Mutation Analysis of a Pakistani Oculocutaneous Albinism Family Identifies a Novel Splice Site Defect in OCA2 Gene

1Department of Biological Sciences, Faculty of Sciences, Gomal University, D.I. Khan, 29050, Pakistan. 2Molecular Neuropsychiatry and Development Lab (MiND), Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, University of Toronto, ON, Canada. 3Gomal Centre of Biochemistry and Biotechnology, Gomal University, D.I. Khan, Pakistan. 4Department of Psychiatry, University of Toronto, Canada. Article Information Received 01 May 2020 Revised 30 July 2020 Accepted 17 October 2020 Available online 09 June 2021


INTRODUCTION
A utosomal recessive non-syndromic oculocutaneous albinism (OCA) is an uncommon condition of defective melanin synthesis. The clinical features of OCA patients comprise of white milky skin, whitish to golden hair and deterioration of retinal cells. Worldwide, OCA affects 1 in 17,000 individuals, which means that ~1 in 70 people are carriers of the defective OCA allele (Gul et al., 2019;Shahzad et al., 2017). Molecular genetic studies have so far reported seven genes (TYR,OCA2,TYRP1,SLC45A2,SLC24A5,C10ORF11,and MC1R) and an uncharacterized locus (OCA5) responsible for causing non-syndromic OCA subtypes. The product of these OCA genes are involved in the melanin bio-synthesis pathway (Khan et al., 2015). Among the known OCA genes, TYR and OCA2 are the most commonly reported defective genes globally, and in Pakistani families as well. Genetic studies have shown that the splice mutation c.1045-15 T>G in OCA2 is the most frequent pathogenic allele (11.3% prevalence) among OCA patients (Gul et al., 2019;Jaworek et al., 2012). Clinical diagnosis of different OCA subtypes is unable to distinguishable between the genes because of its overlapping and variable manifestations. Hence, in this scenario, molecular diagnosis is considered as the gold standard for determining the OCA subtype.
Herein this investigative study, a consanguineous OCA family from Pakistan was analyzed, resulting in the identification of a previously unreported splice donor site defect (NM_000275.2:c.2079+5G>T) within intron-19 O n l i n e

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of the OCA2 gene. This change presumably may either add non-coding nucleotides into the mature mRNA, or may lead to skipping of exon 20, either way resulting in a frame-shift and premature protein truncation.

MATERIALS AND METHODS
The study was approved by the institutional ethical review board of Gomal University, Dera Ismail Khan, Pakistan, and the samples were enrolled after executing the written research ethics agreement and informed written consent by participants. Thereafter, blood samples were obtained for DNA extraction and onward genomic studies. DNA qualitative and quantitative assessment was carried out through NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies, Berlin, Germany). Clinically, the disease was diagnosed through apparent OCA features like whitish skin, un-pigmented hairs, and presence of eye abnormalities (nystagmus, photophobia, and reduced vision).

Homozygosity scanning
HBD mapping (finding Homozygous by Descent regions) was performed through SNP microarray using Illumina Infinium CoreExome-24 Kit following the Infinium HTS Assay (15045738A). Selected individuals were used for whole genome SNP genotyping (III-1, III-2 and III-3). The data were analyzed for finding HBD regions using Homozygosity Mapper (Seelow et al., 2009) and FSuite (Gazal et al., 2014). Each homozygous region was checked at the genotype level to confirm its haplo-identity. This strategy helped in excluding the false positive results, while the remaining regions were selected for further analysis.

Sanger sequencing and mutation analysis
After finding the pathogenic variant, Sanger sequencing was performed to check its segregation with the disease phenotype within the whole family. For Sanger sequencing, the sequencing PCR product was analyzed on ABI 3730XL instrument (Applied Biosystem, USA). For mutation analysis, the Sanger data was analyzed using FinchTV Program, Version 1.4.0 (http://www.geospiza. com). The software was used to read the sequence chromatogram and sequence alignment with human reference genome (hg19). In addition to this, mutations analysis was also performed through BLAT tool of UCSC Genome Browser (http://www.genome.ucsc.edu).

General description of family
The current family was ascertained from among the Pashto ethnic population of Swat District. The family pedigree consists of three generations, with four affected individuals in all generations. The pedigree analysis showed possible autosomal dominant inheritance pattern, although autosomal recessive inheritance was equally plausible given the consanguineous nature of the family. The apparent clinical features of patients include golden hair color, white skin color with reddish pigmentation (Fig. 1). The additional ocular symptoms include brown iris color, photophobia, nystagmus, strabismus and weak eye sight.

Molecular analysis of family under investigation
The current family was previously published in JPMA , wherein the exact genetic mutation was not reported due to low coverage of exome, including splice junctions. Moreover, at that time, microarray based genome-wide SNP genotyping was not performed. Therefore, the family underwent re-sequencing for whole exome including splice junctions with enhanced coverage as well as whole genome homozygosity scan.     25 c.1239+5G>A African-American (King et al., 2003a) West Indian

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Puerto Rican 26 c.1050-3C>G IVS7-3C>G Puerto Rican (Chiang et al., 2009) Genome-wide homozygosity analysis revealed a 1.2 Mb long HBD region on chromosome 15 between markers rs4778147 to rs8036234 (chr15:27752745-28962131bp; hg19) ( Fig. 1). Exome sequence analysis of individual III-2 identified substitution of C by A at position 28171268 (15q13.1) in the 19 th intron of the OCA2 gene that is predicted to affect the donor splice site. Sequence chromatograms of affected and normal siblings of family revealed the homozygous substitution of G nucleotide by T at cDNA position 2079+5, while unaffected sibling was shown to have carrier status of G/T (Fig. 2). The currently identified variant is not reported in genomAD,1000 Genome, the Human Gene Mutation Database and ClinVar. The splicing prediction tool at the BDGP predicts a reduction of the splice donor score from 0.97 to 0.53 (out of 1.0). HSF predicts a reduction from 95.9 to 83.59 (out of 100).

DISCUSSION
OCA is a recessively inherited pigmentation disorder of skin, hair and retinal pigment epithelium, which is caused due to defects in melanin biosynthesis (Mártinez-García and Montoliu 2013). Genetically speaking, defect O n l i n e

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Novel Splice Donor Mutation in OCA2 5 in seven genes have been implicated in OCA, among which TYR and OCA2 have the highest incidence rate. The current study involved the identification of a novel splice site defect NM_000275.2:c.2079+5G>T in OCA2 gene. Epidemiologically, OCA2 is the most widespread form of albinism worldwide; which in South African Black is found to be 1:3600, (Kromberg and Jenkins 1982;Stevens et al., 1997) 1:10 000 in Black and 1:36000 in Caucasians (Witkop et al., 1989), and 1:10 000 in the Irish (Froggatt, 1960). The OCA2 locus is mapped on chromosome 15q11.2-q1211 (Lee et al., 1995). The OCA2 gene encodes an 838 amino-acid long protein that is also known as P protein, which is required for melanin pigment synthesis by mediating Clspecific anion conductance (Bellono et al., 2014;Duffy et al., 2007). Though initially reported as an integral melanosomal protein, OCA2 is potentially associated with transport of some molecules into the melanosome and regulates melanosomal pH (Brilliant, 2001) with an additional possible role of processing and trafficking of melanosomal proteins (Chen et al., 2002;Toyofuku et al., 2002). The OCA2 protein belongs to the Na + /H + antiporter family. It acts as a precursor to melanin synthesis and controls the process of tyrosinase and tyrosinase-related protein, also maintains the pH of the melanosomes (Bellono et al., 2014;Ni-Komatsu et al., 2006). So far, 184 OCA2 mutations have been reported in HGMD (Human Gene Mutation Database, professional 2019.4), including 104 nonsense/missense mutations, 26 splicing mutations, 1 regulatory mutation, 32 indels 20 gross indels, and 1 complex rearrangements.
In this study, the genetic defects underlying OCA2 in two albino patients (III-2, II-2) identified a novel splicing mutation (NM_000275.2:c.2079+5G>T). Before this study, twenty-six (26) splice mutations have been reported globally (Table I). While, literature survey has shown that it is the second splicing mutation being reported in the Pakistani population. Previously, Jaworek et al., 2012 reported one splice site mutation c.1045-15T>G in a consanguineous Pakistani family. The clinical synopsis of current patients is in agreement with previously published OCA2 phenotypes. Hence, the phenotyping in present family will help in genotype-phenotype correlation analysis. Splicing mutations usually result in errors during the splicing process and may lead to improper intron removal and thus causes alteration of the open reading frame.

CONCLUSIONS
Exome sequence analysis in a Pakistani OCA family found a novel splice site defect. The current finding supported the evidence of high incidence of OCA2 gene mutation in Pakistan. Although, variant pathogenicity is strongly supported by in silico analysis, however, functional studies are required to investigate the effect of mutation on mRNA and protein levels.

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Novel Splice Donor Mutation in OCA2