Novel Association of the NOTCH Pathway Regulator MIB1 Gene With the Development of Bicuspid Aortic Valve

Idit Tessler; Juliette Albuisson; Rebeca Piñeiro-Sabarís; Aline Verstraeten; Hatem Elif Kamber Kaya; Marcos Siguero-Álvarez; Guillaume Goudot; Donal MacGrogan; Ilse Luyckx; Shoshana Shpitzen; Galina Levin; Guy Kelman; Noga Reshef; Hugo Mananet; Jake Holdcraft; Jochen D Muehlschlegel; Gina M Peloso; Olya Oppenheim; Charles Cheng; Jean-Michael Mazzella; Gregor Andelfinger; Seema Mital; Per Eriksson; Clarisse Billon; Mahyar Heydarpour; Harry C Dietz; Xavier Jeunemaitre; Eran Leitersdorf; David Sprinzak; Stephen C Blacklow; Simon C Body; Shai Carmi; Bart Loeys; José Luis de la Pompa; Dan Gilon; Emmanuel Messas; Ronen Durst

doi:10.1001/jamacardio.2023.1469

. 2023 Jul 5;8(8):721–731. doi: 10.1001/jamacardio.2023.1469

Novel Association of the NOTCH Pathway Regulator MIB1 Gene With the Development of Bicuspid Aortic Valve

Idit Tessler ^1,^2,^12,^21,^✉, Juliette Albuisson ^3,^4,^5,²², Rebeca Piñeiro-Sabarís ^6,⁷, Aline Verstraeten ^8,²³, Hatem Elif Kamber Kaya ^9,²⁴, Marcos Siguero-Álvarez ^6,⁷, Guillaume Goudot ^4,^10,¹¹, Donal MacGrogan ^6,⁷, Ilse Luyckx ^8,²³, Shoshana Shpitzen ^1,^12,²¹, Galina Levin ^1,^12,²¹, Guy Kelman ^12,²⁵, Noga Reshef ^12,²⁵, Hugo Mananet ^5,²², Jake Holdcraft ¹³, Jochen D Muehlschlegel ¹³, Gina M Peloso ¹⁴, Olya Oppenheim ¹⁵, Charles Cheng ^4,^10,¹¹, Jean-Michael Mazzella ^4,¹⁰, Gregor Andelfinger ¹⁶, Seema Mital ¹⁷, Per Eriksson ¹⁸, Clarisse Billon ^3,⁴, Mahyar Heydarpour ¹⁹, Harry C Dietz ²⁰, Xavier Jeunemaitre ^4,¹⁰, Eran Leitersdorf ^1,^12,²¹, David Sprinzak ¹⁵, Stephen C Blacklow ^9,²⁴, Simon C Body ¹³, Shai Carmi ²¹, Bart Loeys ^8,²³, José Luis de la Pompa ^6,^7,^✉, Dan Gilon ¹, Emmanuel Messas ^4,^10,¹¹, Ronen Durst ^1,^12,²¹

¹Cardiology Department, Hadassah Medical Center, Jerusalem, Israel

²Sheba Medical Center, Ramat Gan, Israel

³Genetics Department, Assistance Publique–Hȏpitaux de Paris, Hôpital Européen Georges Pompidou, National Referral Center for Rare Vascular Diseases, VASCERN MSA European Reference Center, Paris, France

⁴Université Paris Cité, INSERM, U970 PARCC, Paris, France

⁵Platform of Transfer in Cancer Biology, Georges François Leclerc Cancer –UNICANCER, Dijon, France

⁶Intercellular Signaling in Cardiovascular Development & Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain

⁷Ciber de Enfermedades Cardiovasculares, Instituto de Salud Carlos III, Madrid, Spain

⁸Center of Medical Genetics, University of Antwerp and Antwerp University Hospital, Edegem, Belgium

⁹Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts

¹⁰Vascular Medicine Department, Assistance Publique–Hȏpitaux de Paris, Hôpital Européen Georges Pompidou, Paris, France

¹¹French Research Consortium RHU STOP-AS, Rouen, France

¹²Faculty of Medicine, the Hebrew University, Jerusalem, Israel

¹³Department of Anesthesiology, Boston University School of Medicine, Boston, Massachusetts

¹⁴Department of Biostatistics, Boston University School of Public Health, Boston, Massachusetts

¹⁵School of Neurobiology, Biochemistry and Biophysics, George S. Wise Faculty of Life Science, Tel Aviv University, Tel Aviv, Israel

¹⁶Cardiovascular Genetics, Department of Pediatrics, CHU Sainte-Justine, Université de Montreal, Montreal, Quebec, Canada

¹⁷Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada

¹⁸Cardiovascular Medicine Unit, Center for Molecular Medicine, Department of Medicine, Karolinska Institute, Karolinska University Hospital, Solna, Sweden

¹⁹Department of Medicine, Division of Endocrinology, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts

²⁰McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland

²¹Braun School of Public Health and Community Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

²²Genomic and Immunotherapy Medical Institute, Dijon, France

²³Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands

²⁴Department of Cancer Biology, Dana Farber Cancer Institute, Boston, Massachusetts

²⁵The Jerusalem Center for Personalized Computational Medicine, Jerusalem, Israel

Accepted for Publication: April 21, 2023.

Published Online: July 5, 2023. doi:10.1001/jamacardio.2023.1469

Correction: This article was corrected on August 9, 2023, to correct the dates on which study data were analyzed.

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Corresponding Authors: Idit Tessler, MD, MPH, Braun School of Public Health and Community Medicine, The Hebrew University of Jerusalem, Ein Karem PO Box 12272, Jerusalem 9112102, Israel (idit.gabay@mail.huji.ac.il); José Luis de la Pompa, PhD, Intercellular Signaling Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Melchor Fernández Almagro 3, 28029 Madrid, Spain (jlpompa@cnic.es).

Author Contributions: Dr Tessler had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Drs Tessler, Albuisson, and Piñero-Sabarís are considered co–first authors. Drs de la Pompa, Gilon, Messas, and Durst contributed equally to this work.

Concept and design: Tessler, Albuisson, Piñeiro-Sabarís, Siguero-Álvarez, Goudot, Shpitzen, Levin, Kelman, Cheng, Dietz, Blacklow, de la Pompa, Gilon, Messas.

Acquisition, analysis, or interpretation of data: Tessler, Albuisson, Piñeiro-Sabarís, Verstraeten, Kamber Kaya, Siguero-Álvarez, Goudot, MacGrogan, Luyckx, Shpitzen, Kelman, Reshef, Mananet, Holdcraft, Muehlschlegel, Peloso, Oppenheim, Mazzella, Andelfinger, Mital, Eriksson, Billon, Heydarpour, Jeunemaitre, Leitersdorf, Sprinzak, Blacklow, Body, Carmi, Loeys, de la Pompa, Gilon, Durst.

Drafting of the manuscript: Tessler, Albuisson, Piñeiro-Sabarís, Goudot, Shpitzen, Levin, Kelman, Reshef, Mananet, Cheng, Jeunemaitre, Sprinzak, Body, de la Pompa, Gilon, Durst.

Critical revision of the manuscript for important intellectual content: Piñeiro-Sabarís, Verstraeten, Kamber Kaya, Siguero-Álvarez, Goudot, MacGrogan, Luyckx, Kelman, Holdcraft, Muehlschlegel, Peloso, Oppenheim, Mazzella, Andelfinger, Mital, Eriksson, Billon, Heydarpour, Dietz, Leitersdorf, Blacklow, Body, Carmi, Loeys, de la Pompa, Gilon, Messas.

Statistical analysis: Tessler, Piñeiro-Sabarís, Siguero-Álvarez, Luyckx, Kelman, Mananet, Heydarpour, Carmi.

Obtained funding: Tessler, Levin, Dietz, Jeunemaitre, Blacklow, Body, de la Pompa, Gilon.

Administrative, technical, or material support: Tessler, Albuisson, Piñeiro-Sabarís, Verstraeten, Siguero-Álvarez, Goudot, MacGrogan, Shpitzen, Levin, Holdcraft, Muehlschlegel, Cheng, Andelfinger, Billon, Body, Gilon, Messas.

Supervision: Albuisson, Goudot, Kelman, Leitersdorf, Sprinzak, Carmi, Loeys, de la Pompa, Gilon, Durst.

Conflict of Interest Disclosures: Dr Albuisson reported receiving grants from Région Bourogne Franche Comté 2020 outside the submitted work. Dr Peloso reported receiving grants from the National Institutes of Health/National Heart, Lung, and Blood Institute outside the submitted work. Dr Andelfinger reported receiving grants from Banque Nationale Research Excellence Chair in Cardiovascular Genetics and Fondation Leducq during the conduct of the study. Dr Mital reported receiving grants from Bristol Myers Squibb and personal fees from Bristol Myers Squibb outside the submitted work. Dr Sprinzak reported receiving grants from USA-Israel Binational Science Foundation during the conduct of the study. Dr Blacklow reported receiving funding for an unrelated project from Novartis, advisory board fees from ERASCA, advisor fees from MPM Capital, and consultant fees from Odyssey Therapeutics, IFM, Scorpion Therapeutics, and Ayala Pharmaceutical outside the submitted work. Dr Body reported receiving grants from National Institutes of Health during the conduct of the study. No other disclosures were reported.

Funding/Support: This study was supported in part by grants PID2019-104776RB-I00 and CB16/11/00399 (Dr de la Pompa) from the Spanish Ministerio de Ciencia e Innovación (MCIN/ AEI/10.13039/501100011033/); a grant from Hadassah France Association (Drs Gilon and Tessler); a grant from the Center for Interdisciplinary Data Science Research of the Hebrew University of Jerusalem (Dr Tessler); grant R35 CA220340 from the National Institutes of Health (Dr Blacklow), and grants R21HL150373, R01HL114823 (Dr Body); BSF grants 2013269 and 2017245 (Drs. Sprinzak and Blacklow); a consolidator grant from the European Research Council (Genomia – ERC-COG-2017-771945; Dr Loeys); the European Reference Network on rare multisystemic vascular disorders (VASCERN - project ID: 769036 partly cofunded by the European Union Third Health Programme (Drs Loeys and Verstraeten); funding from the Outreach project (Dutch Heart Foundation; Dr Luyckx); funding from Heart and Stroke Foundation of Canada/Robert M Freedom Chair of Cardiovascular Science (Dr Mital); sample biobanking and sequencing from Canada were supported by grants from the Leducq Foundation Transatlantic Networks of Excellence grant, and the Ted Rogers Centre for Heart Research; ISF grant 1053/12 (Dr Durst); and grant R01HL150401 from National Heart, Lung, and Blood Institute (Dr Muehlschlegel).

Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Data Sharing Statement: See Supplement 3.

Additional Contributions: We thank Ilai Ovadia, BA (Bezalel Academy of Art and Design, Jerusalem, Israel) for his contribution in the figures design. No financial compensation was received for this contribution.

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Corresponding author.

PMCID: PMC10323766 NIHMSID: NIHMS1915245 PMID: 37405741

This genetic association study investigates the genetic cause for bicuspid aortic valve development.

Key Points

Question

What genes are associated with the development of bicuspid aortic valve (BAV)?

Findings

By integrating independent human genetic approaches starting with familial segregation and followed by rare and common variants analyses in this genetic association study of 938 patients, the MIB1 gene was identified; MIB1 is an essential regulator of NOTCH ligands signaling and represents a potential new gene in the development of BAV in humans. These data were further supported by mouse models; mice carrying the identified human MIB1 gene variants demonstrated BAV.

Meaning

These findings suggest a role for MIB1 in the pathophysiology of BAV and identify the NOTCH pathway as a potential target in the management of BAV.

Abstract

Importance

Nonsyndromic bicuspid aortic valve (nsBAV) is the most common congenital heart valve malformation. BAV has a heritable component, yet only a few causative genes have been identified; understanding BAV genetics is a key point in developing personalized medicine.

Objective

To identify a new gene for nsBAV.

Design, Setting, and Participants

This was a comprehensive, multicenter, genetic association study based on candidate gene prioritization in a familial cohort followed by rare and common association studies in replication cohorts. Further validation was done using in vivo mice models. Study data were analyzed from October 2019 to October 2022. Three cohorts of patients with BAV were included in the study: (1) the discovery cohort was a large cohort of inherited cases from 29 pedigrees of French and Israeli origin; (2) the replication cohort 1 for rare variants included unrelated sporadic cases from various European ancestries; and (3) replication cohort 2 was a second validation cohort for common variants in unrelated sporadic cases from Europe and the US.

Main Outcomes and Measures

To identify a candidate gene for nsBAV through analysis of familial cases exome sequencing and gene prioritization tools. Replication cohort 1 was searched for rare and predicted deleterious variants and genetic association. Replication cohort 2 was used to investigate the association of common variants with BAV.

Results

A total of 938 patients with BAV were included in this study: 69 (7.4%) in the discovery cohort, 417 (44.5%) in replication cohort 1, and 452 (48.2%) in replication cohort 2. A novel human nsBAV gene, MINDBOMB1 homologue MIB1, was identified. MINDBOMB1 homologue (MIB1) is an E3-ubiquitin ligase essential for NOTCH-signal activation during heart development. In approximately 2% of nsBAV index cases from the discovery and replication 1 cohorts, rare MIB1 variants were detected, predicted to be damaging, and were significantly enriched compared with population-based controls (2% cases vs 0.9% controls; P = .03). In replication cohort 2, MIB1 risk haplotypes significantly associated with nsBAV were identified (permutation test, 1000 repeats; P = .02). Two genetically modified mice models carrying Mib1 variants identified in our cohort showed BAV on a NOTCH1-sensitized genetic background.

Conclusions and Relevance

This genetic association study identified the MIB1 gene as associated with nsBAV. This underscores the crucial role of the NOTCH pathway in the pathophysiology of BAV and its potential as a target for future diagnostic and therapeutic intervention.

Introduction

Bicuspid aortic valve (BAV) is a heritable condition, affecting 1% to 2% of the population^1,2 and is the most common congenital heart valve defect. BAV is heritable, with first-degree family members having a 10-fold increased risk of being affected.^3,4,5,6,7 When seen in families, BAV mostly presents as a dominant inherited trait with incomplete penetrance and a higher prevalence in male individuals (sex ratio of 3:1).^3,4,8,9,10 BAV genetics are complex and can be caused by a combination of mendelian, oligogenic, and polygenic inheritance.⁵ Several candidate genes, often identified in animal models, were reported.^6,7,11,12,13 However, only few genes were shown to cause nonsyndromic BAV (nsBAV) in humans.^14,15 These include NOTCH1, GATA6, and SMAD6, each accounting for 1% to 3% of cases.^3,14,16

The NOTCH pathway, an evolutionarily conserved cell-cell communication signaling pathway involved in multiple developmental processes,¹⁷ is known to be related to BAV. NOTCH signaling regulates aortic valve morphogenesis, and its disruption causes aortic valve diseases in both humans and mice.^18,19,20 MIB1 (MINDBOMB1) is an essential E3-ubiquitin ligase that induces NOTCH ligand ubiquitination and endocytosis, an essential pathway activation step.²¹

Methods

The study was approved by each institution’s review board according to local regulations, and written informed consent was obtained from all participants. Animal studies were approved by the CNIC Animal Experimentation Ethics Committee and the Madrid community. All animal procedures conformed to European Union Directive 2010/63EU and Recommendation 2007/526/EC regarding protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201/2005. This study followed the Strengthening the Reporting of Genetic Association Studies (STREGA) reporting guidelines.

We combined 3 human genetic approaches and animal model functional studies. The discovery cohort was a large cohort of inherited cases from 28 pedigrees of French and Israeli origin. Replication cohort 1 for rare variants analysis included unrelated sporadic cases from various European ancestries. Replication cohort 2 was a second validation cohort for common variants analysis in unrelated sporadic cases from Europe and the US.

Study Cohort

This study did not address a specific race or ethnicity and was based on the population of origin. Data collected included self-reported ancestry to avoid subpopulation stratification error.

Discovery Cohort

In the discovery cohort, we included a large familial cohort from 2 tertiary medical centers. Index cases were included if they had BAV and if 1 or more relatives had BAV or thoracic aortic aneurysm. All patients underwent a comprehensive clinical examination and personal medical and family histories. Inclusion was based on BAV presence as shown by echocardiography, magnetic resonance imaging, or cardiac surgery. Dysmorphic features were evaluated medically, and syndromic conditions were excluded. Patients younger than 18 years were included only as part of a family, not as index cases.

Replication Cohort 1

Patients whose relatives were unaffected (a negative BAV or thoracic aortic aneurysm test) or unavailable were classified as sporadic cases and were included in replication cohort 1, along with additional cases from the Mechanistic Interrogation of BAV Aortopathy (MIBAVA)–Leducq consortium.

Replication Cohort 2

Replication cohort 2 included patients with BAV from Mass General Brigham Hospital who were of European ancestry. In addition, ethnically matched controls from the Framingham Heart Study were also included.⁶

Whole-Exome and Candidate Gene Sequencing

Discovery Cohort

DNA was extracted from the peripheral blood or saliva of study participants using standard protocols. Whole-exome sequencing was performed at the Genomics Core Facility in the Imagine Institute.²² DNA extraction using the QIAmp Blood DNA Mini Kit (Qiagen) was followed by exome capture using the SureSelect Human All Exon Kit (Agilent) and sequencing on a HiSeq2500 system (Illumina). The mean depth of coverage obtained for each sample was greater than 150×, with more than 97% of the exome covered at least 30×.

Replication Cohort 1

Sanger sequencing of the 21 exons and flanking intronic sequences of MIB1 (NM_020774) was performed on the sporadic Israeli and French cohort. Sequences were amplified by polymerase chain reaction with specific primers, sequenced using BigDye Terminator v3.1 cycle sequencing kits (Thermo Fisher Scientific), and run on an ABI Prism 3730XL DNA Analyzer (Life Technologies). DNA variants were identified using Sequencher software (Gene Codes Corporation).

Mechanistic Interrogation of BAV Aortopathy–Leducq Consortium

Whole blood–derived genomic DNA of patients was used for whole-exome sequencing with the Nimblegen SeqCap Exome Enrichment Kit (Roche). Samples were 2 × 100 base pair–end sequenced on a HiSeq1500 instrument (Illumina). Raw data were processed using an in-house developed pipeline,²³ followed by variant calling with the Genome Analysis Toolkit Unified Genotyper (Broad Institute).²⁴ Variants were annotated and filtered with VariantDB, an in-house developed tool.²⁵ All rare variants of MIB1 identified by whole-exome sequencing were confirmed by targeted Sanger sequencing. When samples and phenotypes from relatives were available, segregation analysis of the selected variants was performed by Sanger sequencing as described previously.

Exome Data Analyses and Prioritization in Discovery Cohort

A filtering pipeline was systematically applied for exome sequencing data.²² Variant calling was performed with the Genome Analysis Toolkit Unified Genotyper based on the ENSEMBL 72 database, using standard parameters. Variants that did not pass the quality filters were excluded (read depth <10 × and/or Phred score <30). Only rare variants (minor allele frequency [MAF] <0.1% according to gnomAD, version 2.1.1) with a combined annotation dependent depletion (CADD) score above 20 (using the CADD, version 1.6 framework²⁶) and with coding impact were retained for any further analysis.

We assembled a list of 34 known BAV genes by thorough and critical literature review, identifying the most robust genes associated with syndromic and nsBAV (in humans and/or mice, eTable 1 in Supplement 1). We aimed to exclude cases with predicted deleterious variants in these 34 known genes. The remaining genes carrying variants in the discovery cohort were then subjected to a prioritization and ranking process, aimed to identify a leading candidate gene for further investigation via validation cohorts and functional work based on their roles in heart development pathophysiology (detailed in eFigure 2 and eTable 1 in Supplement 1 and eTable 2 in Supplement 2).^24,25,26,27 The resulting candidate genes were prioritized by in-silico bioinformatics tools (VarElect and Endeavor), based on pathobiology, relevant pathways, and genetic and biological interactions. The top-rated genes were chosen for further qualitative analyses according to (1) biological relevance to BAV (genes were prioritized if known to have a role in the valve development), (2) frequency (the number of families and family members sharing pathogenic variants in the gene), and (3) variant weight (according to the variant type and its predicted deleteriousness).

Rare Variants Burden Testing in Discovery Cohort and Replication Cohort 1

Burden testing was performed for MIB1 rare variants with coding impact in index cases from the discovery cohort and replication cohort 1 against gnomAD, version 2.1.1, publicly available controls using the TRAPD software package (BioTools).²⁸ Only gnomAD variants passing filters from exome data were retained, using the same filtering process as in the discovery step (MAF <0.1%, CADD score >20, read depth ≥10×). Similarly, burden testing was performed on presumably benign, silent variants used as an internal method control. Owing to the various ethnic backgrounds of patients, testing was performed in all patients with BAV vs all gnomAD controls, as well as by ethnically matched subpopulations. A 2 × 2 contingency table was constructed for each of the groups described previously. A 1-sided Fisher exact test was used to estimate the association P values. An AlphaFold model (DeepMind) of MIB1 with BAV variants mapped was constructed to demonstrate the sites of the identified variants.^29,30

Common Single-Nucleotide Variations Genotyping Data Analyses in Replication Cohort 2

Patients of the second replication cohort were recruited from Mass General Brigham, Boston, Massachusetts.⁶ Genotypes were generated using the Omni2.5 chip (Illumina) for cases and the HumanOmni5.0 chip for controls (obtained from the Framingham Heart Study). The Illumina Omni2.5 chip is a subset of the Omni5.0 chip, with a similar set of primers. In the quality control process, only markers with MAF greater than 1% were included, and populations were stratified by principal components–based filtering (eFigure 3 in Supplement 1). After merging cases and controls, we had BAV cases and controls with a set of 24 single-nucleotide variations (SNVs) in MIB1 ([uc002ktp.3] hg19 chr18:19,284,918-19 450 912 region +/−100kb) common to both Illumina arrays,⁶ which were included for final analysis.

PLINK, version 1.9 (Harvard University) was used for SNV association tests in an additive logistic regression model for BAV cases and controls. The false discovery rate method was applied for multiple comparison correction. The coefficient of linkage disequilibrium (D′) between SNVs was calculated using LDlink (National Cancer Institute).³¹ PHASE, version 2.1 (University of Chicago)³² was used for haplotype reconstruction and for performing association tests between haplotypes and BAV status. We used a permutation test with 1000 repeats to test for differences in haplotype frequencies between BAV cases and controls, each time shuffling the case/control status of individuals.

Generation of Mib1^K735R Mouse Line

Clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) sequences were designed using the CRISPOR-TEFOR online tool (Tefor Paris-Saclay). The annealed 2-part synthetic crRNA (Alt-R CRISPR-Cas9 crRNA, 2 nmol [Integrated DNA Technologies]) and tracrRNA (Alt-R CRISPR-Cas9 tracrRNA, 5 nmol; Integrated DNA Technologies) molecules were diluted in microinjection buffer (1mM Tris hydrochloric acid, pH 7.5; 0.1mM EDTA) and incubated with Sp Cas9 nuclease (Integrated DNA Technologies 1081058). To generate the Mib1K735R line, a complementary and asymmetric single-stranded oligodeoxynucleotide (ssODN) was designed³³ as custom synthetic genes (Megamer single-stranded Gene Fragments [Integrated DNA Technologies]) introducing this point variation. The final concentration of components was 0.61 pmol/μL of crRNA and tracrRNA, 30 ng/μL of Cas9 protein, and 10 ng/μL of ssODN. Microinjections were performed at 1-cell stage fertilized C57BL/6 mouse embryos.³⁴ Pups were screened for the targeted gene variation or insertion by polymerase chain reaction analysis and sequencing, and the selected founders were backcrossed to the C57BL/6 background. Detailed information about the microinjection reagents, summary of results, genotyping, and tissue processing^35,36 is given in eTable 3 in Supplement 1. We have described recently the generation of the mice harboring the Mib1^V943F variant.³⁷ Mice heterozygous for Notch³⁸ (denoted as Notch1^+/−) and Rbp³⁹ (denoted as Rbp^+/−) variant lines were used for genetic sensitization studies with Mib1 alleles.

Statistical Analysis

Study data were analyzed from October 2019 to October 2022. In addition to the analysis stated before, we performed the additional statistical analysis using R studio (RStudio). We reported categorical variables as number and percentage and compared variables using the χ² or Fisher exact test. We reported continuous variables as mean and SD and compared variables using the t test or Mann-Whitney U test. All P values were 2-sided (unless otherwise specified), and P <.05 was considered statistically significant.

Results

A total of 938 patients with BAV were included in this study: 69 (7.4%) in the discovery cohort, 417 (44.5%) in replication cohort 1, and 452 (48.2%) in replication cohort 2. Mean (SD) overall age was 49.8 (14.2) years. A flowchart summarizing the study design and main results is displayed in eFigure 1 in Supplement 1. The composition of the study cohorts including participant age and sex data is presented in the Table.

Table. Nonsyndromic Bicuspid Aortic Valve (nsBAV) Discovery Cohort and Replication Cohorts (N = 938).

Cohort	No./total No. of cases	Center	Location	No. of patients with BAV (No. of families)	Male sex, No. (%)	Female sex, No. (%)	Age, mean (SD), y
Discovery cohort	69/938 Familial cases	APHP-Hôpital Européen Georges Pompidou	Paris, France	46 (18)	30 (65.2)	16 (34.8)	39 (16.5)
Discovery cohort	69/938 Familial cases	Hadassah Medical Center	Jerusalem, Israel	23 (11)	17 (73.9)	6 (26.1)	40 (22.7)
Replication cohort 1	417/938 Cases^a	MIBAVA-Leducq	Europe, Canada, US	195	NA	NA	52.2 (11.8)
		APHP-Hôpital Européen Georges Pompidou	Paris, France	164	127 (77.4)	37 (22.6)	51 (15.5)
		Hadassah Medical Center	Jerusalem, Israel	58	40 (68.9)	18 (31.1)	44.8 (18.8)
	62 874 Controls	Genome Aggregation Database	Mixed	NA	NA	NA	NA
Replication cohort 2	452/938 Cases	Mass General Brigham Hospital	Boston, Massachusetts, US	452	336 (74.3)	116 (25.7)	54.4 (11.8)
Replication cohort 2	1834 Controls	The Database of Genotypes and Phenotypes	Framingham Heart Study, US	NA	713 (38.9)	1121 (61.1)	NA

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Abbreviations: AP-HP, Assistance Publique–Hȏpitaux de Paris; MIBAVA, Mechanistic Interrogation of BAV Aortopathy; NA, not available.

^{^a}

Analyzed with 29 familial index cases.

Inherited nsBAV as a Highly Heterogeneous Trait

Sixty-nine familial cases from 29 pedigrees from various ethnic backgrounds were included in the discovery cohort, dedicated to new candidate gene identification (Table). Detailed description of the cohort demographics and clinical characteristics can be found in eTable 4 in Supplement 1 and eTable 5 and eTable 6 in Supplement 2. In most families, 2 members were affected (18 families, 62%); 10 families had 3 affected members (34.5%), and 1 family had 4 (3.5%). Patients with BAV were predominantly male (46 of 69 [66%]).

After conducting exome sequencing, we applied a step-by-step variant- and gene-filtering strategy detailed in eFigure 2 in Supplement 1. We retained only genes harboring rare and predicted damaging protein-altering variants, with MAF less than 1%, and CADD score greater than 20.

First, we excluded known or suspected syndromic and nsBAV genes (34 genes, including NOTCH1, ROBO4, and SMAD6) (eTable 1 in Supplement 1). We then excluded genes that are not expressed during cardiac development or involved in congenital heart valve defect (eTable 2 in Supplement 2).^33,34,35 Following, we used in silico gene prioritization tools (Endeavor and VarElect)^36,37 (eTable 7 in Supplement 2) to further prune the remaining genes. The top 10 ranked genes from each tool were chosen for further qualitative analysis based on population rarity, gene recurrence in families, and known or potential impact of each variant. Ultimately, we identified 4 candidate genes (eTable 8 in Supplement 1).

MIB1 was identified as the leading candidate gene. In addition to the genetic finding (Figure 1),⁴⁰ our selection was based on literature data: (1) MIB1 role in NOTCH pathway, (2) mouse models of Mib1 inactivation have BAV,^18,38 and (3) the identified variant was previously found in various congenital heart valve defect phenotypes including left ventricular noncompaction (LVNC).^39,41,42The index case in our pedigree carrying the MIB1 variant had a complex phenotype combining BAV with a myocardial crypt. Myocardial crypts have been reported in increased prevalence among carriers of cardiomyopathy gene variants with otherwise normal phenotype.⁴¹

Segregation analysis in this pedigree was complex as both parents of the index case had BAV (eFigure 4 in Supplement 1). The index case (III-1) and his affected mother (II-2) both harbored the MIB1 V943F variant. The affected father (II-1) was deceased at the time of the study. The affected brother of the index case (III-3) did not harbor the variant.

Association Between nsBAV and Rare MIB Protein-Altering Variants

We assembled replication cohort 1, including nsBAV sporadic cases (n = 417) from European, Canadian, and Israeli backgrounds (Table and eTable 4 and eTable 9 in Supplement 1) and performed next-generation sequencing or Sanger sequencing targeting MIB1. Using the same variant-filtering criteria as in the discovery cohort, we identified 7 additional rare, predicted deleterious, protein-altering MIB1 variants in 8 patients: 3 nonsense variants and 4 missense variants, of which 1 was found twice (eTable 10 in Supplement 1). Using Genomic Evolutionary Rate Profiling,⁴² all the identified variants were predicted to be under a selective constraint with high positive scores (range, 4.27-5.64). Segregation analysis could be done for the p.D380N missense variant, which was confirmed de novo (parents were healthy and did not harbor the variant). In an in vitro model, we previously showed that this variant induced impaired interaction between MIB1 and its ligand JAG1, leading to defective NOTCH pathway activation.⁴³

The cumulative frequency of rare and predicted deleterious variants in sporadic BAV cases was 2% compared with 0.9% for silent variants. To determine if sporadic BAV index cases from the discovery cohort and replication cohort 1 present enrichment for rare and predicted deleterious variants, a variant association study was performed using TRAPD (Testing Rare Variants using Public Data) burden testing²⁸ against either all controls or ethnically matched controls from gnomAD (Genome Aggregation Database). Enrichment analysis demonstrated association for all cases vs all gnomAD controls (1% vs 0.5% alleles; 2% vs 0.9% genotypes/cases and controls in a dominant model; P = .03) and the Israeli cases vs Ashkenazi Jewish and non-Finland European controls (1% vs 0.5% alleles; 2% vs 0.9% genotypes in a dominant model; P = .03). In contrast, silent variants were not enriched in our cases (eTable 11 in Supplement 1).

Association of MIB1 Locus With Sporadic nsBAV

Replication cohort 2 included 452 patients with BAV from the Mass General Brigham and Women’s Hospital and 1834 controls from the Framingham Heart Study. Demographics and clinical data of BAV cases and controls are detailed in eTable 12 Supplement 1 and eTable 13 in Supplement 2. We considered common SNVs at the MIB1 locus (Figure 2).⁴⁴ Using a logistic regression model with a false discovery rate correction for multiple comparisons, we detected an association for 5 noncoding SNVs (eTable 14 in Supplement 1), 4 of them within coding regions or introns (Figure 2A).⁴⁴ Linkage disequilibrium (LD) assessment demonstrated that all 5 SNVs were part of a delineated LD block, with D′ (coefficient of LD) of 1, indicating that the SNVs were in high LD. (Figure 2B).⁴⁴ Two blocks were apparent from the LD analysis: one with SNVs 1, 2, and 5 (rs7241299, rs79023008, rs11083391) and the other with SNVs 3 and 4 (rs1893384, rs3017041). Haplotype reconstruction allowed us to identify significant differences in haplotype frequencies between cases and controls, identifying 2 risk haplotypes and 1 protective haplotype (permutation test, 1000 repeats; P = .02) (eTable 15 in Supplement 1). This was further supported by permutation tests in a randomly shuffled cohort of cases and controls, which resulted in nonsignificant differences.

Figure 2. — A, SNVs in the *MIB1* locus. The x-axis represents the chromosomal coordinates; the y-axis represents the log10 P value (left) and the combined recombination rate from HapMap (right). The locus spans 166 Kb from chr18:19 284 918 to chr18:19 450 912 in GRCh37/hg19. Each point represents an SNV and is colored on the basis of D′ in relation to the most significant SNV, colored in blue. The dashed horizontal line marks the critical P value of .01, which was set by the false discovery rate multiple comparisons analysis.⁴⁴ B, A heat map of pairwise linkage disequilibrium statistics for the statistically significant SNVs. The x and y dimensions represent the 5 significant SNVs identified, demonstrating linkage disequilibrium′ approaching 1 for all 5, implying high linkage disequilibrium, modified after being created by LDlink.³¹

Mice Harboring Mib1 Missense Variants Show BAV

We introduced the Mib1 p.K735R and the Mib1 p.V943F³⁷ missense variants into the mouse genome using CRISPR-Cas9 genomic edition. Previously, only loss-of-function studies have been performed in mice based on conditional Mib1 inactivation in the heart.^19,45 Genetic studies have suggested a dominant requirement of NOTCH signaling for aortic valve development; thus, NOTCH1 haploinsufficiency might predispose to cardiac outflow tract abnormalities, including BAV.⁴⁶ We found that mice heterozygous or homozygous for the Mib1^K735R variant (n=41 and n=28, respectively), or the Mib1^V943F variant (n=50 and n=24, respectively) displayed normal aortic valves (Figure 3A [1, 4] and Figure 3C; eFigure 5A,B,G in Supplement 1 shows quantification of background controls). To test the sensitivity of the BAV phenotype to NOTCH gene dosage, we introduced Rbp or Notch1 loss-of-function alleles^38,39 into the Mib1^V943F and Mib1^K735R backgrounds. Notch1^+/− mice alone showed only a 9% BAV penetrance (n = 11; P ≤.05 by χ² analysis) (Figure 3B [1, 2], Figure 3C, and eTable 16 in Supplement 1). In contrast, the combination of missense Mib1 variant alleles with Notch1 or Rbp loss-of-function gene variants revealed that Mib1^K735R/+;Notch1^+/− double-heterozygous mice developed BAV and associated valve defects with 44% penetrance (n = 9; P <.001 by χ² analysis) (Figure 3B [3, 4], Figure 3C, and eTable 16 in Supplement 1). All Mib1^KR/+; Notch1^+/− mice showed 100% ventricular septal defect (VSD) and 44% BAV; thus, all BAV was accompanied by VSD, and there is no BAV without VSD in these double-heterozygous mice. Rbp^+/⁻ mice did not show BAV (n = 10) (Figure 3B [5, 6] and Figure 3C). Mib1^V943F/+;Rbp^+/− mice developed BAV with 6% frequency (n = 20) (Figure 3B [7, 8], Figure 3C, and eFigure 5E,F,G in Supplement 1 show background controls). These results suggest that NOTCH signaling attenuation in a double-heterozygous Mib1 and Notch1 variant background was associated with BAV development and valve defects in mice.

Figure 3. — A, Hematoxylin-eosin staining of aortic valves from (1) E16.5 Mib1^K735R/+, (2) Mib1^K735R/K735R, (3) Mib1^V943F/+, and (4) Mib1^V943F/V943F mice showing normal tricuspid morphology (asterisks). B, Aortic valves and transverse heart sections of E16.5 Mib1^+/+;Notch1^+/− (1, 2), Mib1^K735R/+;Notch1^+/− (3, 4), Mib1^+/+;Rbp^+/− (5, 6), and Mib1^V943F/+;Rbp^+/− (7, 8) embryos. The aortic valves leaflets are marked by an asterisk. BAVs are observed in the double heterozygotes (3, 7). Arrowheads mark the defective membranous ventricular septum, which is observed in the Notch1^+/−;Mib1^+/+ (1, 2) and Mib1^K735R/+;Notch1^+/− hearts (3, 4). C, Percentage of mice manifesting BAV and ventricular septal defect (VSD) phenotypes according to Mib1 variant and Notch1 and Rbp sensitization. (1) Mib1^K735R/+ (n = 41), (2) Mib1^K735R/K735R (n = 28), (3) Notch1^+/− (n = 11), (4) Mib1^K735R/+, Notch1^+/− (n = 9), (5) Mib1^V943F/+ (n = 50), (6) Mib1^V943F/V943F (n = 24), (7) Rbp^+/− (n = 10), and (8) Mib1^V943F/+;Rbp^+/− (n = 20). Scale bars, 100 μm for aortic valve sections and 200 μm for transverse heart sections.

^aP ≤ .0001 by χ².

Discussion

Using both human genetics and functional assays, we identified MIB1 as a novel gene associated with nsBAV in humans, with variants present in approximately 2% of BAV index cases in our cohorts. Our study implemented complementary human genetics approaches that allowed for the identification of MIB1 and was further supported by functional analysis (eFigure 1 in Supplement 1). The initial strategy was based on exome sequencing of a large BAV familial cohort (the discovery cohort), in which we identified an MIB1 germline variant (p.V943F) previously shown to cause LVNC.¹⁵ We used a first replication cohort of unrelated BAV cases, finding 7 additional rare variants with high predicted pathogenicity. A rare variant association study with burden testing³⁹ showed enrichment in MIB1 variants among the BAV cohort. A common variant association study identified risk haplotypes in an additional independent cohort of 452 sporadic BAV cases compared with 1849 ethnically matched controls. Finally, we constructed functional models to confirm our findings, demonstrating that 2 genetically modified animal models, carrying the identified Mib1 missense variants in double heterozygous combination with Notch1 or Rbp deficiency, were associated with the development of BAV. These findings are entirely consistent with a mouse model of Mib1 inactivation in which mice developed cardiac abnormalities, including BAV.¹⁹

Very low penetrance and oligogenic architecture are distinct frameworks to interpret inheritance that is neither purely mendelian nor polygenic.^3,8,9 Our data suggest that rare variants are associated with BAV, which evokes oligogenic inheritance involving MIB1. An oligogenic pattern has been demonstrated in mouse models with BAV.^{3,6,47,48,49,50} In the Slit/Robo pathway, the generation of double-variants’ progenies is necessary to increase the penetrance of BAV.⁴⁹ Genetic interaction between NOS3 and NOTCH1 was also demonstrated to increase BAV penetrance.⁵¹ A similar oligogenic dose effect has been suggested for NOTCH pathway variations in outflow tract syndromes such as Alagille syndrome.⁵² Our mouse data show that the Mib1^V943F and Mib1^K735R variants were not associated with BAV in the heterozygous or homozygous condition, but rather when combined with Notch1 or Rbp heterozygous loss-of-function variants, indicating that the BAV phenotype is very sensitive to the combined insufficiency of NOTCH pathway genes (Figure 4). Our data are in full agreement with the results of a report showing that double heterozygous Mib1^V943F/+;Notch1^+/− mice show highly significant BAV.³⁷ Previously, our group and others demonstrated that deleterious MIB1 variants associated with LVNC,^40,53 atrial septal defects and VSDs, and patent ductus arteriosus.⁵⁴ A complex phenotype of BAV and cardiac muscle malformation, as found in 1 case in the discovery cohort, has also been described.^55,56 This phenotypic overlap between congenital heart valve defects, including BAV and other left heart anomalies, is common. The genes GATA4, GATA5, GATA6, ROBO4, and TBX20 show patterns of inheritance in multiple heart defects (such as atrial septal defect, VSD, BAV, patent ductus arteriosus, and mitral valve anomalies).^{57,58,59,60,61,62,63,64} The phenotypic overlap between LVNC and valve anomalies was also observed in a mouse model of Mib1 inactivation,⁴⁰ as well as in a diversity of phenotypes induced by variations in other BAV-associated genes. Genetic sensitization experiments, in which variations in various genes are combined to uncover a dose-sensitive variant phenotype as found in our mice model, are typical of complex functional studies and are essential to identify the full implication of a given signaling pathway (ie, NOTCH) in a developmental process or disease.²⁷

Figure 4. — NOTCH is a local signaling mechanism in which cells are in a close position. Ligand-receptor interaction leads to a series of cleavage events that ultimately lead to the generation of the NOTCH intracellular domain (NICD), which is able to activate gene expression when bound to the appropriate factors. MIB1 and the DELTA and JAGGED ligands are expressed in the signaling cell (gray). The receiving cell (green) expresses receptors from the NOTCH family that are cleaved and glycosylated in the Golgi apparatus (step 1). Once at the membrane (step 2), NOTCH receptor binds to the ligands expressed in a neighboring cell (step 3). After this interaction, the exposed S2 cleavage site in membrane-bound NOTCH is recognized by ADAM metalloproteinases and cleaved (step 4), while MIB1 ubiquitinates the intracellular domain of the ligand in the signaling cell (step 5), eliciting ligand-receptor complex endocytosis and degradation or recycling. This induces the γ-secretase cleavage (step 6) that releases NICD, which is able to translocate to the nucleus (step 7). Once in the nucleus, NICD binds to the repressor RBPJ/RBP/CSL/Su(H), releasing its corepressors and recruiting coactivators as MAML, leading to the activation of a tissue-specific transcriptional program (step 8). MIB1 is essential for NOTCH pathway activation. This image was created using BioRender image creation software.

Ligand ubiquitination by MIB1 is essential for NOTCH pathway activation, although the downstream mechanisms are not fully understood. The key notion is that ubiquitination in the signaling cell of the ligand bound to the extracellular domain of NOTCH drives its endocytosis,⁶⁵ eliciting in the receiving cell further NOTCH receptor processing and signaling activation (Figure 4).⁶⁶

Other promising candidates were identified during our analysis (eTable 7 in Supplement 2). One of the leading candidates is JAG1, another NOTCH1 pathway gene and a crucial substrate of MIB1 during cardiogenesis.^19,67 Interestingly, both Dll4 and Jag1 are expressed in valve endocardium during early valve development (E9.5), whereas only Jag1 is expressed in aortic valve endocardium at later stages (E12.5 onward).^19,67 These 3 ligands depend on MIB1 for their normal function as NOTCH signaling activating ligands⁶⁸ (Figure 3). In our discovery cohort, we identified rare and predicted deleterious missense variants in JAG1 in 2 pedigrees (eTable 7 in Supplement 2). Previous mice models of a cardiac-specific JAG1 variant resulted in BAV in 7 of 15 mice (46.7%).¹⁹ Further studies and collaboration may facilitate the discovery of its role in BAV.

Limitations

Several limitations to our study should be considered. By using a referral center–based cohort, there is a possibility for bias as the hospital clinic patients are characterized by a more severe form of disease compared with the general BAV population. Therefore, the prevalence of the MIB1 variant in the general population of patients with BAV may be lower than that in our cohort. In both replication cohorts, we used public databases for controls, and these lack validated phenotyping and may also include BAV cases, as in the general population. Finally, here we studied only 1 gene in the NOTCH pathway, but further investigation of the entire pathway is warranted, as several genes of the pathway are involved in valve development in mice.⁶⁹

Conclusions

In conclusion, results of our genetic association study combining various human analyses and in vivo functional studies suggest the involvement of MIB1 in the development of nsBAV, highlighting the NOTCH pathway as a potential significant contributor to nsBAV inheritance and pathophysiology. This work also underscores the need for further investigation of NOTCH pathway components as additional candidate genes for nsBAV and as a future therapeutic target.

Supplement 1.

eTable 1. List of 34 Genes Known or Suspected to Cause Syndromic and Nonsyndromic BAV

eTable 3. Detailed Information About the Microinjection Reagents and Genotyping of the Mice Model

eTable 4. Phenotyping of French and Israeli Cases From Discovery and Replication I Cohorts

eTable 8. Candidate Genes Identified After Multistep Variant and Gene Filtering

eTable 9. MIBAVA-Leducq Cohort (Belonging to Replication Cohort I) Characteristics

eTable 10. MIB1 Identified Coding Variants

eTable 11. MIB1 Association Study by Burden Testing Using gnomAD Control Population

eTable 12. Replication Cohort II—Summary of Demographics and Clinical Data of BAV Cases

eTable 14. The 5 Most Significant SNVs in the Common Variants Analysis

eTable 15. Risk Haplotypes at the MIB1 Locus: a Comparison Between BAV Cases and Controls

eTable 16. Results From the Combination of Missense Mib1 K735R Mutant Alleles With Notch1 Loss of Function mutations

eFigure 1. A Flow Chart Summarizing the Design of the Study

eFigure 2. Flow Chart Displaying the Prioritization Process for Candidate Genes and Variants Analysis

eFigure 3. Multidimensional Scaling (MDS) Plot for 452 BAV Cases and 1911 FHS Controls

eFigure 4. Pedigree of Family BAV-003 With MIB1 p.V943F Variant and Bicuspid Aortic Valve (BAV) Phenotype

eFigure 5. Histological Analysis of Mib1+/+, Mib1+/+;Notch1+/+ and Mib1+/+;Rbp+/+ Control Mice

Click here for additional data file.^{(1.1MB, pdf)}

Supplement 2.

eTable 2. List of Known Candidate Genes

eTable 5. French Cohort Main Demographics and Clinical Characteristics

eTable 6. Israeli Cohort Main Demographics and Clinical Characteristics

eTable 7. Candidate Genes Used in the In Silico Analyses

eTable 13. Replication Cohort II Individuals Demographics and Clinical Data

Click here for additional data file.^{(86.5KB, xlsx)}

Supplement 3.

Data Sharing Statement

Click here for additional data file.^{(16.3KB, pdf)}

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