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Genetic association analyses highlight biological pathways underlying mitral valve prolapse

Abstract

Nonsyndromic mitral valve prolapse (MVP) is a common degenerative cardiac valvulopathy of unknown etiology that predisposes to mitral regurgitation, heart failure and sudden death1. Previous family and pathophysiological studies suggest a complex pattern of inheritance2,3,4,5. 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 factor6 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 repair7.

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Figure 1: Plots representing the association of 4.8 million SNPs in the GWAS meta-analysis.
Figure 2: Cardiac regurgitation in zebrafish embryos with morpholino-mediated knockdown of orthologs to the candidate genes at 2q35 in human.
Figure 3: Tensin 1 expression in developing mouse valves and the knockout phenotype at 9 months.
Figure 4: Cardiac regurgitation in zebrafish embryos with morpholino-mediated knockdown of the ortholog of LMCD1 at 3p13 in human.

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Acknowledgements

We acknowledge the major contribution of the Leducq Foundation, Paris for supporting a transatlantic consortium investigating the physiopathology of mitral valve disease, for which this genome-wide association study was a major project (coordinators: R.A.L. and A.A.H.). We thank J. Leyton-Mange, X.-X. Nguyen and M. McLellan for help with the zebrafish experiments and P. Mathieu as one of the main investigators of the PROGRAM (Determinants of the Progression and Outcomes of Organic Mitral Regurgitation) study, from which was ascertained the Canadian MVP case control study. C.D., T.L.T. and J.-J.S. acknowledge a translational research grant on genetics of mitral valve funded by the Nantes Hospital (CHU Nantes). R.A.L. acknowledges grant support from the US National Institutes of Health (NIH; grants K24 HL67434, R01 HL72265 and HL109506). N.B.-N. is recipient of a French young investigator fund (ANR-13-ISV1-0006-0). D.J.M. acknowledges the support of the Hassenfeld Scholar Program and a gift from Michael Zak. P.T.E. is supported by grants from the NIH (HL092577, HL104156, K24HL105780, HL065962), an Established Investigator Award from the American Heart Association (13EIA14220013) and support from the Fondation Leducq (14CVD01). P.P. holds the Canada Research Chair in Valvular Heart Diseases and is supported by grants from the Canadian Institutes of Health Research (CIHR; grants MOP-102737, MOP-114997 and MOP-126072). Y.B. is the recipient of a Junior 2 Research Scholar award from the Fonds de recherche Québec–Santé (FRQS) and is supported by the CIHR (MOP-102481 and MOP-137058). L.F.-F. and J.S. received financial support from the Spanish Society of Cardiology. R.D. was supported by fellowships from the fund for medical discovery of the Massachusetts General Hospital and by a Marie Curie reintegration award from the European Commission R.R.M. and R.A.N. performed their work in a facility constructed with support from the US National Institutes of Health, grant C06 RR018823, from the Extramural Research Facilities Program of the Heart, Lung, and Blood Institute: R01-HL33756 (R.R.M.), COBRE 1P30 GM103342 (R.R.M. and R.A.N.), 8P20 GM103444-07 (R.R.M. and R.A.N.), R01-HL127692 (D.J.M., S.A.S. and R.A.N.) and American Heart Association 15GRNT25080052 (R.A.N.). A.A.H., N.B.-N. and X.J. acknowledge funds from INSERM and the French Society of Cardiology.

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Contributions

C.D., N.B.-N. and X.J. organized and designed the genome-wide association study and the manuscript preparation. A.A.H., R.A.L., S.A.S., H.L.M., P.P., J.S. and L.F.-F. organized and coordinated the network efforts and recruitment of patients. R.D., V.P., T.L.T., F.K., P.P. and Y.B. participated in the recruitment of patients and the interpretation of the echocardiographs. J.-J.S. and X.J. coordinated the collection of patient samples. M.P. and S.L. managed the collection of patient samples and performed DNA extraction and genotyping. C.D. and N.B.-N. conceptualized the statistical analyses. C.D. supervised the statistical analyses. C.D., M.-H.C. and F.S. performed the statistical analyses. D.J.M. conceptualized and supervised the zebrafish experiments. N.T. performed zebrafish experiments, interpreted the data and wrote parts of the manuscript. P.T.E. and E.D. participated in zebrafish experiments and interpretation of data. R.R.M. and R.A.N. conceptualized and supervised the mouse studies and interpreted the data. K.T. performed immunohistochemistry staining of mouse organs. S.H.L. generated the mice. F.N.D., E.J.B., D.Z., M.L., S.H., R. Roussel, F.B., R. Redon, P.F. and R.S.V. conducted the epidemiological studies in control cohorts and/or contributed samples to the GWAS and/or follow-up genotyping. P.B. supervised valve tissues DNA extraction. S.A.S. contributed patient samples and organized follow-up genotyping and interpreted the GWAS and follow-up data. C.D., N.B.-N. and X.J. wrote the manuscript. F.N.D., R.A.N., D.J.M. S.A.S., R.A.L., J.-J.S. and A.A.H. edited the manuscript, and all authors approved its content.

Corresponding authors

Correspondence to Christian Dina, Nabila Bouatia-Naji or Xavier Jeunemaitre.

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Additional information

A full list of members is provided in the Supplementary Note.

A full list of members is provided in the Supplementary Note.

Integrated supplementary information

Supplementary Figure 1 Study design with cohorts in discovery and follow-up and filtration strategy of SNPs.

Supplementary Figure 2 Igfbp5 expression in mouse developing heart.

Cardiac expression of Igfbp5 (red) was analyzed at embryonic (E13.5), fetal (E17.5) and adult time points. At the time points investigated, very little Igfbp5 protein was detected in the valves. Expression was observed in the myocardium of the left and right atria and the primary atrial septum (PAS) at E13.5 and E17.5 (arrows) as well as the coronary endothelium in the adult (arrow heads). AL, PL, IVS, LV = anterior and posterior mitral leaflets, interventricular septum, and left ventricle, respectively. Green = MF20 (sarcomeric myosin-myocytes), Blue = Hoescht (nuclei).

Supplementary Figure 3 In vivo assessment of mitral valve phenotypes in Tns1 total knockout mice using echocardiographs.

Tensin1+/+, wildtype; Tensin1-/-, Tns1 knockout mice. Images were obtained in a four-chamber view of the heart.

Supplementary Figure 4 In situ hybridizations of tns1 and lmcd1 mRNA distribution in zebrafish embryos.

Top panel shows tns1 expression pattern at 96 hours post fertilization. Expression is found throughout the myocardium, with highest levels in the outflow tract (right). Lower panel shows lmcd1 expression at 72 hours post fertilization (hpf). Expression is found in the entire myocardium, with enhanced expression in the ventricular chamber.

Supplementary Figure 5 In situ hybridizations for developmental markers of valvulogenesis in lmcd1 and tns1 knockdown embryos.

Top panel shows anti-notch1b probe labeling the developing valve in 72-hpf embryos. Lower panel shows anti-bmp4 probe labeling the valve and surrounding myocardium in control (CN) and lmcd1 knockdown embryos. Arrows in both panels point to the approximate location of the AV valve.

Supplementary Figure 6 Pitpnb expression in mouse developing heart.

Cardiac expression of Pitpnb (red) was analyzed at embryonic (E13.5), fetal (E17.5) and adult time points. Pitpnb was detected in valve endothelial and interstitial cells within the mitral leaflets at each of the time points investigated. Expression was also observed in the epicardium (arrow head) and coronary endothelium (arrow) at E17.5. AL, PL, IVS, LV = anterior and posterior mitral leaflets, interventricular septum, and left ventricle, respectively. Green = MF20 (sarcomeric myosin-myocytes), Blue = Hoescht (nuclei).

Supplementary Figure 7 Assessment of cardiac regurgitation in zebrafish morpholino knockdown for genes on Chr17p13.

(a) Genomic context of the association signal observed in the GWAS meta-analysis. (b) Morpholino-mediated knockdown efficacy. Efficacy for smg6 and sgsm2 in embryonic zebrafish was measured by RT-PCR. (c) Fold change in observed mitral regurgitation in 72-hpf zebrafish embryos after morpholino-mediated knockdown. All results are relative to clutchmate controls. n = number of biological replicates per morpholino. (d) Brightfield micrographs displaying gross morphology of 72-hpf embryos following smg6 or sgsm2 knockdown. Scale bar represents 1 mm. CN = control morpholino-injected embryos.

Supplementary Figure 8 Multidimensional scale–based principal-component analysis.

Cases and controls positions on first and second components axis are presented for the two discovery samples (GWAS 1 and GWAS 2). C1, first principal component. C2, second principal component. All individuals plotted are those included in the discovery GWAS who are all French with European ancestry origin. Cases and controls with non-European ancestry based on the principal component analyses were excluded before association tests.

Supplementary Figure 9 Representative gel images from analysis of morpholino efficacy.

rsID indicates the sentinel SNP at the analyzed locus. CN indicates samples amplified from control-injected embryos, whereas MO indicates amplicons from samples obtained following microinjection of gene-specific morpholino. All samples were obtained from 72-hpf embryos.

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Dina, C., Bouatia-Naji, N., Tucker, N. et al. Genetic association analyses highlight biological pathways underlying mitral valve prolapse. Nat Genet 47, 1206–1211 (2015). https://doi.org/10.1038/ng.3383

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