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

International audienceFamilial 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

Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility.

Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel Na1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on Na1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings

Critical Structural Defects Explain Filamin A Mutations Causing Mitral Valve Dysplasia

Mitral valve diseases affect approximately 3% of the population and are the most common reasons for valvular surgery because no drug-based treatments exist. Inheritable genetic mutations have now been established as the cause of mitral valve insufficiency, and four different missense mutations in the filamin A gene (FLNA) have been found in patients suffering from non-syndromic mitral valve dysplasia (MVD). The FLNA protein is expressed, in particular, in endocardial endothelia during fetal valve morphogenesis and is key in cardiac development. The FLNA-MVD causing mutations are clustered in the N-terminal region of FLNA. How the mutations in FLNA modify its structure and function, have mostly remained elusive. In this study, using NMR spectroscopy and interaction assays, we investigated FLNA-MVD causing V711D and H743P mutations. Our results clearly indicated that both mutations almost completely destroy the folding of the FLNA5 domain, where the mutation is located, and also affect the folding of the neighboring FLNA4 domain. The structure of the neighboring FLNA6 domain was not affected by the mutations. These mutations also completely abolish FLNA’s interactions with protein tyrosine phosphatase (PTP) non-receptor type 12 (PTPN12), which has been suggested to contribute to the pathogenesis of FLNA-MVD. Taken together, our results provide an essential structural and molecular framework for understanding the molecular bases of FLNA-MVD, which is crucial for the development of new therapies to replace surgery.peerReviewe

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

International audienceThe 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

International audienceBACKGROUND 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 \textless0.1%). 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

0077 : DOCK1 a new candidate gene in inherited form of mitral valve prolapse

Mitral valve prolapse (MVP) affects 2-4% of the general population and remains one of the most frequent indications for valvular surgery. So far, the only gene described in MVP, with an X-linked form of inheritance, is FLNA but only represents a small part of MVP.To this respect, we investigated a french MVP pedigree to uncover new molecular insights associated to the disease. We focus on a family of 5 affected patients characterized by a dysmorphic myxomatous phenotype with 2 operated patients.From 4 MVP patients, we performed a Whole Exome Sequencing screening. From bioinformatics analysis, we focused on rare (MAF<0.1%) functional variants shared by the 4 affected patients. Among 25 variants of interest, 11 were novel. One of them, found in a highly conserved residue (GERP=4.88), in DOCK1 (c.4646G>A; p. R1549Q) co-segregates in all affected members in the family. DOCK1 is highly expressed in the mitral valve as evidenced by RNA sequencing experiments using human mitral valve tissue.DOCK1, encodes an atypical Rac exchange factor, Dock180, which acts as a guanine exchange factors (GEF) for small Rho family G proteins. Interestingly, Sanematsu et al. (2010) described a major role of Dock180 during cardiovascular development. Mitral valves of Dock1 depleted mice are thickened and lead to blood retention in left atrium. We currently investigate repercussions of p. R1549Q (located in GEF activity domain of Dock180) on Dock180 activity in the cellular adhesion phenotype (XCELLigence; IF assays) and GEF activity (Pull-Down assays) using a heterologous transfection system. Finally, a cohort of 285 MVP affected patients is screened to evaluate the prevalence of DOCK1 in the disease. This study reports a familial approach, coupled to an exome sequencing strategy which identifies a novel DOCK1 missense mutation associated with MVP phenotype. In vivo previous studies and cellular mechanisms learning will allow us to better understand mechanisms involved in the MVP pathogenesis

0131: A phenotypic study of ARHGAP24 mitral valve prolapse suggests a genetic origin for fibro elastic deficiency

Mitral valve prolapse (MVP) by Barlow disease is recognized a genetic disease. Fibro-elastic deficiency (FED)-MVP is considered a pure degenerative condition. FLNA, the first gene involved in MVP, encodes for Filamin-A, a cytoskeleton associated protein. It interacts with a protein named Filgap, encoded by ARHGAP24 (Chr. 4), in the mechanical transduction. We hypothesized that ARHGAP24 mutations could elicit MVP with same pathway.Four probands with ARHGAP24 mutations were identified among 96 MVP. By a familial echocardiographic screening we enrolled 19 adults of whom 13 had an ARHGAP24 mutation. The mutated group was matched with a control group of 39 healthy adults. Anterior (AML) and posterior (PML) mitral leaflets length and thickness were measured. The coaptation point position was the ratio of coaptation height on the systolic annulus diameter. MVP (displacement>2mm above the annulus line), minimal systolic displacement (MSD, displacement<2mm) and abnormal antero-posterior coaptation position (AAC, ratio<60%) were assessed.The conjunction of MSD and AAC defines a MVP prodromal form (MVP-prod).There was no difference between the two groups on baseline characteristics. Leaflets were thin in the mutated group (PML: 2.7±1 vs 2.2±0.5mm in control, P=0.21, AML: 2.5±0.9 vs 2.1±0.4mm, P=0.25), as reported in FEDMVP. Only the PML was elongated (8.2±1.6 vs 6.0±1.2mm/m 2, P=0.0003) in the mutated group, leading to an anterior displacement of the coaptation point (51±11 vs 66±7%, P=0.0003). Abnormal mitral phenotype (70% of MVP, 23% of MVP prod) and mitral regurgitation (93 vs 38%, P=0.0007) were frequent in the mutated group. Two probands were operated for severe MR related to chordal rupture; histological examination confirmed the leaflets thinness.ARHGAP24 is the first gene for autosomal dominant inherited MVP. Our limited series of patients exhibit typical features of FED-MVP. Our results could change the paradigm of a pure degenerative disease for FED-MVP

Fine-scale human genetic structure in Western France

International audienceThe 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

Servius

Il reste le commentateur de Virgile le plus célèbre. Grâce à ses écrits, les Bucoliques, les Géorgiques et l’Énéide ont pu être appréhendées comme les trois volets d’un projet poétique sans exemple, dont l’unité souterraine ne demandait qu’à être révélée. Il laisse, plus encore qu’une élucidation précise et pénétrante des chefs-d'œuvre de la littérature augustéenne, une encyclopédie du monde antique, qui ne traite pas seulement de grammaire, de style et de poétique, mais aussi de mythologie, d’histoire, de politique et philosophie. L’œuvre de Servius s’offre comme l’irremplaçable reliquaire d’un savoir perdu et d’une sagesse oubliée. Pourtant, elle a été lue, méditée, appréciée et célébrée pendant des siècles, au point que les hommes du Moyen Âge et de la Renaissance confondaient ce qu’ils héritaient de Virgile et ce que leur léguait son commentateur : les poèmes et les scolies se déployaient comme un seul discours continu, d’une richesse et d’une bigarrure inépuisables. Certains poètes humanistes empruntent indifféremment aux vers de l’un et à la prose de l’autre. Lorsque, néanmoins, le texte de Servius était étudié comme un ensemble autonome, il apparaissait comme le modèle indépassable de tout commentaire. Examiner les structures de son discours, suivre la tradition qu’il inaugure, percevoir les échos qu’il fait entendre dans les gloses de la Pharsale ou des Métamorphoses, dans le Roman d’Eneas, dans les écrits d’Isidore de Séville, de Boccace ou de Lorenzo Valla, c’est comprendre quelles ont été les pratiques exégétiques, du IVe au XVIe siècle, et comment elles ont ensemencé la création poétique. Malgré l’intérêt porté aujourd’hui à Servius dans de nombreux pays, il n’existe encore aucune grande synthèse sur son œuvre. C’est pour pallier ce manque qu’un échange a eu lieu, à Rennes, en 2009, entre des chercheurs internationaux qui se sont confrontés aux questions primordiales que soulèvent le texte de Servius et sa réception, de l’Antiquité à la Renaissance. Les trente-et-une études ici regroupées sont le reflet de ces échanges