Population-based variation in cardiomyopathy genes.

Abstract

Hypertrophic cardiomyopathy and dilated cardiomyopathy arise from mutations in genes encoding sarcomere proteins including MYH7, MYBPC3, and TTN. Genetic diagnosis of cardiomyopathy relies on complete sequencing of the gene coding regions, and most pathogenic variation is rare. The 1000 Genomes Project is an ongoing consortium designed to deliver whole genome sequence information from an ethnically diverse population and, therefore, is a rich source to determine both common and rare genetic variants.
We queried the 1000 Genomes Project database of 1092 individuals for exonic variants within 3 sarcomere genes MHY7, MYBPC3, and TTN. We focused our analysis on protein-altering variation, including nonsynonymous single nucleotide polymorphisms, insertion/deletion polymorphisms, or splice site altering variants. We identified known and predicted pathogenic variation in MYBPC3 and MYH7 at a higher frequency than what would be expected based on the known prevalence of cardiomyopathy. We also found substantial variation, including protein-disrupting sequences, in TTN.
Cardiomyopathy is a genetically heterogeneous disorder caused by mutations in multiple genes. The frequency of predicted pathogenic protein-altering variation in cardiomyopathy genes suggests that many of these variants may be insufficient to cause disease on their own but may modify phenotype in a genetically susceptible host. This is suggested by the high prevalence of TTN insertion/deletions in the 1000 Genomes Project cohort. Given the possibility of additional genetic variants that modify the phenotype of a primary driver mutation, broad-based genetic testing should be employed.

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391
Original Articles
C
myopathy (DCM) each have a significant heritable compo-
nent.1 Although debated, the estimated prevalence of HCM in
the general population is 1:500.2–5 DCM, classified as left ven-
tricular dilatation and systolic dysfunction in the absence of
other causes of cardiomyopathy, has an estimated prevalence
of 1:2500.5–7 Mutations in MHY7, the gene encoding β-myosin
heavy chain, and MYBPC3, the gene encoding myosin-
binding protein C, cause both HCM and DCM.1,3,5 Mutations
in the TTN gene, encoding the giant protein titin, have been
associated with multiple forms of cardiomyopathy, including
DCM and arrhythmogenic right ventricular cardiomyopa-
thy.8-10 The number and size of cardiomyopathy genes have
limited the sensitivity of genetic testing since most testing has
not included TTN. Improvements in sequencing technology
now facilitate access to cardiomyopathy genes, including TTN
with its 363 exons.11
ardiomyopathy is a major cause of heart failure, and
hypertrophic cardiomyopathy (HCM) and dilated cardio-
Clinical Perspective on p 399
The genetic diagnosis of cardiomyopathy relies on com-
pletely sequencing the coding regions of cardiomyopathy
genes since most pathogenic variation is rare or private.
Common variation is atypical in inherited cardiomyopathy,
although common variants may modify disease phenotype.
Genomic variants are defined as rare if present at a minor
allele frequency (MAF) of <0.5% or as low frequency if
present at an MAF of 0.5% to 5%.12 Since allele frequen-
cies differ among ethnic and racial groups, interpretation of
individual genetic variation is limited by the ethnic makeup
of available genome databases. The 1000 Genomes Project
is an ongoing consortium designed to deliver whole genome
sequence from an ethnically varied population, with the goal
of making publically available the genomes of 2500 individu-
als. In 2010, the 1000 Genomes pilot project was published,
providing data from low-coverage (≈2-fold to 6-fold) whole-
genome sequencing of 179 individuals, high-coverage (aver-
age 42-fold) whole-genome sequencing of 6 individuals in 2
trios, and exon- targeted sequencing (more than 50-fold cov-
erage) of 8140 exons in 697 individuals.12 Since that time,
sequentially released data from each phase of the project has
been made available. The most recent release, in February
2012, contained variant calls and phased genotypes across
1092 individuals, with an average coverage of >4-fold per
August 2012
Received December 21, 2011; accepted June 20, 2012.
From the Department of Medicine and Department of Human Genetics, The University of Chicago, Chicago, IL.
The online-only Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.111. 962928/-/DC1.
Correspondence to Elizabeth McNally, The University of Chicago, 5841 S Maryland Ave, MC 6088, Room G611, Chicago, IL 60637. E-mail
emcnally@uchicago.edu
© 2012 American Heart Association, Inc.
Circ Cardiovasc Genet is available at http://circgenetics.ahajournals.org DOI: 10.1161/CIRCGENETICS.112.962928
Population-Based Variation in Cardiomyopathy Genes
Jessica R. Golbus, BA; Megan J. Puckelwartz, PhD; John P. Fahrenbach, PhD;
Lisa M. Dellefave-Castillo, MS, CGC; Don Wolfgeher, BS; Elizabeth M. McNally, MD, PhD
Background—Hypertrophic cardiomyopathy and dilated cardiomyopathy arise from mutations in genes encoding sarcomere
proteins including MYH7, MYBPC3, and TTN. Genetic diagnosis of cardiomyopathy relies on complete sequencing of
the gene coding regions, and most pathogenic variation is rare. The 1000 Genomes Project is an ongoing consortium
designed to deliver whole genome sequence information from an ethnically diverse population and, therefore, is a rich
source to determine both common and rare genetic variants.
Methods and Results—We queried the 1000 Genomes Project database of 1092 individuals for exonic variants within
3 sarcomere genes MHY7, MYBPC3, and TTN. We focused our analysis on protein-altering variation, including
nonsynonymous single nucleotide polymorphisms, insertion/deletion polymorphisms, or splice site altering variants.
We identified known and predicted pathogenic variation in MYBPC3 and MYH7 at a higher frequency than what would
be expected based on the known prevalence of cardiomyopathy. We also found substantial variation, including protein-
disrupting sequences, in TTN.
Conclusions—Cardiomyopathy is a genetically heterogeneous disorder caused by mutations in multiple genes. The
frequency of predicted pathogenic protein-altering variation in cardiomyopathy genes suggests that many of these variants
may be insufficient to cause disease on their own but may modify phenotype in a genetically susceptible host. This is
suggested by the high prevalence of TTN insertion/deletions in the 1000 Genomes Project cohort. Given the possibility
of additional genetic variants that modify the phenotype of a primary driver mutation, broad-based genetic testing should
be employed. (Circ Cardiovasc Genet. 2012;5:391-399.)
Key Words: cardiomyopathy ◼ hypertrophic cardiomyopathy ◼ myosin heavy chain ◼ myosin-binding subunit ◼ titin

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392 Circ Cardiovasc Genet August 2012
individual in those sequences released as part of the full
project.12
Phenotypic data are unavailable from the subjects whose
genomes were determined in the 1000 Genomes Project.
Thus, interrogation of genetic variation in this cohort should
be considered to include normal individuals and those with
disease, with an expected prevalence of cardiomyopathy
mirroring that of the population at large. Analysis of the
1000 Genomes Project dataset now allows for estimates of
variant prevalence. Unlike the HapMap project, which pro-
vided information on common variation, the 1000 Genomes
Project provides information on rare or private variation.13
Herein, we queried the 1000 Genomes Project database to
interrogate genetic variation in 3 sarcomeric genes (MHY7,
MYBPC3, and TTN) to demonstrate how this database can
be used to estimate genetic variation in the population. We
focused our analysis on protein-altering variation (PAV), or
those DNA deviations that predict a distinct protein from the
referent genome sequence. PAVs are mainly single nucleo-
tide polymorphisms (SNPs) that are nonsynonymous, or mis-
sense, in nature. PAV also includes insertion/deletions (indel)
in coding regions, splice site altering variants, and nonsense
polymorphisms. We found substantial variation, including
protein-disrupting sequences, in TTN. We also identified
previously reported and predicted pathogenic variation in
MYBPC3 and MYH7 in the 1000 Genomes Project database
at a frequency substantially higher than what would be pre-
dicted based on population-based studies of DCM and HCM
prevalence. This data can be used to inform estimates of
baseline genetic variation and, importantly, suggests that at-
risk genotypes are more common in the population at large
than previously expected.
Methods
Variant Discovery for Sarcomeric and
Nonsarcomeric Genes
Exonic boundaries for MYBPC3 (ENST00000545968), MYH7
(ENST00000355349), CAMK2D (ENST00000342666), KCNQ1
(ENST00000155840), KCNH2
(ENST00000337851), SGCG (ENST00000218867), and LMNA
(ENST00000368300) were downloaded from the Ensemble Genome
Browser (www.ensemble.org). Exonic variants were extracted from
the 1000 Genomes Project February 2012 updated genotypes for
the integrated phase 1 release (No. ICHG2011) using the online
data slicer available through the 1000 Genomes Project browser
(http://browser.1000genomes.org). Variants were filtered based on
their Phred quality score, discarding those below 29. Variants were
subsequently submitted to the web site’s Variant Effect Predictor
(VEP), allowing for the analysis of potentially splice site altering
variation within exons. The default settings were adjusted so as to
return National Center for Biotechnology Information (NCBI)
terms for variant consequences and to yield Sorts Intolerant From
Tolerant (SIFT), Polyphen 2 (PP2), and Condel predictions and
scores.14–16 Variant predictions corresponding to the aforementioned
transcripts were extracted. Variants were compared with those pres-
ent in the National Institutes of Health National Heart, Lung, and
Blood Institute (NHLBI) Exome Sequencing Project (ESP) (http://
evs.gs.washington.edu/EVS/) using the Compare two Datasets tool
available through the University of Pennsylvania Galaxy server
(http://main.g2.bx.psu.edu/).
(ENST00000262186),
SGCD
Variant Discovery for TTN
The 3 primary isoforms of titin (N2-BA, N2-A, and N2-B) result from
differential splicing of the I-band encoding region of titin. The N2-
BA isoform encodes the full-length protein and contains blocks of
sequence that are specific to either N2-B or N2-A titin.17 Both N2-BA
and N2-B titin are expressed in the heart. The DNA sequence encod-
ing the N2-BA isoform does not exist in the Ensemble or University
of California Santa Cruz (UCSC) databases but instead must be de-
duced from the N2-B and N2-A isoforms.
The FASTA sequence for N2-A titin (NP_596869.4) was down-
loaded from the NCBI-Gene database and aligned with the Uniprot
titin sequence Q8WZ42 using Uniprot’s online sequence alignment
tool (http://www.uniprot.org). The 927 amino acids absent from
the N2-A isoform were extracted and run through Blat, a sequence
alignment tool publically available through the UCSC Genome
Browser (http://genome.ucsc.edu/cgi-bin/hgBlat?command=start). The
amino acids were mapped to positions 179 603 868 to 179 606 648
on chromosome 2 and were found to correspond to exon 45 in the
N2-B isoform of titin (ENST00000460472) through cross-refer-
encing of its exon boundaries on the Ensemble Genome Browser.
Exonic boundaries encoding the N2-A TTN isoform
(ENST00000342992) and exon 45 of N2-B were downloaded
from Ensemble. Exonic variants were extracted from the 1000
Genomes Project February 2012 updated genotypes for the inte-
grated phase 1 release, using the online data slicer. Variants were
filtered based on their Phred-scaled quality score, retaining only
those variants with a score ≥29.18,19 Exonic variants were then
submitted to the Variant Effect Predictor (VEP), using the previ-
ously described settings. Variant consequences corresponding to
ENST00000342992 and exon 45 of ENST00000460472 were ex-
tracted to yield all variation within N2-BA TTN as defined by the
Uniprot consensus sequence Q8WZ42. Amino acid positions from
the N2-A transcript were shifted from position 4380 onward to
give their amino acid positions within N2-BA titin. TTN variants
were compared with those present in the NHLBI ESP, as described
above. Indels from the 1000 Genomes Project were identified in
the low-coverage, whole-genome project. Indels are not available
from the NHLBI ESP.
Variant Mapping to Protein Domains
Variants in MYBPC3 and TTN were mapped to their respective loca-
tions within the protein using Uniprot sequence identifiers Q14896
and Q8WZ42. Variants in MYH7 were mapped to their location
within the protein using the boundaries outlined in Blair et al.20,21
Pathogenic variants were identified through comparison with those
listed on the online Human Gene Mutation Database (HGMD)
Professional release 2011.3 (http://www.hgmd.org/).
Ethnic Distribution of TTN Variants
Subjects were matched to their corresponding ethnic groups (African,
American, Asian, European) using the supplement provided with the
integrated phase 1 release (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/
release/20110521/phase1_integrated_calls.20101123.ALL.panel).
Variants were eliminated from this analysis in cases where the minor
allele frequency exceeded that of the reference allele. The number of
total PAVs per individual was assessed within each ethnic group for
MYH7, MYBPC3, and TTN.
Statistical Analysis
A Kruskal-Wallis one-way analysis of variance was performed in
PRISM to test for differences in variant load between ethnic groups.
Posttest analysis was performed using Dunn’s multiple comparison
test.

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Golbus et al Prevalence of Cardiomyopathy Mutations 393
Results
Extracting Protein-Coding Variants From
Cardiomyopathy-Associated Genes From the 1000
Genomes Project Database
The variants used for this analysis were extracted from the
1000 Genomes Project February 2012 updated genotypes for
the integrated phase 1 release. This call set derives from low-
coverage, whole-genome, and exome sequencing data and con-
tains phased genotype calls across 1092 individuals for SNPs,
indels, and larger deletions. We queried the dataset for SNPs
in 3 genes associated with cardiomyopathy: MYH7, encoding
the major myosin heavy chain in the heart; MYBPC3, encod-
ing myosin-binding protein C3; and TTN, encoding the giant
protein titin.21 TTN and MYBPC3 SNPs were identified in data
derived from low-coverage genome sequencing, as they were
not included in the targeted exome analysis performed as part
of the pilot project. MYH7 SNPs were identified in both the
low-coverage, whole-genome sequence data and the higher-
coverage, targeted exome sequence. Of the SNPs in MYH7,
48.8% were found in both whole-genome and exome sequenc-
ing, 45.2% in exome sequencing alone, and 6.0% in whole-
genome sequencing alone.
Variant Discovery in 3 Sarcomere Genes
Of the 11 153 total variants in MYH7, MYBPC3, and TTN,
1136 were SNPs and 17 were indels, the later of which were
all found in TTN. Indels ranged in size from 1 to 3bps, with
the addition of 1 larger 18-bp deletion. Of the 1153 coding
SNPs, 386 were synonymous, 15 of which were exonic SNPs
predicted to alter a splice site, and 726 resulted in missense
changes. Three nonsense variants were detected, all of which
were in TTN (online-only Data Supplement Table I). Of these
protein-altering variants (PAVs), 79.0% were defined as rare
(MAF <0.5%), and 14.6% were low-frequency (defined as
a MAF of 0.5% to 5.0%). Thus, 93.6% of variants with the
potential to alter protein structure or function are found in a
small fraction of the population, underscoring the contribution
of infrequent variation to genotype diversity.
Distribution of MYH7 and MYBPC3 Variants
Figure 1 depicts the protein structure encoded by MYH7 and
displays the position of PAV detected in 1092 individuals
from the 1000 Genomes Project database. Twenty-one dif-
ferent PAVs were found in the 1000 Genomes Project cohort
(Table 1). Eleven fall within the S1 and S2 domains of the
β-myosin heavy chain protein, whereas the remaining 10
are within the rod domain. Of the 21 PAVs in MYH7, 8 were
reported by both PP2 and SIFT to be damaging (online-only
Data Supplement Table II).14,16 All 21 variants were present in
the 1000 Genomes Project database as rare or low-frequency
variants.
Figure 2 shows those PAVs detected in MYBPC3 in the
1000 Genomes Project database. Twenty-two different PAVs
were found (Table 2). Eight PAVs were reported by both PP2
and SIFT to be damaging (Table 2). All PAVs were present at
a rare or low frequency.
Spatial Distribution of TTN Variants
Within TTN, 711 PAVs were detected in the 1000 Genomes
Project database from 1092 individuals. Because of the large
number of nonsynonymous SNPs (nsSNPs), we focused our
analysis on indels. All indels had a Phred-scaled quality score
of ≥29, indicating a base-call accuracy of almost 99.9%.
Figure 3 shows the 17 indels detected, including 1 large 18-bp
deletion, I11443-E11449del, found in 62 individuals with a
MAF of 0.03 (Table 3). This 18-bp deletion falls within the
PEVK region that regulates extensibility of titin.22 Two indi-
viduals had multiple TTN indels, the implications of which
depend on both phase and effect on protein frame. One had a
frameshift combined with an in-frame deletion, and the sec-
ond had a frameshift and 2 in-frame deletions. One individual
was homozygous for the 18-bp deletion, and no individuals
were homozygous for a frameshifting variant in TTN. Of the
17 indels in TTN, 7 were found in the Ig-like domains and
none within the fibronectin type III domains. Of these 17
indels, 6 are in-frame deletions, and 11 are frameshifts that
disrupt titin’s carboxyl-terminus. Considering only the 11
indels that disrupt TTN, 3.2% of the population (35/1092) has
a frameshift in TTN.
Variation in Sarcomere Genes Compared With
Nonsarcomere Genes
The complexity of and requirements for sarcomere assembly
are highly intricate. Because of the multiple protein-protein
interactions required for proper assembly of the sarcomere,
it has been suggested that sarcomere proteins, especially
myosin, may be less tolerant of protein-coding variation.23
To assess this, we queried both common and rare variation
in the 3 sarcomere genes (MYH7, MYBPC3, and TTN) and
compared this to several genes encoding proteins important to
cardiac function. We evaluated CAMK2D, encoding calcium/
calmodulin-dependent protein kinase 2 subunit δ; KCNQ1 and
KCNH2, encoding voltage-gated potassium channels linked
Figure 1. Missense variants identified
in the MYH7 gene in 1000 Genomes
Project. The position of 21 rare missense
variants from the 1000 Genomes Project
February 2012 updated genotypes for
the integrated phase 1 release. Variants
also reported as pathogenic in the Human
Genome Mutation Database (HGMD)
are underlined. Adenosine trisphosphate
(ATP) and actin refer to the ATP and
actin-binding sites respectively.

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394 Circ Cardiovasc Genet August 2012
to hereditary long QT syndrome; SGCD and SGCG, encod-
ing the dystrophin-associated proteins δ-sarcoglycan and
γ-sarcoglycan; and LMNA, encoding the nuclear membrane
protein Lamin A (online-only Data Supplement Table 1).
The latter 3 genes have been linked to cardiomyopathy.24
When considering PAVs/1 kb of coding sequencing, sar-
comeric genes averaged 5.77 variants/kb compared with
6.35 variants/kb for nonsarcomeric genes (online-only Data
Supplement Table 3). The range of PAV/1 kb of coding region
varied considerably, from 2.29 to 14.84, suggesting that sar-
comeric genes fall within this range. Among the 3 sarcomere
genes, MYH7 had the greatest amount of total variation but the
least amount of PAV, with only 26.6% of the total exonic SNPs
altering protein in MYH7 compared with 73.3% and 68.2%
PAV in MYBPC3 and TTN.
Known or Reported Disease-Associated Variants in
1000 Genomes Project
Twenty-two SNPs detected in the 1000 Genomes Project
database were also found in the Human Genome Mutation
Database (HGMD), a comprehensive online database of
inherited disease mutations (www.hgmd.org) (online-only
Data Supplement Table 4). Of the 22 SNPs in the 1000
Genomes Project also identified in the HGMD, 11 were in
MYBPC3, 7 in MYH7, and 4 in TTN. These SNPs represent a
small portion of pathogenic variants reported in the HGMD,
3.6%, 3.2%, and 17.4% of published disease-causing muta-
tions in MYBPC3, MYH7, and TTN, respectively. Within the
1000 Genomes Project population, 18 of the 22 mutations
also present in the HGMD appeared as rare variants while 3
others were present at a low frequency. One variant, S236G
Table 1. MYH7 Protein-Altering Variants Found in the 1000 Genomes Project
AA Change Domain
SIFT Prediction
(Score) PP2 Prediction (Score)
MAF (No. of Occurrences
From n=2184 Alleles)
A26V*
V133 M
N306D
R442C*
L620Q
R858H*
R869H*
M982T
R1045C*
M1046I
R1079W
Q1267H
R1289W
Q1458E
N1486D
S1491C*
R1500L
I1558F
I1558S
R1832G
E1902Q
S1domain
S1 domain
S1 domain
S1 domain
S1 domain
S2 domain
S2 domain
S2 domain
S2 domain
S2 domain
S2 domain
S2 domain
LMM
LMM
LMM
LMM
LMM
LMM
LMM
LMM
LMM
T (0.25)
D (0.04)
T (0.52)
D (0)
D (0.03)
D (0.02)
T (0.07)
D (0)
D (0)
T (0.2)
D (0)
T (0.06)
D (0)
T (0.24)
T (0.07)
T (0.1)
D (0)
D (0.02)
T (0.05)
D (0)
T (0.16)
Benign (0.017)
Probably damaging (0.998)
Possibly damaging (0.235)
Probably damaging (1)
Probably damaging (0.992)
Benign (0.002)
Possibly damaging (0.496)
Benign (0.06)
Benign (0.012)
Benign (0.002)
Possibly damaging (0.682)
Benign (0.001)
Probably damaging (0.997)
Benign (0.037)
Possibly damaging (0.539)
Benign (0.001)
Probably damaging (0.982)
Probably damaging (0.99)
Probably damaging (0.99)
Possibly damaging (0.831)
Benign (0.166)
0.0032 (7)
0.0005 (1)
0.0005 (1)
0.0005 (1)
0.0005 (1)
0.0005 (1)
0.0005 (1)
0.0005 (1)
0.0005 (1)
0.0005 (1)
0.0005 (1)
0.0009 (2)
0.0005 (1)
0.0005 (1)
0 (0)
0.01 (17)
0 (0)
0.0005 (1)
0.0009 (2)
0.005 (1)
0.0009 (2)
SIFT indicates Sorts Intolerant From Tolerant; PP2, polyphen 2; MAF, minor allele frequency; T, tolerated in SIFT; and D, deleterious
in SIFT.
*Reported in the Human Gene Mutation Database (HGMD).
Figure 2. Missense variants identified in
the MYBPC3 gene in the 1000 Genomes
Project. Cardiac myosin-binding protein
C, with depiction of 22 missense variants,
one of which alters a splice site from the
1000 Genomes Project February 2012
updated genotypes for the integrated
phase 1 release. Variants present in the
Human Genome Mutation Database
(HGMD) are underlined.

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Golbus et al Prevalence of Cardiomyopathy Mutations 395
in MYBPC3, was present at a higher frequency (MAF=0.09;
n=189) and therefore is not likely an HCM-associated muta-
tion, given its high prevalence.
To address the possibility of multiple variants within indi-
viduals, we evaluated the sarcomeric mutations found in
both the HGMD and the 1000 Genomes Project (online-only
Data Supplement Table 4). MYBPC3 S236G was excluded
from this analysis, given its frequency (MAF=0.09, n=189).
Ninety-nine individuals had at least 1 pathogenic variant,
and, of these, 93 individuals were heterozygous for a single
variant. Three individuals from European and American
cohorts were homozygous for MYBPC3 mutations (V896 M,
Q998E). One European individual was homozygous for the
mutation S1491C in MYH7. Two individuals had 2 patho-
genic variants each; 1 in MYH7 and 1 in MYBPC3 (R1138H
MYBPC3 plus M982T MYH7 and S217G MYBPC3 plus
Table 2. MYBPC3 Protein-Altering Variants in the 1000 Genomes Project
AA ChangeSIFT Prediction
(Score)
PP2 Prediction (Score) MAF (No. of Occurrences
From n=2184 alleles)
V158 M
R160W*
S217G*
S236G*
A259V
G278E*
R326Q*
R382W
G440D
G507R*
N515S
A522T*
A833V*
I867I g->a synSS
E872K
V896 M*
Q998E*
R1002W
R1036C
T1075S
Q1103H
R1138H*
R1190H
D (0.01)
D (0)
D (0.01)
T (1)
T (0.3)
T (0.14)
D (0.01)
D (0.02)
T (1)
D (0)
T (0.34)
T (0.2)
T (0.52)
n/a
T (0.05)
T (0.05)
T (0.11)
D (0)
D (0)
T (0.05)
D (0)
D (0)
T (0.05)
Possibly damaging (0.269)
Probably damaging (1)
Benign (0.154)
Benign (0)
Benign (0.002)
Possibly damaging (0.637)
Benign (0.02)
Probably damaging (0.936)
Benign (0.021)
Probably damaging (1)
Probably damaging (0.994)
Benign (0.143)
Probably damaging (0.888)
n/a
Probably damaging (0.994)
Benign (0.005)
Possibly damaging (0.799)
Probably damaging (1)
Probably damaging (0.931)
Benign (0.01)
Probably damaging (0.997)
Probably damaging (1)
Probably damaging (1)
0.05 (101)
0.0014 (3)
0.0005 (1)
0.09 (189)
0.0023 (5)
0.0046 (10)
0.0018 (4)
0.01 (31)
0.0005 (1)
0.0027 (6)
0.0018 (4)
0.0014 (3)
0.01 (14)
0.02 (34)
0.0005 (1)
0.0032 (7)
0.01 (17)
0.0027 (6)
0.0009 (2)
0.0005 (1)
0.0005 (1)
0.0009 (2)
0.0005 (1)
SIFT indicates Sorts Intolerant From Tolerant; PP2, polyphen 2; MAF, minor allele frequency; D, deleterious in SIFT; T,
tolerated in SIFT; and synSS, synonymous splice site.
*Reported in the Human Gene Mutation Database (HGMD). The exon-encoded predicted splice variant cannot be
computed by SIFT or PP2.
Figure 3. Frameshifting mutations identified in TTN in the 1000 Genomes Project. The titin isoform N2-BA is shown. Titin is linearly
depicted with its 152 Ig-like domains in red and 132 fibronectin type III domains in blue. Depicted are the 17 indels identified in the
1000 Genomes Project February 2012 updated genotypes for the integrated phase 1 release. Six of 17 indels do not disrupt reading
frame. One (underlined) that deletes 6 amino acids in the PEVK region is found in 5% of the population. Variants are shown relative to
the titin Uniprot Sequence identifier Q8WZ42. The dashed lines below the protein schematic indicate the location of variants within the
sarcomere.

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396 Circ Cardiovasc Genet August 2012
S1491C MYH7). Thus, 5 of 1092 individuals in the 1000
Genomes Project carry multiple reported pathogenic muta-
tions in sarcomeric genes. This number also exceeds popula-
tion estimates of cardiomyopathy prevalence, suggesting that
these variants may be insufficient to cause disease but may
modify phenotype in the context of a primary driver mutation.
We employed 3 protein prediction algorithms as part of
our analysis: SIFT, PP2, and Condel, a normalized composite
of the prior 2 programs.12 We used Condel to analyze previ-
ously reported mutations now identified in the 1000 Genomes
Project dataset and found that it predicted 15 of the 17 pos-
sible variants to be pathogenic. Using this same approach,
Condel predicted that 83.7% (36/43) of the missense variants
detected in MYH7 and MYBPC3 would be pathogenic.
Comparison to the NHLBI ESP
We queried the NHLBI ESP25 for total SNPs in MYH7,
MYBPC3, and TTN. This is a database of high-coverage
exonic sequence that includes 5379 samples drawn from
African American and European American individuals.
Average coverage in the NHLBI ESP across the 3 sarcomeric
genes MYH7, MYBPC3, and TTN is higher (114.32-, 55.42-,
and 108.73-fold, respectively) than the lower coverage 1000
Genomes Project, but the ESP lacks indel calls at this time;
51.2%, 60.6%, and 63.5% of all SNPs identified in the 1000
Genomes Project in MYH7, MYBPC3, and TTN respectively
were also identified in the NHLBI ESP. Those SNPs in the 1000
Genomes Project but not in the NHLBI ESP were rare (pres-
ent at an average frequency of 0.0020, 0.0037, and 0.0010 for
MHY7, MYBPC3, and TTN, respectively.) Those SNPs found
in the NHLBI ESP but not the 1000 Genomes Project were
similarly rare (present at an average frequency of 0.00018,
0.00020, and 0.0049, respectively). The similar frequency of
rare alleles in the low-coverage 1000 Genomes Project and the
high-coverage NHLBI ESP argues that these findings are not
false-positives related to low-coverage sequence. The differ-
ent ethnic groups that constitute each sequenced cohort likely
account for the difference in observed rare alleles.
Distribution of Variants by Race and Ethnicity
A Kruskal-Wallis one-way analysis of variance was used to test
for differences in PAV in MYH7, MYBPC3, and TTN among
ethnic groups (African, American, Asian, European) in the
1000 Genomes Project cohort. Five variants were removed
before this analysis, as their MAF exceeded that of the refer-
ence allele. The frequency of variants differed significantly
across the 4 groups in MYH7 [H(3)=148.1, P<0.0001], MYBPC3
[H(3)=10.05, P=0.0181], and TTN [H(3)=262.9, P<0.0001],
(Figure 4). On posttest comparisons, Africans were shown to
have significantly more variation in MYH7 than all other eth-
nic groups at 0.68 variants/individual while Asians were shown
to have significantly more variation in TTN at 28.76 variants/
individual. The latter differed from the results of the 1000
Genomes pilot project in which African individuals were reported
to have the greatest variation across the genome, as measured by
the number of total and novel variants within each ethnic group.12
Thus, variant frequencies differ between ethnic groups at the
level of individual genes, and, notably, the extent and direction of
these ethnic differences changes between genes.
Discussion
Pathogenic Variation in the 1000 Genomes Project
Is Higher Than Expected; Implications for Testing
The 1000 Genomes Project launched in 2008 with the goal
of creating an ethnically diverse public reference database for
DNA polymorphisms. Unprecedented in its scope, the proj-
ect is the first to approximate the breadth of human genetic
Table 3. TTN Insertion/Deletion Polymorphisms
Location Reference/ Alternate AlleleAA ChangeDomain
MAF (No. of Occurrences
in n=2184 Alleles)
179666885–179666886
179664626–179664628
179658211–179658212
179640461–179640462
179570049–179570051
179554281
179549673–179549674
179548796
179514942–179514959
179505311–179505312
179501447–179501449
179495075–179495076
179428912–179428913
179439949
179414950–179414952
179395288
179398424–1179398426
G/GT
ACTT/A
C/CT
C/CT
CTCT/C
GT/G
A/AG
TC/T
Thr92fs
Glu198del
Ala486fs
Glu2044fs
Glu9503del
Thr10313fs
Ala10522fs
Lys10535fs
Ile11443_Glu11449del
Thr11920fs
Pro12089del
Glu13084fs
Leu25675fs
Gly21996fs
Lys28898del
Ser33711fs
Lys32665del
Ig-like 1 0.0037 (8)
0.0009 (2)
0.0027 (6)
0.0009 (2)
0.0009 (2)
0.0005 (1)
0.0018 (4)
0.0005 (1)
0.03 (62)
0.0005 (1)
0.0018 (4)
0.0014 (3)
0.0005 (1)
0.0005 (1)
0.0005 (1)
0.0032 (7)
0.0005 (1)
Z-repeat 2
Poly-Glu
PEVK
PEVK
PEVK
PEVK
PEVK
Ig-like 80
TTTTCCTCTTCAGGAGCAA/T
A/AG
CAGG/C
TTC/T
A/AG
GC/G
TTAA/T
GA/G
ACTT/A
Ig-like 125
Ig-like 115
Ig-like 133
Ig-like 148
Ig-like 144
MAF indicates minor allele frequency.

Page 7

Golbus et al Prevalence of Cardiomyopathy Mutations 397
variation and provide a quantitative framework in which to
predict potentially relevant variants. We queried the database
of 1092 individuals for 3 sarcomeric genes, as they were
thought to contain little sequence variation.21,23,26 This cohort,
in the absence of phenotypic data, should be viewed as a rep-
resentative sample of the population at large, containing both
healthy and diseased individuals. Within the 1000 Genomes
Project database, we identified variants that were previously
reported as pathogenic in the HGMD. Although reported as
mutations, some of these polymorphisms are not sufficient
to cause cardiomyopathy, as several have been reported in
normal populations. We also found variants not reported in
the HGMD that are predicted to be pathogenic using compu-
tational algorithms. Summing these with the observed TTN
frameshifts yields a frequency that substantially exceeds pop-
ulation estimates of the combined HCM and DCM prevalence.
It is likely that some of these variants are positioned to mod-
ify disease outcome and favors a comprehensive sequence
analysis. Supporting this idea, compound and double muta-
tions have been noted in younger, more affected individuals.27
Therefore, we favor comprehensive sequencing analysis espe-
cially in younger individuals.
Currently, assessing gene variants involves the use of
prediction tools, segregation studies within families, and
animal/cellular models to predict pathogenicity. As cardio-
myopathy genetic testing panels become more expansive and
the number of variants increases, costly and labor-intensive
tools for analysis will prove challenging. Improved predic-
tion tools will be necessary to better anticipate the effect
of a given PAV. Better algorithms, along with comprehen-
sive testing, should improve the information obtained from
genetic analysis.
Indels in TTN
We also uncovered a high frequency of indels in TTN, all of
which have a base call accuracy of almost 99.9%. In light of the
high-cumulative frequency of TTN indels in the general popula-
tion (9%), TTN may be positioned to modify phenotype. Just
over 5% of the general population has an 18-bp in frame dele-
tion in the proline rich region of titin. This region of titin has
been closely linked to the elastic properties of titin and its ability
to sense and respond to sarcomere length.22 This allele will be
important to track in studies of cardiac modifiers given its preva-
lence and potential functional role. Notably, 3% of the popula-
tion has a protein-truncating TTN allele. This estimate is similar
to the recent findings from Herman et al where protein-truncating
TTN mutations were found in 25% of DCM subjects and in 3%
of a normal control population.28 Our analysis, unlike Herman et
al, focused only on indels as protein-truncating mutations in TTN
and did not include predicted TTN intronic splice-site mutations.
Thus, our analysis is expected to underestimate the population
prevalence of protein-truncating mutations in TTN.
Likely Underestimates of Pathogenic and
Predicted Pathogenic Variation
With increasing sequencing coverage, the ability to detect
private variation increases.29 High-coverage sequence of
MYH7 yielded additional variants that were not detected in
the low-coverage sequencing. The variation described in
TTN and MYBPC3 derives only from low-coverage whole-
genome sequencing, and, therefore, our study probably
underestimates variation. Overall, it is unlikely that these
variants represent false-positives SNP. As of last year’s
reporting, the pilot project low-coverage genome sequence
is estimated to have a <5% false discovery rate for SNPs and
a 1% to 3% genotype error rate.12 Our analysis also did not
Figure 4. Protein-altering varia-
tion (PAV) within the 1000 Genomes
Project cohort differs by cohort. The
histograms above show the percent-
age of individuals within each cohort
(AFR=African; AMR=American;
ASN=Asian; EUR=European) having a
specified number of PAVs per gene. This
analysis included all PAV, except those
5 instances in which the MAF exceeded
that of the reference allele. The amount
of PAV in specified genes differed by
racial groups, and this difference was sig-
nificant (MYH7, [H(3)=148.1, P<0.0001];
MYBPC3, [H(3)=10.05, P=0.0181]; and
TTN, [H(3)=262.9, P<0.0001]; P values
are from nonparametric analysis of vari-
ance [ANOVA]). On posttest comparisons,
African individuals had more variation
in MYH7 than all other groups, whereas
Asians had significantly more variation in
TTN.

Page 8

398 Circ Cardiovasc Genet August 2012
query variation in intronic sequence, including potentially
pathogenic splice-site altering sequence, thus providing an
additional source for pathogenic variation that is not repre-
sented in our baseline estimates of genetic variation. At pres-
ent, the current 1000 Genomes Project database contains data
on 1092 individuals and will extend sequence coverage to
2500 individuals to provide further information on rare and
low-frequency variation.
Interpreting Rare Variation in
Cardiomyopathy Genes
Given the extent of rare PAV in both sarcomeric and nonsar-
comeric genes, the mere presence of rare variation does not
imply pathogenicity. Segregation of a rare variant within a
family or demonstration of impaired protein function within
a biological context is often used to interpret the findings
of genetic testing; however, these in vitro studies are often
impractical, and not all family structures support this analy-
sis, especially in families with diseases that limit lifespan. The
continued development of analyses that better predict vari-
ant pathogenicity will be required. This analysis highlights
the challenges that accompany analyzing population-based
genetic information and applying findings to the individual
patient with cardiomyopathy. The 1000 Genomes Project
dataset will aid in interpreting genetic testing information,
although its use is limited by lack of phenotypic correlates. In
case-control studies of genotype and phenotype, control sub-
jects should be carefully considered if the subjects have not
been clinically evaluated or especially if they are too young to
yet manifest disease
Sources of Funding
Supported by the Doris Duke Charitable Foundation, the Sarnoff
Foundation, and NIH R01 HL092443 and NIH HL61322.
Disclosures
None.
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Golbus et al Prevalence of Cardiomyopathy Mutations 399
CLINICAL PERSPECTIVE
The 1000 Genomes Project is an international consortium designed to provide full genomic sequence information from an
ethnically varied population. The clinical history of 1000 Genomes Project participants is unknown, so information from the
project is best used to determine population estimates of sequence variation. We queried the 1000 Genomes Project database
for genetic variation in three cardiomyopathy genes, MYH7 (β myosin heavy chain), MYBPC3 (myosin binding protein C),
and TTN (titin). We focused our analysis on rare variation that was either reported previously as pathogenic or was predicted
to be pathogenic by computational algorithms. These 3 genes encode sarcomere proteins, and mutations in these genes cause
familial dilated and hypertrophic cardiomyopathy. We identified predicted and previously reported pathogenic variation at
a much higher frequency than the incidence of both dilated and hypertrophic cardiomyopathy. Specifically, we found 9% of
genomes had insertion/deletion polymorphisms in TTN. The functionality of this genetic variation is not known given the
absence of clinical information from 1000 Genomes Project participants; however, these variants may be important genetic
modifiers that increase the risk for developing heart failure in the general population. For patients with a history of familial
cardiomyopathy, these findings favor comprehensive genetic testing to identify both primary mutations and secondary dis-
ease modifiers.
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