delegation à
la recherche clinique AP-HP (EMUL and PHRC n°AOM95082).
The study control group consisted of 433 healthy subjects of European origin recruited in
Centres for Preventive Medicine all over metropolitan France as part of the FITENAT study 2.
Their parents had to be born in France and their four grandparents had to be born in Europe.
Controls were selected to match the distribution of cases for sex and age at diagnosis of
cases.
EUROGENE study (EHF). All cases3 were patients of European origin (all born in Europe, from
parents and grand-parents born In France or neighbouring countries) with a diagnosis of
idiopathic DCM, i.e. left ventricle end-diastolic volume/diameter >117% of predicted value
according to age and body surface area on echocardiography and low ejection fraction
(<45%) confirmed over a three-month period, in the absence of causal factors such as
coronary artery disease (coronary angiography or coronary CT scan was mandatory if DCM
occurred after 35 years of age) or intrinsic valvular disease, documented myocarditis,
systemic disease, sustained rapid supraventricular arrhythmia or congenital malformation.
Recruitment was performed in 11 hospitals in seven European countries from September
2000 to February 2005. Only apparently sporadic DCM cases without additional (first degree)
relatives with DCM were selected from EUROGENE for the GWAS, and only patients from
France (3 centres in Paris-Ile de France), Italy (1 centre in Pavia) and Germany (3 centres in
Regensburg, Marburg and Munster) were selected to have a sufficient number per country.
A total of 463 DCM patients were included (382 males and 81 women, only 1 patient had
undergone a cardiac transplantation at inclusion). Mean age of patients was 51 ± 13 years,
mean ejection fraction was 30 ± 10%, mean end-diastolic diameter was 67 ± 11 mm. The
study was supported by grants from the “Fondation LEDUCQ”.
Controls for the German cases of the EUROGENE study were recruited from the German MI
Family Study at the University Hospital Regensburg and had no medical history for coronary
artery disease, myocardial infarction or dilated cardiomyopathy. A total of 281 white
European males with a mean age of 59.0 ± 9.6 years were included.
Controls for the French cases of the EUROGENE study were selected among the French
controls of the ECTIM Study (Etude Cas-Témoin sur l'Infarctus du Myocarde)4. These were
subjects aged 35-64 who had been randomly selected using electoral rolls in the areas of Lille,
Strasbourg and Toulouse covered by the WHO-MONICA (Multinational MONItoring of trends
and determinants in CArdiovascular disease) Project registers. Their parents had to be born in
the same regions and their four grand-parents had to be born in Europe.
Control subjects were also selected from healthy consultants or hospital professional
2
workers in clinical centres in Italy (72 controls).
PHRC study. DCM cases were French patients of European origin (all born in France, from
parents and grand-parents born in France or neighbouring countries; some patients of
Maghreb origin were retrospectively excluded) with a diagnosis of idiopathic DCM (enlarged
left ventricle end-diastolic volume/diameter >117% of predicted value according to age and
body surface area on echocardiography and low ejection fraction (<45%) clinically stable
over a three-month period, in the absence of causal factors such as coronary artery disease
(coronary angiography or coronary CT scan was mandatory if DCM occurred after 35 years of
age) or intrinsic valvular disease, documented myocarditis, or congenital malformation).
Only apparently sporadic DCM cases without additional (first degree) relative with DCM
were included. Recruitment was performed in eight hospitals in six regions in France (Lille,
Lyon, Nantes, Nice, Paris-Ile de France, and Tours) from October 2005 to November 2008. A
total of 292 DCM patients were included (232 males and 60 women, no patients had
undergone a cardiac transplantation at inclusion). Mean age of patients was 57 ± 12 years,
mean ejection fraction was 29 ± 9%, mean end-diastolic diameter was 70 ± 9 mm. The study
was supported by grants from “Programme Hospitalier de Recherche Clinique” (PHRC
n°AOM 04141).
Controls were selected among the French controls of the ECTIM Study as described above.
German replication study. German idiopathic DCM cases were recruited in at the German
Heart Institute Berlin and were of white European origin. Inclusion criteria for DCM cases
were the following: reduced systolic function (left ventricular ejection fraction (LVEF) <45 %),
after exclusion of major coronary artery disease (by angiography), significant (>grade 2)
valvular heart disease, hypertensive heart disease, congenital heart disease, myocarditis or
other secondary forms of heart failure. Patients with a positive family history were also
excluded. A total of 723 DCM patients were included (617 males, 106 females, mean age
45.6 ± 11.3 years, mean left ventricular ejection fraction 24.6 ± 9.3%).
German controls of European origin were recruited from two studies at the University
Hospital Regensburg (German MI Family Study and GoKard) and had no medical history for
coronary artery disease, myocardial infarction or DCM. A total of 726 controls were included
(606 males, 120 females, mean age 56.3 ± 12.2 years).
British replication study. DCM samples (442) were assembled from two cohorts of DCM
patients with either end-stage DCM requiring transplantation or with DCM confirmed both
clinically and by cardiac MRI scanning. The first group of patients (337 Caucasian cases) were
selected from a larger group of all patients who had been tissue-typed in the workup for
heart transplantation at the Harefield Hospital, Royal Brompton and Harefield NHS
Foundation Trust, London. Cases were selected based on a clinical diagnosis of DCM in the
absence of significant coronary artery disease, valvular heart disease or congenital heart
disease. Analysis of a subset of patients where ejection fraction (EF) by MUGA was available
showed an average EF of 21% ± 10%. Mean age of patients was 43±27 years. The second
group of patients (104 Caucasian cases) were selected from patients who had undergone
cardiac MRI phenotyping at the Royal Brompton Hospital. Cases were selected based on a
clinical and MRI diagnosis of DCM in the absence of significant coronary artery disease,
valvular heart disease or congenital heart disease. In this group of patients the mean age
was 55 ± 34 years, mean ejection fraction was 40%, mean end-diastolic diameter was 250 ±
487 mm. Age-matched controls were selected from the UK Blood Service panel 1 as
3
described in the WTCCC5 A total of 576 controls were included (277 males, 299 females,
mean age 41 ± 24 years).
Selection of patients with familial DCM for molecular screening of BAG3. Patients (n=168)
and their relatives gave written informed consent to participate to the study in accordance
with the protocol approved by the local ethics committee. The diagnostic criteria for DCM
were as described by Mestroni et al. 6. Patients were enrolled from the Paris registry of DCM
or from European countries participating in the Eurogene Heart Failure (EHF) program 3. The
familial origin of the disease was accepted if the index case had at least one affected firstdegree relative based on clinical examination. Included familial cases had no identified
mutation in the genes known to be mutated in familial DCM and screened in previous
studies: MYH7 and ANKRD1 for all participant, and ACTC, DES, VCL, LMNA, SGCD, TNNT2,
PLN, MYPN for the Paris registry only.
Methods
Constitution of DNA pools. Stock DNA of each sample was first quantified using a
spectrophotometer, checked for degradation on agarose gel and then adjusted to 15 ng/µl
and quantified 3 times by picogreen (Invitrogen). If the difference between the three
measures was greater than 10%, a fourth picogreen quantification was done. The average of
all picogreen measures was taken to determine the DNA concentration of each sample. The
volume of each sample added to a pool was calculated according to this concentration and
the number of samples in the pool which was defined a priori in the pooling scheme. The
DNA pools were stratified on population as well as gender and age when the numbers
permitted. We required that at least 25 samples were mixed in a single pool.
(Supplementary Table 1). The whole process of concentration adjustment of individual
samples and aliquoting to constitute the pools was automatized on a robot (Beckman
Biomek 3000) to reduce technical variability. Each pool was constituted twice and each pool
replicate was analysed on 2 arrays independently.
No correction for differences in allelic signal intensities when computing odds-ratios. To
estimate allele frequencies from DNA pools It has been proposed to introduce a correction
factor k which is specific of each SNP, to compensate for systematic differences in signal
intensities (p(x)=x/(x+ky))7. The coefficient k may be obtained from the ratio of allelic signals
in heterozygotes by genotyping a sample of individuals using the same array as for the poolsGWAS. However, k may be difficult to estimate reliably8. In the present analysis, we were not
interested in allele frequencies per se, but in association statistics (odds ratio), which are not
affected by k, under the assumption that the difference in signal intensity is not related to
disease status. As a consequence, despite some loss of precision due to a larger standard
error of the association statistic, we did not include a k correction.
BAG3 exonic sequencing in patients with familial DCM. Each of the exonic coding regions of
the gene was PCR amplified and directly sequenced by big dye chemistry on an ABI 3100
capillary sequencing apparatus (Applera). PCR primers (Supplementary Table 9) were also
used for sequence priming. Sequences were analysed with the CodonCode aligner software.
All variants were confirmed on both strands on an independent PCR amplification from a
second aliquot of DNA. When available, DNAs from other family members were genotyped
4
for the familial variant by PCR and sequencing. We also sequenced BAG3 in DNAs from 364
individuals of European descent without known cardiac disease. They were all sequenced on
one strand for the exons 2, 3 and 4 allowing detailed SNPs identification and frequency
determination in the control population (Supplementary Table 6). Moreover, 95 controls of
North African origin and 45 controls from Turkey were also sequenced to check respectively
for variants P115S in exon 2 and P380S in exon 4, respectively, as they were identified in
index cases originating from these 2 geographical regions.
Individual level validation by TaqMan 5’nuclease assay technology. For each SNP needing
validation at the individual level, 2 primers and 2 dual-labelled probes (5’FAM-3’TAMRA or
5’TET-3’TAMRA) were designed with the Primer Express software (Applied Biosystems).
Twenty ng of each DNA were amplified on a 96-well GeneAmp PCR System 9700 (Applied
Biosystems) in 7µl final volume with 900nM of each primer, 200nM of each probe, dNTP, 0.2
unit of BIOTAQ DNA Polymerase (Bioline) and a passive fluorescence reference Rox
(Euromedex). Before and after the amplification, the fluorescence of the reaction was
checked on an ABI Prism 7000 Real-Time PCR System (Applied Biosystem). The primers and
probes sequences used and the reaction conditions are provided in Supplementary Table 8.
References for supplementary material
1.
Charron P, Tesson F, Poirier O, Nicaud V, Peuchmaurd M, Tiret L, Cambien F, Amouyel
P, Dubourg O, Bouhour J, Millaire A, Juilliere Y, Bareiss P, André-Fouët X, Pouillart F, Arveiler
D, Ferrières J, Dorent R, Roizès G, Schwartz K, Desnos M, Komajda M. Identification of a
genetic risk factor for idiopathic dilated cardiomyopathy. Involvement of a polymorphism in
the endothelin receptor type A gene. CARDIGENE group. Eur Heart J 1999; 20:1587–1591.
2.
Mazoyer E, Ripoll L, Gueguen R, Tiret L, Collet J-P, dit Sollier CB, Roussi J, Drouet L.
Prevalence of factor V Leiden and prothrombin G20210A mutation in a large French
population selected for nonthrombotic history: geographical and age distribution. Blood
Coagul Fibrin 2009; 20:503–510.
3.
Duboscq-Bidot L, Charron P, Ruppert V, Fauchier L, Richter A, Tavazzi L, Arbustini E,
Wichter T, Maisch B, Komajda M, Isnard R, Villard E. Mutations in the ANKRD1 gene
encoding CARP are responsible for human dilated cardiomyopathy. Eur Heart J 2009;
30:2128–2136.
4.
Parra HJ, Arveiler D, Evans AE, Cambou JP, Amouyel P, Bingham A, McMaster D,
Schaffer P, Douste-Blazy P, Luc G. A case-control study of lipoprotein particles in two
populations at contrasting risk for coronary heart disease. The ECTIM Study. Arteriosclerosis
and Thrombosis 1992; 12:701–707.
5.
WTCCC Consortium. Genome-wide association study of 14,000 cases of seven
common diseases and 3,000 shared controls. Nature 2007; 447:661–678.
6.
Mestroni L, Maisch B, McKenna WJ, Schwartz K, Charron P, Rocco C, Tesson F, Richter
A, Wilke A, Komajda M. Guidelines for the study of familial dilated cardiomyopathies.
Collaborative Research Group of the European Human and Capital Mobility Project on
Familial Dilated Cardiomyopathy. Eur Heart J 1999; 20:93–102.
7.
Sham P, Bader JS, Craig I, O'Donovan M, Owen M. DNA Pooling: a tool for large-scale
association studies. Nature Rev Genet 2002; 3:862–871.
8.
Macgregor S, Visscher PM, Montgomery G. Analysis of pooled DNA samples on high
5
density arrays without prior knowledge of differential hybridization rates. Nucleic Acid Res
2006; 34:e55.
6
Supplementary Table 1. Constitution of DNA pools
Study
Sub-study
gender
Age
Number
of DNA
Sub-study
samples
/pool
CASES
gender
Number
of DNA
Age
samples
/pool
CONTROLS
EHF-FRA
Male
<46
41
ECT-STR
Male
<55
71
EHF-FRA
Male
>46
41
ECT-STR
Male
>55
99
PHR-FRA
Male
<52
113
ECT-TOU
Male
<55
94
PHR-FRA
Male
>52
112
ECT-TOU
Male
>55
59
CARDIGENE CAR-FRA
FRANCE CAR-FRA
Male
< 52
171
FIT-FRA
Male
<46
156
Male
> 52
166
FIT-FRA
Male
>46
151
PHR-FRA
Female
<52,5
29
FIT-FRA
Female
<52
37
CAR-FRA
Female
< 58
43
PHR-FRA
Female
>52,5
27
FIT-FRA
Female
>52
44
CAR-FRA
Female
> 58
43
<46,5
139
CON-GER
Male
<60
138
>46,5
139
CON-GER
Male
>60
135
EHF-ITA Male+Female
<45
51
CON-ITA
Male
<47
35
EHF-ITA Male+Female
>45
52
CON-ITA
Male
>47
36
PHRC +
EHF
FRANCE
FEMALE/
FRANCE
EHF-GER Male+Female
EHF
GERMANY EHF-GER Male+Female
EHF ITALY
EHF: European Heart Failure study, PHRC: Programme Hospitalier de Recherche Clinique
Overall, DNA from 1167 patients and 1055 control subjects contributed to the pools. These
numbers are slightly smaller than those reported in table 1 because some samples could not
be included in the pools constitution for technical reasons but could be individually
genotyped.
The same set of controls from ECTIM/Strasbourg and ECTIM/Toulouse was used for the
French male cases included in the EHF and PHRC studies. The pools from German and Italian
cases included both males and females and the comparison groups were constituted only of
males. As a consequence in the statistical analysis of the pools-GWAS, no adjustment on
gender was possible in the German and Italian samples. On the other hand, in the analyses
based on individual genotyping, adjustment on population and gender accounted for the
presence of women in the German and Italian samples.
7
Supplementary Table 2. Allele frequencies and association statistics for SNPs found
associated with DCM in the pools-GWAS and confirmed by individual genotyping
study
cases contr.
MAF
cases
MAF P(HW) P(HW)
contr. cases contr.
OR 95%CI
P (OR)
rs10927875
CARDIGENE MEN
337
308
0.264
0.351
1.00
0.95
0.662
0.520
0.843
8.1E-04
EHF FRA MEN
297
312
0.258
0.337
0.24
0.40
0.700
0.549
0.891
3.8E-03
EHF GER MEN
221
274
0.265
0.349
0.69
0.86
0.673
0.510
0.888
5.2E-03
EHF ITA MEN
87
71
0.293
0.338
0.33
0.18
0.787
0.465
1.331
3.7E-01
EHF WOMEN
224
85
0.286
0.300
1.00
1.00
0.915
0.620
1.350
6.5E-01
REPLIC GER MEN
614
603
0.269
0.324
0.95
0.68
0.806
0.671
0.968
2.1E-02
REPLIC GER WOMEN
106
120
0.274
0.300
0.41
0.94
0.844
0.528
1.349
4.8E-01
REPLIC UK MEN
337
275
0.274
0.305
0.84
0.56
0.832
0.644
1.073
1.6E-01
REPLIC UK WOMEN
101
299
0.292
0.329
0.34
0.04
0.834
0.582
1.197
3.2E-01
rs16983785
CARDIGENE MEN
332
307
0.096
0.078
0.33
1.00
1.269
0.851
1.892
2.4E-01
EHF FRA MEN
296
320
0.106
0.056
0.26
1.00
2.068
1.335
3.203
1.1E-03
EHF GER MEN
222
274
0.079
0.040
0.01
1.00
1.904
1.124
3.225
1.7E-02
EHF ITA MEN
87
71
0.167
0.070
0.54
1.00
2.889
1.308
6.378
8.7E-03
EHF WOMEN
225
83
0.107
0.060
0.95
1.00
1.858
0.916
3.768
8.6E-02
REPLIC GER MEN
611
603
0.066
0.061
0.99
0.33
1.100
0.795
1.522
5.6E-01
REPLIC GER WOMEN
104
120
0.072
0.067
1.00
1.00
1.096
0.513
2.341
8.1E-01
REPLIC UK MEN
335
276
0.058
0.034
1.00
1.00
1.749
0.993
3.078
5.3E-02
REPLIC UK WOMEN
101
298
0.059
0.047
1.00
1.00
1.300
0.634
2.664
4.7E-01
rs2234962
CARDIGENE MEN
334
304
0.129
0.188
0.96
0.69
0.636
0.467
0.866
4.1E-03
EHF FRA MEN
300
317
0.142
0.216
0.50
0.78
0.599
0.443
0.810
8.8E-04
EHF GER MEN
223
273
0.114
0.209
0.37
1.00
0.480
0.333
0.693
8.8E-05
EHF ITA MEN
87
71
0.075
0.190
1.00
0.46
0.307
0.145
0.652
2.1E-03
EHF WOMEN
224
85
0.125
0.259
0.04
0.60
0.385
0.240
0.617
7.5E-05
REPLIC GER MEN
613
602
0.153
0.196
0.20
0.37
0.732
0.588
0.909
4.9E-03
REPLIC GER WOMEN
106
120
0.189
0.138
0.59
0.09
1.608
0.924
2.798
9.3E-02
REPLIC UK MEN
327
266
0.180
0.214
0.70
0.83
0.809
0.603
1.087
1.6E-01
REPLIC UK WOMEN
101
297
0.188
0.204
0.96
0.08
0.902
0.592
1.374
6.3E-01
MAF: minor allele frequency, P(HW): test of deviation from Hardy-Weinberg equilibrium.
The proportion of samples successfully genotyped for rs10927875, rs16983785 and
rs2234962 were 0.983, 0.981 and 0.978 respectively.
£ OR statistics were computed from the regression coefficients of the population specific
logistic model. Meta analysis summary effects: rs2234962 (BAG3) OR: 0.66 (95% CI 0.530.82), rs10927875 (ZBTB17) OR: 0.76 (0.70-0.84), rs16983785 (TAK1L) OR: 1.53 (1.25-1.89)
were computed using function meta.summaries of the rmeta R package.
8
Supplementary Table 3. Pools-GWAS SNPs at the ZBTB17/ HSPB7/CLCNKA locus and
association p-value with DCM
SNP
rs848189
rs848194
rs10927875
rs698894
rs4661681
rs1739822
rs1739828
rs1763613
rs1763601
rs1544131
rs3754323
rs945425
rs1010069
rs10927888
rs1805152
rs883867
Gene
ZBTB17
ZBTB17
ZBTB17
C1orf64
C1orf64
HSPB7
HSPB7
CLCNKA
CLCNKA
CLCNKA
CLCNKA
CLCNKA
P
.1237
.0005
1.33 x 10-7
.9333
.0086
.8797
.4723
.5073
.0001
.6066
.3733
.0003
.0006
.0542
5.61 x 10-6
.2393
Region
INTRONIC
INTRONIC
INTRONIC
INTERGENIC
INTERGENIC
INTERGENIC
UPSTREAM
INTRONIC
DOWNSTREAM
UOSTREAM
INTERGENIC
UPSTREAM
INTRONIC
INTRONIC
NON_SYNONYMOUS_CODING
INTRONIC
In bold are shown the DCM-associated SNPs that were tested at the individual level.
9
Supplementary Table 4. Haplotype analysis of 5 SNPs at the ZBTB17/HSPB7/CLCNKA locus
Haplotype
SNP (rs number)
Frequencies
Haplo
Controls
N=985
Cases
N=1137
0.995
0.508
0.603
T
0.954
0.026
0.022
C
T
0.998
0.088
0.080
C
T
A
0.908
0.032
0.024
T
G
C
T
0.898
0.025
0.023
G
C
T
A
0.995
0.301
0.230
10927875
1763601
945417
945425
1048261
-typic
R2
C
T
G
C
T
C
T
C
C
C
G
C
C
G
T
T
Haplotypic ORs
[95%CI]*
reference
0.730 [0.479 – 1.112]
p = 0.142
0.745 [0.592 – 0.937]
p = 0.012
0.655 [0.448 – 0.958]
p = 0.029
0.805 [0.521 – 1.244]
p = 0.3239
0.641 [0.553 – 0.742]
p = 3.22 10-9
Global Test for Differences in Haplotypic Frequencies: 2 with 5 df = 39.78, p = 1.65 10-7
* ORs were adjusted for gender and study.
10
Supplementary Table 5. Associations between cis-SNPs and the expression of SPEN, ZBTB117, HSPB7, CLCNKA and CLCNKB in monocytesderived macrophages – Cardiogenics expression study.
Probe ID
ILMN_1802611
Gene
sequence
start-end
Within
gene
number of genotypes
ILMN_1711048
ILMN_2200836
ILMN_1787576
ILMN_2364072
SPEN
ZBTB17
HSPB7
CLCNKA
CLCNKA
1604694616139537
1614095316175101
1621311216217538
1622107316233131
1622107316233131
snp
position
t
P
t
P
t
P
t
P
t
P
rs12026701
15995786
CC:195
CT:317
TT:102
-
-
-
-
-5.11
4.4E-07
-
-
-
-
rs11589957
15998794
TT:328
CT:240
CC:46
-
-
rs2013019
16001907
TT:178
CT:293
CC:143
-4.91
1.2E-06
-
-
11.94
1.2E-29
7.57
1.4E-13
7.28
1.0E-12
-4.07
5.4E-05
8.94
4.7E-18
4.93
1.1E-06
4.52
7.3E-06
rs2862162
16015547
CC:271
AC:274
AA:69
-3.49
5.1E-04
-
-
11.27
7.3E-27
6.28
6.6E-10
6.41
2.9E-10
rs4661661
16034423
AA:467
AG:136
GG:11
-
-
-3.03
2.6E-03
-
-
-
-
-
-
rs6701290
16061268 SPEN
AA:497
AG:110
GG:7
-
-
-
-
-
-
-
-
-
-
rs7552077
16069346 SPEN
AA:210
AG:289
GG:115
2.94
3.5E-03
4.95
9.9E-07
-9.33
2.0E-19
-6.15
1.4E-09
-5.93
5.2E-09
rs904910
16072750 SPEN
TT:307
CT:250
CC:57
-2.69
7.2E-03
-
-
10.60
3.4E-24
6.93
1.1E-11
7.29
9.9E-13
rs16852052
16074089 SPEN
GG:514
GT:98
TT:2
-
-
-
-
3.09
2.1E-03
-
-
-
-
rs598371
16085326 SPEN
AA:212
AG:287
GG:115
2.96
3.1E-03
4.95
9.5E-07
-9.46
7.0E-20
-6.31
5.4E-10
-5.96
4.2E-09
rs12046015
16114409 SPEN
CC:536
CT:68
TT:10
-
-
-
-
-
-
-
-
-
-
rs10927873
16124487 SPEN
GG:536
AG:75
AA:3
-
-
-
-
-
-
-
-
-
-
rs848206
16125583 SPEN
AA:508
AG:100
GG:6
2.59
9.8E-03
-
-
-
-
-
-
-
-
rs848208
16128231 SPEN
GG:508
AG:100
AA:6
2.59
9.8E-03
-
-
-
-
-
-
-
-
rs848210
16132400 SPEN
GG:212
AG:287
AA:115
2.96
3.1E-03
4.95
9.5E-07
-9.46
7.0E-20
-6.31
5.4E-10
-5.96
4.2E-09
rs848212
16136039 SPEN
CC:210
CT:288
TT:115
2.95
3.3E-03
4.98
8.3E-07
-9.32
2.2E-19
-6.15
1.4E-09
-5.92
5.5E-09
rs848215
16139734
CC:507
CT:101
TT:6
-
-
-
-
-
-
-
-
-
-
rs848189
16150234 ZBTB17
GG:406
AG:190
AA:18
-
-
-3.10
2.0E-03
-
-
-
-
-
-
rs848194
16157929 ZBTB17
AA:215
AG:284
GG:115
-
-
4.94
1.0E-06
-9.44
7.7E-20
-6.29
6.0E-10
-6.09
2.0E-09
11
rs10927875
16171899 ZBTB17
CC:275
CT:279
TT:60
-3.56
3.9E-04
-
-
14.72
3.6E-42
8.77
1.8E-17
8.79
1.5E-17
rs698894
16182875
CC:536
CT:73
TT:4
-
-
-
-
-
-
-
-
-
-
rs4661681
16194110
TT:496
CT:113
CC:4
-
-
-
-
-
-
-
-
-
-
rs1739822
16196820
AA:495
AG:112
GG:7
-
-
-
-
-
-
-
-
-
-
rs1739828
16200009
AA:412
AG:182
GG:20
-
-
-
-
-4.09
4.9E-05
-2.78
5.7E-03
-
-
rs1763613
16204496
CC:450
AC:150
AA:14
-
-
-
-
-3.49
5.2E-04
-2.60
9.7E-03
-
-
rs1763601
16213047
TT:221
GT:300
GG:93
-
-
-
-
13.45
2.9E-36
9.40
1.1E-19
8.99
3.1E-18
rs1544131
16218322 HSPB7
CC:535
CT:75
TT:4
-
-
-
-
-
-
-
-
-
-
rs3754323
16219044
TT:522
GT:89
GG:3
-
-
-
-
-3.12
1.9E-03
-
-
-
-
rs945425
16220999
CC:283
CT:275
TT:56
-
-
-
-
17.72
6.1E-57
11.15
2.2E-26
9.87
2.1E-21
rs1010069
16225524 CLCNKA
GG:196
AG:289
AA:129
-
-
4.33
1.7E-05
-9.72
7.2E-21
-7.32
8.0E-13
-6.34
4.5E-10
rs1805152
16229088 CLCNKA TT:195
CT:299
CC:120
-
-
-
-
12.25
5.6E-31
8.74
2.4E-17
7.73
4.6E-14
rs883867
16230672 CLCNKA
CC:473
AC:135
AA:6
-
-
-3.27
1.1E-03
-2.96
3.2E-03
-
-
-
-
rs10803410
16240911
CC:171
CT:302
TT:141
-
-
-
-
8.37
4.1E-16
5.88
6.8E-09
5.81
9.9E-09
rs6683445
16240990
AA:171
AC:305
CC:138
-
-
-
-
8.26
8.9E-16
5.77
1.3E-08
5.79
1.2E-08
rs5257
16245711
GG:382
AG:205
AA:26
-
-
-4.09
4.8E-05
-
-
-
-
-
-
rs945403
16246917
GG:544
AG:66
AA:2
-
-
-
-
-
-
-
-
-
-
rs5252
16250802
GG:461
AG:143
AA:8
-
-
-3.67
2.6E-04
-
-
-
-
-
-
rs10803414
16253169
GG:171
AG:320
AA:123
-
-
-
-
-6.78
2.8E-11
-4.15
3.9E-05
-3.97
8.0E-05
rs9442235
16265944
GG:142
GT:333
TT:139
-
-
-
-
-6.94
9.9E-12
-3.79
1.7E-04
-3.28
1.1E-03
rs10927902
16267734
CC:368
CT:220
TT:26
-
-
-3.12
1.9E-03
-
-
-
-
-
-
This table is based on the analysis of Cardiogenics expression study data. We selected all SNPs located on the sequence of chromosome 1
encompassing SPEN, ZBTB17, HSPB7, CLCNKA and CLCNKB (as well as 50KB upstream and downstream). DCM-associated SNPs are shown in
bold. Note that no eQTL for CLCNKB was detected.
12
The observed associations with HSPB7 and CLCNKA expression must be interpreted with caution because the ILMN_2200836 probe (HSPB7)
carries a SNP (rs1048334) which is located in the middle of the probe and is in strong LD with rs945417 (r²=0.85). Recent data from the
“Thousand genomes” low coverage pilot study on 60 CEU individuals (http://browser.1000genomes.org/) indicate that the ILMN_1787576 and
ILMN_2364072 also appear to target polymorphic sequences of CLCNKA. Although we have no precise information on the LD of these CLCNKA
polymorphisms with the other SNPs we investigated in the region, it cannot be excluded that the associations observed with probe expression
at the HSPB7 and CLCNKA loci are the consequence of the polymorphism of the sequences targeted by the probes and are therefore not true
eQTLs.
13
Supplementary Table 6. Identified SNPs and mutations in the coding sequences of BAG3 in 168
index cases with familial form of DCM
variant name
protein
effect
nature of
variant
dbSNP
reference
frequency
in index
cases,
n/336
alleles (%)
121429394
212G>A
R71Q
missense
rs35434411
9 (0.026)
14 (0,020)
2
121429412
536C>T
P77L
missense
0
1 (0,001)
2
121429462
586A>T
I94F
missense
1 (0.003)
0
2
121429525
649C>T
P115S
missense
1 (0.003)
0
2
121429633
757T>C
C151R
missense
rs2234962
30 (0.089)
143 (0,206)
2
121429645
769G>A
A155T
missense
rs61756328
0
4 (0,006)
3
121431946
993G>A
Q229Q
synonymous
0
1 (0.001)
3
121432011
1058delA
251RfsX56
frame shift
1 (0.003)
0
3
121432036
1083C>G
P259P
synonymous
0
1 (0.001)
3
121432147
1194C>T
His296His
synonymous
0
1 (0.001)
4
121435991
1231C>T
R309X
nonsense
1 (0.003)
0
4
121436068
1308T>G
P334P
synonymous rs3858339
43 (0.128)
64 (0.092)
4
121436204
1444C>T
P380S
missense
1 (0.003)
0
4
121436219
1459_1466
delTCTTCCC
385QfsX56
frame shift
1 (0.003)
0
4
121436246
1486_1487 delGA
395GfsX48
frame shift
1 (0.003)
0
4
121436286
1526C>T
P407L
missense
43 (0.128)
66 (0.095)
4
121436362
1602A>G
V432V
synonymous rs196295
69 (0.205)
146 (0.210)
4
121436429
1669G>A
E455K
missense
1 (0.003)
0
4
121436468
1708G>A
V468M
missense
1 (0.003)
0
genomic
Exon position
2
rs3858340
frequency
in
controls,
n/694
alleles (%)
Among identified variants, 6 were known SNPs referenced in dbSNP and 4 were present in the control
group of 347 healthy individuals of European descent in which they were tested. The 9 remaining variants
were found once and none of them was present in the control group. All carriers of these mutations were
heterozygous. The identified variants included three insertions/deletions resulting in a truncation of the
encoded protein sequence (Q251RfsX56, R396GfsX48, S385QfsX56), a substitution creating a premature
stop codon (p.R309X) (Supplementary Fig. 3) and 5 missense mutations leading to single amino acid
changes (I94F, P115S, P380S, E455K, V468M).
Further analyses excluded 3 missense variants as possibly disease causing: I94F, despite perfect
interspecies conservation, was not segregating in one affected relative of the family; P115S was not
conserved (Fig. 5d) and was found in one control subject of North African origin out of 95 genotyped (the
index case carrying this variant was of North African origin, explaining why a control group of similar origin
was genotyped for this variant); P380S was not conserved (Fig. 5d). Software prediction of mutation
consequences using Polyphen, SIFT and SNAP also showed that only E455K and V468M among the 5
missense mutants were predicted to impair the protein function.
14
Supplementary Table 7: Clinical features of DCM patients and BAG3 mutation carriers (at genetic inquest) from familial forms of DCM
Subject
Age (y)
/sex
Age at
diagnosis (y)
NYHA ECG
class
LVEDD EF
(mm) (%)
IVS
(mm)
Muscular Clinical
clin./CPK status #
Comments #
Pedigree A (S385QfsX56 mutation)
I.1
(no DNA)
death at
48y /M
NA
NA
NA
NA
NA
NA
NA
Possibly
Affected
cardiomegaly, no additional data,
cardiac death at 48y
II.2
(no DNA)
non
cardiac
death/F
60
NA
AF and
bradycardia
63
19
NA
NA
Affected
Death (non-cardiac cause) before
genetic inquest
II.3
67/F
-
III
SR,
52
intermittent
atrial
fibrillation
50% 10
NL/NA
Possibly
affected
LVEDD>112% + atrial fibrillation +
congestive heart failure
II.4
69/F
-
I
SR, LVH
63*
54% 7
NL/NA
Possibly
affected
LVEDD>117% + congestive heart
failure
II.6
67/F
60
I
SR
64*
40% 9
NL/NA
Affected
non sustained VT at 63y, atrial
fibrillation at 69y, pace maker at
73y; cramps in legs and hands at
67y
III.1
(no DNA)
death at
34y /M
29
IV
SR, iBBG
71*
25
NA /
elevated
CPK
Affected
DCM known at 29y; sustained VT
and congestive heart failure
leading to death although
circulatory support; death before
genetic inquest
15
III.2
32/M
- (no
systolic
dysfunction)
II
SR
66*
59
8
NL/NA
Affected
LVEDD>117% + nsVT + Dyspnoea
III.3
33 /F
-
I
SR, RBBi
52
59
8
NL/NA
Not affected mutation carrier
III.4
34 /M
-
I
SR
56,5*
59
9
NL/NA
Possibly
affected
LVEDD>117%; mutation carrier
III.7
28/M
18
IV
RS, systolic
LVH
69*
10
NA
NL/NL
Affected
Index case, diagnosis of DCM at
18y, fulminant evolution with
Heart Transplant at 18y
Pedigree B (V468M mutation)
II.2
46/M
45
II
AF
66*
26
12
NL/NL
Affected
III.4
26/M
26
I
AF
59*
44
11
NL/NL
Affected
Pedigree C (R309X mutation)
II.1
24 /F
24
II
SR, RBBB
67*
36
7
NL/NL
Affected
III.1
47 /F
47
II
SR
67*
37
8
NL/NL
Affected
Pedigree D (R395GfsX48 mutation)
I.2
52 /F
51
III
SR
53*
44
8
NL/NL
Affected
LVEDD>117% + LVEF<50%
II.1
26 /F
26
IV
SR,
abnormal T
waves
72*
17
7
NL/NL
Affected
Index case, Heart transplantation
at 26 y
16
Pedigree E (Q251RfsX56 mutation)
II.1
57 /F
I.1
death at
(no DNA) ?/M
58
III
SR, cRBBB
61*
26
6
NL/NL
Affected
Index case
NA
NA
NA
NA
NA
NA
NA/NA
Affected
DCM, death before genetic
inquest
Affected
DCM diagnosed before death
(congestive heart failure), death
before genetic inquest
Pedigree F (E455K mutation)
II.1
death at
(no DNA) 53 y/M
NA
NA
NA
NA
NA
NA
II.2
death at
(no DNA) 49 y/ F
NA
IV
NA
NA
NA
NA
NA
Possibly
affected
Death due to heart failure, before
genetic inquest (no DNA) but
obligate carrier
II.3
death at
(no DNA) 51 y/F
NA
NA
NA
NA
NA
NA
NA
Affected
DCM diagnosed before death
(congestive heart failure), awaiting
for HT, death before genetic
inquest
II.4
death at
(no DNA) 45 y/ M
NA
NA
NA
NA
NA
NA
NA
Possibly
affected
Sudden death at 45y, before
genetic inquest, previous history
of cancer
III.2
death at
(no DNA) 36 y/ M
NA
NA
NA
NA
NA
NA
NA
Affected
DCM diagnosed before death
(congestive heart failure), awaiting
for HT, death before genetic
inquest
III.5
47
I
SR
59*
40
9
NB/NA
Affected
Sustained VT (47 y) and DCM
48/F
17
III.7
45 /M
44
III
SR, iLBBB
80*
16
7
NL/NL
Affected
Index patient with DCM and nsVT,
HT at 45y (3 months after
genetics)
IV. 8
25/F
13
IV
NA
NA
NA
NA
NA
Affected
DCM diagnosed at 13 y, with HTx
in emergency
IV.10
17/F
16
I
SR
50*
46
6
NL/NA
Affected
LVEDD>112% (33mm/ m²) and
LVEF<50% at inclusion, then LVEF
decreased to 36%
IV. 11
07/M
-
1
SR
47
69
7
NL/NA
not affected mutation carrier
Age is age at genetic inquest; NYHA: New York Heart Association functional class; LVEDD : left ventricular end diastolic diameter; * is for LVEDD
above theoretical value accounting for age and body surface area; EF: ejection fraction; IVS: inter-ventricular septum thickness
(echocardiography); muscular clin.: abnormality in clinical muscular testing; CPK: elevated serum creatine kinase level; M: male; F: female; SR:
sinus rhythm; iLBB or cLBB: incomplete or complete left bundle branch block: rBBB: right bundle branch block; nsVT: non sustained ventricular
tachycardia; LVH: left ventricular hypertrophy on ECG NL: normal; NA: not available; HT: heart transplantation; IAD: inter-atrial defect; †:no
DNA available for genotyping but obligated carrier. AF: atrial fibrillation. #: Clinical status according to Mestroni et al., Eur. Heart J 20, 93-102
(1999)
18
Supplementary Table 8: primers and probes sequences and reaction conditions for Taqman genotyping
SNP
rs16983785
rs7328410
rs2234962
rs1991914
rs13176432
rs10491858
rs5970164
rs856003
rs10927875
rs11543052
F
Forward primer sequence (5'-3')
WT
probe sequence (5'Fam - 3' Tamra)
Anne
aling
R
Reverse primer sequence (5'-3')
M
probe sequence (5'Tet - 3'Tamra)
Temp.
F
TGA CCC CCA GGA TTT TTC TGT
T
ACA AGT TTG AGC ATG TAA TTT GCA GTG CTT C
R
GTT TTT CAC TGC TAT TCT TGC ACA A
G
CAA GTT TGA GCA TGT AAT TTG CAG GGC TT
F
AAC ATT TTT ATC AAC AAG GAT AAA GGA AA
T
TCC CCC AAC CTC AAT TGT AAG CAA AAA AT
R
TCC AGA AAG GGC AAA TAA TAT AGA CC
G
CCC CCA ACC TCA ATT GTA AGC AAA CAA T
F
TCC CAG TCA CCT CTG CGG
T
CCA GAA ACC ACT CAG CCA GAT AAA CAG TGT
R
CTC TCC TTA CCT CAG GTC CGT G
C
ACC ACT CAG CCA GAT AAA CAG CGT GG
F
TTT ATA AAA ACT ACC TAC CAA CAT TG
A
AGA AAA TTC TAC ACC CAC AAA CTT TGA G
R
TTC CTT GTA CTT AAA GTG TAC AGT CT
C
AGA AAA TTC TAC ACC CAC ACA CTT TGA
F
CCG CAG GGC TCA TCC TT
T
ATG CTT TGA CCA GGC CTG AAG GTC TC
R
AAT CCG CTC ATC AGA TAC TGC TT
G
ATG CTT TGA CCA GGC CTG ACG GT
F
CCA TGA GAG GCT GTA ATA TTG CAG
T
TGT GCT GGC ATA GTG GAA AGG TTC ATC
R
AGC TGT AGT GTA TCA AGA AAC GGG TAG
C
TGC TGG CAT AGT GGA AAG GTC CAT CT
F
GTT GGC TGC ATC ACG AGA AGT AA
C
ACT GGC TTA CAT GCT ACA TGA CGC CAT T
R
TGG AAG TGT TCT CTC TCC CTT TCA
T
ACT GGC TTA CAT GCT ACA TGA TGC CAT TC
F
TGG CGT TTG TAA AAA AAC
A
ATT GAT ATA TTT GGA TTC AAA TCT ACC A
R
GAA CTG ATA GAA TGG GAA AA
G
ATT GAT ATA TTT GGA TTC AAG TCT ACC A
F
GTT TGG TTG GGA AAT TGT TGA TCT
C
CAC TGG GCA ATG CTC ACG CCT T
R
TGG ACT GTG TAC TTC CAG AAT GTT AAG
T
TGA TCA CTG GGC AAT GCT CAC ACC
F
GGA TTT AGA AGT GAA GCA TGC ATT TT
T
CAG AGC AGT GTG TGG ATT ACA CTA TCA CTG GA
R
GTC TTT TCC TTC TTC TCA ATT CGT ATT TT
C
AGA GCA GTG TGT GGA TTA CAC TAT CAC CGG
69 °C
67 °C
67 °C
62 °C
65 °C
67 °C
69 °C
57 °C
67 °C
69 °C
19
rs1353456
rs2832070
rs1378796
rs2290906
rs1763601
rs945417
rs945425
rs1048261
rs3858340
rs35434411
F
TCT AGG GAA ACG CAG CTG G
A
TAC AAC AGA GAA ACG TCG CTA TAG AAC TGC A
R
AGT CGC CAA GGT ACA AGT GAA A
C
AAC AGA GAA ACG TCG CTA TAG ACC TGC ATT
F
GCT AAC TAT AAT CTT GTA TTG ACT TAC TGG CT
T
TTT GCA AAT TAT TGG ATT TGT CAT TGC CTT T
R
GGA AAC TTT TTG GCT GAT TTG G
C
TGC AAA TTA TTG GAT TTG TCA TTG CCC TT
F
TTG CCA TCT GTC CAG TGA GGT A
A
CAA AGC CAC TGG CTC ATA GAC TGC TAT CTC
R
TTT GAA GGA TTC AAC CCA TAA TGA C
C
CAA AGC CAC TGG CTC ATA GCC TGC
F
CTC CCC GCC ATC ATC TCA
T
AAT GCC ACG CTG CCT TCT TCG AG
R
CAA CAC TAG GTG CGG ATG CA
C
AAT GCC ACG CTG CCT TCC TCG
F
GCC TGC CTG GAG TTC TCA AA
T
AGG GCT GGA AAC CCT GAG AGA AAA AGA
R
CCA TCC CCA AAT CTC AAA GAA G
G
AGG GCT GGA AAC CCT GAG AGA AAC AG
F
TGG AAT GTC AGG CTG TGA GCT
G
TCT GGG CCA TGG ACA GGC CT
R
ACT GTC CCT GGG CTT GGG
C
CTC TGG GCC ATG GAG AGG CCT
F
GAC AGG TAT ATG CTC TAC CAA TCA TCA CT
C
CGG CCG GGG TTT ACC GTG G
R
TTG GTT TGT CAA GGA CGT TTT TAA TC
T
CGG CCG GGG TTT ACC ATG GA
F
ACA ATC TCC ATT TTC ATT AAC GGG
T
TCT GAA CCC AGG GTG TCA ACA GCT G
R
GGA AAT ACT CCT CAG TGC CCA G
A
TCT GAA CCC AGG GTG TCA ACT GCT G
F
CAC TTT CAG CAC TCC TGG ATG TT
C
TCG GCT TCT CCT GGT TTT GGA GGT
R
GGC TAC AGA AGA GAG GGC AGC
T
TCG GCT TCT CCT GGT TTT GGA AGT G
F
CTG TGT TTC TCC ACT TTT TAT TTC AGG
G
CCA ATG GCC CTT CCC GGG AG
R
GGG TGG CCT TCC CTA GCA
A
TGC CAA TGG CCC TTC CCA GG
68 °C
69 °C
67 °C
67 °C
69 °C
66°C*
68 °C
67 °C
67 °C
67 °C
The PCR program used was: 95°C 10min followed by 40 cycles : [ 92°C - 15sec / annealing Temp - 1 min]. Nucleotides for wild-type (WT, define as the more
frequent allele) and mutant (M; define as the less frequent allele) are indicated in the 4 th column.
* for this SNP, 5% DMSO was added in the PCR reaction
20
Supplementary Table 9. PCR primers and conditions for sequencing of BAG3 and HSPB7
BAG3 exon
primers (5'>3')
name
5' position (ref hg19) PCR: size;Tm;MgCl2;DMSO
1
cgcgattatagccgatgact
BAG3-exon1F
121410858
gcctgccgtcgaggt
BAG3-exon1R
121411420
aggagggttcacttcccagt
BAG3-exon2F
121429306
atgccctgcatgtgaacag
BAG3-exon2R
121429805
ggagtcatttgtggggtcat
BAG3-exon3F
121431722
ccctggagacataccaccat
BAG3-exon3R
121432219
caatttctgtgactttcagtcagtt
BAG3-exon4.1F
121435918
tttgtcagtcttcttgccttca
BAG3-exon4.1R
121436413
gccatcctggagaaggtaca
BAG3-exon4.2F
121436345
tttgcctccacccaagttac
BAG3-exon4.2R
121436927
cagggacagtcggccttat
Hspb7-exon1F
16344582
cagacgtcccctccctgt
Hspb7-exon1R
16344207
atgcaaggcctgctccat
Hspb7-exon2F
16343748
gtctggggtcccaggatg
Hspb7-exon2R
16343472
ggggttagaatggggagaag
Hspb7-exon3F
16342343
ggctagaacctgggctgag
Hspb7-exon3R
16341970
2
3
4.1
4.2
562bp;62°C;2mM;10%
499bp;65°C;2mM;none
497bp;55°C;2mM;none
495bp;65°C;2mM;none
582bp;55°C;1mM;10%
HSPB7 exon
1
2
3
376bp;TD65-55°C;2mM;10%
271bp;TD65-55°C;2mM;10%
374bp;60°C;2mM;none
The PCR program used was: 95°C 5 min followed by 35 cycles : [ 95°C – 15 sec / Tm (see table) – 30 sec/ elongation Temp – 30 sec]
21
Supplementary Figure 1. Association plot of SNP with DCM around the lead
SNP rs16983785 (legend in figure 2)
22
Supplementary Figure 2. Ideogram from UCSC Genome Browser (assembly
GRCh37/hg19) presenting the associated region on chromosome 1 and gene
structure and 5’->3’ orientation. Position of each of the five SNPs in LD
defining DCM associated haplotypes are indicated.
rs10927875
rs1763601
rs1048261
rs945417 rs945425
23
Supplementary Figure 3. Truncation mutations in the BAG3 gene identified in
patients with familial DCM
24
Supplementary Figure 4. Families of index cases with DCM in whom a
mutation in the BAG3 gene was detected
25