Simon Lecointe

Common variants at SCN5A-SCN10A and HEY2 are associated with Brugada syndrome, a rare disease with high risk of sudden cardiac death

Brugada syndrome is a rare cardiac arrhythmia disorder, causally related to SCN5A mutations in around 20% of cases. Through a genome-wide association study of 312 individuals with Brugada syndrome and 1,115 controls, we detected 2 significant association signals at the SCN10A locus (rs10428132) and near the HEY2 gene (rs9388451). Independent replication confirmed both signals (meta-analyses: rs10428132, P = 1.0 × 10−68; rs9388451, P = 5.1 × 10−17) and identified one additional signal in SCN5A (at 3p21; rs11708996, P = 1.0 × 10−14). The cumulative effect of the three loci on disease susceptibility was unexpectedly large (Ptrend = 6.1 × 10−81). The association signals at SCN5A-SCN10A demonstrate that genetic polymorphisms modulating cardiac conduction can also influence susceptibility to cardiac arrhythmia. The implication of association with HEY2, supported by new evidence that Hey2 regulates cardiac electrical activity, shows that Brugada syndrome may originate from altered transcriptional programming during cardiac development. Altogether, our findings indicate that common genetic variation can have a strong impact on the predisposition to rare diseases.

Genetic association analyses highlight biological pathways underlying mitral valve prolapse

Nonsyndromic mitral valve prolapse (MVP) is a common degenerative cardiac valvulopathy of unknown etiology that predisposes to mitral regurgitation, heart failure and sudden death. Previous family and pathophysiological studies suggest a complex pattern of inheritance. We performed a meta-analysis of 2 genome-wide association studies in 1,412 MVP cases and 2,439 controls. We identified 6 loci, which we replicated in 1,422 cases and 6,779 controls, and provide functional evidence for candidate genes. We highlight LMCD1 (LIM and cysteine-rich domains 1), which encodes a transcription factor and for which morpholino knockdown of the ortholog in zebrafish resulted in atrioventricular valve regurgitation. A similar zebrafish phenotype was obtained with knockdown of the ortholog of TNS1, which encodes tensin 1, a focal adhesion protein involved in cytoskeleton organization. We also showed expression of tensin 1 during valve morphogenesis and describe enlarged posterior mitral leaflets in Tns1−/− mice. This study identifies the first risk loci for MVP and suggests new mechanisms involved in mitral valve regurgitation, the most common indication for mitral valve repair.

Fine-scale human genetic structure in Western France

The difficulties arising from association analysis with rare variants underline the importance of suitable reference population cohorts, which integrate detailed spatial information. We analyzed a sample of 1684 individuals from Western France, who were genotyped at genome-wide level, from two cohorts D.E.S.I.R and CavsGen. We found that fine-scale population structure occurs at the scale of Western France, with distinct admixture proportions for individuals originating from the Brittany Region and the Vendée Department. Genetic differentiation increases with distance at a high rate in these two parts of Northwestern France and linkage disequilibrium is higher in Brittany suggesting a lower effective population size. When looking for genomic regions informative about Breton origin, we found two prominent associated regions that include the lactase region and the HLA complex. For both the lactase and the HLA regions, there is a low differentiation between Bretons and Irish, and this is also found at the genome-wide level. At a more refined scale, and within the Pays de la Loire Region, we also found evidence of fine-scale population structure, although principal component analysis showed that individuals from different departments cannot be confidently discriminated. Because of the evidence for fine-scale genetic structure in Western France, we anticipate that rare and geographically localized variants will be identified in future full-sequence analyses.

New insights into mitral valve dystrophy: A Filamin-A genotype-phenotype and outcome study

Aims Filamin-A (FLNA) was identified as the first gene of non-syndromic mitral valve dystrophy (FLNA-MVD). We aimed to assess the phenotype of FLNA-MVD and its impact on prognosis. Methods and results We investigated the disease in 246 subjects (72 mutated) from four FLNA-MVD families harbouring three different FLNA mutations. Phenotype was characterized by a comprehensive echocardiography focusing on mitral valve apparatus in comparison with control relatives. In this X-linked disease valves lesions were severe in men and moderate in women. Most men had classical features of mitral valve prolapse (MVP), but without chordal rupture. By contrast to regular MVP, mitral leaflet motion was clearly restricted in diastole and papillary muscles position was closer to mitral annulus. Valvular abnormalities were similar in the four families, in adults and young patients from early childhood suggestive of a developmental disease. In addition, mitral valve lesions worsened over time as encountered in degenerative conditions. Polyvalvular involvement was frequent in males and non-diagnostic forms frequent in females. Overall survival was moderately impaired in men (P = 0.011). Cardiac surgery rate (mainly valvular) was increased (33.3 ± 9.8 vs. 5.0 ± 4.9%, P < 0.0001; hazard ratio 10.5 [95% confidence interval: 2.9-37.9]) owing mainly to a lifetime increased risk in men (76.8 ± 14.1 vs. 9.1 ± 8.7%, P < 0.0001). Conclusion FLNA-MVD is a developmental and degenerative disease with complex phenotypic expression which can influence patient management. FLNA-MVD has unique features with both MVP and paradoxical restricted motion in diastole, sub-valvular mitral apparatus impairment and polyvalvular lesions in males. FLNA-MVD conveys a substantial lifetime risk of valve surgery in men.

The alternatively spliced LRRFIP1 Isoform-1 is a key regulator of the Wnt/β-catenin transcription pathway

The GC-rich Binding Factor 2/Leucine Rich Repeat in the Flightless 1 Interaction Protein 1 gene (GCF2/LRRFIP1) is predicted to be alternatively spliced in five different isoforms. Although important peptide sequence differences are expected to result from this alternative splicing, to date, only the gene transcription regulator properties of LRRFIP1-Iso5 were unveiled. Based on molecular, cellular and biochemical data, we show here that the five isoforms define two molecular entities with different expression profiles in human tissues, subcellular localizations, oligomerization properties and transcription enhancer properties of the canonical Wnt pathway. We demonstrated that LRRFIP1-Iso3, -4 and -5, which share over 80% sequence identity, are primarily located in the cell cytoplasm and form homo and hetero-multimers between each other. In contrast, LRRFIP1-Iso1 and -2 are primarily located in the cell nucleus in part thanks to their shared C-terminal domain. Furthermore, we showed that LRRFIP1-Iso1 is preferentially expressed in the myocardium and skeletal muscle. Using the in vitro Topflash reporter assay we revealed that among LRRFIP1 isoforms, LRRFIP1-Iso1 is the strongest enhancer of the β-catenin Wnt canonical transcription pathway thanks to a specific N-terminal domain harboring two critical tryptophan residues (W76, 82). In addition, we showed that the Wnt enhancer properties of LRRFIP1-Iso1 depend on its homo-dimerisation which is governed by its specific coiled coil domain. Together our study identified LRRFIP1-Iso1 as a critical regulator of the Wnt canonical pathway with a potential role in myocyte differentiation and myogenesis.

Identification of novel APOB mutations by targeted next-generation sequencing for the molecular diagnosis of familial hypobetalipoproteinemia

BACKGROUND AND AIMS Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder characterized by decreased plasma levels of LDL-cholesterol and apolipoprotein B (ApoB). Currently, genetic diagnosis in FHBL relies largely on Sanger sequencing to identify APOB and PCSK9 gene mutations and on western blotting to detect truncated ApoB species. METHODS Here, we applied targeted enrichment and next-generation sequencing (NGS) on a panel of three FHBL genes and two abetalipoproteinemia genes (APOB, PCSK9, ANGPTL3, MTTP and SAR1B). RESULTS In this study, we identified five likely pathogenic heterozygous rare variants. These include four novel nonsense mutations in APOB (p.Gln845*, p.Gln2571*, p.Cys2933* and p.Ser3718*) and a rare variant in PCSK9 (Minor Allele Frequency <0.1%). The affected family members tested were shown to be carriers, suggesting co-segregation with low LDL-C. CONCLUSIONS Our study further demonstrates that NGS is a reliable and practical approach for the molecular screening of FHBL-causative genes that may provide a mean for deciphering the genetic basis in FHBL.

0216 : Involvement of the receptor-type tyrosine-protein phosphatase F gene PTPRF, a cell adhesion-like molecule, in Mitral Valve Prolapse (MVP)

Our objectives were to identify MVP causative genes using familial approach, to assess MVP phenotype and to determine the molecular and cellular mechanisms involved in the pathology. The family was identified after one member underwent valve surgery for Barlow-type MVP. Exome sequencing in 3 affected coupled to IBD analysis identified a rare variant (MAF G (p.Ile328Met) in PTPRF gene. Out of 16 mutated patients, 12 (75%) had a MVP (7 bileaflet and 5 posterior leaflet MVP) and 3 (19%) abnormal anterior coaptation. Phenotype assessed by echocardiography showed elongated and significantly thickened mitral leaflets. PTPRF alternative splicing gives rise to several isoforms in humans. The longest isoform encodes the LAR receptor involved in neuronal development and axon guidance. LAR is a repressor of cell migration and proliferation, participates in adherens junctions, modulates small GTPases activity by interacting with the Rho/Rac regulator Trio and inhibits EGF/FGF signaling. It also interacts with Heparan Sulfates Proteoglycans (HSPGs) involved in cell-cell and cell-matrix adhesion. PTPRF thus appeared as a good MVP causing candidate gene. However, the identified mutation (I328M) only targets the short isoform (sPTPRF) whose structure, expression profile and functions remain unknown. We showed here by RT-PCR in mitral valve tissue that sPTPRF is expressed in human mitral valve together with the longest PTPRF isoform encoding LAR. Re-expression study in Hek293 cells revealed the sPTPRF isoform interacts with HSPGs. PTPRF protein stability experiments evaluated by western blotting after cycloheximide treatment showed that the I328M mutation decreases the protein stability.Our results suggest a potential loss of function mutation in PTPRF-short isoform in autosomal dominant Barlows disease. The precise role of the mutation on cell proliferation, cytokine (EGF-TGFβ) signaling and cell matrix interaction remains to be elucidated. The author hereby declares no conflict of interest

0493: Mutations in the gene encoding FilGAP as a cause for mitral valve prolapse

The mitral valve prolapse (MVP) is a common cardiac disorder which affects 2-4% of the population and remains one of the most frequent indications for valvular surgery. The familial nature of MVP has been proposed for many years and so far, FLNA remains the only identified gene. Recently, it has been shown that FLNA mutations deregulate the RhoA/ Rac1 GTPases balance and provided evidences for a role of the Rac1 specific GTPase activating protein, FilGAP, in this network. FilGAP is a recognized FlnA-binding RhoGTPase-activating protein. Giving the tight interactions of FlnA and FilGAP, we first tested, using a candidate gene approach, the hypothesis that FilGAP, encoded by ARHGAP24 , could be involved in MVP. We have sequenced ARHGAP24 in 95 MVP operated patients and identified 3 rare missense mutations in highly conserve residues (FilGAP p.R95Q; p.P417H and p.T481M). One mutation was novel and the 2 others present a minor allele frequency lower than 0.1% in EVS. Moreover, p.T481M co-segregates with the pathology in a family with 3 affected patients. We then investigated the impact of these mutations in HEK293 cells. The role of FilGAP is to decrease Rac1 activity and thus to regulate cell processes involved in actin cytoskeleton properties as adhesion, protrusion and intracellular dynamics. From pull-down assays, we have shown that FilGAP mutations alter Rac1 GTPase activity and significantly decrease the FilGAP interaction with the active form of Rac1 (p ARHGAP24 variants are loss-function mutations. Moreover, we demonstrate that FilGAP mutations alter the downstream signaling pathway by two different mechanisms. FilGAP p.P417H and p.T481M decrease the interaction with FlnA while p.R95Q impacts the plasma membrane anchorage. This work reinforces the involvement of GTPases pathway in MVP pathogenesis.