Whole exome sequencing identifies a troponin T mutation hot spot in familial dilated cardiomyopathy

Abstract

Dilated cardiomyopathy (DCM) commonly causes heart failure and shows extensive genetic heterogeneity that may be amenable to newly developed next-generation DNA sequencing of the exome. In this study we report the successful use of exome sequencing to identify a pathogenic variant in the TNNT2 gene using segregation analysis in a large DCM family. Exome sequencing was performed on three distant relatives from a large family with a clear DCM phenotype. Missense, nonsense, and splice variants were analyzed for segregation among the three affected family members and confirmed in other relatives by direct sequencing. A c.517T C>T, Arg173Trp TNNT2 variant segregated with all affected family members and was also detected in one additional DCM family in our registry. The inclusion of segregation analysis using distant family members markedly improved the bioinformatics filtering process by removing from consideration variants that were not shared by all affected subjects. Haplotype analysis confirmed that the variant found in both DCM families was located on two distinct haplotypes, supporting the notion of independent mutational events in each family. In conclusion, an exome sequencing strategy that includes segregation analysis using distant affected relatives within a family represents a viable diagnostic strategy in a genetically heterogeneous disease like DCM.

Figure 1. Pedigrees of families (A) AD-FDC1 and (B) AD-FDC27.

The family structures of both TNNT2 mutation families are shown. The proband is indicated by an arrow. Males and females are depicted as squares and circles, respectively. Affected individuals are identified by shading. Presence or absence of the Arg173Trp variant confirmed by Sanger sequencing is indicated by plus (‘+’) and minus (‘−’) signs, respectively. Paired numbers beneath individuals represent the numbered haplotypes according to (Table 4); double-numbers represent recombinant haplotypes further detailed in the Table S3. Individuals studied by exome sequencing are indicated by present of black underline beneath haplotype numbering.

Figure 2. Venn diagram reflecting variant overlap between and among patients.

A Venn diagram depicts the number of variants after bioinformatics filtering to identify nonsynonymous, nonsense and splice site variants that were located in conserved regions (by ANNOVAR), novel (absent in 1000 Genomes and NHLBI Exome Sequencing Project datasets), and were predicted to be damaging by in silico analyses. The numbers and overlap regions in the Venn diagram show the variants unique to or shared among three individuals (IV:4, IV:7, and IV:14) from family AD-FDC1.

Figure 3. Visualization of NGS alignment and chromatogram from Sanger sequencing confirming the TNNT2 Arg173Trp variant.

The alignment and Sanger sequencing profiles of the TNNT2 R173W variant are shown. A) C>T variant alignment reads of Arg173Trp variant B) (inset) Chromatogram of C>T variant of Arg173Trp variant from Sanger sequencing; arrow depicts the c.517T C>T (chr1∶201,332,477) position.