Gene 511 (2012) 470474

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Gene
journal homepage: www.elsevier.com/locate/gene

Short Communication

Molecular basis of albinism in India: Evaluation of seven potential candidate genes and some new ndings
M. Mondal a, 1, M. Sengupta a, 1, S. Samanta b, A. Sil c, K. Ray a,
a b c

Molecular & Human Genetics Division, CSIRIndian Institute of Chemical Biology, Kolkata700032, India Department of Ophthalmology, Calcutta National Medical College and Hospital, Kolkata700014, India Netra Niramay Niketan, Vivekananda Mission Ashram, Chaitanyapur, Purba Medinipur721645, India

a r t i c l e

i n f o

a b s t r a c t
Albinism represents a group of genetic disorders with a broad spectrum of hypopigmentary phenotypes dependent on the genetic background of the patients. Oculocutaneous albinism (OCA) patients have little or no pigment in their eyes, skin and hair, whereas ocular albinism (OA) primarily presents the ocular symptoms, and the skin and hair color may vary from near normal to very fair. Mutations in genes directly or indirectly regulating melanin production are responsible for different forms of albinism with overlapping clinical features. In this study, 27 albinistic individuals from 24 families were screened for causal variants by a PCR-sequencing based approach. TYR, OCA2, TYRP1, SLC45A2, SLC24A5, TYRP2 and SILV were selected as candidate genes. We identied 5 TYR and 3 OCA2 mutations, majority in homozygous state, in 8 unrelated patients including a case of autosomal recessive ocular albinism (AROA). A homozygous 4-nucleotide novel insertion in SLC24A5 was detected in a person showing with extreme cutaneous hypopigmentation. A potential causal variant was identied in the TYRP2 gene in a single patient. Haplotype analyses in the patients carrying homozygous mutations in the classical OCA genes suggested founder effect. This is the rst report of an Indian AROA patient harboring a mutation in OCA2. Our results also reveal for the rst time that mutations in SLC24A5 could contribute to extreme hypopigmentation in humans. 2012 Elsevier B.V. All rights reserved.

Article history: Accepted 6 September 2012 Available online 23 September 2012 Keywords: Albinism TYR OCA2 SLC24A5 SILV TYRP2

1. Introduction Albinism represents a heterogeneous group of inherited disorders manifested by complete or partial lack of melanin in the skin, hair, and eyes. While oculocutaneous albinism (OCA) involves both ocular and cutaneous features, in ocular albinism (OA) the phenotype is conned primarily to the eyes.

Mutations in the tyrosinase (TYR), oculocutaneous albinism type 2 (OCA2/P gene), tyrosinase related protein 1 (TYRP1) and solute carrier family 45, member 2 (SLC45A2) genes cause the 4 classical types of OCA, viz. OCA type 1 (MIM: 203100 and MIM: 606952; Spritz et al., 1989), type 2 (MIM: 203200; Rinchik et al., 1993), type 3 (MIM: 203290; Boissy et al., 1996), and type 4 (MIM: 606574; Rundshagen et al., 2004), respectively. In all four cases the disease is transmitted

Abbreviations: A, adenosine; Ala, alanine; Arg, arginine; AROA, autosomal recessive ocular albinism; Asn, asparagine; C, cytidine; CSIR, Council of Scientic and Industrial Research; Cterminal, carboxy terminal; Cys, cysteine; DCT, DOPAchrome tautomerase; db SNP, The Single Nucleotide Polymorphism database; Del, deletion; DHICA, 5,6 dihydroxyindole carboxylic acid; DNA, deoxyribonucleic acid; DOPA, dihydroxyphenylalanine; E. coli, Escherichia coli; et al., et alia (and others); ExoSap, exonucleaseshrimp alkaline phosphatase; G, guanosine; Glu, glutamic acid; Gly, glycine; ID, identication/identity; Het, heterozygous; Hom, homozygous; i.e., id est, that is; Ins, insertion; Ile, isoleucine; Leu, leucine; Lys, lysine; MAF, minor allele frequency; MATP, membrane associated transporter protein gene; Met, methionine; MIM, Mendelian inheritance in man; ml, milliliter; MLP, Major Lab Project; mRNA, messenger ribonucleic acid; l, microliter; NA, not applicable; ng, nanogram; OA, ocular albinism; OCA, oculocutaneous albinism; OCA2, oculocutaneous albinism type 2 (as well as the protein coded by OCA2 gene); OCA2, oculocutaneous albinism type 2 gene; PCR, polymerase chain reaction; Phe, phenylalanine; PMEL17, premelanosome protein; PolyPhen, polymorphism phenotyping; Pro, proline; Prof, professor rsrefSNP; P gene, human homologue to the mouse pinkeyed dilution locus; Ser, serine; SIFT, sorting intolerant from tolerant; SILV, silver protein; SILV, silver gene; SIP, Supra Institutional Project; Slc24a5, mouse homologuous gene for SLC25A5; SLC45A2, solute carrier family 45, member 2 protein; SLC45A2, solute carrier family 45, member 2 protein gene; SLC24A5, solute carrier family 24, member 5; SLC24A5, solute carrier family 24, member 5 gene; Sl. no., serial number; SNPs, single nucleotide polymorphisms; Thr, threonine; T, thymidine; Tyr, tyrosine; TYR, tyrosinase protein; TYR, tyrosinase gene; TYRP1, tyrosinase related protein 1; TYRP1, tyrosinase related protein 1 gene; TYRP2, tyrosinase related protein 2; TYRP2, tyrosinase related protein 2 gene; Tyrp2, mouse homologuous protein for TYRP2; USA, United States of America; UTR, untranslated region; Val, valine; viz., videlicet (it is permitted to see). Corresponding author at: Molecular & Human Genetics Division, CSIRIndian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata700032, India. Tel.: +91 33 2483 1984; fax: +91 33 2473 5197. E-mail addresses: maitreyee.bonny@gmail.com (M. Mondal), sengupta.mainak@gmail.com (M. Sengupta), swapansamanta53@gmail.com (S. Samanta), asimsil@sify.com (A. Sil), kunalray@gmail.com, kray@iicb.res.in (K. Ray). 1 The rst two authors contributed equally to the work. 0378-1119/$ see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.09.012

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as an autosomal recessive trait. Incidentally, the gene causing OCA type 2 is also known as OCA2 and the other name, P gene, is not commonly used in the current literature related to albinism. Tyrosinase (TYR) acts as the key enzyme in melanin biosynthesis, catalyzing the rate limiting steps that convert L-tyrosine to L-DOPA and L-DOPA to DOPAquinone (Lerner et al., 1950). TYRP1, also a member of the tyrosinase-gene family, encodes TYRP1 protein that catalyzes the oxidation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into indole-5,6-quinone-2-carboxylic acid in the melanin biosynthesis pathway. The OCA2 protein (homologous to the mouse pink-eyed dilution gene, p), present in the large granular fraction of melanocytes (Rinchik et al., 1993), appears to be an integral membrane protein of melanosomes and is thought to be a member of a transporter family that includes an Escherichia coli Na +/H + antiporter. Considering its role in disease causation, it is likely that OCA2 might act as a tyrosine transporter (Lee et al., 1995). Human SLC45A2 (also known as MATP, i.e. membrane associated transporter protein) has structural homology to plant sucrose-proton symporters. Presumably being located in the melanosomal membrane, it probably functions as a membrane transporter directing the trafc of melanosomal proteins and other substances to the melanosomes (Fukamachi et al., 2001). However, the precise role of neither OCA2 nor SLC45A2 proteins has yet been elucidated. Two distinct forms of OA have also been recognized: the X-linked recessive NettleshipFalls form (OA1; MIM: 300500; Bassi et al., 1995) and autosomal recessive ocular albinism (AROA) caused by mutations in TYR, OCA2 and possibly TYRP1, and represent a phenotypically milder variant of OCA (Hutton and Spritz, 2008a). In many of the observed albinism cases, however, mutations remain unidentied, emphasizing the need to screen other potential candidate genes to better understand the molecular basis of the albinistic condition. In this context, SLC24A5, TYRP2 and SILV have been regarded as important candidates for albinism. SLC24A5 (solute carrier family 24, member 5), a putative sodium/ potassium/calcium exchanger protein, has previously been implicated in hypopigmentation. In fact, Slc24a5-null mice have been reported to have albinistic features (Vogel et al., 2008). It has also been shown in a genome wide association study of skin pigmentation variation, using 1,620,742 SNPs in a population of 737 individuals of South Asian ancestry that SLC24A5 SNP rs1426654 (Ala111Thr) is associated with skin pigmentation (Stokowski et al., 2007). The TYRP2 (tyrosinase related protein 2; orthologous to the murine slaty locus) functions as a DOPAchrome tautomerase (DCT), converting DOPAchrome to 5,6 dihydroxyindole carboxylic acid (DHICA) in the eumelanotic pathway. A defect in TYRP2 could result in a lack of eumelanin and could also lead to a decreased level of total melanin. Also, non-synonymous variants identied in mouse Tyrp2 have been reported to be responsible for a signicant decrease in DOPAchrome tautomerase activity, as well as the slaty coat color in mice (Budd and Jackson, 1995; Jackson et al., 1992). SILV (also known as PMEL17) encodes a melanosomal matrix protein, whose expression is closely correlated with cellular melanin content (Bailin et al., 1996). It forms a brillar sheet structure in melanosomes on which precursors of melanin synthesis are deposited and concentrated; i.e., it might serve to accelerate melanin production (Leonhardt et al., 2011). SILV protein is also thought to be directly involved in the biogenesis of premelanosomes (Berson et al., 2001) and has been analyzed as a candidate OCA gene in multiple studies (Hutton and Spritz, 2008a, 2008b). However, to date, no pathogenic variant has been reported in any of these genes to explain ocular or cutaneous albinistic features. OCA is one of the major causes of childhood blindness in India (Gothwal and Herse, 2000), which underscores the importance of identifying mutations causal to OCA in the different ethnic groups of the country followed by genetic counseling. Mutations identied in Indian patients so far are included in Indian Genetic Disease Database (Pradhan et al., 2011) with free accessibility (http://www.igdd.iicb. res.in/IGDD/home.aspx). In this study, we looked for genetic variants

in TYR, OCA2, TYRP1, SLC45A2, SLC24A5, TYRP2 and SILV in Indians presenting with ocular and/or cutaneous albinistic features. 2. Materials and methods 2.1. Collection of blood samples and genomic DNA preparation from patients and controls Twenty seven albinistic individuals from 24 families were enrolled in this study, mostly from the eastern and southern parts of India. Twelve of these families had been previously screened for defects in TYR, OCA2, TYRP1, SLC45A2 and SLC24A5 (Chaki et al., 2006; Sengupta et al., 2007, 2010), with no detectable mutations in any of these genes. In this study, the same families were screened for mutations in SILV and TYRP2 only. One of the patients (OCA109) presented with albinistic eye features only, while his skin tone was very fair but certainly not albinistic, thereby representing an ocular albino (OA) patient. Another individual (OCA97) showed extreme hypopigmentation of the skin, but not the eyes and hair. However, detailed clinical data was unavailable to examine the ocular abnormalities, if any. The rest of the individuals enrolled in the study presented with both ocular and skin hypopigmentation. Detection of OCA and OA involved ophthalmologic examinations by our clinical collaborators (SS and AS), including testing of nystagmus, visual acuity and fundoscopy. Ethnically matched controls without any history of ocular disease were later selected from the general population, majorly from eastern India, based on the fact that barring one all of the potential novel mutations were identied in the eastern Indian patient cohort. Approximately 10 ml of peripheral blood was drawn with informed consent from the donors. Genomic DNA was prepared by the salting-out method, or by using the QIAGEN PAXgene Blood DNA kit according to the manufacturer's protocol. 2.2. Polymerase chain reaction, DNA sequencing and in silico analysis of the variants The exons, splice-sites and UTRs of the relevant genes were amplied by touchdown PCR. However, exon 19 of OCA2 was not analyzed since it generates a small proportion of an alternatively spliced mRNA containing an in-frame stop codon resulting in a nonfunctional OCA2 protein (Duffy et al., 2007; Hutton and Spritz, 2008a; Lee et al., 1995). PCR was done for a total volume of 20 l using 2050 ng of DNA, 20 pmol of primers (the sequence of the primers used are available on request) and 10 l Taq Premix (GeNet Bio). The amplicons were puried with ExoSap (USB) and bidirectional-sequencing was performed in the ABI Prism 3130 DNA sequencer (ABI). Haplotyping with SNPs and microsatellite markers was done following our previous reports (Chaki et al., 2006; Sengupta et al., 2010). For genotyping with microsatellite markers in case of TYR gene, 3 CA-repeat markers within (GDB: 11511691) and anking (GDB: 11511689 and GDB: 11511690) the TYR locus were used. The markers were amplied from the affected and immediate family members using uorescently labeled primers and subjected to Genescan analysis in an ABI Prism3100 DNA Sequencing System using the 500 TAMRA Size Standard (Applied Biosystems, California, USA) (Chaki et al., 2006). For haplotype analysis in OCA2 locus, six polymorphic SNPs were genotyped in the members of the affected families (Sengupta et al., 2010). Relevant nucleotide changes were analyzed using SIFT (http://sift.jcvi.org/), PolyPhen-2 (http:// genetics.bwh.harvard.edu/pph2/) and InterProScan (http://www.ebi. ac.uk/Tools/pfa/iprscan/). 3. Results and discussions Among the 27 albinistic individuals from 24 unrelated families, 13 patients belonging to 12 different OCA-affected families, were screened for variants in TYR, OCA2, TYRP1, SLC45A2 and SLC24A5 in our previous studies (Chaki et al., 2006; Sengupta et al., 2007, 2010) but no potential

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disease causing mutation was detected. Screening the remaining 14 patients for variants in these genes in the current study suggested the presence of 9 mutations (Table 1) in 11 cases only but none in 3 patients. Thus, 16 patients from 15 unrelated families 13 from previous studies and 3 from the current study who apparently did not contain any suspected mutation in these genes were examined for variants in two other candidate genes TYRP2 (DOPAchrome tautomerase) and SILV. 3.1. TYR mutations in 5 Indian OCA families Five different TYR mutations were identied in 5 patients with unrelated family backgrounds. Relevant information concerning the mutations identied is furnished in Table 1. Three individuals from 2 unrelated families (Patient IDs: OCA99, 100 and 117) from eastern India were homozygous for the frequently reported p.Arg278* mutation (Chaki et al., 2005). A homozygous p.Arg402* mutation was detected in another proband (OCA101), also from eastern India. This variant, although identied in various populations (Hutton and Spritz, 2008a; Zahed et al., 2005), has not been reported in the Indian population to date. A previously reported non-synonymous mutation (p.Gly419Arg) was also detected in the homozygous state in the proband (OCA119) of a western Indian family, as well as in his affected daughter (OCA120). It has already been demonstrated that this tyrosinase variant lacks enzyme activity, and is retained in the endoplasmic reticulum (Chaki et al., 2010). In all the homozygous cases, common haplotypes for a particular mutation

suggested a common origin and thereby, a possible founder effect. A single patient (OCA113) from an eastern Indian family harbored two novel mutations in a compound heterozygous state viz. (a) c.1065delG, resulting in a mutant protein containing a total of 369 amino acids with 14 residues unrelated to TYR at the C-terminal end, and (b) c.827G>A; p.Cys276Tyr. In silico analyses (SIFT and PolyPhen-2) predicted p.Cys276Tyr to be deleterious (Table 1). InterPro scan analysis suggested that the putative transmembrane region of TYR would be lost due to the c.1065delG mutation. None of the novel changes were present in the 50 controls analyzed. All of the above-mentioned patients presented with a complete lack of melanin in their eyes, hair and skin, had nystagmus, photophobia and transparent iris (Table 1). While screening the patient samples for TYR mutations, we detected 2 reported SNPs in both anking regions of the gene (Supplementary Table 1). 3.2. OCA2 mutations in 2 Indian OCA families Three novel OCA2 mutations including 1 small insertion and 2 non-synonymous variants were detected in 2 families from eastern India (Table 1). A single individual (OCA96) was detected with two novel changes: (a) c.2020C> G; p.Leu674Val, and (b) c.775_776insG, resulting in a mutant protein containing a total of 292 amino acids with 34 residues unrelated to OCA2 at the C-terminal end. InterPro scan analysis demonstrated that 11 out of 12 predicted transmembrane regions of OCA2 would be lost due to the c.775_776insG mutation. Interestingly, the patient showed relatively milder symptoms,

Table 1 Mutations detected in TYR, OCA2 and SLC24A5 genes in Indians manifesting albinistic phenotypes. OCA families (no. of patients and patient ID) TYR (eastern India) Group 1 (2; OCA99,100) Mutation (amino acid change) Allele 1 Allele 2 Haplotypes Remarks Phenotype

c.832C>T (p.Arg278*)

c.832C>T (p.Arg278*)

181-C-T-C-C-G-101157 181-C-T-C-C-G-101157

Reported mutation in exon 2

Group 2 (1; OCA117)

c.832C>T (p.Arg278*) c.1204C>T (p.Arg402*) c.827G>A (p.Cys276Tyr)

c.832C>T (p.Arg278*) c.1204C>T (p.Arg402*) c.1065delG

181-C-T-C-C-G-101157 181-C-T-C-C-G-101157 173-C-T-A-C-A-96161 173-C-T-A-C-A-96161 Not done

Group 3 (1; OCA101)

Group 4 (1; OCA113)

Presence of nystagmus and iris transillumination, transparent iris; white hair; pinkish-white skin with freckles Reported mutation in exon 2 Presence of nystagmus and iris transillumination, transparent iris; white hair; white skin Reported mutation in exon 4 Presence of nystagmus and iris transillumination, transparent iris; white hair; white skin Presence of nystagmus and iris Both novel variants; located in exon 2 and exon 3 resp. SIFT: 0; transillumination, transparent iris, PolyPhen-2: 1.00 (for Cys276Tyr) 6/60 visual acuity; white hair; white skin

TYR (western India) Group 1 (2; OCA119,120) c.1255G>A (p.Gly419Arg)

c.1255G>A (p.Gly419Arg)

179-C-T-C-C-G-101157 179-C-T-C-C-G-101157

Reported mutation in exon 4

Presence of nystagmus and iris transillumination, transparent iris; white hair (OCA120 had yellowish-white hair); white skin

OCA2 (eastern India) Group 1 (1; OCA96)

c.775_776insG

c.2020C>G (p.Leu674Val) c.1580T>G (p.Leu527Arg) c.2020C>G (p.Leu674Val)

Not done

Group 2 (1; OCA121) Group 3 (1; OCA109)

c.1580T>G (p.Leu527Arg) c.2020C>G (p.Leu674Val)

A-A-A-G-C-G A-A-A-G-C-G G-A-A-G-C-G G-A-A-G-C-G

Both novel variants; located in exons 7 and 20 resp. SIFT: 0, PolyPhen2: 1.00 (for Leu674Val) Novel variant; located in exon 15. SIFT: 0, PolyPhen-2: 0.994 Novel variant; located in exon 20. SIFT: 0.04, PolyPhen-2: 1.00

Presence of nystagmus, hazel iris; light golden-brown hair; pinkish-white skin Brown iris; light golden-white hair; white skin No apparent nystagmus, iris transillumination, brown iris; silky-brown hair; very fair pinkish skin

SLC24A5 (eastern India) Group 1 (1; OCA97)

c.569_570insATTA c.569_570insATTA Not done

Novel variant; located in exon 5.

Brown iris (no apparent ocular defects); dark brown hair; pinkish-white skin

TYR markers used for haplotyping: microsatellite marker no. 1 (GDB: 11511689), rs5021654, rs4547091, rs1799989, rs1042602, rs12804012, microsatellite marker no. 2 (GDB: 11511691) and microsatellite marker no. 3 (GDB: 11511690) (Chaki et al., 2005). OCA2 markers used for haplotyping: rs1800404, rs10852218, rs1800410, rs1900758, rs1800411 and rs12592307 (Sengupta et al., 2010). SIFT score > 0.05 indicates a tolerant change whereas PolyPhen-2 score > 0.454 denotes a damaging change.

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with golden brown hair, hazel eyes manifesting nystagmus and a slightly pinkish skin (Table 1). The other proband (OCA121) with whitish skin and golden white hair was homozygous for the novel mutation c.1580T>G (p.Leu527Arg). Haplotype analyses with 6 SNPs (Sengupta et al., 2010) suggested a common origin of that mutation. In silico analyses also predicted both of the non-synonymous changes to be deleterious (Table 1). Incidentally, the patient carrying the p.Leu527Arg variant was also homozygous for a novel innocuous variant viz. c.954G> A; p.Met318Ile (SIFT score: 0.18; PolyPhen-2 score: 0.027; benign) (Supplementary Table 1). None of these novel variants were present in the 50 controls, examined by sequencing the relevant regions of the gene. The reported SNPs and novel polymorphisms detected in OCA2 are shown in Supplementary Table 1. 3.3. OCA2 mutations in one autosomal recessive ocular albinism case A male patient (OCA109) clinically diagnosed to have albinistic eye features had very fair skin and silky brown hair. The features were similar to ocular albinism (OA) and not OCA. However, no mutation was detected in OA1, whereas screening of the classical OCA genes revealed a homozygous p.Leu674Val mutation in OCA2 (Table 1). Therefore, the case represents autosomal recessive OA (AROA) that has been regarded as a less severe form of OCA, and not a typical OCA type 2 case. The nding that p.Leu674Val is predicted to be a relatively milder mutation by SIFT analysis (Table 1) is consistent with the observed restricted pathology of the patient only involving the eyes. In fact, p.Leu674Val might contribute towards the less severe manifestation of OCA type 2 in OCA96, also harboring this change in a compound heterozygous state with an insertion mutation. However, according to PolyPhen-2, it is a severely damaging change. In silico analysis is therefore insufcient to evaluate the tness of the protein with great condence. Functional characterization will be essential to decipher the nature of the mutation, and determine the extent of its biological impairment. To our knowledge, this is the rst report of a mutation identied in an AROA patient from India. 3.4. SLC24A5 mutations in an individual with extreme cutaneous hypopigmentation SLC24A5 has been proposed to encode a trans-Golgi network protein with potassium-dependent sodiumcalcium exchange activity that regulates human epidermal melanogenesis (Ginger et al., 2007). A novel homozygous insertion mutation (c.569_570insATTA) in SLC24A5 was detected in an eastern Indian male (OCA97) with extreme hypopigmentation of the skin. The insertion of a base causes a frameshift, leading to a premature stop codon 3 bases downstream of the insertion. The truncated mRNA codes for a 192 amino acid peptide instead of the 500 amino acid present in the wild type protein. InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/) results revealed that the mutant protein is completely devoid of one of the two predicted sodium/calcium exchanger membrane regions of the wild type protein. The skin tone of the individual was totally different from either of the parents, who were dark pigmented. His brother also shared a similar phenotype. However, neither of the parents nor his brother was ready to donate blood or share their pictures. Detailed clinical data was not available either to examine the eye abnormalities, if any. The eye pigmentation of the individual was dark as was his hair color. To our knowledge, this type of an atypical cutaneous phenotype relating to SLC24A5 has not yet been reported in humans. Incidentally, we did not detect any genetic defects in the patient in the four known classical OCA genes. Thus, the occurrence of this SLC24A5 (c.569_570insATTA) mutation in an OCA patient suggests that the gene could represent a candidate for albinism. In fact, our observation is not surprising since a Slc24a5-null mouse model has been shown to have albinistic features (Vogel et al., 2008), and

Table 2 TYRP2 and SILV variants detected in Indian OCA patients. Sl. Nucleotide no. change Variants in TYRP2 1 c.132delG Amino acid change NA Location No. of alleles Remarks in gene (Hom/Het) 5UTR 0/1 Novel change; MAF in controls: 0.01 rs9584235 rs12877849 rs12864325 rs12876569 Novel change; MAF in controls: 0; SIFT: 0.07; PolyPhen-2: 0.807 rs3215804 rs71111527

2 3 4 5 6

c.154A>G c.696 + 71G>A c.696 + 77A>G c.696 + 206C>G c.853G>C

NA NA NA NA Val285Leu

5UTR Intron 3 Intron 3 Intron 3 Exon 4

0/1 0/6 0/6 0/6 0/1

7 8

c.1560 + 263delC NA c.1560 + 310delAa NA

3UTR 3UTR

1/4 14/0

Variants in SILV 1 c.148C>T

p.Pro50Ser

Exon 2

0/1

2 3

c.741C>T c.1020G>A

p.Pro247Pro p.Ala340Ala

Exon 6 Exon 6

2/6 0/2

c.1956T>C

p.Asn652Asn Exon 11

0/2

Novel change; MAF in controls: 0; SIFT:0.01; PolyPhen-2: 0.998 rs1052165 Novel change; MAF in controls: 0 rs1052206

The patients screened were not found to contain any suspected disease causing mutation in TYR, OCA2, TYRP1, SLC45A2 and SLC24A5. NA: not applicable; MAF: minor allele frequency; SIFT score more than 0.05 demarcates a tolerant change whereas PolyPhen-2 score above 0.454 denotes a damaging change. a delA represents major allele in our Indian population cohort.

SLC24A5 SNP rs1426654 (p.Ala111Thr) has been reported to be associated with fairer skin tone (Stokowski et al., 2007). 3.5. Status of TYRP2 and SILV variants in Indian OCA patients We screened TYRP2 and SILV in those OCA patients for whom we could not detect any pathogenic mutations in TYR, OCA2, TYRP1, SLC45A2 and SLC24A5. While 8 variants were detected in TYRP2 including 2 novel changes, a total of 4 variants, 2 of which were novel, were identied in SILV (Table 2). A non-synonymous change c.853G>C; p.Val285Leu was detected in TYRP2 in 1 south Indian patient (OC131) in heterozygous condition. PolyPhen-2 predicted the change to be potentially harmful but according to SIFT it is not a severe mutation (Table 2). No other affected family member was available to assess the segregation pattern in the family. A potentially pathogenic non-synonymous mutation was also identied in heterozygous condition in a south Indian patient (OC26) in the SILV gene viz. c.148C>T; p.Pro50Ser. However, the change was not found in the affected sibling but was present in an unaffected brother, thereby suggesting that SILV is not the causal gene in this case. Except for a variation (c.132delG) in the 5UTR of TYRP2 gene, neither of the novel non-synonymous variants identied in TYRP2 and SILV were found in the 50 control individuals examined. This study is consistent with a previous report (Sengupta et al., 2010) suggesting that TYR and OCA2 mutations are the major cause of OCA among Indians. Identication of the same non-synonymous change (p.Leu674Val) in OCA2 in two patients manifesting OCA2 and AROA emphasizes the need to investigate the molecular basis of phenotypic heterogeneity. Our observation of a potential mutation in TYRP2 highlights the need for further investigation using a much larger cohort of individuals with albinistic phenotypes lacking defects in the major OCA loci. Most importantly, this study brings forth the role of SLC24A5 in albinistic phenotypes in humans.

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Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.gene.2012.09.012. Acknowledgments This study was supported by grants SIP-007 and MLP-0016 from the Council of Scientic and Industrial Research (CSIR), India. MM and MS are supported by fellowships from CSIR. The authors are grateful to all the members of the albinism-affected families who participated in the study. Prof. Partha P. Majumder generously provided the southern Indian samples. We thank Ms. Ananya Ray (University of Wisconsin, USA) for critically proof reading the manuscript. References
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