Complex Molecular Mechanisms Underlie Primary Congenital Glaucoma Due to the Involvement of Multiple Biochemical Pathways

Abstract

Purpose : The molecular etiology underlying autosomal recessive primary congenital glaucoma (PCG) is poorly understood as the known candidate genes (CYP1B1, LTBP2, TEK, MYOC and FOXC1) contribute to less than half of these cases worldwide. In order to understand the molecular pathogenesis of PCG, we undertook a comprehensive approach involving gene discovery, followed by in silico characterization of mutations and functional validations.

Methods : A large cohort of clinically well characterized PCG cases (n=535) and ethnically matched normal controls (N=1781) were screened by a combination of whole exome and targeted sequencing using the Ion Ampliseq chemistry. All the samples had an average depth of 500X and identification of rare variants and pathogenic characterizations (SIFT, PolyPhen2, MutationTaster) were incorporated in our data analysis pipelines using the GATK and Ion Reporter softwares. Pathway analysis of interacting genes was undertaken by the Ingenuity software. The genetic interactions were confirmed by the corresponding physical interactions with recombinant epitope-tagged versions of these proteins in HEK293 cells. Photopic negative responses (pHNR) were assessed in PCG cases with different mutation profiles.

Results : Our comprehensive analysis of 75 target genes revealed cumulative mutant allele frequencies of 49.27% (95%CI, 39.68%-59.21%) in the PCG-associated candidate genes and 22.62% (95%CI, 15.52%-31.74%) in genes involved in the anterior segment, ciliary dysfunctions and DNA damage. Co-occurrences of mutant heterozygous alleles were observed in 18/75 genes indicating genetic interactions in 120/535 PCG cases. Pathway analysis revealed 3 novel functional clusters of the interacting genes. The HEK293 cells co-transfected with plasmids encoding GFP-CYP1B1 (a candidate gene in PCG) and MYC-CEP164 (ciliary centrosomal protein) provided evidence of physical interaction. PCG patients harboring mutant alleles had reduced pHNR function compared to those without mutations.

Conclusions : Our study expands the mutation spectrum of PCG with the discovery of novel genes along with their genetic and physical interactions. This collectively accounts for 22.42% (95%CI, 19.09%-26.15%) of additional PCG cases devoid of mutations in the known genes and needs further replications. The novel gene clusters provide additional insights into their roles in PCG pathogenesis.

This is a 2020 ARVO Annual Meeting abstract.