Utilization of Whole Exome Sequencing to Identify Causative Mutations in Familial Congenital Heart Disease

Abstract

Background: Congenital heart disease (CHD) is the most common type of birth defect with family- and population-based studies supporting a strong genetic cause for CHD. The goal of this study was to determine whether a whole exome sequencing (WES) approach could identify pathogenic-segregating variants in multiplex CHD families.

Methods and results: WES was performed on 9 kindreds with familial CHD, 4 with atrial septal defects, 2 with patent ductus arteriosus, 2 with tetralogy of Fallot, and 1 with pulmonary valve dysplasia. Rare variants (<1% minor allele frequency) that segregated with disease were identified by WES, and variants in 69 CHD candidate genes were further analyzed. These selected variants were subjected to in silico analysis to predict pathogenicity and resulted in the discovery of likely pathogenic mutations in 3 of 9 (33%) families. A GATA4 mutation in the transactivation domain, p.G115W, was identified in familial atrial septal defects and demonstrated decreased transactivation ability in vitro. A p.I263V mutation in TLL1 was identified in an atrial septal defects kindred and is predicted to affect the enzymatic functionality of TLL1. A disease-segregating splice donor site mutation in MYH11 (c.4599+1delG) was identified in familial patent ductus arteriosus and found to disrupt normal splicing of MYH11 mRNA in the affected individual.

Conclusions: Our findings demonstrate the clinical utility of WES to identify causative mutations in familial CHD and demonstrate the successful use of a CHD candidate gene list to allow for a more streamlined approach enabling rapid prioritization and identification of likely pathogenic variants from large WES data sets.

Clinical trial registration: URL: https://clinicaltrials.gov; Unique Identifier: NCT0112048.

Keywords: ductus arteriosus, patent; exome; genetic testing; heart septal defects, atrial; tetralogy of Fallot.

Figure 1

GATA4 Gly115Trp (G115W) mutation in Family C with atrial septal defects. (A) Pedigree of family C with autosomal dominant inheritance of ASD. (B) Table showing cardiac phenotypes of affected family members. (C) Sequence chromatogram of GATA4 exon 1 in affected individual II-5 displays a heterozygous nucleotide change 343G>T, causing a glycine to tryptophan change at amino acid residue 115 as compared to an unaffected, unrelated control subject (D). (E) Cross-species alignment of GATA4 protein sequence demonstrating highly conserved glycine at codon 115 (arrow). NCBI accession numbers that were utilized for GATA4 alignment are as follows: Human: NP_001295022.1, Cow: NP_001179806.1, Rat: NP_653331.1, Mouse: NP_032118.2, Chicken: NP_001280035.1, Frog: NP_001084098.1, Zebrafish: NP_571311.2. (F) Gly115Trp is located adjacent to the first GATA4 transactivation domain (TAD1). TAD2, transactivation domain 2; NZf, n-terminal zinc finger; CZf, c-terminal zinc finger; NLS, nuclear localization sequence. (G) Decreased luciferase activity in HeLa cells transfected with Gata4 G115W plasmid when compared to wildtype Gata4. Similar results were noted with both alpha myosin heavy chain (α-MHC) and atrial natriuretic factor (ANF) luciferase reporters. (H) Western blot showing expression of Gata4 wildtype or G115W mutant protein. GAPDH is shown as a loading control. Four independent experiments were performed and statistical comparisons were done utilizing the student’s t-test. Two tailed p-value <0.05 was considered statistically significant. * p value <0.05.

Figure 2

TLL1 Ile263Val (I263V) mutation in Astacin-Like Domain of TLL1 in family D with atrial septal defects. (A) Pedigree of autosomal dominant inheritance of ASD in family D. (B) Phenotypes of affected family members are shown in Table. (C) Sequence chromatogram of affected patient II-1 displays a heterozygous nucleotide change 787A>G in TLL1, predicting a isoleucine to valine mutation at amino acid position 263, as compared to unaffected family member II-6 (D). (E) Cross-species alignment of protein sequence of TLL1 demonstrating highly conserved isoleucine at position 263 (arrow). NCBI accession numbers that were utilized for GATA4 alignment are as follows: Human:NP_036596.3, Cow:NP_001180043.1, Rat:NP_001099551.1, Mouse:NP_033416.2, Chicken:NP_990034.2, Frog:NP_001083894.1, Zebrafish: NP_571085.1. (F) Ile263Val (*) is located in the astacin-like metalloprotease domain of TLL1. ZnMc: astacin-like metalloprotease domain; CUB: Complement C1r/C1s, Uegf, Bmp1 domain; EGF: epidermal growth factor domain.

Figure 3

Single Nucleotide Deletion in MYH11 (c.4599+1delG) in Family F with patent ductus arteriosus. (A) Pedigree showing autosomal dominant inheritance of PDA in family F. (B) Table showing phenotypes of affected family members. (C) Sequence chromatogram of affected family member II-2 shows a heterozygous deletion of the +1 splice site of exon 33, leading to a frameshift mutation as compared to unaffected individual, II-1 (D). (E) Schematic representation of the MYH11 protein and with deletion of 71 amino acids within myosin tail. (F) Sequence chromatogram of cDNA obtained from dermal fibroblasts of affected individual II-2 that shows loss of exon 33 as compared to control. SH3: SR3 homology domain, Myosin Head: myosin head motor domain, Myosin Tail: myosin coiled-coil rod like tail domain.