Targeted analysis of whole genome sequence data to diagnose genetic cardiomyopathy

Abstract

Background: Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of >50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift toward comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy.

Methods and results: Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused on 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1 to 14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and segregation analysis, where available. Three of 3 previously identified primary mutations were detected by this analysis. In 6 subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and had additional pathological correlation to provide evidence for causality. For 2 subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects.

Conclusions: These pilot data demonstrate that ≈30 to 40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.

Keywords: cardiomyopathies; genetics; genomics; humans.

Figure 1

Variant analysis pipeline. A. Missense variant analysis. The Super Gene-set includes genes linked to cardiomyopathy. ~3.7 million variants were identified, restricting to the Super Gene-set reduces the number of variants to ~11.5K. Missense SNVs from these genes were analyzed using PolyPhen-2, SIFT, PhastCons, and GERP.^- Variants were retained if predicted to be probably or possibly damaging by PolyPhen-2 or if damaging by two of the three remaining programs. Cutoff scores of 0.95 and 3 were considered damaging by PhastCons and GERP, respectively. If no prediction was made by at least three of four programs, the variant was retained for further analysis. Variants were then analyzed by Panther and ConSeq.^, Variants with a Panther subSPEC score of < −2 or a ConSeq score < 0 were retained. If neither program was able to make a prediction, the variant was retained. This filtering reduces the number of variants to ~167 per genome. Variants were analyzed for frequency using three databases: the 1000Genomes Project, NHLBI Exome Sequencing Project (ESP 5400), and dbSNP 135/136. Variants present at a frequency of ≤0.01 were retained, resulting in 0-11 nsSNPs per genome. Nonsense SNVs within the myopathy super gene-set were filtered using frequency. B. Splice site variant analysis. Analysis was restricted to intronic SNVs within 10bps of an exon, reducing variant lists from ~3.7M to 50 per genome. Variants were analyzed using MaxENT. The 1000Genomes Project and dbSNP 135/136 were used to determine frequency. Variants present at a frequency of ≤0.01 were deemed potentially deleterious, resulting in 0-2 variants per genome. (SNV=Single nucleotide variant)

Figure 2

SCN5A G1318V detected by WGS. WGS was used to assess the genome of DCM-BH01 with DCM, left bundle branch block and nonsustained VT. A) 12 lead EKG from proband. B) Variants were filtered through the analysis pipeline identifying three potentially pathogenic variants. FKTN and HPS6 were excluded as these genes are linked to recessive, syndromic disease.^, The SCN5A G1318A variant was considered pathogenic since SCN5A gene mutations are known to affect the cardiac conduction system in addition to causing DCM. C)SCN5A G1318A variant was identified in the proband’s offspring with DCM.

Figure 3

WGS identified a desmin (DES) genemutation, R127P. A) DCM-AAL01 pedigree reveals DCM and sudden cardiac death (SCD). The proband (*) underwent cardiac transplantation at the age of 42. B) Seven variants were identified as potentially deleterious, threewere relevant to this individual’s phenotype. MYH11 is linked to muscle phenotypes and therefore was excluded.^, LDB3 I558V may be a contributing variant. The DES R127P variant was further tested. C) The explanted heart from the proband demonstrated desmin aggregates on immunohistochemistry (brown staining, left panel), feature pathognomonic for desmin related myopathies. EM revealed granulofilamentous material consistent with desmin aggregates (right panel). D) When expressed in myogenic C2C12 cells, the DES R127P variant formed aggregates (right panel, arrowhead) while wildtype desmin (left panel) did not. Expressed desmin was tagged with the Xpress epitope tag (green). Nuclei are stained with DAPI.

Figure 4

WGS identified primary and secondary pathogenic sequence variation. A) DCM-AAB03 Pedigree. The TPM1 D230N variant segregates with cardiomyopathy in all family members. Several members of this family had earlier onset disease. Individual II-3 presented at age 20 with heart failure and was found to be hemizygous for the X-linked GLA R118C variant. B) DCM-BI01 Pedigree. The TNNT2 K210del variant has been described previously in cardiomyopathy.^, The two younger members who required cardiac transplantation had an additional TTN mutation predicted to disrupt the splice site and truncate TTN.