Background The underlying molecular aetiology of congenital heart defects is largely unknown. The aim of this study was to explore the genetic basis of non-syndromic severe congenital valve malformations in two unrelated families.
Methods Whole-exome analysis was used to identify the mutations in five patients who suffered from severe valvular malformations involving the pulmonic, tricuspid and mitral valves. The significance of the findings was assessed by studying sporulation of yeast carrying a homologous Phospholipase D (PLD1) mutation, in situ hybridisation in chick embryo and echocardiography and histological examination of hearts of PLD1 knockout mice.
Results Three mutations, p.His442Pro, p.Thr495fs32* and c.2882+2T>C, were identified in the PLD1 gene. The mutations affected highly conserved sites in the PLD1 protein and the p.His442Pro mutation produced a strong loss of function phenotype in yeast homologous mutant strain. Here we show that in chick embryos PLD1 expression is confined to the forming heart (E2–E8) and homogeneously expressed all over the heart during days E2–E3. Thereafter its expression decreases, remaining only adjacent to the atrioventricular valves and the right ventricular outflow tract. This pattern of expression follows the known dynamic patterning of apoptosis in the developing heart, consistent with the known role of PLD1 in the promotion of apoptosis. In hearts of PLD1 knockout mice, we detected marked tricuspid regurgitation, right atrial enlargement, and increased flow velocity, narrowing and thickened leaflets of the pulmonic valve.
Conclusions The findings support a role for PLD1 in normal heart valvulogenesis.
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Contributors AT-S, KZ and ES contributed equally to the article. AT-S, AS, ZP, AJJTR and OE led patients' recruitment, clinical phenotyping and genetic analysis. AT-S, KZ, ES, AZ, DS-M, DS, MF, BN, NR and AN performed and analysed all functional studies. All authors assisted in the assembly and editing of the manuscript.
Funding Lowy Foundation via AUSiMED (Australia/Israel Medical Research) research fellowship grant to AT-S, NIH grant R01 GM072540 to AN, Deutsche Forschungsgemeinschaft (grant Ni556/8-1) to BN, and Deutsche Forschungsgemeinschaft (SFB688, TPA22) to AZ; National Health and Medical Research Council Australia Fellowship and ARC Stem Cells Australia funding to NR. The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government.
Competing interests None declared.
Patient consent Obtained.
Ethics approval Ethical committees of Hadassah Medical Center and the Israeli Ministry of Health.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement FASTQ files are available upon request.
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