Genetic Dissection of Pakistani Families Segregating Autosomal Recessive Albinism

Abstract

Oculocutaneous albinism (OCA) is an uncommon genetic disorder of defective melanin pathway. The condition is characterized by hypopigmentation of hair, dermis, and ocular tissues. Genetic studies have reported seven non-syndromic OCA genes, which includes TYR, TYRP1, OCA2, SLC45A2, SLC24A5, C10ORF11, MC1R, and an uncharacterized OCA5 locus. The product of these OCA genes are responsible for either synthesis or trafficking of melanin pigment. Mutation studies on cohort of Pakistani families have shown TYR and OCA2 genes as the most prevalent genetic factor with prevalence rate of 45.7% and 31.9% respectively (Shahzad et al., 2017). So far, mutations in TYR, OCA2, TYRP1, SLC45A2 genes and an uncharacterized OCA5 locus have been identified to cause OCA in Pakistan. The present genetic study entitled “Genetic dissection of Pakistani families segregating autosomal recessive albinism” was performed to investigate the genetic spectrum of OCA in Pakistani population. Herein the present report, we ascertained and enrolled seventeen OCA families from different localities of Pakistan, after obtaining IRB approvals and informed written consents. Genetic analysis was performed through Human Core Exome single-nucleotide polymorphism (SNP) genotyping (for homozygosity mapping), whole exome sequencing (for pathogenic variant detection in protein coding gene), Sanger sequencing (for validation and segregation analysis) and qPCR (for CNV validation). In homozygosity mapping (considering recessive genetics of OCA disorder), SNPs data were analyzed manually by choosing common homozygous alleles (haplo-identical allele) in affected individuals that were heterozygous or non-haplo-identical in normal persons. Similarly, in case of exome data analysis, homozygous non synonymous or indel variants were manually filtered and then their pathogenicity was determined through bioinformatics tools like Polyphen-2 and Mutationtaster. The molecular investigation in five families identified four novel mutations in different genes that include a novel homozygous deletion mutation of entire TYRP1 gene, deletion of OCA2 exon 19, a novel missense mutation (NM_000275.2:c.1075G>C; p.Gly359Arg) and a splice mutation (NM_000275.2:c.2079+5G>T) in OCA2 gene. While, ten families found previously reported homozygous mutations in TYR (NM_000372.4:c.1255G>A; p.Gly419Arg), (NM_000372.4:c.1424G>A;p.Trp475*), (NM_000372.4:c.895C>T; p.Arg299Cys), (NM_000372.4:c.346C>T; p.Arg116*) OCA2 (NM_000275.2:c.1045-15T>G), (NM_000275.2:c.1046G>T; p.Asp486Tyr) and SLC45A2 (NM_016180:c.1532C>T; p.Ala511Val). Moreover, in further two families (F and G) compound heterozygous TYR variants were identified. In family F a novel variant (NM_000372.4:c.248T>G; p.Val83Gly) and a previously reported variant (NM_0+00372.4:c.1255G>A; xv p.Gly419Arg) was identified while in Family G (NM_000372.4:c.1037-7T>A) and (NM_000372.4:c.1255G>A; p.Gly419Arg) were found. CNV analysis in two families (family K and L) did not show any copy of exon 19 of OCA2 in all affected individuals, while half copies in carrier and two copies of exon 19 was observed in normal individuals while the breakpoints mapping analysis found gross deletion of 69.1 Kb between physical coordinate Chr15:28,196,848 to 28,127,719 within OCA2 gene, and an insertion of 14 base pair fragment in between the deletion point. CNV analysis in another family (family M) did not reveal any copy of TYRP1 gene in affected individuals, while phenotypically normal sibling showed single copy of TYRP1 gene indicating their carrier status. Hence, validated SNP analysis suggested ~1.0 Mb deletion between markers rs4741178 to rs2209273 harboring whole TYRP1 gene. According to the statistical estimates in current study, around 70.5% families have shown linkage to TYR and OCA2 genes. Our study is one among the large cohort studies for exploring the genetics of OCA in Pakistani population, which further extends the evidence of TYR and OCA2 as the most prevalent genes causing OCA. Conclusively, genetic screening of present OCA cases may contribute towards the development of molecular diagnostic test, genetic counseling and personalized healthcare of OCA in Pakistan. In addition to this, mapping of novel genes will further elaborate the melanin biosynthesis pathway and hence disease etiology. Most of the presented work in the current thesis has been published in peer reviewed journals. The list of articles published is in Appendix V at page 144 for reference.