Rare GATA5 sequence variants identified in individuals with bicuspid aortic valve

Abstract

Background: Bicuspid aortic valve (BAV) is the most common type of congenital heart disease (CHD) and has a proposed genetic etiology. BAV is categorized by cusp fusion, with right-left (R-L) cusp fusion being associated with additional CHD, and right-noncoronary cusp (R-NC) fusion being associated with aortic valve dysfunction. Loss of murine Gata5, which encodes a cardiac transcription factor, results in a partially penetrant R-NC BAV, and we hypothesize that mutations in GATA5 are associated with R-NC BAV in humans.

Methods: A cohort of 78 BAV patients (50 with isolated BAV and 28 with associated aortic coarctation) was analyzed using Sanger sequencing to identify GATA5 sequence variants. Biochemical assays were performed to identify functional deficits of identified sequence variants.

Results: We identified two rare heterozygous nonsynonymous variants, p.Gln3Arg and p.Leu233Pro, for a frequency of 2.6% (2/78). Both individuals with nonsynonymous variants had BAV and aortic coarctation, one R-L and one R-NC subtype. Of the nonsynonymous variants, only p.Gln3Arg demonstrated decreased transcriptional activity in vitro.

Conclusion: Rare sequence variants in GATA5 are associated with human BAV. Our findings suggest a genotype-phenotype correlation in regards to associated CHD but not cusp fusion.

Figure 1. Rare non-synonymous sequence variants identified in subjects with BAV

(A) Family pedigree demonstrating p.Gln3Arg is inherited from unaffected mother and sequence chromatogram showing A to G transition which predicts the non-synonymous amino acid substitution, p.Gln3Arg. (B) Family pedigree, which demonstrates p.Leu233Pro is a de novo mutation, and sequence chromatogram shows heterozygous T to C transition, predicting non-synonymous amino acid substitution, p.Leu233Pro, in proband only. In pedigrees shown in (A, B), unshaded shapes indicate no known cardiac phenotype (no echocardiograms available), while shaded shapes indicate affected individuals; squares represent males while circles are females; +, presence of mutation; -, absence of mutation; ^a indicates nucleotide variant position ^b indicates amino acid variant position. (C) Alignment of human GATA5 protein sequence with orthologues from multiple species. The NCBI GenBank Accession Numbers that were utilized for the alignment are as follows: Human-NP_536721.1; Cow-NP_001029393.1; Rat-NP_001019487.1; Mice-NP_032119.2; Chicken-NP_990752.1; Zebrafish-NP_571310.2; Frog-NP_001081962.1.

Figure 2. In vitro functional analysis of GATA5 sequence variations

(A) Transactivation assays in HeLa cells transfected with 100 ng of murine Gata5 p.Leu240Pro and murine Gata5 p.Gln3Arg along with co-transfection of ANF-luciferase reporter and Renilla control reporter (pRL-SV40 plasmid). The p.Gln3Arg protein demonstrated reduced activation ability as compared to wildtype Gata5 while p.Leu240Pro activation ability was unchanged. Luciferase activity is normalized to Renilla. Experiments were performed in triplicate and means and standard deviations are shown. *, P<0.05. (B) Immunoblotting demonstrates equal expression of Gata5 p.Leu240Pro and p.Gln3Arg when compared to wildtype.