AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 75:31-35 (1988) Immunoglobulin (GM and KM) Allotypes in the Sikh Population of India L. LEIGH FIELD, SIRA SURJE, AND AJIT K . RAY Division of Medical Genetics, Department of Pediatrics, University of Calgary and Alberta Children’s Hospital, CaZgary T2T 5C7 (L.L.83 and Department ofAnthropobgy, University of Toronto, Toronto M5S I A l (A.K. R.),Canada KEY WORDS Sikhs, Gm, Km, Population genetics ABSTRACT The populations of India are genetically diverse, both within and between geographic regions; immunoglobulin (GM) allotypes provide important information on genetic differences between populations, since the frequencies of combinations of allotypes (termed “haplotypes”) vary dramatically among ethnic groups. As part of a project to assess genetic diversity among defined Indian populations, we have examined eight GM allotypes in a sample of 101 unrelated Sikhs who have migrated to Toronto, Canada: Glm(1, 2, 3, 17) and G3m (5, 15, 16, 21). Sikhs are a religious group that arose in the Punjab about 1500 A.D.; most of the original converts are believed to have been middle to upper-middle caste Hindus. Gm allotyping showed that six Gm haplotypes occurred at polymorphic frequencies (>0.01) in Sikhs: Gm3;5, ,nm1,17;21 ~ ~ 1 , 2 , 1 7 ; 2 ~1 ~ 1 , 1 7 ;~5~ 1 , 1 7 ; 1 5 , 1 6 , , and Gm1>3;5. These haplotypes have all been previously reported in Indian populations. The frequencies of the first four haplotypes resembled the published frequencies for lower-caste Hindus of NW India more than upper-caste Hindus. However, the last two haplotypes have been found only in upper-caste Hindus. The frequency of one of these, Gn1,17;15316was higher in Sikhs (0.09) than has been reported in any Indian population with the exception of Parsis (who are descended from Iranians). We speculate that the high frequency of this haplotype may have been characteristic of some of the Hindu castes in the Punjab from which Sikhs are descended. 5 7 The populations of India are genetically diverse, both within and between geographic regions. Endogamous mating is the rulethat is, spouses are chosen only from within cultural groups (primarily defined by religion), and often only from within specific subgroups of these (for example, Hindu castes). As part of an ongoing project to assess the genetic diversity among strictly defined Indian populations, we previously reported the GM immunoglobulin allotype frequencies of upper-caste Hindus from Bengal in northeast India (Ray and Field, 1981). We now report GM and KM allotype frequencies in the Sikh population of northwest India. Sikhs are a religious sect that arose in the Punjab about 1500 A.D. Most of the original converts are believed to have been middle to upper-middle caste Hindus, and they 0 1988 ALAN R. LISS. INC. now comprise more than 10 million people (Singh, 1983). GM allotypes are genetically determined variants in the structure of the heavy (gamma) chains of the IgG immunoglobulin class, and KM allotypes are variants in the structure of the kappa light chains of all immunoglobulin classes. There are only two common KM allotypes [Km(l, 311, compared to 18 GM allotypes. Four of the GM allotypes occur on gamma-1 chains [Glm(l, 2, 3, 17)], one on gamma-2 chains [G2m(23)],and 13 on gamma-3 chains [G3m(5,6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28)1. The locations of the allotypes are indicated by the prefixes Glm, G2m, and G3m for gamma-1, gamma-2, and Received April 2, 1987; revision accepted July 21, 1987. 32 L.L. FIELD, S. SURJE, AND A.K. RAY gamma-3 chains, respectively. Genes encoding allotypes at each of the three gammachain loci are inherited together in units, called GM haplotypes. The frequencies of these GM haplotypes vary dramatically among ethnic groups (Steinberg and Cook, 1981); and within each ethnic group, only some of all possible haplotypes that could arise by recombination between the three loci actually occur, suggesting that the distribution of haplotypes may reflect the long-term operation of natural selection. Further support for this suggestion is provided by the many studies that have shown associations between GMKM allotypes and susceptibility to specific diseases, or immune responsiveness to specific antigens (for review, see Whittingham and Propert, 1986).In the present study, we were interested in GM allotypes as indicators of population genetic diversity. Table 1 shows the most common GM haplotypes around the world and the region with which they are primarily associated. Four of these haplotypes are polymorphic (frequency greater than 1%) throughout India: Gm3;5, ~ ~ 1 , 1 7 ; 2~1~ 1 2 , 1 7 ; 2and 1 ~ ~ 1 , 1 7 3 field and Kirk, 1981).The frequencies of these vary in a clinal manner from the northwest to the southern part of the subcontinent, with ,nm1,17;21 and Gm1*2,17;21increasing in frequency, and Gm3;5 and Gm1.17;5 decreasing in frequency, as one moves south. The NE Asian haplotype Gm1,17;15,16 and the SE Asian haplotype Gm193;5are found a t lower frequencies and are not found in all populations sampled; the frequency of Km’ does not show significant variation across India (Schadield and Kirk, 1981). grants were from the middle-to-upper economic classes of Indian Sikhs. Since Sikhs are very conservative in their religious practices, the chance of genetic admixture of nonSikhs into the Indian Sikh population is negligible. We examined eight GM allotypes, Glm (1,2, 3, 17) and G3m (5, 15, 16, 21), and one KM allotype, Km(l), in these individuals using a standard agglutination-inhibition technique (Steinberg, 1962) adapted for microtitre plates. Allotyping reagents were purchased from the Netherlands Red Cross. Sera that displayed agglutinating antibodies were heat-inactivated a t 63°C for 10 min and retyped. Maximum likelihood estimates of GM haplotype frequencies were calculated by the computer program MAXLIK (Reed and Schull, 19681, kindly provided to us by Dr. T.E. Reed. The gene frequency of Km(1) was calculated as 1 minus the square root of the frequency of Km(1) negative individuals. RESULTS Two individuals possessed agglutinating antibodies that could not be heat-inactivated and were therefore omitted from the analyses. Table 2 shows the GM and KM phenotypes of the remaining 101 individuals. It also shows the most probable genotypek for each phenotype, given the results of the haplotype frequency estimation (see below). In order to estimate haplotype frequencies efficiently, one must decide which haplotypes are likely to be present in the sample. The first five phenotypes listed in Table 2 suggested the presence of the three haplotypes that are common in Caucasian populations: Gm3;5 ~ ~ 1 , 1 7 ; 2~1~ 1 , 2 , 1 7 ; (Table 21 1). This is consistent with Indian populations being part of a larger “Inao-European” ethnogeoMATERIALS AND METHODS graphic group. The next three phenotypes Serum samples were obtained from 103 un- provided ample evidence for the presence of related Sikhs who have recently migrated the “northeast Asian” haplotype Gm1,17;15,16. from India to Toronto, Canada. These immi- The phenotypes Gm(1,3,17;5), Gm(1,2,17;5, 9 TABLE 1. GM haplotypes common in various regions GM haplotype’ ~,1,17;5,6,11,24,26 Abbreviation Predominant region Europe Asia, Europe Asia, Europe Asia (NE Asia) Asia (SE Asia) Africa Africa ‘Defined by all known gamma-1 and gamma-3 allotypes (G3m(28) excepted), which are separated by a semicolon 33 GM AND KM ALLOTYPES IN SIKHS TABLE 2. GM and KMphenotype frequencies in 101 Sikhs Phenotype' No. Probable genotype 21 27 14 Gm (3;5) Gm (1,3,17;5,21) Gm (1,2,3,17;5,21) Gm (1,17;21) Gm (1,2,17;21) Grn (1.3.17:5.15.16) Gm (1,17;15,16,21) Grn (1,2,17;15,16,21) Gm (1,3,17;5) Gm (1,17:5,21) Gm (1,2,17;5,21) Grn (1.17:5) Gm (1;17~5,15,16) Gm (1,17;15,16) Grn (1,3;5) I , I 5 9 8 , 7 3 2 0 2 0 1 0 2 TABLE 3. GM haplotype and KM gene frequencies in 101 Sikhs Haplotypellgene Estimated frequency Standard error 0.459 0.252 0.149 0.094 0.025 0.021 0.061 0.037 0.031 0.026 0.011 0.021 0.015 Grn3z5 ~~1~17.21 ~~1,2,17;21 ~~1,17;15,16 ~~1.17:5 ~ ~ 1 3 . 5 Krn' - ~ 'Defined by tested gamma 1 and gamma-3 allotypes, which are separated by a semicolon. 21), and Gm(1,17;5,15,16)suggested the presence of the "African" haplotype Gm1J7;5, while the phenotype Gm(1,3;5) indicated the presence of the "southeast Asian" haplotype Gm1,3;5. All six haplotypes suggested by the observed phenotypes have been previously reported in Indian populations. We therefore estimated haplotype frequencies allowing for the presence of these haplotypes. The results are presented in Table 3; the six GM haplotypes were all polymorphic in this sample of Sikhs. The observed GM phenotype frequencies (Table 2) did not differ significantly from the phenotype frequencies expected at HardyWeinberg equilibrium in a population having the estimated haplotype frequencies (x2(9, 6.8,P= .66). DISCUSSION There is only one other published study of immunoglobulin allotype frequencies in populations of northwestern India that examined as many antigens as the present study (Schanfeld and Kirk, 1981). The investiga- tors typed 163 Hindus from the Punjab, Haryana, Delhi, and Western Uttar Pradesh, broken down into four caste groups: 1) Brahmin (priests), 2) Kshtrya (warriors), which includes Arora, Khatri, and Rajputs, 3)Vaish (farmers and merchants), and 4) Shudra (craftsmen). They suggested that there were GM differences between castes that mirrored the north-south frequency gradients of the subcontinent, with higher castes having higher frequencies of Gm3;5 and Gm1317;5 and lower frequencies of Gm1j17;21 and Gm1,2717;21 Since their sample sizes were small, we have merged the first two castes into a "highercaste" group, and the last two into a "lowercaste" group, for purposes of comparison with our Sikh sample (Table 4). Sikhs have a higher frequency of Grn172,17;21 and lower frequencies of Gm3;5 and Gm1,17;5 than either of these Hindu samples. Thus, with respect to these haplotypes, Sikhs resemble the lower-caste Hindus more than the uppercaste Hindus in the study by Schanfield and Kirk (1981). Daveau et al. (1980) examined L.L. FIELD, S. SURJE, AND A.K. RAY 34 TABLE 4. GM hadotvue and KM gene freouencies in Sikhs and other uouulations o f Northwestern India Sikhs (n = 101) (present study) !,17;21 ~~1,17;15.16 ~ ~ 1 . 1 7 ; 5 ,459 ,252 ,149 ,094 ,025 ~ ~ 1 , 3 ; 5 ,021 Km’ ,061 Hindus from NW India’ Higher caste Lower caste (n = 98) (n = 53) ,560 ,191 ,058 ,005 ,158 ,029 .079 “Punjabis” (religion not stated) (n = 101)~ ,520 ,327 ,067 0 ,086 0 .087 ,396 ,279 ,142 0 ,011 ,149 .056 ‘Haplotype frequencies for the “higher” and “lower” caste groups were calculated as the mean (weighted by sample size) of the haplotype frequencies reported in Schanfield and Kirk (1981). ‘Daveau et al. (1980). GM allotypes in “Punjabis” (religion not specified),but they did not type the antigens Glm(17)and G3m(15,16),which help to differentiate the three non-Caucasian haplotypes from each other (Gm1,’7;15,16, Gm1,17;5,-15-16 when heterozygous with and ~~1,3,-17;5,-15-16) the most common haplotype, Gm3;5. Thus, we do not consider their estimates of the frequencies of these three haplotypes to be as reliable as those of Schanfield and Kirk (1981) or the present study. However, their frequencies for the common Caucasian haplotypes (Grn3i5, Gm1917i21,and Grn1,2,17;21)and for Kml were remarkably similar to those we obtained for Sikhs (Table 4). The presence of the “African” haplotype GmlJ7;5 in Sikhs deserves comment. While this haplotype is most common in Africa, it has been found in previous studies of Indian populations (Schanfield and Kirk, 1981; Ray and Field, 1981; van Loghem et al., 1985). However, since the Gm1?17;5haplotype in India appears to be most often associated with the Am’ allele at the closely linked AM locus (Schadield and Kirk, 1981; Ray and Field, 1981),while that in Africa is usually associated with the Am2 allele, the adjective “African” for Gm’317;5may be a misnomer. The presence of this haplotype in India, and in populations to the north of India (Schanfield et al., 1980), may be very ancient and not indicative of “admixture” with Africans, as is sometimes stated. Schanfeld and Kirk (1981) suggested that in their samples, the Asian haplotypes Gm1,17;15,’6and Gm’>3;5appeared to be confined to higher-caste Hindus (Brahmin in the case of Gm1,17;15716 and Kshtrya in the case of Gm173;5).The presence of Gm’73;5 in Sikhs may thus support the historical tradition that the original converts to Sikhism included Rajputs (Kshtrya caste). The frequency of the “northeast Asian” haplotype Gm1,17;15,16 in Sikhs (0.094) is the highest reported frequency of that haplotype in any Indian population, with the exception of Parsis, who are descended from Iranians and whose Gm1717;15916 frequency was 0.121 in a study by Steinberg and colleagues (1973). As noted above, Schanfield and Kirk (1981) found this haplotype in Hindus only amongst the Brahmin caste. It is not very likely that the high frequency of GmlJ7;15,16 in Sikhs was created by a founder effect, since the number of original converts to Sikhism was not small. However, it is possible that the high frequency of this haplotype may have been characteristic of some of the Hindu castes in the Punjab from which Sikhs are descended. Larger samples from the various Hindu caste groups in that region are needed to provide more information relevant to that possibility. ACKNOWLEDGMENTS We thank Rose Tobias for performing the immunoglobulin allotyping and Carol Sproule for preparing the manuscript. LITERATURE CITED Daveau, M, Rivat, L, Lalouel, JM,Langaney, A, Roberts, DF, and Simons, MJ (1980)Frequencies of Gm and Km allotypes in the population of Singapore, Sri Lanka and Punjabis in North India. Hum. Hered. 30:237-244. Ray, AK, and Field, LL (1981) Immunoglobulin (Gm) allotypes in a sample of upper-caste Hindus from Bengal, India. Hum. Hered. 31:358-362. Reed, TE, and Schull, WJ (1968) A general maximum likelihood estimation program. Am. J. Hum. Genet. 20579-580. Schanfield, MS, and Kirk, RL (1981) Further studies on GM AND KM ALLOTYPES IN SIKHS the immunoglobulin allotypes (Gm, Am, and Km) in India. Acta Anthropogenet. 6:l-21. Schanfeld, MS, Alexeyeva, TE, Crawford, MH (1980) Studies on the immunoglobulin allotypes of Asiatic populations. VIII. Immunoglobulin allotypes among the Tuvinians of the USSR. Hum. Hered. 30:343-9. Singh, H (1983) The Heritage of the Sikhs. Columbia, MO: South Asia Books. Steinberg, AG (1962) Progress in the study of genetically determined human y-globulin types (the Gm and Inv groups). Prog. Med. Genet. 2 - 3 3 . Steinberg, AG, and Cook, CE (1981) The Distribution of 35 the Human Immunoglobulin Allotypes. Oxford Oxford University Press. Steinberg, AG, Undevia, JV,and Tepfenhart, MA (1973) Gm and Inv studies of Parsis and Irani in India: Report of a new polymorphic haplotype Grn'93921. Am. J. Hum. Genet. 25:302-9. Van Loghem, E, Tauszik, T, Hollan, S, and Nijenhuis, LE (1985) Immunoglobulin allotypes in Hungarian gypsies. J. Immunogenet. 12131-137. Whittingham, S, and Propert, DN (1986) Gm and Km allotypes, immune response, and disease susceptibility. Monogr. Allergy 19:52-70.