|Molecular Vision 2005;
Received 1 March 2005 | Accepted 2 July 2005 | Published 19 July 2005
Higher prevalence of OCA1 in an ethnic group of eastern India is due to a founder mutation in the tyrosinase gene
Moumita Chaki,1 Arijit Mukhopadhyay,1 Shamba
Chatterjee,1 Madhusudan Das,2 Swapan Samanta,3
1Human Genetics & Genomics Division, Indian Institute of Chemical Biology, Kolkata, India; 2Department of Zoology, University of Calcutta, Kolkata, India; 3B. C. Roy Children Hospital, Kolkata, India
Correspondence to: Dr. Kunal Ray, Human Genetics and Genomics Division,
Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road,
Jadavpur, Kolkata-700 032, India; Phone: (91) 33-2473-0350; FAX: (91)
33-2473-5197 and (91) 33-2472-3967; email: email@example.com
Dr. Mukhopadhyay is now at the Department of Human Genetics, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands
Purpose: Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by deficient synthesis of melanin pigment and associated with common developmental abnormalities of the eye. It is one of the major causes of childhood blindness in India. The disease is common among an ethnic group (Tili) of Eastern India, which represents about 12.56% of the Bankura district population (approximately 0.4 million) of West Bengal. The purpose of the study was to investigate the molecular lesions causing OCA within this ethnic group for the unequivocal diagnosis of the carriers and attempt to decipher the cause for the high prevalence of OCA.
Methods: Fourteen OCA-affected Tili families consisting a total of 161 individuals, including 26 patients, were recruited for the study. A lack of tyrosinase (TYR) activity among all the patients was ascertained by the tyrosinase hair bulb assay. Mutation screening in the tyrosinase gene (TYR) was done by single strand conformational polymorphism (SSCP) and DNA sequencing. The restriction fragment length polymorphism (RFLP) assay was carried out to determine the frequency of the pathogenic changes among the normal individuals. Haplotype analysis was performed at the TYR locus using a set of informative microsatellite and SNP markers.
Results: All the patients were homozygous for a null mutation (c.832C>T, Arg278stop) in TYR exon 2, which might cause a complete loss of enzyme activity. The mutation occurred in the same haplotype background. The frequency of the disease in this ethnic group was estimated to be significantly higher than the world average.
Conclusions: OCA1 in the Tili population is due to the occurrence of a founder mutation in the TYR as indicated by haplotype analysis. Higher prevalence of the mutation in the population group is due to marriage within the same community. The diagnostic RFLP assay can be utilized for genetic counseling and thereby will help to reduce the disease load on the population.
Oculocutaneous albinism (OCA) is a heterogeneous group of autosomal recessive disorders that often results in the reduction or complete absence of melanin in the skin, hair, and eyes. It is also associated with common developmental abnormalities of the eye. At least 16 different genes have been identified that, when mutated, result in different types of albinism . All forms of OCA are represented with photophobia, strabismus, moderate to severe visual impairment, and nystagmus.
Oculocutaneous albinism type 1 (OCA1; OMIM 203100), results from mutations in the tyrosinase gene (TYR, 11q14-q21; OMIM 606933). The clinical phenotype resembling OCA1 is caused by the absence of (OCA1A) or residual catalytic activity of (OCA1B) tyrosinase . TYR with 5 exons spanning more than 65 kb of DNA  encodes a 58 kDa glycoprotein of 529 amino acids .
Despite a large number of reported TYR mutations (Albinism database), very little is known about the molecular basis of OCA in Indian patients although the disease is quite prevalent in some of the geographical locations. In the Bankura district of West Bengal, OCA is common among the Tili ethnic group. We initially reported the presence of a TYR mutation (R278X) among the Tili . A subsequent study confirmed the presence of the same mutation and reported two frameshift mutations in four affected families representing an ethnic group, which was termed "either Tili or Tamli" . These family samples were collected from the same region of India. It is important to note that Tili and Tamli are two distinct ethnic groups and hence may not have the same genetic basis for occurrence of OCA. Tamli, also known as Tamuli or Tambuli, mostly live in the Burdwan and Hooghly districts of West Bengal. The available literature regarding their origin suggests that they are an offshoot of one of the trading castes . However, Tili or Teli, is a community whose name derives from the Sanskrit word talika or taila, referring to the oil extracted from sesame and mustard. Risley  suggested that they must have been recruited from a different class of Hindu society.
In contrast to the recent report  on "Tili or Tamli" our study based on 188 Tili individuals suggested a lack of any mutation other than R278X among Tili, the ethnic group which represents about 12.56% of the Bankura population (0.4 million). Therefore, we argue that the genetic basis of the disease in the population group should be investigated thoroughly so that advice regarding genetic screening and subsequent counseling is based on clearly established facts for the relevant ethnic or population group. Here we report results of our investigation based on OCA affected families, carefully selected by interviewing the local Tili population group and using experiments designed to provide unequivocal answers. The mutation occurred in the same haplotype background suggesting a founder effect. Our data suggest that R278X is the major, if not exclusive, mutation within the Tili ethnic group.
Fourteen OCA affected Tili families, consisting of a total of 161 individuals including 26 patients, were recruited for the study. Approximately 10 ml of peripheral blood samples were collected in EDTA, from 48 Tili individuals including 19 patients, 29 family members with the normal phenotype, and an OCA-affected boy and his normal brother belonging to the Tamli (also known as Tambuli) ethnic group, with their informed consent. The study protocols adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board. Apart from albinism, the diagnosis involved ophthalmologic examinations including the testing of abnormal ocular movement (nystagmus and strabismus), a visual acuity test, fundoscopy, and tests for other ocular involvement such as cataract, glaucoma, or retinal diseases. Controls were selected from the general Tili population without any family history of ocular disease or albinism to determine the frequency of the carriers for the TYR mutation. Prior to collection of blood samples, a government-registered local organization for the welfare of the Tili community was contacted for information on the ethnic group. Next, one of us (SS), a clinician working in the Bankura district for last 25 years and having good knowledge about the community, visited individual households of Tili families, interviewed household members to ensure their ethnicity along with other relevant information regarding OCA, and collected the blood samples with the consent of the donors.
A tyrosinase hair bulb assay was performed to identify potential OCA1 cases . The coding sequence and splice site junctions of TYR were amplified from the genomic DNA of the patients and controls, and the mutation was identified by direct DNA sequencing of the samples showing band-shift in SSCP. While exons 1-3 were amplified following standard protocol (Table 1), exons 4 and 5 were amplified using gene specific primers to avoid co-amplification of the pseudogene TYRL (OMIM 191270) based on our recently described method . Three CA-repeat markers (GDB:11511689, GDB:11511691, and GDB:11511690), identified within the TYR locus and its flanking regions, were used to determine the genotypes of the patients and their parents. Genescan analysis was done in the ABI Prism 3100 DNA Sequencing System using 500 ROX Size Standard (Applied Biosystems, Foster City, CA). The mutation identified by DNA sequencing was analyzed in the additional control Tili samples with BslI restriction enzyme (New England BioLabs, Beverly, MA). The mutant allele was indicated by the loss of the BslI site.
Results & Discussion
In our sample pool, all the patients seemed to be affected with OCA1 due to mutations in the TYR, as indicated by the tyrosinase hair bulb assay. On clinical examination, these patients were found to have the pigment deficiency in the peripheral retina along with other associated changes to the visual system including decreased visual acuity (usually diminished to as low as 6/60) secondary to foveal hypoplasia, photophobia, iris transillumination, nystagmus, strabismus, and loss of binocular vision.
SSCP analysis revealed that all the Tili patients had the same characteristic band shift (Figure 1A) of the amplicon containing the sequences for the second exon of TYR. The DNA sequence analysis (Figure 1B) of multiple patient samples revealed the same homozygous change (c.832C>T) that would create a premature stop codon (Arg278stop) resulting in a truncated and completely inactive enzyme lacking one potential copper binding region (OCA1A). The mutation occurred in a CpG context and reported previously in different population groups, for example, Japanese , Guayanan , Morroccan Jewish , and Indo-Pakistani  populations. To examine whether the prevalence of this mutation in the Tili population was due to a founder effect, we did haplotype analysis using the newly identified microsatellite markers (GDB:11511689, GDB:11511691, and GDB:11511690) within the TYR locus and its flanking regions. All 19 patients were found to have the same haplotype (179-101-157). The same result was obtained using a set of SNP markers: 1-533G->C, 1-301C->T, 1-199C->A, c.575C->A (S192Y), c1037-201G->A(data not shown).
To determine the carrier frequency of this mutation within this ethnic group, an RFLP-based diagnostic assay was performed (Figure 1C) on the DNA samples from 27 unrelated normal individuals of the same population group, who on specific inquiry informed us that they did not have any OCA affected members in their families. As a larger number of such donors were difficult to obtain, based on the limited samples, the initial carrier frequency of the mutation was found to be 11.11% (3/54 alleles) among the Tili and the disease frequency was estimated to be significantly higher than the world average .
In a recent paper , in addition to R278X, two frameshift mutations have also been reported in exons 2 and 5, to cause OCA in the "Tili or Tamli" ethnic groups of West Bengal, India. To investigate the occurrence of these two mutations in the Tili and Tamli populations (these are not the same groups as mentioned earlier), we directly sequenced exons 2 and 5 from OCA patients of both these ethnic groups. We amplified TYR exon 5 (avoiding co-amplification of the pseudogene TYRL) as we recently described . Our investigation clearly showed that while the Tamli patient was a compound heterozygote for Arg278stop and c.1379delTT (data not shown), none of the Tili individuals (patients, their family members, or unrelated controls) contained the deletion mutation in exon 5 of TYR. We did not detect a deletion mutation in Exon 2 of any individual of our cohort, consisting of 14 large Tili families and one Tamli family. The R278X mutant chromosome in the Tamli family has the same haplotype as in the Tili mutation suggesting a common origin for both. It is likely that the mutation was introduced into the Tamli lineage by the marriage of a person of this community to a Tili individual harboring the mutation. However, such marriages are relatively uncommon.
The authors thank all the members of OCA-affected families and normal individuals belonging to Tili ethnic group who participated in the study. The study has been partially supported by the Council of Scientific and Industrial Research (CSIR), India. MC and AM are supported by predoctoral fellowship from University Grant Commission (UGC) and CSIR, respectively. The valuable advice of Prof. P. P. Majumder (Indian Statistical Institute, Kolkata, India) for this study is gratefully acknowledged. The authors are also thankful to Ms. A. Ray (Calcutta International School, Kolkata, India) for proofreading the manuscript.
1. Tomita Y, Suzuki T. Genetics of pigmentary disorders. Am J Med Genet C Semin Med Genet 2004; 131C:75-81.
2. Passmore LA, Kaesmann-Kellner B, Weber BH. Novel and recurrent mutations in the tyrosinase gene and the P gene in the German albino population. Hum Genet 1999; 105:200-10. Erratum in: Hum Genet 2001; 218:208.
3. Giebel LB, Strunk KM, Spritz RA. Organization and nucleotide sequences of the human tyrosinase gene and a truncated tyrosinase-related segment. Genomics 1991; 9:435-45.
4. Shibahara S, Tomita Y, Tagami H, Muller RM, Cohen T. Molecular basis for the heterogeneity of human tyrosinase. Tohoku J Exp Med 1988; 156:403-14.
5. Chaki M, Mokhopadhyay A, Das M, Samata S, Ray K. Identification of a founder tyrosinase mutation causing oculocutaneous albinism in an endogamous caste population of west Bengal. 13th Annual Meeting of the Indian Eye Research Group; 2004 August 20-22; Chennai, India.
6. Sundaresan P, Sil AK, Philp AR, Randolph MA, Natchiar G, Namperumalsamy P. Genetic analysis of oculocutaneous albinism type 1 (OCA1) in Indian families: two novel frameshift mutations in the TYR Gene. Mol Vis 2004; 10:1005-10 <http://www.molvis.org/molvis/v10/a119/>.
7. Risley HH. The tribes and castes of Bengal. Calcutta: Bengal Secretariat Press; 1891.
8. King RA, Witkop CJ Jr. Hairbulb tyrosinase activity in oculocutaneous albinism. Nature 1976; 263:69-71.
9. Chaki M, Mukhopadhyay A, Ray K. Determination of variants in the 3'-region of the Tyrosinase gene requires locus specific amplification. Hum Mutat 2005; 26:53-8.
10. Matsunaga J, Dakeishi-Hara M, Miyamura Y, Nakamura E, Tanita M, Satomura K, Tomita Y. Sequence-based diagnosis of tyrosinase-related oculocutaneous albinism: successful sequence analysis of the tyrosinase gene from blood spots dried on filter paper. Dermatology 1998; 196:189-93.
11. Spritz RA. Molecular genetics of oculocutaneous albinism. Semin Dermatol 1993; 12:167-72.
12. Gershoni-Baruch R, Rosenmann A, Droetto S, Holmes S, Tripathi RK, Spritz RA. Mutations of the tyrosinase gene in patients with oculocutaneous albinism from various ethnic groups in Israel. Am J Hum Genet 1994; 54:586-94.
13. Tripathi RK, Bundey S, Musarella MA, Droetto S, Strunk KM, Holmes SA, Spritz RA. Mutations of the tyrosinase gene in Indo-Pakistani patients with type I (tyrosinase-deficient) oculocutaneous albinism (OCA). Am J Hum Genet 1993; 53:1173-9.
14. King RA, Hearing VJ, Creel DJ, Oetting WS. Albinism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. 8th edn. New York: McGraw-Hill; 2001. p. 5587-627.