Genetic studies of TYRP1 and SLC45A2 in Pakistani patients with nonsyndromic oculocutaneous albinism.

TO THE EDITOR Autosomal recessive oculocutaneous albinism (OCA) is a genetic disorder characterized by the partial or complete loss of pigmentation in the skin, hair, and iris (Tomita and Suzuki, 2004). OCA is associated with a number of vision problems and can severely affect an individual’s quality of life (Oetting and King, 1999; Tomita and Suzuki, 2004; Chaki et al., 2011). Many of the molecular components that are necessary for the development, trafficking, and maintenance of melanin pigment have been discovered by identifying the genes for the nonsyndromic and syndromic forms of OCA in humans. Identifying the genetic causes of OCA, conducting genetic testing, detecting carriers, and improving patients’ understanding of the implications and consequences of the disorder through genetic counseling are essential, especially in populations that commonly practice inbreeding (Hussain and Bittles, 1998). The Pakistani population is one such example; however, genetic information regarding OCA in this population is limited (Jaworek et al., 2012). To the best of our knowledge, only one allele of OCA3 (TYRP1) and no mutations in OCA4 (SLC45A2) have been documented in the Pakistani population (Forshew et al., 2005; Jaworek et al., 2012). Here, we report the identification of four Pakistani albinism mutations, including three SLC45A2 alleles and one 22-nucleotide deletion in TYRP1, segregating in four large families (Figure 1). After obtaining institutional review board approval and written informed consent from all participating individuals and the parents of minor subjects, consistent with the tenets of the Declaration of Helsinki, we enrolled 75 large, multigeneration families segregating nonsyndromic OCA (i.e., without any other apparent clinical phenotypes) from various regions of Pakistan (see Supplementary Materials online) and conducted a comprehensive molecular analysis of TYRP1 and SLC45A2. Mutations in TYRP1 rarely cause OCA, and, to date, only nine alleles have been detected in individuals of Southern African, African-American, Caucasian, Indian, or Pakistani descent (Boissy et al., 1996; Manga et al., 1997; Forshew et al., 2005; Rooryck et al., 2006; Hutton and Spritz, 2008; Rooryck et al., 2008; Chiang et al., 2009). Mutations in TYRP1 typically result in reddish brown or golden hair with orange highlights (Boissy et al., 1996; Manga et al., 1997; Forshew et al., 2005; Rooryck et al., 2006; Hutton and Spritz, 2008; Rooryck et al., 2008). A sequence analysis of TYRP1 in our cohort revealed a 22-base-pair deletion (c.647_668del) in the coding region in family PKAB131 (Figure 1 and Supplementary Figure S1 online). Deletion of these nucleotides is predicted to cause a frameshift in the reading frame that will result in a truncated (p.Glu216GlyfsX42) TYRP1 protein (Table 1). The affected individuals of family PKAB131 had golden hair with orange highlights, brown eyes with foveal hypoplasia, and nystagmus (Figure 1 and Table 1). All of the affected individuals had white skin at birth that gradually darkened on the body parts exposed to sunlight, such as the face, arms, and hands. However, pigment deficiency was still observable on the body parts not directly exposed to sunlight. To date, this is the second mutation of TYRP1 that causes an OCA3 phenotype in a family of Pakistani origin. A sequence analysis of SLC45A2 revealed three variants that are likely to be pathogenic in three families (Figure 1, Table 1 and Supplementary Figure S2 online). Two missense substitutions were identified: p.Leu84Pro (c.251T>C) and p.Ala511Val (c.1532C>T) in families PKAB053 and PKAB051, respectively (Figure 1). Both missense mutations affected amino-acid residues that are conserved among the SLC45A2 orthologs, and these mutations are predicted to be deleterious (Table 1 and Supplementary Figure S2 online). Molecular modeling of wild-type and mutant SLC45A2 proteins suggested that the p.Leu84Pro mutation alters the hydrogen bonding pattern in the alpha helix; specifically, the replacement of leucine with proline eliminates three potential hydrogen bonds between neighboring amino acids that are critical for the stabilization of the alpha helix (Supplementary Figure S3 online). Proline is considered to be a more rigid amino acid and can create a kink in the chain; however, this effect may not be noticeable in the model, because other chains may help maintain the overall structure. The alanine residue at position 511 forms three hydrogen bonds within a distance of 2.5 Å (Supplementary Figure S3 online). Replacement of this alanine with valine creates an additional cryptic hydrogen bond (Supplementary Figure S3 online). Furthermore, valine is bulkier than alanine and may cause steric strain against the neighboring alpha helix. An alanine to glutamate mutation at amino acid position 511 (p.Ala511Glu) has been previously documented (Rooryck et al., 2008). Our molecular modeling predicted that glutamate at position 511 could potentially create a hydrogen bond (in the same manner as valine) within a distance of 2.5 Å (Supplementary Figure S3 online), which would affect the folding and secondary structure of mutant SLC45A2. A sequence analysis of SLC45A2 also revealed a putative splice-site mutation in intron 3 (c.889-6T>G) segregating with nonsyndromic OCA in family PKAB059 (Figure 1). To determine whether c.889-6T>G alters the normal splicing of SLC45A2 mRNA, we performed an exon-trapping assay. In this assay, the wild-type construct produced a PCR product of 321 bp (pSPL3+exon 4) when amplified with vector primers (Supplementary Figure S2 online). The construct with a mutated 5′ splice site (c.889-6T>G) produced a band of similar size; however, sequencing analysis revealed an insertion of 5 bp from intron 3 directly upstream of exon 4 (Supplementary Figure S2 online). The results of the exon-trapping assay demonstrate that the c.889-6T>G mutation created a cryptic splice acceptor site, leading to aberrant splicing and the insertion of a 5-bp intron sequence into the spliced mRNA product. If a similar splicing effect occurs in affected individuals homozygous for the c.889-6T>G mutation, this will lead to a shift in the reading frame and a predicted stop codon in exon 4 (p.Thr297PhefsX6), resulting in an mRNA that presumably undergoes nonsense-mediated decay (Maquat, 2004). However, the effect of the c.889-6T>G mutation on in vivo splicing of SLC45A2 pre-mRNA is not known. All of the affected individuals from the OCA4 families exhibited a loss of pigmentation in their skin, hair, and eyes, regardless of sex or age (Figure 1 and Table 1). Intra-familial variation in hair color was noted among individuals and ranged from white to honey blonde or brown (Table 1). All of the affected individuals were photophobic, exhibited nystagmus and foveal hypoplasia, and their visual acuity was affected to varying degrees (Table 1). In summary, SLC45A2 mutations accounted for 4% (3 of 75 families; 95% confidence interval (CI), 1.45–11.1%) of OCA in our Pakistani cohort study, whereas only one pathogenic mutation in TYRP1 (1.3%; 95% CI, 0.32–7.1%) was found. This knowledge, in combination with data from previous studies, will improve our understanding of the molecular epidemiology of OCA in Pakistan. The results of our study will be important for future OCA diagnoses, genetic counseling, and functional studies of the TYRP1 and SLC45A2 proteins. The authors state no conflict of interest. We thank the families for their participation and cooperation in our study. We also thank Dr S. Riazuddin for a critique of the manuscript. The work in Pakistan was supported by a research grant (HEC-1262) from the Higher Education Commission, to R.S.S. This work was also supported by intramural research funds granted by the Cincinnati Children’s Hospital Research Foundation (CCHMC) to Z.M.A. and by a Career Development Award from the Research to Prevent Blindness Foundation. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper