Functional analysis of OCA4 mutant sequences using under white mouse melanocytes

Dear Sir, Oculocutaneous albinism (OCA) is a group of autosomal recessive hypopigmentary disorders of the skin, hair and eyes. The four types of OCA (OCA1–4) are the result of single gene mutations in tyrosinase (TYR; OCA1 [MIM 203100]), P (P; OCA2 [MIM 203200]), tyrosinase-related protein 1 (TYRP1; OCA3 [MIM 203290]) and Solute Carrier Family 45, member 2 (SLC45A2; OCA4 [MIM 606574]). The SLC45A2 gene (formerly membrane-associated transporter protein; MATP) encodes a melanosomal protein, the function of which remains unknown (Newton et al., 2001). Its homolog in medaka fish, b, encodes a transporter that mediates melanin synthesis and exhibits structural homology to plant sucrose–proton symporters (Fukamachi et al., 2001). OCA4 is a major OCA type in the Japanese population ( 30%), but is rare in Caucasians (Inagaki et al.,2004; Newton et al., 2001; Suzuki and Tomita, 2008). The clinical phenotype of OCA4 patients varies depending on their mutant genotypes. While some resemble OCA1A (tyrosinase-negative OCA) cases and have no pigmentation, others exhibit some generalized pigmentation with brown hair and irides without nystagmus. Moreover, some patients show improvement during the first decade of life (Inagaki et al., 2004; Rundshagen et al., 2004). The human SLC45A2 protein is a 530-amino acid polypeptide that contains 12 putative transmembrane domains, and is only expressed in melanocytes (Newton et al., 2001). More than thirty-five different mutations have been reported in Turkish, German, Korean, Japanese, Indian, French, Dutch and Belgian OCA4 patients (Inagaki et al., 2004, 2006; Newton et al., 2001; Rooryck et al., 2008; Rundshagen et al., 2004; Sengupta et al., 2007; Suzuki et al., 2005). Two of these mutations, the p.Asp157Asn (p.D157N) and p.Gly188Val (p.G188V) alleles, were the first and second most frequently found variants in Japanese patients, with allele frequencies of 0.39 (14 ⁄ 36) and 0.19 (7 ⁄ 36), respectively (Inagaki et al., 2004). Judging from the relationship between patient phenotypes and genotypes, we previously reported that the p.D157N variant might be non-functional, or have very low functional activity, in melanogenesis. There is also evidence that the p.G188V allele might produce a protein with low functional activity (Inagaki et al., 2004). The function of mutant SLC45A2 proteins was evaluated by comparing the ability of the wild-type versus the mutant polypeptide to produce melanin in melanocytes (under white cells; uw) established from an OCA4 mouse model (Costin et al., 2003). We report here the differential complementation of defective melanin biosynthesis and the hypopigmentation of uw cells by transfection with wild and mutant human SLC45A2 cDNA sequences. We constructed wild-type and mutant SLC45A2 (p.D157N and p.G188V) cDNA constructs. Full-length wild-type human SLC45A2 cDNA was cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA, USA). Sitedirected mutagenesis was used to introduce substitutions for the G at nucleotide positions (n.p.) 469 and 563 with A in p.D157N and T in p.G188V. The cDNA products were sequenced to confirm accuracy and inserted into mammalian expression plasmid pIREShyg3 (BD Biosciences, San Jose, CA, USA), which contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV), creating pIREShyg3-SLC45A2 wild, pIREShyg3-SLC45A2mutant-p.D157N and pIREShyg3-SLC45A2mutant-p.G188V. Following an initial 24-hr culture period, uw melanocytes were transfected with 1.6 lg of one of the three constructs or pIREShyg3 for mock-transfected cells (per 4cm flask) using Lipofectamine 2000 Reagent (Invitrogen). Stable transformants were selected in culture media containing 500 lg ⁄ ml of hygromycin B (Invitrogen). Six independent clones established from each transformant were harvested separately from each plate by trypsinization. Expression of SLC45A2 mRNA in each of the six clones was confirmed with RT-PCR (Figure 1). For melanin assays, each suspension was pelleted and incubated at 95 C for 1 h after resuspension in 100 ll of 1 N NaOH. After a 100· dilution, the OD475 was measured and