Study of Genetic Variants for inherited ocular disorders in Sindhi Inbred Pedigrees

Abstract

The inherited ocular disorders are the leading cause of vision impairment, eye degeneration, and dysfunction worldwide. The most of the ocular diseases are inherited as autosomal recessive traits and are in populations where endogamous marriages are common. Identification of disease causing gene and pathogenic variants are of great importance for screening of carriers and management of the disease, moreover, it is fundamental to understand the underlying mechanisms of diseases and thus pave the path for successful therapeutic approaches to cure the disease. This study was aimed to find genetic and molecular basis of enrolled consanguineous pedigrees affected with inherited ocular disorders. Fifty four consanguineous families affected with oculo cutaneous albinism, Retinitis pigmentosa and congenital glaucoma were enrolled from various areas of Sindh province. Detailed family history was recorded and pedigrees were drawn. Genomic DNA was extracted from whole blood for genetic studies. Homozygosity mapping was performed for the most common loci and genes. Frequent mutations were screened through ARMS assays. The disease causing and candidate genes were Sanger sequenced. The whole exome sequencing was carried out on selected unlinked families, data were analyzed and desired variants were filtered out. In-slico bioinformatics tools were used to access the pathogenicity of the novel variants and protein structures were modeled to compare wild and mutant proteins. Disease causing genes were identified in twenty eight families, whereas twenty six families were remained unresolved. Mutations in three genes OCA2, TYR and MC1R, associated with albinism, were found in 14 (14/19) families. Four novel variants c.1056A>C, p.Arg352Ser, c.1322A>G, p.Asp441Gly, c.987C>AGA, p.Gln339Aspfs*2, c.1951 +4A>G were found in OCA2 gene including three reported variants. One novel c.1037 -18 T>G and three reported variants were found in TYR gene, whereas one reported variant was found in the MC1R gene. Moreover 10 families with congenital glaucoma were screened. Four PCG causing mutation were found in CYP1B1 gene, including 1 novel allele, c.1048 C>A, p.Pro350Thr and three known, variants. c.1572G>A, p.Arg390His was remained the frequent PCG causing mutation in CYP1B1 gene. Twenty five families affected with non-syndromic RP and 6 with syndromic RP were analyzed. Five disease causing variants were found in Bardet Biedl Syndrome associated genes in 6 10 syndromic RP families. One novel nonsense variant was detected in c.223C>T, p.Arg75*, in BBS9 gene and four reported variants in MKKS ,BBS1 and BBS2 . Homozygosity mapping was done for common RP loci in the nineteen families with non-syndromic retinitis pigmentosa and were found unlinked. Two families were selected for the whole exome sequencing, and two different candidate genes were identified in each. The pathogenic variant 1708C>G, p.Arg570Gly in MARK3 was found responsible for low eye vision and phthisis phenotype in the VI-12 family. The functional studies of MARK3 gene on animal model support the findings in humans. The second family LURP-33 revealed the pathogenic variant, c.75C>A, p.Asp25Glu in novel chloride like gene, CLCC1, segregated in five affected individuals. The Cl channels play important function in the retinal structure in mice and its dysfunction leads to cause retinal degeneration. In-slico functional studies indicate the pathogenic nature of the CLCC1 variant; however, detailed study of CLCC1 knock out animals may further help to understand its function and role in causing RP. In the present study, OCA2 37% (7/19) was the frequently mutated gene causing OCA in Sindhi population followed by TYR mutations 31% (6/19). CYP1B1 was the most common gene causing PCG with a recurrent mutation, p.R390H in patients having different ethnic groups. Furthermore, Retinitis pigmentosa showed genetic heterogeneity as compared to other disorders studied here. Just MKKS gene mutation was found twice in two families with BBS, whereas, 68% (17/25) RP families were remained unlinked to common RP genes. Identification of two novel candidate gene in Sindhi consanguineous pedigrees causing RP and phthisis indicate genetic heterogeneity and may help to explore novel mechanisms of normal vision and pathophysiology of the phenotype caused by the mutation in MARK3 and CLCC1 genes. The findings may help to provide genetic counseling to affected families and may facilitate to develop cost effective, rapid diagnostic procedures for carrier screening for common mutations.