Coding Sequence Mutations Identified in MYH7, TNNT2, SCN5A, CSRP3, LBD3, and TCAP from 313 Patients with Familial or Idiopathic Dilated Cardiomyopathy

Abstract

Introduction

In the past 10 years substantial progress has been made in identifying genes that cause dilated cardiomyopathy. Yet determining the mutation frequencies of these genes in patients with idiopathic or familial dilated cardiomyopathy (IDC or FDC) has been challenging for several reasons. Gathering large cohorts of carefully phenotyped FDC/IDC probands and their family members is time and effort intensive. In addition, locus heterogeneity (more than 20 genes have been described) ¹ , ² and allelic heterogeneity (mutations can involve any coding nucleotides) greatly complicate investigational strategies. ¹ For these reasons there is a lack of comprehensive gene mutation reports of large FDC cohorts, a deficit that prevents the field from achieving a greater understanding of the genetics of dilated cardiomyopathy.

We utilized the resequencing service provided by the National Heart, Lung, and Blood Institute (NHLBI) to obtain mutational data from a large cohort of probands with FDC or IDC. Direct DNA sequencing is the gold standard in mutation detection; however, it involves considerable cost and effort. We therefore selected six of the more than 20 known FDC genes using a strategy designed to maximize the yield of genetic variants. The six genes included β‐myosin heavy chain (MYH7), cardiac troponin T (TNNT2), the cardiac sodium channel (SCN5A), titin‐cap or telethonin (TCAP), LIM binding domain 3 (LBD3), and muscle LIM protein (CSRP3). We present here the protein‐altering variants identified in the disease cohort.

Methods

Results

Bidirectional sequencing of six genes implicated in IDC/FDC was carried out on DNA specimens from 313 unrelated probands with IDC or FDC, 302 of whom have been previously described. ³ Protein‐altering variants, none of which were present in 253 control specimens, were identified in 36 of 313 probands (11.5%) (Table 2). ¹⁰ , ¹¹ , ¹⁸ , ³⁴ , ³⁵ , ³⁶ , ³⁷ , ³⁸ , ³⁹ , ⁴⁰ , ⁴¹ , ⁴² , ⁴³ Most were missense mutations, most altered highly conserved amino acids, and several had previously been reported in association with dilated cardiomyopathy or other cardiovascular disease (Table 2).

Of the six genes examined, the most commonly mutated was MYH7, with variants identified in 13 of the 313 probands (4.2%). All MYH7 variants were considered possibly or likely disease‐causing (Table 2). One mutation (Arg1500Trp), observed to segregate with disease (A.8), had been previously reported in a patient with IDC. ³⁴ One proband (A.11) demonstrated a single mutant allele (Gly1808Ala) and no wild‐type allele, indicating the subject was either homozygous or hemizygous for the mutation. Family history indicated no consanguinity, but parental DNAs were not available for analysis. Proband A.1 had both a possibly disease‐causing novel MYH7 variant, Arg237Trp and a possibly disease‐causing LDB3 mutation.

Six troponin T alterations were identified in nine probands (Table 2). In seven of the probands there was evidence to suggest that the variants were likely disease‐causing. The variant in individual B.1 (Arg134Gly) segregated with the disease in other affected family members and was therefore considered likely disease‐causing. IDC patient B.2, whose parents were second cousins, appeared to be homozygous for an Arg151Cys alteration. Individual B.4's Arg205Trp mutation had been previously reported in association with DCM, and was therefore considered likely causative. ¹⁸ A previously described disease‐causing Lys210del ¹⁰ , ¹¹ , ¹⁸ was observed in four unrelated probands (B.5, B.6, B.7, B.8). The remaining two TNNT2 nucleotide alterations were considered possibly disease‐causing. The Arg159Gln alteration in B.3 predicted the replacement of a conserved amino acid, as did the Glu244Asp variant in individual B.9, which was previously reported in a patient with hypertrophic cardiomyopathy. ³⁸ However, in each of the latter two cases, there were not enough family members to establish segregation with disease.

Seven different protein‐altering variants were identified in SCN5A, the cardiac sodium channel, among eight probands (Table 2). In six of the eight cases, the nucleotide changes were considered possibly or likely disease‐causing. Mutations in SCN5A have been well established as a cause of the long QT syndrome Type III, ⁴⁴ as well as of dilated cardiomyopathy. ¹⁵ , ¹⁶ Four SCN5A alterations (one of which was carried by two probands, C.1 and C.2) were previously reported in association with the long QT syndrome, and one, Arg526His, was previously associated with the Brugada syndrome (Table 2). All seven SCN5A variants changed conserved amino acids. Two novel variants (in individuals C.3 and C.7) segregated with FDC in their respective families and were considered likely disease‐causing. However, the Arg526His alteration (C.4) reported in association with the Brugada syndrome did not segregate with DCM, and the Ala572Asp variant (in C.5) previously reported in association with the long QT syndrome did not alter a highly conserved amino acid nor did it segregate with DCM in the proband's large family. Therefore, the C.4 and C.5 variants were considered unlikely to be disease‐causing in these families. No evidence of the Brugada or the long QT syndrome was present in the eight probands.

Three protein‐altering variants were identified in TCAP, also known as titin‐cap or telethonin, a part of the titin complex. Mutations in TCAP have been previously associated with DCM ⁹ and HCM. ⁹ , ⁴⁵ One variant identified (D.3) did not segregate with disease, and this proband was also known to carry a likely disease‐causing lamin A/C mutation (data not shown). The other two variants were considered possibly disease‐causing.

Two nucleotide alterations were identified in LDB3, the LIM domain binding 3 gene, which encodes the ZASP (Z band alternatively spliced PDZ motif‐containing) protein that functions in cytoskeletal assembly. Mutations in LDB3 have previously been reported in association with IDC/FDC and left ventricular noncompaction. ¹⁴ One nucleotide change identified in an FDC proband (E.1) from whom no familial DNA specimens were available, occurred in an amino acid that was not well conserved. The identified mutation, Ala371Thr, predicted the replacement of the wild‐type alanine with a threonine, which is the wild‐type amino acid in the mouse. Thus, the Ala371Thr LDB3 variant was considered an unlikely cause of the proband's FDC. The second LDB3 protein‐altering variant, Ala698Thr, was identified in two unrelated probands (E.2 and E.3), and was predicted to change a highly conserved amino acid. Notably, proband E.3 also carried an MYH7 mutation in addition to the LDB3 mutation, and therefore the relative contribution of the LDB3 and MYH7 mutations to the phenotype of proband E.3 could not be determined; for this reason the Ala698Thr mutation was considered possibly disease‐associated. None of the LDB3 mutation‐carrying probands showed evidence of left ventricular noncompaction.

One protein‐altering variant, Gly72Arg, was identified in CSRP3, a gene encoding cysteine rich protein 3 (also called cardiac LIM domain protein, or MLP), part of mechanical stretch sensing. This protein colocalizes with TCAP, a titin‐associated protein. ¹⁷ The Gly72Arg variant predicted the replacement of a conserved amino acid and was therefore considered possibly disease‐causing.

Eighteen of 183 FDC probands (9.8%) and 14 of 130 IDC probands (10.8%) carried possibly or likely disease‐causing mutations. Because of the finding that possibly or likely disease‐causing mutation frequencies in FDC and IDC probands were similar, we subdivided the FDC category into those with confirmed FDC and probable FDC, and subdivided the IDC category into those with IDC and those with possible FDC, according to our research database and as previously described. ³ The results were similar: for confirmed FDC there were 12 mutations in 134 probands (9.0%); for probable FDC there were six mutations in 49 probands (12.2%); for possible FDC, six mutations in 73 probands (8.2%); and IDC, eight mutations in 57 probands (14.0%).

Discussion

A longstanding issue in cardiovascular medicine has been the underlying cause of nonischemic dilated cardiomyopathy after all known and detectable causes have been excluded. This entity, known as idiopathic dilated cardiomyopathy (IDC), is at times present in two or more closely related family members; in such cases the assignment of familial dilated cardiomyopathy (FDC) has been made. It has recently been shown that genetic disease underlies some proportion of FDC, ¹ , ² but few studies have examined multiple genes in large cohorts to obtain preliminary frequency estimates.

We now present the frequency of possibly and likely disease‐causing mutations in the coding regions and intron‐exon boundaries of six genes implicated in FDC and IDC within a cohort of 313 probands with FDC or IDC. In DNA samples from these 313 probands we identified 36 subjects (11.5%) carrying a total of 31 unique protein‐altering variants that were not observed in 253 control individuals. The genetic variants carried by 32 of the subjects (10.2%) were considered possibly or likely disease‐causing mutations based upon their absence in control DNA samples, alteration of a conserved amino acid, segregation with disease, presence in two or more unrelated probands, and/or prior reports of the mutation in association with dilated cardiomyopathy.

Only a minority of the mutations showed segregation in families with multiply affected members or were previously published in association with FDC; therefore, most mutations were categorized as possibly disease‐causing. We selected this conservative approach because in this multigene survey we were unable to provide functional data to substantiate the putative pathologic effects of these mutations. We note, however, that variations of the type here referred to as “possibly disease‐causing” (i.e., rare nonsynonymous mutations not found in appropriately matched controls) would in some clinical genetic testing situations be considered likely causative.

We also observed similar mutation frequencies between IDC and FDC probands. Of the 183 probands with FDC, 18 carried likely or possibly disease‐causing mutations (9.8%) similar to the 14 of 130 probands with IDC (10.8%). Similar results were observed by subdividing FDC and IDC into confirmed (9.0%), probable (12.2%), and possible (8.2%) FDC and IDC (14.0%) categories in accordance with our research database. ³ Although great care was taken in establishing and assigning probands to these categories, these data suggest that the clinical and family history‐based distinctions between IDC, possible FDC, probable FDC, and confirmed FDC may not be meaningful at the molecular level.

Another recent study from our group (which analyzed 324 probands, 304 of whom are in this study) identified 19 probands with likely disease‐causing mutations in the coding region of LMNA, the gene encoding lamin A/C. ³² When these 19 probands carrying LMNA mutations are added to the 32 probands with possible or likely disease‐causing mutations identified in this study, the overall frequency of putative genetic causation is 15.7% (51 of 324) within these seven FDC genes.

The 10.2% of IDC/FDC mutations identified in this study (or 15.7% including the lamin A/C data) likely underestimate the overall impact of genetic influence on IDC/FDC, as the coding regions of only a fraction of the 20+ known genes were examined. Further, it is likely that not all FDC‐associated genes have been discovered. It is also possible that additional disease‐associated genetic variation resides in regulatory areas (promoters, 5’ and 3’ untranslated regions) or introns of these genes, which would not have been detected in this study.

So while it can be argued that these studies have only scratched the surface toward discovering the full impact of gene variation on dilated cardiomyopathy, this preliminary evidence of the similar mutation frequencies for FDC and IDC (9.8% and 10.8%, respectively) for these genes suggests that the frequency of genetic causation in IDC, usually considered to be a sporadic, nongenetic disease, may in fact be similar to that of FDC. The similar frequencies of mutations observed in IDC and FDC, if substantiated in larger cohorts with more genes, suggest that the likelihood of genetic causation may be comparable with either an IDC or FDC diagnosis. Similar frequencies of genetic causation for familial and sporadic hypertrophic cardiomyopathy (HCM) ³⁵ have been observed, and HCM is now principally considered a genetic disease. ⁴⁶ Of the 197 HCM probands in one study, 172 had familial disease. The overall frequency of genetic disease was 63% in the familial cases and 60% in the sporadic cases. ³⁵ By analogy, it is possible that a similar paradigm may emerge, in which IDC and FDC will be different points on a single spectrum of genetic disease. Resolution of these issues will require evaluation of these and other genes in larger cohorts of patients.

Acknowledgments

References