Next Generation Sequencing Reveals A Novel Gene Mutation in Primary Congenital Glaucoma Patients

Abstract

Purpose: Primary congenital glaucoma (PCG) is a potentially-blinding disease due to a mal-developed anterior segment structure. Three major genes have been implicated -CYP1B1, LTBP2, and MYOC- but do not account for all cases. The study aim was to determine novel gene sequence variants in PCG families using whole exome sequencing (WES).

Methods: DNA samples from 13 families negative for known PCG gene mutations underwent WES. WES was performed using the NimbleGen v2 exome capture kit and Illumina HiSeq2000 platform. SNP and Variation Suite 7.5 software was used to filter single nucleotide variants (SNVs) and insertions/deletions (indels). Public databases and internal whole exome controls were used to filter out known variants. Filtered novel SNVs were validated by Sanger sequencing to confirm familial segregation, and was performed in 32 additional PCG cases. In silico prediction tools (Polyphen/SIFT and FoldX) were used to determine mutational impact on protein function and structure.

Results: An average 50X coverage depth for all coding regions was achieved. Two affected individuals in 1 family inherited a novel, heterozygous stop codon amino acid change, p.Tyr307*, in the tyrosine kinase, endothelial, (TEK) gene. Sequencing revealed TEK mutations in 2 additional families, one as a heterozygous mutation (p.Lys294Asn), and the other as a compound heterozygous (p.Pro346Gln and p.Tyr611Cys) mutation. The p.Tyr307* causes TEK protein truncation, resulting in unstable protein formation, as several inter-domain interactive hydrophobic residues become accessible to water. For p.Lys294Asn and p.Pro346Gln, replacement of the positively charged Lys with a polar Asn molecule perturbs hydrophilic interactions, while the substitution of non-polar Pro with a polar Gln affects hydrophobic interactions causing flexible loop region instability. FoldX predictive effects on protein structure note an insignificant average free energy change for p.Lys294Asn, while that of p.Tyr611Cys and p.Pro346Gln had significant destabilization. cDNA expression panels validated TEK presence in relevant human ocular tissues.

Conclusions: In all, TEK SNVs were present in 3/35 PCG families (6.67%). The absence of the kinase domain due to the stop codon clearly indicates that p.Tyr307* has deleterious effects on TEK function. WES is useful in causal gene discovery for rare ocular disorders.

Keywords: 604 mutations • 440 candidate gene analysis • 539 genetics