Molecular outcomes, clinical consequences, and genetic diagnosis of Oculocutaneous Albinism in Pakistani population

Abstract

Nonsyndromic oculocutaneous Albinism (nsOCA) is clinically characterized by the loss of pigmentation in the skin, hair, and iris. OCA is amongst the most common causes of vision impairment in children. To date, pathogenic variants in six genes have been identified in individuals with nsOCA. Here, we determined the identities, frequencies, and clinical consequences of OCA alleles in 94 previously unreported Pakistani families. Combination of Sanger and Exome sequencing revealed 38 alleles, including 22 novel variants, segregating with nsOCA phenotype in 80 families. Variants of TYR and OCA2 genes were the most common cause of nsOCA, occurring in 43 and 30 families, respectively. Twenty-two novel variants include nine missense, four splice site, two non-sense, one insertion and six gross deletions. In vitro studies revealed retention of OCA proteins harboring novel missense alleles in the endoplasmic reticulum (ER) of transfected cells. Exon-trapping assays with constructs containing splice site alleles revealed errors in splicing. As eight alleles account for approximately 56% (95% CI: 46.52-65.24%) of nsOCA cases, primarily enrolled from Punjab province of Pakistan, hierarchical strategies for variant detection would be feasible and cost-efficient genetic tests for OCA in families with similar origin. Thus, we developed Tetra-primer ARMS assays for rapid, reliable, reproducible and economical screening of most of these common alleles.

Figure 1. Subcellular distribution of wild type and mutant tyrosinase and tyrosinase-like proteins in human melanocytes.

eGFP-tagged TYR and TYRP1 wild type and mutant constructs (green) were transiently transfected in melanocytes grown at 37 °C. Calregulin (red) and DAPI (blue) were used as markers for the endoplasmic reticulum and nucleus, respectively. Merged images show the co-localization of only tyrosinase variants with calregulin, which indicated ER retention. The scale bar represents 20 μm for all panels.

Figure 2. Subcellular distribution of wild type and mutant OCA2 in HEK293 cells.

eGFP-tagged OCA2 wild type and mutant constructs (green) were transiently transfected in HEK293 cells grown at 37 °C. Calregulin (red) and DAPI (blue) were used as markers for the endoplasmic reticulum and nucleus, respectively. Merged images show partial co-localization of OCA2 variants with calregulin, which indicated increased ER retention. The scale bar represents 20 μm for all panels.

Figure 3. Functional analysis of splice site variants.

Schematic representation and Sanger sequencing chromatograms of minigene assays for TYR and OCA2 novel splice site variants revealed aberrant splicing products, supporting their pathogenic nature.

Figure 4. Gross genomic exonic deletions identified in six OCA families.

(A) Compare the exome sequencing data (green-blue peaks) of control sample wild type sample, the exon 4 and 5 (indicated by red arrows) were deleted in DNA samples from PKAB64 and PKAB168 families. Breakpoints for genomic deletions are given according to the human genome build hg19/GRCh37. (B) Gross genomic deletions observed in OCA2 gene as compared to control samples are shown either by red arrows or red lines.

Figure 5. Prevalence of albinism genes and their alleles in a Pakistani population.

(A–C) Relative distribution of variants in albinism genes in Pakistani families. (A) The distribution of OCA1-4 genes in 94 families screened in the current study. Number of families and their percentage contribution are given in parentheses. (B) Total pool of albinism genes in our cohort. (C) Frequencies of recurrent alleles of nsOCA genes in Pakistanis. (D) Results of tetra-primer ARMS assays for detection of recurrent variants. The top band in each gel represents the positive control amplimer in all samples generated using the outer primers. Nested allele-specific primers were used to generate the wild type (Wild) or variant harboring (Mutant) PCR products. IC: inner control product.