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. Author manuscript; available in PMC: 2013 Apr 1.
Published in final edited form as: Circ Cardiovasc Genet. 2012 Feb 15;5(2):167–174. doi: 10.1161/CIRCGENETICS.111.961805

Evaluating pathogenicity of rare variants from dilated cardiomyopathy in the exome era

Nadine Norton 1, Peggy D Robertson 2, Mark J Rieder 2, Stephan Zuchner 3, Evadnie Rampersaud 3, Eden Martin 3, Duanxiang Li 1; NHLBI GO Exome Sequencing Project, Deborah A Nickerson 2, Ray E Hershberger 1
PMCID: PMC3332064  NIHMSID: NIHMS364763  PMID: 22337857

Abstract

Background

Human exome sequencing is a recently developed tool to aid in the discovery of novel coding variants. Now broadly applied, exome sequencing datasets provide a novel opportunity to evaluate the allele frequencies of previously published pathogenic rare variants.

Methods and Results

We examined the exome dataset from the NHLBI Exome Sequencing Project (ESP) and compared this dataset with a catalog of 197 previously published rare variants reported as causative of dilated cardiomyopathy (DCM) from familial and sporadic cases. Of these 197, 33 (16.8%) were also present in the ESP database, raising the question of whether they were uncommon polymorphisms. Supporting functional data has been published for 14 of the 33 (42%), suggesting they are unlikely to be false positives. The frequencies of these functional variants in the ESP dataset ranged from 0.02–1.33% (median 0.04%), which when applied as a cut-off to filter variants in a DCM pedigree identified an additional DCM candidate gene. A greater proportion of sporadic DCM cases had variants that were present in the ESP dataset vs novel variants (i.e. not in ESP; 44% vs 21%), p=0.002), suggesting some of the variants identified as disease causing in sporadic DCM are either false positives or low penetrance alleles in human populations.

Conclusions

Rare nonsynonymous variants identified in DCM subjects also present at very low frequencies in public databases are likely relevant for DCM. Allele frequencies >0.04% are of less certain pathogenicity, especially if indentified in sporadic cases, although this cut-off should be viewed as preliminary.

Keywords: cardiomyopathy, genetics, genes


One of the key challenges of medical genetics has always been to assess the pathogenic potential of rare variants in coding sequence. This is highly relevant, since assigning ‘disease causing’ to any variant may be useful for predictive testing, early disease intervention, and with further progress, specific treatment of the underlying variants or their disease pathways. The Human Genome Mutation database contains >100,000 mutations from ~4000 genes attributed to human inherited diseases, and of these, approximately 65% are single base substitutions in coding sequence or splice junctions.1

Criteria for reporting a nonsynonymous variant as pathogenic in Mendelian disease has relied upon several criteria (Table 1),24 including its novel identity. In earlier Mendelian studies, variants were attributed as novel based on their absence in ~200–400 unaffected chromosomes5 and more recently in larger numbers (usually 500–1000).6,7 A current method for assessing the novelty of a variant has been to determine its presence or absence in dbSNP. However, regardless of approach used, the lack of large numbers of clinically defined and ethnically matched control samples has been a confounding factor for many analyses. Further, while the recent rapid advances and application of next generation sequencing (NGS) has nearly doubled the amount of publicly available variant data in the past year alone, dbSNP is becoming increasingly “contaminated” with disease mutations that are not sufficiently annotated. Thus, variants determined as novel in earlier study designs may now be hidden in dbSNP amongst a pool of rare variants present in the general population, some of which may be benign and some pathogenic or deleterious.

Table 1.

Proposed Criteria for Evaluation of Rare Variants in Mendelian Exome Sequencing Studies

Existing criteria Proposed criteria / comments
Rarity Usually not identified in 300–1000 chromosomes Disease specific iterative approach: Utilization of ESP or other large population based datasets to empirically determine frequencies of known pathogenic variants. Apply these frequencies to filtering criteria for exome data in each disease.
Racial/ethnic matching Same as above - usually not identified in 300–1000 racially and ethnically matched chromosomes Utilization of ESP and other large population based datasets will help to resolve false positives due to insufficient ethnic matching.
Conservation PhastCon, GERP scoring Conservation scores do not distinguish between ‘novel’ and ‘very rare’ pathogenic variants, but in general are high. These scores may be used for ranking or prioritization.
Type of variant Missense, nonsense, splice variant Functional classification allows for prioritization and hypothesized effect (see Functional data below). This may be impacted in the era of whole genome sequencing, when many rare non-coding variants will also be identified and conservation scores or other metrics would be needed.
Segregation Present in all affected family members No immediate change; however, with greater insight, the 'autosomal dominant' genetic paradigm may be enlarged to account for more complexity (e.g. modifiers and penetrance). New rare variant pathway analyses will highlight novel genes with rare variants in multiple sporadic cases.
Functional data Pathologic change in animal or cellular model The stringency for emphasizing relevant pathologic changes in an appropriate model will only increase. This is congruent with interest in using iPS cells to create an investigational tissue source from a subject harboring the variant that has the same genetic background.

Our own work in a rare variant Mendelian disease has focused on idiopathic dilated cardiomyopathy (DCM). DCM is genetically heterogeneous, typically showing autosomal dominant transmission with variable age of onset and reduced penetrance.4 Rare variants in >30 previously reported DCM genes account for ~35–40% of familial cases, while the remaining genetic cause remains to be determined.4 We have contributed 95 DCM variants to the literature813 but unaffected individuals for these studies were limited. The Exome Sequencing project (ESP), funded by the National Heart, Lung and Blood Institute (NHLBI), recently released a searchable public database containing genetic variants identified in the human exome for >2,400 individuals. Since exome sequencing is now commonly applied in discovery of rare variants in Mendelian disease,14 we catalogued the 197 discrete variants from 235 DCM cases (Supplementary Table 1) and screened for their presence in the exome sequences of 1,351 European and 1,088 African-descent individuals. While most of these variants were not present in the ESP database, those that were present provide insight into preliminary guidance for future use of large publicly available genomic resources.

Methods

Data collection

We collated single nucleotide substitutions or small insertion/deletion allelic variants reported in the literature as ‘mutations’ or ‘rare variants’ in 30 autosomal DCM genes (LMNA, MYH6, MYPN, TNNT2, SCN5A, MYBPC3, RBM20, TMPO, LAMA4, VCL, LDB3, TCAP, PSEN1, PSEN2, ACTN2, CRYAB, TPM1, ABCC9, ACTC, PDLIM3, ILK, TNNC1, TNNI3, PLN, DES, SGCD, CSRP3, TTN, ANKRD1, BAG3) as previously published in a review4 and subsequent publications.8,15

Genomic positions (Hg19) and the reference and alternate allele on the forward strand were determined for each variant. Conservation for single base variants and prediction of functional effects was assessed with PhastCons,16 GERP17 and Grantham scores,18 using SeattleSeq SNP annotation (http://snp.gs.washington.edu/SeattleSeqAnnotation131/) and PolyPhen2.19

Search for published DCM variants in Exome Sequencing project data

Details of the ESP dataset are described in supplemental methods. Exome sequencing was performed at the Broad Institute or the University of Washington. Single base variant data for these analyses are available on the ESP Exome Variant Server (EVS- snp.gs.washington.edu/EVS/).

Genomic positions of DCM single base variants were searched against exome data for 2,439 individuals, of which 1,351 were of European and 1,088 were of African descent respectively. For small insertion deletions, genomic positions 15 bases either side of the reported variant were searched against VCF files for 1,200 ESP controls of which 1,028 were of European and 167 were of African descent and 5 were of unknown ancestry, respectively. Of 1,200 ESP individuals, ~600 overlapped with those used for single base analysis.

Exome sequencing of DCM pedigree

The exomes of three related individuals with DCM were sequenced as previously described.8

Statistical analysis

Wilcoxon two-sample tests to determine statistical difference of Grantham, PhastCon and GERP scores between DCM variants present and absent in the ESP dataset were performed in SAS v. 9.2.

Results

ESP data across DCM genes and variant positions

Only one of 30 autosomal DCM genes, RBM20, was not captured in this version of the ESP dataset, eliminating its nine known DCM variants from consideration.11,20 The remaining 29 genes were included in the ESP target sequence with an average sequence coverage exceeding 60x and with a minimum of >80% of the target sequence having coverage exceeding 20x (Supplementary Table 2).

DCM allelic variation

Allelic variants from 29 genes reported as either ‘mutations’ or ‘rare variants’ causative of DCM, and in one case as a ‘disease modifier,’ from 53 published studies are summarized (Figure 1). A total of 235 DCM cases with variants attributed as causative of disease were reported, of which 197 variants were discrete. Of these 197, 87 were reported in our studies 813 and eleven were reported by more than one study. Most variants (88%) were ‘private’ to a single family with the remaining occurring in two to four DCM probands; 89% were single base substitutions (93% missense, 5% nonsense and 2% occurring in consensus splice sites). The remaining 11% of variants were small insertion/deletions (indels) ranging from one to four bases in size, of which 86% resulted in frameshifts within the coding regions. Most variants (73.6%) occurred in familial dilated cardiomyopathy, 24.4% occurred in sporadic cases, and 2% were observed in both familial and sporadic DCM cases.

Figure 1.

Figure 1

DCM variant data collected from 53 publications. ESP, Exome Sequencing Project.

Known DCM allelic variants present in dbSNP build 131

52 of 176 single base substitution variants were present in dbSNP, of which eighteen have no reported population frequency data, 20 have reported frequency of <0.1%, two have frequencies between 0.1 and 1% and seven have frequencies > 1% in at least one population. Only 22 of 52 (42%) of known DCM variants present in dbSNP were tagged as ‘clinically associated.’ Eight of 21 small indels were also present in dbSNP and seven of eight were flagged as ‘clinically associated’. Seven of eight indels had no population frequency data and one (TNNT2 Lys210del) was not identified in 243 individuals from European, Asian, Hispanic and Yoruban populations.

Known DCM allelic variants present in >2000 ESP exomes

A total of 31 single base substitutions (17.6%) reported in the literature as ‘mutations’ or ’rare variants’, and in one case, (LDB3 Lys204Arg),21 as a ‘disease modifier’ were also present in the ESP dataset (Table 2). These variants occurred in seventeen of the 29 DCM genes captured in the ESP data set (Table 3). Frequencies in ESP individuals ranged from 0 to 0.59 % in Europeans, and from 0 to 4.64% in Africans. Two variants occurred at frequency >1% in ESP individuals of African ancestry: LDB3 Lys204Arg (4.64%) and LDB3 Asp117Asn (1.33%). These variants were highly conserved with PhastCons scores of 1 and 0.997 and GERP scores of 5.55 and 3.48, respectively, although Grantham scores for these variants were not suggestive of structural impact, (26 and 23 respectively, where a score >50 is suggestive of impact). Lys204Arg was predicted to be ‘probably-damaging’ by PolyPhen2, while the discovery study observed this variant in 6 DNAs from African American control individuals and suggested it was a disease modifier or polymorphism.21 Asp117Asn was predicted to be benign by PolyPhen2, but despite these predictions, cells transfected with this particular variant showed cytoskeleton disarray.21

Table 2.

DCM Rare Variants Present in the ESP Data Set

Gene Variant Type* rs Number PhastCon GERP Clinical
Assoc,
dbSNP*
N, DCM
families
%, all
ESP*
%
European
ESP*
%
African
ESP*
DCM
Functional
Data
Refs
ABCC9 Leu1524fs fs NA NA 1 0.04 0.05 0 yes 32
ACTN2 Gln9Arg m 1 3.17 1 0.04 0.08 0 yes 26
ANKRD1 Thr116Met m 1 5.47 1 0.02 0 0.05 40
ANKRD1 Arg66Gln m 1 5.71 1 0.1 0.19 0 40
CRYAB Arg157His m 1 NA 1 0.02 0.04 0 yes 24
CSRP3 Trp4Arg m rs45550635 1 5.67 yes 2 0.35 0.59 0.05 yes 10, 2526, 35
CSRP3 Gly72Arg m rs45552933 1 5.29 1 0.02 0.04 0 10
DES Asp312Asn m rs34337334 0.241 4.44 yes 1 0.06 0 0.14 yes 30
LDB3 Ser196Leu m rs45487699 0.002 2.48 1 0.02 0.04 0 21
LDB3 Asp117Asn m 0.997 3.48 2 0.73 0.37 1.33 yes 21
LDB3 Lys204Arg m rs34423165 1 5.55 1 2.15 0.15 4.64 21
LDB3 Ala698Thr m rs45577134 0.992 5.07 2 0.04 0.07 0 10
MYBPC3 Glu619Lys m 0.961 3.2 1 0.06 0.1 0 41
MYBPC3 Arg326Gln m rs34580776 1 3.48 2 0.15 0.23 0 41
MYBPC3 Asp605Gly m 1 4.14 1 0.03 0.05 0 9
MYBPC3 Gly5Arg m 0.964 4.13 1 0.06 0.09 0 9
MYBPC3 Gly490Arg m 0.954 4.28 1 0.09 0.14 0 9
MYBPC3 Ala833Thr m 0.873 4.16 3 0.17 0.28 0 9
MYH6 Ala1004Ser m 1 4.84 4 0.06 0.11 0 9, 42
MYH6 Ala1440Pro m 0.847 4.51 1 0.02 0.04 0 9
MYH6 Arg568Cys m 0.878 4.44 1 0.04 0.04 0.05 9
MYH7 Val964Leu m rs45496496 1 4.86 1 0.04 0.07 0 10
MYPN Val1195Met m rs71534280 0.924 4.87 1 0.06 0 0.14 yes 22
MYPN Pro1112Leu m rs71534278 1 5.03 1 0.14 0.19 0.09 yes 22
PSEN2 Ser130Leu m rs63750197 0.021 3.26 yes 1 0.02 0.04 0 yes 12
SCN5A Thr220Ile m rs45620037 1 3.95 1 0.03 0.05 0 yes 28, 43
SCN5A Ser216Leu m rs41276525 1 3.95 2 0.06 0.09 0 10
SCN5A Pro2005Ala m rs45489199 0.133 −9.85 1 0.12 0.18 0 10
SCN5A Ile1835Thr m rs45563942 1 4.27 1 0.2 0 0.54 yes 10, 31
SGCD Lys238 del In fr del 1 0.3 0 1.8 44
TMPO Arg690Cys m rs17028450 0.956 0.893 1 0.03 0.04 0 yes 29
TNNT2 Glu244Asp m rs45466197 0.996 2.28 1 0.02 0 0.05 yes 23, 45
VCL Ala934Val m rs16931179 0.998 4.74 1 0.04 0.04 0.05 yes 27
*

fs, frameshift; m, missense; in fr del, in-frame deletion; Clin Assoc dbSNP, clinical association denoted in the dbSNP database; % all ESP, % European or African ESP, all reflect the frequency expressed in percent against the 2,439 exomes of the entire dataset, the 1,351 European or 1,088 African samples in the ESP. Refs; references

Table 3.

DCM variants by gene

Gene Number
reported DCM
variants (%)
Number
variants in ESP
Freq ESP % Number observations
in ESP
LMNA 43 (20.57) 0 0 0
MYH7 28 (13.40) 1 0.07 2
MYBPC3 16 (7.66) 6 0.05–0.28 1–6
BAG3 13 (6.25) 0 0 0
TNNT2 12 (5.74) 1 0.05 1
SCN5A 10 (4.78) 4 0.05–0.54 1–7
TPM1 9 (4.31) 0 0 0
MYH6 9 (4.31) 3 0.05–0.11 1–3
RBM20* 9 (4.31) NA NA NA
LDB3 7 (3.35) 4 0.04–4.64 1–101
DES 6 (2.87) 1 0.14 3
TNNC1 5 (2.39) 0 0 0
ANKRD1 5 (2.39) 2 0.05–0.19 1–5
TNNI3 4 (1.91) 0 0 0
MYPN 4 (1.91) 2 0.14–0.19 3–5
PLN 3 (1.44) 0 0 0
TCAP 3 (1.44) 0 0 0
VCL 3 (1.44) 1 0.05 1
CSRP3 3 (1.44) 2 0.04–0.59 1–16
ACTC1 2 (0.96) 0 0 0
TTN 2 (0.96) 0 0 0
ABCC9 2 (0.96) 1 0.05 1
SGCD 2 (0.96) 1 1.8 6
ILK 1 (0.48) 0 0 0
LAMA4 1 (0.48) 0 0 0
PSEN1 1 (0.48) 0 0 0
ACTN2 1 (0.48) 1 0.08 2
CRYAB 1 (0.48) 1 0.04 1
TMPO 1 (0.48) 1 0.04 1
PSEN2 1 (0.48) 1 0.04 1

The number of variants per DCM gene that also occurred in the ESP dataset are shown with the highest allele frequency and highest number of observations in a single ESP population. Where more than one variant per gene is observed in the ESP population, the range of frequency and occurrence is shown.

*

RBM20 was not captured in the ESP dataset, but is included here to demonstrate its frequency in the DCM in context to other known genes.

Eighteen of 31 single base substitutions present in the ESP dataset were also present in dbSNP (three of which were flagged as clinically associated). Thirteen variants not present in dbSNP but present in ESP individuals had frequencies ranging from 0.04 to 1.33% in at least one population. Published functional data was available for fifteen of these 31 variants, with thirteen of fifteen showing some functional deficit12, 2130 while two of fifteen showed no significant functional effect. In the case of SCN5A Ile1835Thr, observed in the ESP individuals of African origin at a frequency of 0.54% (absent in those of European ancestry), sodium current was significantly altered but only in the presence of a constitutively expressed SCN5A transcript variant, Q1077del.31

Among the 21 DCM indels, an in-frame deletion of a single amino acid (SGCD Lys238del) was detected in the ESP individuals, occurring in six of 134 chromosomes at this position (1.8%) from individuals of African descent but not present in any individuals of European descent. A second DCM indel was identified in a single heterozygous individual of European descent within the ESP data as insertion A at chr12: 21958185. The DCM variant at this locus was reported as a complex deletion of ATT and an insertion of AAAT resulting in a frameshift in ABCC9, with a functional deficit supported by cell based assays showing an abnormal ATP-sensitive potassium channel phenotype32 (chr12:21958186–21958188).

Of the DCM variants present in ESP individuals, five were present only in samples of European origin, whilst none were unique to samples of African American origin. This is likely a reflection of the initial DCM discovery samples which were predominantly of European origin. The exact composition of the DCM discovery samples is not reported in several of the published manuscripts but for our own published cohort813 which contributed 44% of variants in the study, patients is 93% European, 5% African American, and 2% other origin.

Lack of discriminating features of DCM variants present versus those absent in the ESP dataset

For single base substitutions, we compared DCM variants present in the ESP dataset against those that were absent (novel), under the assumption that DCM variants identified in the general population may be less conserved, and thus less pathogenic, than novel variants unique to DCM. However, conservation scores at the nucleotide (GERP) or amino acid level (PhastCons and Grantham scores) and PolyPhen2 classification did not discriminate between DCM variants present in the ESP dataset and those that were absent. Of those present in the ESP dataset, seven were classed as ‘benign’, sixteen were classified as ‘probably damaging’ and eight as ‘possibly damaging’. Wilcoxon two-sample tests of Grantham and GERP scores for variants present in the ESP dataset verses those that were novel were not significantly different, p-values 0.26 and 0.20 (1-tailed) respectively. PhastCons scores between the two groups showed weak evidence for statistical difference, p=0.049 (1-tailed), average 0.951, compared with 0.858 in DCM variants that were present in the ESP dataset, although this was not significant after correction for multiple testing.

We further stratified those DCM variants present in the ESP against those that were novel by whether they occurred in familial or sporadic DCM cases and the amount of supporting functional data. Forty-four percent of DCM variants present in the ESP dataset occurred in sporadic cases compared to 21% of the novel variants, p=0.002 (2-tailed). One possibility for the excess of sporadic DCM cases with variants that were also observed in the ESP dataset is that these variants were false positives for disease causation. An alternate possibility is that these rare variants were low penetrance or susceptibility alleles that could be resolved to some extent by functional assays. Six of fifteen variants present in both sporadic DCM cases and the ESP dataset showed some functional deficit, suggesting that they were low penetrance alleles. Furthermore, no significant difference was observed between the amount of supporting functional data in those variants present and those that were absent in the ESP data, p=0.43 (2-tailed).

The true picture may become clearer once functional assays are available for many more variants. Based on the assumption that DCM variants with functional data that have also been observed in the ESP dataset are indeed pathogenic but with reduced penetrance, we propose an empirically derived frequency cut-off to aid in determining pathogenicity. In this dataset there were fourteen DCM variants with supporting functional data that were also observed in the 2,439 ESP individuals. In the ESP dataset the median frequency of these variants was 0.04% (2/4878 alleles), which we suggest may be a useful cut point for lower penetrance alleles when filtering exome data.

Application of a 0.04% cut point to exome sequencing data in a multiply affected DCM pedigree

To further illustrate the potential utility of the ESP dataset and evaluate our proposed cut point of allele frequency of 0.04%, we performed exome sequence analysis on three DCM cases within the same pedigree, known to be point mutation negative for sixteen known DCM genes, 813 (Figure 2, Table 4). After filtering against dbSNP and 1000 Genomes Project, an average of 368 nonsynonymous or splice site variants remained per individual (Table 4). We then screened the remaining variants against the ESP data and categorized these into two discrete categories: novel variants (absent from ESP data) and very rare variants (present one and two times in the ESP dataset, a frequency of 0.04%. This reduced the number of novel variants to ~100 per individual, and sharing across first cousins, reduced this number to eighteen. Sharing across all three affected individuals reduced the number of novel variants to eight genes (OTOP1, FSIP1, LOC100130086, FAM115C, CCL3L3, FRG1, CDC27 and EPB41L3). Ten additional rare variants were shared between first cousins that also occurred between one and two times in the ESP data, and this was reduced to two variants (COLQ at chr3:15507925 and OR11H12 at chr14:19378189) when sharing across all three affected members. Upon further examination of DCM transcriptome data, previously described8 only FRG1, EPB41L3 and COLQ were expressed in heart tissue. FRG1 (Facioscapulohumeral muscular dystrophy Region Gene 1) is a valid candidate, given that other DCM genes such as DMD1, LMNA and BAG3 are also causative of muscular dystrophy.3334 However, this variant resulted from paralagous sequence (unpublished data). The remaining variants occurred in EPB41L3, (erthyrocyte protein band 4.1) and COLQ (Collagenic tail of endplate acetylcholinesterase), the former being a novel variant (chr18:5423409 T>C) and the latter (chr3:15507925 C>G) occurring in 2/2702 ESP alleles of European ancestry and in 0/2174 ESP alleles of African ancestry. On the basis of our findings with known DCM variants occurring at very low frequency in the ESP dataset, we consider COLQ to be an equally valid candidate as EPB41L3. Further investigation is underway.

Figure 2.

Figure 2

DCM family with exome sequencing. The number of variants in affected individuals A, B and C. AB, AC, BC, and ABC are shown in Table 3 and reflect the variants in common between these combinations of patients. Relationships are AB, first cousins; BC aunt/niece, and AC are first cousin/first cousin once removed.

Table 4.

Exome sequence variants in a DCM pedigree (see Figure 2), mutation negative for 16 known DCM genes

A B C AB AC BC ABC
Total Variants: Nonsense,
Missense, or Splice Site
6878 6887 6851 4524 4429 4799 3708
Above, not in dbSNP, 1000
genomes, other local
databases
357 379 369 98 61 137 48
novel very
rare
novel very
rare
novel very
rare
novel very
rare
novel very
rare
novel very
rare
novel very
rare
Above, compared to ESP: 101 36 98 54 101 56 18 10 11 2 26 19 8 2
Expressed in heart 39 15 40 24 43 19 6 7 2 2 11 6 2 1

Novel means not identified in the ESP dataset; very rare means identified only 1 or 2 times.

Discussion

Exome sequencing is a recently developed approach with the potential to uncover pathogenic mutations for Mendelian diseases.14 Because of its comprehensive nature, the ESP exome dataset (ESP2500) provides a new coding reference database (http://snp.gs.washington.edu/EVS/). Using this resource, we analyzed DCM, a rare variant Mendelian adult onset disease, for the number of reported DCM variants present among the ESP dataset and dbSNP. Our results raise several issues.

A filtering approach based on removing variants found in exome data from unaffected individuals and variants in dbSNP/1000 Genomes Project has proved effective in many Mendelian diseases,14 but these likely represent extremely rare diseases from highly penetrant mutations. When this approach is applied to other disease models with variable penetrance and very rare allele frequency (0.1%) it will likely be less effective in discerning causative alleles. The development of NGS technology has rapidly increased the amount of publicly available sequence variant data. As we report herein for DCM, 16.8% of variants previously reported as disease causative are present in the sequenced individuals within the 2,439 ESP samples. It is possible that a fraction of these rare variants were false positives, although the supporting functional data suggested they may actually be lower penetrance disease-causing alleles. Importantly, prior to large scale datasets such as the ESP it has been nearly impossible to accurately assess the level of penetrance due to the lack of large population-based estimates of variant allele frequencies relative to their disease frequencies.

This raises a second issue - how to distinguish pathogenic variants present at very low frequency in the general population from benign rare variants. Understandably, an oversimplified filtering approach could result in discarding variants that are truly pathogenic. Fourteen of the 33 (42%) DCM variants that were also present in ESP individuals had functional effects in either heterologous cell-based assays or in animal models. In an effort to derive guidance from this previously published data, we suggest an allele frequency of 0.04% as a conservative initial cut point, which is the median allele frequency of variants present in the ESP but also with supporting functional data. In our own example of exome sequencing of a DCM pedigree (table 4), this analysis yielded an additional candidate gene.

Ideally, functional assays would be performed for all variants, as the use of any cut-point has caveats. CSRP3 Trp4Arg is a pertinent example, as it has been observed in multiple DCM families from independent studies,10, 2526 and was observed within the ESP individuals at a frequency of 0.35% in individuals of European and 0.05% in individuals of African descent, respectively. The CSRP3 Trp4Arg variant specifically affects the ability of Csrp3 to interact with T-cap,35 and in a knock-in mouse model resulted in an age- and gene dosage-dependent hypertrophic cardiomyopathy and heart failure phenotype.25

Further discussion is deserved for the SCN5A variant, Ile1835Thr, also present in dbSNP (rs45563942) with no clinical association. This variant was observed at a frequency of 0.54% (7/1292 alleles) in the ESP individuals of African ancestry and absent in those of European origin. In a cell based assay, Ile1835Thr significantly reduced peak sodium current but the effect was dependent on genetic background in the context of transcriptional variation and combination with a common polymorphism, SCN5A H558R (rs1805124).31 Two constitutively expressed SCN5A transcript variants (not allelic variants) exist at frequencies of 65% and 35% expression in heart.36 These transcripts are identical with the exception that the more highly expressed transcript has a deletion of a single amino acid, Q1077. The functional change in sodium current due to the rare DCM variant, Ile1835Thr was specifically observed in cDNA constructs for the Q1077deletion transcript in combination with the common H558R variant and no significant change was observed on the Q1077 background.

These levels of complexity are likely just one example of genetic interactions, and DCM is but one paradigm that the ESP data can illuminate. Supporting evidence for our preliminary cut-point is derived from the example of the long-QT syndrome (LQTS). Although as with DCM, inheritance is predominantly autosomal dominant, a study of 147 discrete variants from two LQTS genes in 753 families suggested that long-QT alleles are more frequently transmitted to daughters than to sons.37 In this example, careful matching of unaffected individuals by gender could also be important. However, only one variant, KCNQ1 Pro320His, is present in the ESP dataset, frequency 0.037% in individuals of European origin, falling within our suggested cutpoint.

Traditionally, Mendelian disease has been viewed as caused by one unique or very rare variant in an individual or pedigree. However, this is likely an over simplification, as other genetic disease paradigms are possible.38 We note that DCM has been shown to result principally from autosomal dominant transmission, commonly understood to result in gain of function mutations, even though loss of function, at least for calcium handling contractile proteins in DCM, also commonly occurs.39 Much greater understanding of disease mechanisms may be relevant, including the possibility of other more complex genetic mechanisms such as di- or multi-genic ‘recessive’ mechanisms where multiple dysfunctional protein cascades interact, all of which leads to the final phenotype of DCM.38

Limitations

Use of unaffected individuals from the same ethnicity and geographical region is highly important. The ESP dataset consists of European and African American ancestries, and a small proportion of the DCM cases are of other origins. This is a potential bias in our study, most strongly highlighted by the failure to observe the LMNA Leu85Arg variant in the ESP data. This variant is present in dbSNP, has a reported frequency >1% in Asian and Sub-Saharan Africans and >20% in Maasai.

Our proposed allele frequency cut point of 0.04% is the median of a small number of observations, and thus with larger sample sizes and more functional data will likely need to be revised. This is the first empiric evidence derived from an exome dataset that provides any guidance, although it may differ for other phenotypes.

Summary

In the exome era, traditional boundaries between Mendelian and more complex diseases will be tested, as illustrated in this example of DCM. This will contribute a degree of uncertainty in clinical interpretation of Mendelian gene variants. The importance of careful annotation of variants cannot be underestimated and through this we predict the level of evidence to assign pathogenicity will be enhanced.

Supplementary Material

01

Acknowledgements

We thank the National Heart, Lung, and Blood Institute GO Exome Sequencing Project and its ongoing studies that produced and provided exome variant calls for comparison: the Lung Cohorts Sequencing Project (HL-102923), the WHI Sequencing Project (HL-102924), the Heart Cohorts Sequencing Project (HL-103010), the Broad Institute Sequencing Project (HL-102925), the Northwest Genomics Center Sequencing Project (HL-102926; D.A.N., M.J.R.), and the Family Studies Project Team.

Funding sources. This work was supported by National Institute of Health awards HL58626 (R.E.H.), 5U54NS065712-02 and 1R01NS072248-01 (S.Z.).

Footnotes

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Disclosures. None

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