Primary congenital glaucoma (PCG) occurs in children due to developmental abnormalities in the trabecular meshwork and anterior chamber angle. Previous studies have implicated rare variants in CYP1B1, LTBP2, and TEK and their interactions with MYOC, FOXC1, and PRSS56 in the genetic complexity and clinical heterogeneity of PCG. Given that some of the gene-encoded proteins are localized in the centrosomes (MYOC) and perform ciliary functions (TEK), we explored the involvement of a core centrosomal protein, CEP164, which is responsible for ocular development and regulation of intraocular pressure. Deep sequencing of CEP164 in a PCG cohort devoid of homozygous mutations in candidate genes (n = 298) and controls (n = 1757) revealed CEP164 rare pathogenic variants in 16 cases (5.36%). Co-occurrences of heterozygous alleles of CEP164 with other genes were seen in four cases (1.34%), and a physical interaction was noted for CEP164 and CYP1B1 in HEK293 cells. Cases of co-harboring alleles of the CEP164 and other genes had a poor prognosis compared with those with a single copy of the CEP164 allele. We also screened INPP5E, which synergistically interacts with CEP164, and observed a lower frequency of pathogenic variants (0.67%). Our data suggest the potential involvements of CEP164 and INPP5E and the yet unexplored cilia-centrosomal functions in PCG pathogenesis.

Bone morphogenetic protein7 (BMP7), an important member of the TGFβ superfamily, is known to be vital for embryonic growth and development. To decipher the role of BMP7 in congenital cardiac malformations, genetic screening of 285 CHD cases along with 400 healthy controls individuals was performed by Sanger sequencing method. Five missense variants were identified in 5 unrelated CHD probands with distinct phenotypes. Three novel missense (p.D85V, p.R175W, and p.A283T) variants in the pro-peptide region and two other variants (p.M315I and p.N321S) in the mature domain were documented. In vitro functional analysis revealed WT as well as all five mutant BMP7 proteins localized within ER compartment, which was confirmed by ER-Tracker and SERCA2 staining. Western blot analysis demonstrated enhanced phosphorylation of SMAD1/5 associated with these variants. Furthermore, transactivation assays showed increased activity of BMP-responsive promoters- Id1-luc, Id3-luc, Tlx2-luc, and p(SBE)4-luc with a synergistic effect observed upon AKL2 co-expression. Additionally, overexpression of downstream targets, namely Bmp2, Bmp7, Nkx2.5, Gata4, Irx4, Smad1, Smad4, and Smad5, alongside downregulation of left-right patterning genes (Nodal and Pitx2) and the BMP antagonist Chordin, was observed. Likewise, EdU staining revealed increased cellular proliferation associated with all these variants. Moreover, modeling of secondary and tertiary structures also suggested that these variants might induce conformational changes in BMP7, potentially strengthening its binding affinity with the receptor and thereby amplifying SMAD signaling. Collectively, in vitro and in silico analyses suggest that these BMP7 variants exhibit gain-of-function (GoF) activity, disrupting normal BMP signaling pathways and thereby contributing to the development of CHD.

Bone morphogenic protein2 (BMP2), a member of TGF-β super-family, known to play a wide range of roles during embryonic development, particularly in the formation of bone/skeleton, differentiation of neurons, skeletal muscle, and development of cardiac valve septa and outflow tract. BMP2 haploinsufficiency is reported to cause multiple congenital malformations including cardiac defects mainly endocardial cushion formation and chamber specification. To investigate the functional relevance of BMP2 variations in isolated CHD cases, we performed genetic screening of BMP2 in 285 CHD probands along with 400 healthy controls by Sanger’s method. Five non-synonymous variants namely, an already known variant p.Ser37Ala in N-terminal region, one nonsense variant, p.Lys241X in pro-peptide region and three missense variants p.His321Leu, p.Glu328Lys and p.Ser351Cys in mature domain, were identified in 8 unrelated CHD cases. In vitro functional analysis by western blotting depicted an increase in phosphorylation of SMAD1/5 due to all five variants. Furthermore, overexpression of cardiac-specific downstream target genes namely Smad1, Smad4, Smad5, Nkx2 . 5, Gata4 and Irx4 of the BMP pathway was observed in response to all the variants. Luciferase assay also validated the enhanced expression of multiple downstream promoters Id1-luc, Id3-luc, Tlx2-luc, and p(SBE) ₄ -luc . Additionally, computational analysis of RNA structural features and protein secondary and tertiary structural changes also highlighted the increased activity of mutants, possibly due to enhanced interactions of mutant proteins with their binding partners owing to more stable structures. Overall, this is the first study which characterized the functional association of BMP2 variants with the pathogenesis of CHD by in vitro and in silico methods.