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

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.

Figure 1.. MIB1 Rare Variants in Nonsyndromic Bicuspid Aortic Valve (nsBAV)

A, Cardiac magnetic resonance imaging (MRI) of a patient with the MIB1 p.V943F variant and a combined valve and muscle phenotype, sagittal section, showing a myocardial crypt in the left ventricle free wall. B, Sagittal section of the same study; showing BAV. C, A graphical representation of the MIB1 domain organization and the location of the identified MIB1 variants. The asterisk indicates a nonsense variant. D, An AlphaFold model (DeepMind) of MIB1 with BAV variants mapped. The model is rendered as a cartoon (colored on a rainbow scale from blue at the N-terminus to red at C-terminus) with sites of BAV variants indicated and rendered in ball-and-stick format. E, A phylogenetic analysis showing high interspecies conservation of the amino acid. ANK indicates ankyrin repeats 1 to 9; MZM, MIB-Herc2 domain 1 + ZZ finger domain + MIB-Herc2 domain 2; REP, MIB repeats 1 and 2; RNG, Ring domains 1 to 3.

Figure 2.. MIB1 Common Single-Nucleotide Variations (SNVs) Associated With Bicuspid Aortic Valve (BAV)

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. 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.

Figure 3.. Association of Heterozygous Mib1K735R and Mib1V943F Variants With Bicuspid Aortic Valve (BAV) in a NOTCH-Sensitized Mouse Genetic background

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.

Figure 4.. Schematic Presentation of the Main Players in the NOTCH Pathway

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.