Multi-Omics Approach Delineates Functional Interactions of Novel Genes and Proteins in Primary Congenital Glaucoma

Abstract

Purpose : Autosomal recessive primary congenital glaucoma (PCG) is attributable to multiple genes with varying magnitudes of effect. Using a multi-omics approach, we aimed to functionally characterize interactions of novel genes and their corresponding proteins.

Methods : Clinically well characterized PCG cases (n=586) devoid of homozygous mutations in the candidate genes along with geographically and ethnically matched normal controls (n=1758) were screened through a combination of targeted and whole exome sequencing (WES) using the Ion Ampliseq chemistry. Based on standard quality control measures and data analysis pipelines (using GATK and VARSEQ softwares), novel genes and their interacting partners were identified and functionally characterized. Proteins were extracted from surgically excised trabecular meshwork (TM) tissues of these patients and subjected to mass spectrometry. Network analysis (using Ingenuity Pathway Analysis) identified pathways that were common to the mutated genes and their corresponding differentially expressed proteins. Genotype-phenotype correlation was undertaken on the presenting and post-surgical clinical parameters and photopic negative responses (phNR) of patients.

Results : Deep sequencing revealed novel rare mutations (as per revised ACMG guidelines) in genes comprising ranscription factors involved in development, crystallins, structural proteins, anterior segment and other forms of glaucoma contributing to 34.12% (95%CI, 30.40%-38.06%) of all PCG cases that were either absent or rarely observed in our controls and gnomAD database. Muti-allelic inheritance based on co-occurrences of these alleles were observed in 31.82% (95%CI, 28.10-35.62%) of cases. Proteomics analysis of patients' TM revealed 305 unique proteins across different mutation categories that enriched focal adhesion, extracellular matrix-receptor interaction, endocytosis, complement-coagulation cascades, cholinergic synapse, actin cytoskeleton, PI3K/Akt, chemokine and estrogen signalling pathways. Cases harboring mutations indicated the strongest genotype-phenotype correlation for presenting intraocular pressure (IOP) with the “a-wave” implicit time (0.98) and for corneal diameter with phNR at minimum (0.99).

Conclusions : Dysfunctions of key proteins in the developmental pathways along with mutations in the corresponding genes could be contributing to PCG pathogenesis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.