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Subhabrata Chakrabarti, Sriparna Ganguly, Meha Kabra, Anil Mandal, Sirisha Senthil, Inderjeet Kaur, Partha Majumder; Runs of Homozygosity Across the Whole Genome Suggests two Novel Genes in Primary Congenital Glaucoma. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4499.
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© ARVO (1962-2015); The Authors (2016-present)
Primary congenital glaucoma (PCG) is largely attributed to mutations in the CYP1B1 gene that are globally structured on intragenic haplotypes. These mutations account for 20-50% of cases in different populations worldwide while the contributions of other genes in the remaining cases are yet unknown. Based on the premise of large regions of homozygosity harboring the CYP1B1 mutations, we aimed to identify the missing genetic determinants in the CYP1B1-negative PCG cases using runs of homozygosity (ROH) across the whole genome.
Initially, an algorithm (based on ‘R’ program) was devised to identify regions within each chromosome where the homozygote genotype frequencies differed significantly across the 90,000 single nucleotide polymorphisms (SNPs) between the PCG cases devoid of CYP1B1 mutations (n=131) and ethnically matched normal controls (n=130). Genotyping of these SNPs were accomplished by a combination of microarray and resequencing. Test of proportions were calculated for the consecutive markers harboring the associated SNPs that exhibited homozygosity. Further, haplotypes were generated with the flanking SNPs to the associated variant observed through ROH and analyzed using the Haploview software (version 4.2) that uses an EM algorithm. Using online databases (NCBI and Seattle SNPs) putative gene(s) harboring the risk haplotypes were identified.
We identified multiple genomic regions across different chromosomes, wherein stretches of 7, 6 and 5-loci were significant (Z score>=1.96). Further, test of proportions among cases and controls indicated that only 7 regions were significant (p<0.01) with uninterrupted stretches of consecutive SNPs. Of these, two regions on chromosomes 19 (33 kb) and 21 (4.25 kb) exhibited the largest stretches that were associated with PCG. Haplotype analysis using 10,000 permutation tests indicated major risk haplotypes on chromosomes 19 (C-C-G-G-G-A; p=0.006) and 20 (C-C-A-C-A-T; p=0.001). Bioinformatic analysis further demonstrated that the ROH on chromosomes 19 and 20 lay within the KLHL26 and the TSHZ2 genes, respectively.
Based on ROH approach, we identified two novel candidate genes in PCG that have been earlier implicated in regulating aqueous humor outflow (KLHL26) and embryonic development (TSHZ2).
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