Exome sequencing in multiplex families with left-sided cardiac defects has high yield for disease gene discovery

Abstract

Congenital heart disease (CHD) is a common group of birth defects with a strong genetic contribution to their etiology, but historically the diagnostic yield from exome studies of isolated CHD has been low. Pleiotropy, variable expressivity, and the difficulty of accurately phenotyping newborns contribute to this problem. We hypothesized that performing exome sequencing on selected individuals in families with multiple members affected by left-sided CHD, then filtering variants by population frequency, in silico predictive algorithms, and phenotypic annotations from publicly available databases would increase this yield and generate a list of candidate disease-causing variants that would show a high validation rate. In eight of the nineteen families in our study (42%), we established a well-known gene/phenotype link for a candidate variant or performed confirmation of a candidate variant's effect on protein function, including variants in genes not previously described or firmly established as disease genes in the body of CHD literature: BMP10, CASZ1, ROCK1 and SMYD1. Two plausible variants in different genes were found to segregate in the same family in two instances suggesting oligogenic inheritance. These results highlight the need for functional validation and demonstrate that in the era of next-generation sequencing, multiplex families with isolated CHD can still bring high yield to the discovery of novel disease genes.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. The CASZ1 p.R25C variant in family 16 causes mislocalization of the protein and reduced transcriptional transactivation.

A. The pedigree of family 16, showing the two affected individuals, proband [38] and his affected mother [39], for whom ES was performed. B. IF staining for FLAG in HEK293T (top) and H9c2 (bottom) cells transfected with either FLAG-tagged CASZ1 WT or FLAG-tagged CASZ1 p.R25C. Note the predominately nuclear staining in both cell types with CASZ1 WT and the increased cytoplasmic staining in cells with CASZ1 p.R25C. C. Western blot analysis of unfractionated and fractionated protein extracts from HEK293T cells transfected with either FLAG-tagged CASZ1 WT or CASZ1 p.R25C, and probed with anti-FLAG antibody to detect tagged CASZ1, a-tubulin as a cytoplasmic marker, and histone H3 (HH3) as a nuclear marker. A majority of CASZ1 WT was present in the nuclear fraction. The CASZ1 p.R25C signal was higher in the cytoplasmic fraction, consistent with the IF staining results. D. A luciferase assay, using the tyrosine hydroxyylase (TH) promoter as a target on a luciferase expression construct in HEK293T cells, showed that cotransfection of CASZ1 WT increased luciferase reporter signal by over 10-fold, while CASZ1 p.R25C did not significantly increase luciferase signal above background. The effect of CASZ1 WT was dosage sensitive (compare 0.5 mg vs 0.25 mg), and cotransfection of CASZ1 WT and p.R25C was not significantly different from CASZ1 WT alone. The graphed data represent the combined results from three independent experiments in which each condition was performed in triplicate. (Error bars indicate SEM, * p = 0.013, ** p = 0.004).

Fig 2. The heterozygous ROCK1 p.Lys695* stop gain variant in family 154 caused reduced expression of ROCK1 in the proband.

A. Pedigree of family 154 showing the proband (517) and his affected identical twin brother (514) and their affected mother (515). ES was performed on individuals marked with an asterisk. B. A western blot of whole cell extracts from proband-derived lymphoblastoid cell lines (LCLs) and LCLs from seven unaffected individuals probed with anti-ROCK1 and anti-α-tubulin antibodies. Quantitation of ROCK1 signal relative to α-tubulin signal in each sample showed a ~50% reduction in ROCK1 in the proband relative to the mean value for the unaffected samples. The mean ROCK1/α-tubulin ratio for the unaffected samples was 0.53, SD±0.08, while the ratio in the proband sample was 0.23, greater than three standard deviations lower than the mean of the control samples.

Fig 3. Rare variants in SMYD1 and BMP10 identified in family 346 are functionally damaging.

A. ES was performed on two BAV patients from family 346 (asterisks). Both individuals were heterozygous for the SMYD1 p.R441W and BMP10 p.R209C variants. B. In a luciferase assay, using a reporter construct driven by the SV40 promoter (pGL3-SV40), cotransfection of a SMYD1 WT expression construct resulted in a 50% reduction in luciferase activity, demonstrating that SMYD1 represses transcription from the SV40 promoter. Transfection of the SMYD1 p.R441W variant protein caused significantly greater repression (~70%) of the reporter activity, to the same level as that of the SMYD1 DCTD variant that lacks the C-terminal autoinhibitory domain of SMYD1. These results indicate that SMYD1 p.R441W is a gain-of-function variant. The data shown represent the mean of values from 3 independent experiments in which each of the samples was assayed in triplicate (error bars indicate standard deviation, **** p<0.0001, *** p<0.001). C. Western blots of whole cell extracts or filtered culture medium from HEK293T cells stably transfected with BMP10 WT or BMP10 p.R209C expression constructs showed reduced levels of both the cellular (prodomain) and secreted form (growth factor domain) of the p.R209C variant protein.