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1.
Cardiovasc Res ; 119(3): 759-771, 2023 05 02.
Article in English | MEDLINE | ID: mdl-36001550

ABSTRACT

AIMS: Degenerative mitral valve dystrophy (MVD) leading to mitral valve prolapse is the most frequent form of MV disease, and there is currently no pharmacological treatment available. The limited understanding of the pathophysiological mechanisms leading to MVD limits our ability to identify therapeutic targets. This study aimed to reveal the main pathophysiological pathways involved in MVD via the multimodality imaging and transcriptomic analysis of the new and unique knock-in (KI) rat model for the FilaminA-P637Q (FlnA-P637Q) mutation associated-MVD. METHODS AND RESULTS: Wild-type (WT) and KI rats were evaluated morphologically, functionally, and histologically between 3-week-old and 3-to-6-month-old based on Doppler echocardiography, 3D micro-computed tomography (microCT), and standard histology. RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC-seq) were performed on 3-week-old WT and KI mitral valves and valvular cells, respectively, to highlight the main signalling pathways associated with MVD. Echocardiographic exploration confirmed MV elongation (2.0 ± 0.1 mm vs. 1.8 ± 0.1, P = 0.001), as well as MV thickening and prolapse in KI animals compared to WT at 3 weeks. 3D MV volume quantified by microCT was significantly increased in KI animals (+58% vs. WT, P = 0.02). Histological analyses revealed a myxomatous remodelling in KI MV characterized by proteoglycans accumulation. A persistent phenotype was observed in adult KI rats. Signalling pathways related to extracellular matrix homeostasis, response to molecular stress, epithelial cell migration, endothelial to mesenchymal transition, chemotaxis and immune cell migration, were identified based on RNA-seq analysis. ATAC-seq analysis points to the critical role of transforming growth factor-ß and inflammation in the disease. CONCLUSION: The KI FlnA-P637Q rat model mimics human myxomatous MVD, offering a unique opportunity to decipher pathophysiological mechanisms related to this disease. Extracellular matrix organization, epithelial cell migration, response to mechanical stress, and a central contribution of immune cells are highlighted as the main signalling pathways leading to myxomatous MVD. Our findings pave the road to decipher underlying molecular mechanisms and the specific role of distinct cell populations in this context.


Subject(s)
Mitral Valve Prolapse , Mitral Valve , Adult , Humans , Rats , Animals , Infant , Mitral Valve/metabolism , Filamins/genetics , Filamins/metabolism , Transcriptome , X-Ray Microtomography , Mitral Valve Prolapse/pathology , Phenotype
2.
Biophys J ; 117(8): 1467-1475, 2019 10 15.
Article in English | MEDLINE | ID: mdl-31542223

ABSTRACT

Mitral valve diseases affect ∼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 nonsyndromic mitral valve dysplasia (MVD). The filamin A (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 has 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 destroyed 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 nonreceptor type 12, 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.


Subject(s)
Filamins/chemistry , Mitral Valve Prolapse/genetics , Mutation, Missense , Protein Folding , Binding Sites , Filamins/genetics , Filamins/metabolism , Humans , Molecular Dynamics Simulation , Protein Binding , Protein Tyrosine Phosphatase, Non-Receptor Type 12/metabolism
3.
Structure ; 27(1): 102-112.e4, 2019 01 02.
Article in English | MEDLINE | ID: mdl-30344108

ABSTRACT

Filamin A (FLNa), expressed in endocardial endothelia during fetal valve morphogenesis, is key in cardiac development. Missense mutations in FLNa cause non-syndromic mitral valve dysplasia (FLNA-MVD). Here, we aimed to reveal the currently unknown underlying molecular mechanism behind FLNA-MVD caused by the FLNa P637Q mutation. The solved crystal structure of the FLNa3-5 P637Q revealed that this mutation causes only minor structural changes close to mutation site. These changes were observed to significantly affect FLNa's ability to transmit cellular force and to interact with its binding partner. The performed steered molecular dynamics simulations showed that significantly lower forces are needed to split domains 4 and 5 in FLNA-MVD than with wild-type FLNa. The P637Q mutation was also observed to interfere with FLNa's interactions with the protein tyrosine phosphatase PTPN12. Our results provide a crucial step toward understanding the molecular bases behind FLNA-MVD, which is critical for the development of drug-based therapeutics.


Subject(s)
Filamins/chemistry , Heart Valve Diseases/genetics , Mutation, Missense , Binding Sites , Filamins/genetics , Filamins/metabolism , Humans , Mitral Valve/pathology , Molecular Dynamics Simulation , Protein Binding , Protein Tyrosine Phosphatase, Non-Receptor Type 12/metabolism
4.
Circ Genom Precis Med ; 12(10): e002617, 2019 10.
Article in English | MEDLINE | ID: mdl-32141789

ABSTRACT

BACKGROUND: Calcific aortic valve stenosis (CAVS) is a frequent and life-threatening cardiovascular disease for which there is currently no medical treatment available. To date, only 2 genes, LPA and PALMD, have been identified as causal for CAVS. We aimed to identify additional susceptibility genes for CAVS. METHODS: A GWAS (genome-wide association study) meta-analysis of 4 cohorts, totaling 5115 cases and 354 072 controls of European descent, was performed. A TWAS (transcriptome-wide association study) was completed to integrate transcriptomic data from 233 human aortic valves. A series of post-GWAS analyses were performed, including fine-mapping, colocalization, phenome-wide association studies, pathway, and tissue enrichment as well as genetic correlation with cardiovascular traits. RESULTS: In the GWAS meta-analysis, 4 loci achieved genome-wide significance, including 2 new loci: IL6 (interleukin 6) on 7p15.3 and ALPL (alkaline phosphatase) on 1p36.12. A TWAS integrating gene expression from 233 human aortic valves identified NAV1 (neuron navigator 1) on 1q32.1 as a new candidate causal gene. The CAVS risk alleles were associated with higher mRNA expression of NAV1 in valve tissues. Fine-mapping identified rs1800795 as the most likely causal variant in the IL6 locus. The signal identified colocalizes with the expression of the IL6 RNA antisense in various tissues. Phenome-wide association analyses in the UK Biobank showed colocalized associations between the risk allele at the IL6 lead variant and higher eosinophil count, pulse pressure, systolic blood pressure, and carotid artery procedures, implicating modulation of the IL6 pathways. The risk allele at the NAV1 lead variant colocalized with higher pulse pressure and higher prevalence of carotid artery stenosis. Association results at the genome-wide scale indicated genetic correlation between CAVS, coronary artery disease, and cardiovascular risk factors. CONCLUSIONS: Our study implicates 3 new genetic loci in CAVS pathogenesis, which constitute novel targets for the development of therapeutic agents.


Subject(s)
Alkaline Phosphatase/genetics , Aortic Valve Stenosis/genetics , Aortic Valve/pathology , Calcinosis/genetics , Interleukin-6/genetics , Microtubule-Associated Proteins/genetics , Cohort Studies , Genetic Predisposition to Disease , Genome-Wide Association Study , Humans , Polymorphism, Single Nucleotide
5.
Eur Heart J ; 39(15): 1269-1277, 2018 04 14.
Article in English | MEDLINE | ID: mdl-29020406

ABSTRACT

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.


Subject(s)
Filamins/genetics , Mitral Valve Prolapse/genetics , Mitral Valve Prolapse/pathology , Mitral Valve/pathology , Adolescent , Adult , Echocardiography , Female , Genotype , Humans , Male , Middle Aged , Mitral Valve/diagnostic imaging , Mutation/genetics , Phenotype , Prognosis , Retrospective Studies , Risk Factors , Young Adult
6.
Biochim Biophys Acta Mol Cell Res ; 1864(7): 1142-1152, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28322931

ABSTRACT

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 ß-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.


Subject(s)
RNA-Binding Proteins/metabolism , Wnt Signaling Pathway , Alternative Splicing , Animals , Cells, Cultured , HEK293 Cells , Humans , Male , Mice , Muscle, Skeletal/metabolism , Myocardium/metabolism , Protein Domains , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Rats , Rats, Sprague-Dawley , Wnt Proteins/genetics , Wnt Proteins/metabolism , beta Catenin/genetics , beta Catenin/metabolism
7.
Atherosclerosis ; 250: 52-6, 2016 07.
Article in English | MEDLINE | ID: mdl-27179706

ABSTRACT

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.


Subject(s)
Apolipoprotein B-100/genetics , Hypobetalipoproteinemias/genetics , Mutation , Abetalipoproteinemia/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Cholesterol, LDL/genetics , Codon, Nonsense , Gene Library , Genetic Variation , Heterozygote , High-Throughput Nucleotide Sequencing , Humans , Hypobetalipoproteinemias/diagnosis , Middle Aged , Pedigree , Phenotype , Proprotein Convertase 9/genetics , Reproducibility of Results , Sequence Analysis, DNA , Young Adult
8.
Nat Genet ; 47(10): 1206-11, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26301497

ABSTRACT

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.


Subject(s)
Mitral Valve Prolapse/genetics , Animals , Case-Control Studies , Genome-Wide Association Study , Humans , Mice
9.
Eur J Hum Genet ; 23(6): 831-6, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25182131

ABSTRACT

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.


Subject(s)
Genome, Human , Polymorphism, Genetic , Population/genetics , France , HLA Antigens/genetics , Humans , Lactase/genetics
10.
Basic Res Cardiol ; 109(6): 446, 2014.
Article in English | MEDLINE | ID: mdl-25341504

ABSTRACT

Brugada syndrome (BrS) is characterized by ST-segment elevation in the right precordial leads and is associated with increased risk of sudden cardiac death. We have recently reported families with BrS and SCN5A mutations where some affected members do not carry the familial mutation. We evaluated the involvement of additional genetic determinants for BrS in an affected family. We identified three distinct gene variants within a family presenting BrS (5 individuals), cardiac conduction defects (CCD, 3 individuals) and shortened QT interval (4 individuals). The first mutation is nonsense, p.Q1695*, lying within the SCN5A gene, which encodes for NaV1.5, the α-subunit of the cardiac Na(+) channel. The second mutation is missense, p.N300D, and alters the CACNA1C gene, which encodes the α-subunit CaV1.2 of the L-type cardiac Ca(2+) channel. The SCN5A mutation strictly segregates with CCD. Four out of the 5 BrS patients carry the CACNA1C variant, and three of them present shortened QT interval. One of the BrS patients carries none of these mutations but a rare variant located in the ABCC9 gene as well as his asymptomatic mother. Patch-clamp studies identified a loss-of-function of the mutated CaV1.2 channel. Western-blot experiments showed a global expression defect while increased mobility of CaV1.2 channels on cell surface was revealed by FRAP experiments. Finally, computer simulations of the two mutations recapitulated patient phenotypes. We report a rare CACNA1C mutation as causing BrS and/or shortened QT interval in a family also carrying a SCN5A stop mutation, but which does not segregate with BrS. This study underlies the complexity of BrS inheritance and its pre-symptomatic genetic screening interpretation.


Subject(s)
Brugada Syndrome/genetics , Calcium Channels, L-Type/genetics , Mutation , NAV1.5 Voltage-Gated Sodium Channel/genetics , Adult , Aged, 80 and over , Animals , COS Cells , Chlorocebus aethiops , Female , Humans , Male , Middle Aged , Pedigree , Young Adult
11.
J Cardiovasc Transl Res ; 4(6): 748-56, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21773876

ABSTRACT

Myxomatous dystrophy of the cardiac valves is a heterogeneous group of disorders, including syndromic diseases such as Marfan syndrome and isolated valvular diseases. Mitral valve prolapse, the most common form of this disease, is presumed to affect approximately 2% to 3% of the population and remains one of the most common causes of valvular surgery. During the past years, important effort has been made to better understand the pathophysiological basis of mitral valve prolapse. Autosomal-dominant transmission is the usual inheritance with reduced penetrance and variable expressivity. Three loci have been mapped to chromosomes 16p11-p12, 11p15.4 and 13q31-32, but the underlying genetic defects are not currently known. An X-linked recessive form has been originally described by Monteleone and Fagan in 1969. Starting from one large French family and three smaller other families in which MVP was transmitted with an X-linked pattern, we have been able to identify three filamin A mutations p.Gly288Arg and p.Val711Asp and a 1,944-bp genomic deletion coding for exons 16 to 19. In this review, we describe the genetic, echocardiographic and functional aspects of the filamin-A-related myxomatous mitral valve dystrophy.


Subject(s)
Contractile Proteins/genetics , Genetic Diseases, X-Linked/genetics , Microfilament Proteins/genetics , Mitral Valve Prolapse/genetics , Mitral Valve , Mutation , Animals , Contractile Proteins/metabolism , Echocardiography , Filamins , Genetic Diseases, X-Linked/diagnostic imaging , Genetic Diseases, X-Linked/metabolism , Genetic Diseases, X-Linked/physiopathology , Genetic Predisposition to Disease , Heredity , Humans , Microfilament Proteins/metabolism , Mitral Valve/diagnostic imaging , Mitral Valve/metabolism , Mitral Valve/physiopathology , Mitral Valve Prolapse/diagnostic imaging , Mitral Valve Prolapse/metabolism , Mitral Valve Prolapse/physiopathology , Pedigree , Phenotype
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