Association of IL-1ß +3953 C and HLA-DRB1*15 with Coronary Artery and Rheumatic Heart Diseases in South India.

Among the various candidate genes predisposing for cardiovascular diseases, HLA-DRB1* and IL-1ß +3953C/T alleles have been implicated repeatedly. To test these in South India, we carried out a case control study of 323 Coronary Artery Disease (CAD) patients, 56 Rheumatic Heart Disease (RHD) patients and 254 endemic controls. The polymorphisms were studied by PCR - SSP and ARMS-PCR methods and results analyzed for various clinical and demographic parameters. In CAD, HLA-DRB1*14 allele showed significant predisposition (OR: 2.19; 95% CI: 1.04-4.58; p value=0.023), particularly in male patients (OR: 4.07; 95% CI: 1.20-13.81; p value=0.01) and further in males with Triple Vessel Disease (OR: 5.49; 95% CI: 1.45-20.60; p value=0.007). On the other hand, HLA-DRB1*15 predisposed for RHD (OR: 3.56; 95% CI: 1.87-6.78; p value=0.001) in both the genders. Population stratification showed this higher risk association in Vanniyar caste (OR: 5.00; 95% CI: 1.27-19.59; p value=0.022). Among the IL1-ß +3953C/T polymorphism, the ancestral allele 'C' showed a significant high risk association with CAD (OR: 1.83; 95% CI: 1.24-2.70; p value=0.001), particularly in Mudaliar (OR: 6.07; 95% CI: 1.77-20.74; p value=0.003; AF=0.7) and Vanniyar castes (OR: 3.67; 95% CI: 0.92-14.57; p value=0.05; AF=0.660). Two different cardiac ailments studied, RHD & CAD thus showed varied associations in this South Indian cohorts. RHD having an infectious aetiology shared a HLA-DRB1*15 high risk association, while HLA-DRB1*14 and IL-1ß +3953C predisposed for CAD, an inflammatory disorder, reiterating the diverse genetic predisposition of the two cardiac ailments studied.

Recombination gives a new insight in the effective population size and the history of the old world human populations.

The information left by recombination in our genomes can be used to make inferences on our recent evolutionary history. Specifically, the number of past recombination events in a population sample is a function of its effective population size (Ne). We have applied a method, Identifying Recombination in Sequences (IRiS), to detect specific past recombination events in 30 Old World populations to infer their Ne. We have found that sub-Saharan African populations have an Ne that is approximately four times greater than those of non-African populations and that outside of Africa, South Asian populations had the largest Ne. We also observe that the patterns of recombinational diversity of these populations correlate with distance out of Africa if that distance is measured along a path crossing South Arabia. No such correlation is found through a Sinai route, suggesting that anatomically modern humans first left Africa through the Bab-el-Mandeb strait rather than through present Egypt.

HLA diversity among Nadars, a primitive Dravidian caste of South India.

South India is one of the oldest geophysical regions mainly occupied by Dravidian language-speaking people. Here a random panel of 61 unrelated Nadar healthy individual from Tamil Nadu State were analyzed and compared with other populations of India and the world. HLA-A, B and C alleles frequencies and their haplotype frequencies were determined by high-resolution typing of genomic DNA. The analysis revealed that the Nadar caste of South India have several characters shared with East Asian populations consistent with the demographic history of South India, as well as specific features including several unique alleles such as A*03011, A*31011, B*15011, B*3501, B*51011, Cw*02022. In addition, haplotypes such as A*31011-Cw*02022-B*3501, A*03011-Cw*04011-B*4406 and A*2402101-Cw*04011-B*51011 are of high frequency in both these populations but are rare or absent in other populations of India and the world. The study suggests that a comparatively lesser degree of genetic admixture occurred between the South Indian and North Indian racial groups than that between South Indian and East Asian groups.

Mycobacterium bovis BCG scar status and HLA class II alleles influence purified protein derivative-specific T-cell receptor V beta expression in pulmonary tuberculosis patients from southern India.

Purified protein derivative (PPD) RT23-recalled T-cell receptor (TCR) V beta expression was studied in the peripheral blood of 42 pulmonary tuberculosis patients and 44 healthy controls from southern India, a region where tuberculosis is endemic. Forty-eight-hour whole-blood cultures in the presence or absence of PPD-RT23 were set up, and at the end of the culture period total RNA was extracted and cDNA was synthesized. Expression of various TCR V beta families was assessed by using family-specific primers. PPD-specific expression (usage) of TCR V beta families 4, 6, 8 to 12, and 14 was found in more controls than patients. Among the responders (individuals who showed PPD-specific expression), endemic controls had significantly higher responses than the patients had for TCR V beta families 2, 3, 7, 13, and 17. The majority of the patients did not show usage of most of the TCR V beta families, and this was attributed to T-cell downregulation. A four-way nested classification analysis revealed that TCR V beta family 1, 5, 9, 12, and 13 usage in the context of HLA class II high-risk alleles (DRB1*1501, DRB1*08, and DQB1*0601) and Mycobacterium bovis BCG scar status were the determining factors in susceptibility and resistance to tuberculosis. The healthier status of controls was attributed to the wider usage of many TCR V beta families readily recalled by PPD, while the disease status of the patients was attributed to TCR V beta downregulation and the resultant T-cell (memory cell?) unresponsiveness. Host genetics (HLA status) and BCG vaccination (scar status) seem to play important roles in skewing the immune response in adult susceptibility to pulmonary tuberculosis through TCR V beta usage.

HLA-DRB1*, -DQB1* in Piramalai Kallars and Yadhavas, two Dravidian-speaking castes of Tamil Nadu, South India.

Two Dravidian-speaking castes of Tamil Nadu, Piramalai Kallars (PKs, n = 205) and Yadhavas (YDs, n = 239) and a random panel (84) were studied for HLA-DRB1* and -DQB1* polymorphisms by DNA-SSOP typing methods. XI and XII International Histocompatibility primers and non-radioactive-labelled oligo probes were employed to identify the alleles. Results revealed that PKs possessed >0.1 allele frequencies of HLA-DRB1*15011, 0301, -DQB1*0201, 0501 and 0601; YDs, HLA-DRB1*0301, 0401, 07 and -DQB1*0601; and the random panel, DRB1*15021, 0401, 07, -DQB1 0201, 0301, 0302 and 0501. The highest frequency of DRB1*1501 in the world (GF = 0.225) was found in PKs. The most frequent two-locus haplotype (>500/10,000) in all the study samples was DRB1*10-DQB1*0501, while 1501-0601 was frequent in PKs and YDs. Comparison of the HLA-DRB1* data with Eastern European and South-East Asian populations suggested migration as the prime cause of the observed diversity in DRB1* allele frequencies. Nonetheless, the heterozygocity test and Watterson's homozygosity test indicated that balancing selection still operates on HLA-DRB1* locus, in this endemic region of various infectious diseases. This and spatial autocorrelation analysis support the view that selection may be a cause of "generating" new variants and allelic diversity in different ancient settlements. The study suggested that South Indian, inbred, endogamous, sympatrically isolated castes or similar well-defined breeding isolates around the world, living under the same milieu-epidemiology, may be ideal models to test the immunogenetic basis of disease susceptibility.

Genetic basis of tuberculosis susceptibility in India.

Tuberculosis is a complex disease resulting from the responses of immunological, genetic and environmental factors to the chronic infectious agent, Mycobacterium tuberculosis. Several genetic factors have been implicated in host disease susceptibility and the prevalence of a disease in a population may be equal to the product of the frequencies of the susceptible alleles present in the population living in an endemic area. The endogamous, sympatrically isolated gene pools, exposed to the highly infectious environmental load of India, is an ideal model to study tuberculosis susceptibility. Our recent studies in this endemic region have reiterated the association of HLA-DRB1*02 and its subtype DRB1*1501 with tuberculosis susceptibility and have identified an IL-10 associated disease susceptibility in HLAnon-DRB1*02, BCG scar negative individuals and a skewed usage of TCR Vb in BCG scar negative, HLA high risk allele carrying individuals. This indicates that there may be several pathways leading to disease. Tuberculosis susceptibility is not thus a one-gene one product manifestation but multifactorial and epistatic influences of various factors finally lead to the disease. We review the factors that has been explored under Indian context in tuberculosis susceptibility.

A sensitive micro-platelet ELISA technique for screening anti-HLA antibodies.

An enzyme-linked immunosorbant assay (ELISA) in a Terasaki plate (Micro-Platelet ELISA), using 30000 platelets per well, 2 microL primary antibody (anti HLA antiserum) and 5 microL of secondary antibody (1:2000) are described. Platelets from 30 selected HLA tissue typed cell panel individuals were used to characterize anti HLA A and B antibodies. The first half of the Terasaki tray had platelets in sequence to characterize anti HLA antibodies, while the second half contained anti HLA B antibodies. Results revealed that the HLA specificities of the sera identified by micro-platelet ELISA and microlymphocytotoxicity were concordant. Moreover, split antigens of broader specificities were identified in the Platelet ELISA technique. The advantages of micro-platelet ELISA technique were: (i) it does not require viable/frozen lymphocytes, (ii) reading is very simple and macroscopic, (iii) specificity of the serum is identified with accuracy within the same day, (iv) avoids inter-cell, inter-day variations, (v) complement is not required, (vi) requires only 1/50 volume of the reagents required by conventional ELISA in microtitre plates, and (vii) using platelets isolated from 5 mL of peripheral blood, fifteen thousand sera can be tested. This technique is, thus, very simple, cost effective, and very much suitable for any developing HLA laboratory, which is in the process of developing indigenous HLA reagents.

Castes, migration, immunogenetics and infectious diseases in south India.

It has been said that the grandest genetic experiment of nature has been conducted in south India in the name of the caste system. One can expect the frequency of an infectious disease to be equal to the product of the frequencies of various indicated loci/alleles, whether physiological, hormonal or immunological, in an endemic area. The sympatrically isolated caste and sub-caste populations of southern India, with differing origins, migration patterns and breeding habits, differ significantly in their HLA and other immune repertoire and are ideal models to study and test this hypothesis. The prevalence of a number of major infectious diseases, including TB and leprosy, are reviewed in different communities in the light of their genetic history.

The Eurasian heartland: a continental perspective on Y-chromosome diversity.

The nonrecombining portion of the human Y chromosome has proven to be a valuable tool for the study of population history. The maintenance of extended haplotypes characteristic of particular geographic regions, despite extensive admixture, allows complex demographic events to be deconstructed. In this study we report the frequencies of 23 Y-chromosome biallelic polymorphism haplotypes in 1,935 men from 49 Eurasian populations, with a particular focus on Central Asia. These haplotypes reveal traces of historical migrations, and provide an insight into the earliest patterns of settlement of anatomically modern humans on the Eurasian continent. Central Asia is revealed to be an important reservoir of genetic diversity, and the source of at least three major waves of migration leading into Europe, the Americas, and India. The genetic results are interpreted in the context of Eurasian linguistic patterns.

Association of interleukin-10 cytokine expression status with HLA non-DRB1*02 and Mycobacterium bovis BCG scar-negative status in south Indian pulmonary tuberculosis patients.

HLA DRB1*02 and its subtypes predispose individuals for a far-advanced sputum-positive pulmonary tuberculosis transcending ethnic boundaries. Mycobacterium bovis BCG does not afford the desired protection against adult pulmonary tuberculosis, and a spectrum of immune reactivity exists in controls and hospital contacts. All of these findings have been identified and demonstrated in areas of endemicity. Skewing of immunity from protective to pathogenic may involve a shift in the Th1-Th2 paradigm. To elaborate these ideas, we studied gamma interferon (IFN-gamma), interleukin-4 (IL-4), and IL-10 cytokine expression in 71 adult pulmonary tuberculosis patients and 74 controls from areas of endemicity in south India by 48-h microculture and reverse transcription-PCR. Most of the patients and controls expressed IFN-gamma de novo, and in the presence of purified protein derivative (PPD), all of them expressed significantly higher levels of IFN-gamma, suggesting a PPD-specific recall memory. HLA DRB1* allele-dependent IFN-gamma expression was identified only in controls, suggesting a skewing of the immune response in patients. In contrast to the case for IFN-gamma, only some patients and controls expressed IL-4 or IL-10 (Th2 profile); thus, the Th1 profile was identifiable only by a nonexpression of IL-4 or IL-10 in this area of endemicity. The Th2 profile was associated with HLA non-DRB1*02 and BCG scar-negative status in patients, attributing a significant risk (odds ratio = 2.074; 95% confidence interval = 0.612 to 7.07). It is possible that Mycobacterium tuberculosis (PPD)-specific IL-10 is expressed preemptively in unvaccinated (BCG scar-negative) individuals with a non-DR2 genetic background by chronic exposure in this area of endemicity and leads to pulmonary tuberculosis of adults.

A major susceptibility locus for leprosy in India maps to chromosome 10p13.

Leprosy, a chronic infectious disease caused by Mycobacterium leprae, is prevalent in India, where about half of the world's estimated 800,000 cases occur. A role for the genetics of the host in variable susceptibility to leprosy has been indicated by familial clustering, twin studies, complex segregation analyses and human leukocyte antigen (HLA) association studies. We report here a genetic linkage scan of the genomes of 224 families from South India, containing 245 independent affected sibpairs with leprosy, mainly of the paucibacillary type. In a two-stage genome screen using 396 microsatellite markers, we found significant linkage (maximum lod score (MLS) = 4.09, P < 2x10-5) on chromosome 10p13 for a series of neighboring microsatellite markers, providing evidence for a major locus for this prevalent infectious disease. Thus, despite the polygenic nature of infectious disease susceptibility, some major, non-HLA-linked loci exist that may be mapped through obtainable numbers of affected sibling pairs.

Associations of HLA-DRB1, DQB1 and DPB1 alleles with pulmonary tuberculosis in south India.

SETTING: Tuberculosis is endemic in south India: sputum positive pulmonary tuberculosis is predisposed by HLA-DR2 in south India and few other populations of the world. OBJECTIVE: To study HLA-DRB1, DQB1, DQA1 and DPB1 allelic polymorphism in pulmonary tuberculosis patients and endemic controls from south India. DESIGN: One hundred and twenty-six, sputum positive pulmonary tuberculosis patients and 87, endemic controls, from Madurai were studied for MHC class II allelic polymorphism by PCR-SSOP method. XI IHWC primers and probes and non-radioactive probing methods were employed. RESULTS: HLA DRB1*1501 and DQB1*0601 predisposed for pulmonary tuberculosis (DRB1*1501: odds ratio (OR) = 2.68, 95% confidence interval (CI) = 1.30-5.89, P value (P) = 0.013, aetiological fraction (EF) = 0.17; DQB1*0601: OR = 2.32, CI = 1.29-4.27, P = 0.008, EF = 0.26). Haplotype DRB1*1501-DQB1*0601 was higher in patients (1324 per 10,000, X2 = 27.07) than controls (F = 404/10,000, X2 = 8.84). In a subset of 63 caste matched samples, DPB1*04 was preventive (OR = 0.45, CI = 0.21-0.95, P = 0.036, PF = 0.26): the distributions of DRB1*1501-DQB1*0601-DPB1*04 phenotypes were different between patients and controls (P = 0.0092). These alleles were predominant in patients and controls of T5SU caste. CONCLUSION: HLA-DRB1*1501 and DQB1*0601 predisposed to sputum positive pulmonary tuberculosis, and DPB1*04 was preventive and epistatic to this risk. Caste T5SU is an ideal model to study immunology of tuberculosis.

Sociobiology and HLA genetic polymorphism in hill tribes, the Irula of the Nilgiri hills and the Malayali of the Shevroy hills, south India.

Two endogamous tribes of Tamil Nadu, South India, the Irula of the Nilgiri hills and the Malayali of the Shevroy hills, were studied for their sociobiology and HLA polymorphism. For sociobiological studies 166 marriages in the Irula and 368 marriages in the Malayali were recorded. The number and spatial distribution of patrilineal clans and their marriageable range (number of clans from which the brides came) were studied. Eight clans in the Irula and 16 clans in the Malayali were identified. Of these the Kuppar of the Irula and the Malayan of Malayali were the largest clans, and both of them had the greatest marriageable range. The numerical strength and the resultant spatial distribution correlated well with the marriageable range. HLA-A, B, and DR polymorphism was studied on 191 Irula and 42 Malayali following standard procedures. HLA typing revealed high frequencies (> 10%) of alleles HLA-A2, A9, A11, B17, B35, B40, DR2, and DR7 in both tribes, but the Irula had elevated HLA-A10, B8, and DR8 frequencies and the Malayali had elevated HLA-A31, B7, DR4, and DR5 frequencies. Two-locus haplotypes A10-B8 and A2-B5 were identified in both tribes, but A11-B40 and A2-B53 were present only in the Irula and A33-B44 and B15-DR6 were present only in the Malayali. The sociobiology of the Irula was correlated to the HLA genetic profile. The Irula sample was stratified based on clan and HLA data; The Kuppar clan was closer to the Kalkatti, the second largest clan, than to the Pungar and the Sambar clans. Thus the numerical strength and spatial distribution of various exogamous clans, presumably a result of migration during different periods of history, is reflected in the marriageable range and thus in the genetic distance. In studying HLA or any other genetic polymorphism of an endogamous tribe or caste, one needs to consider the social structure, spatial distribution, and marriageable range.

HLA affinities of Iyers, a Brahmin population of Tamil Nadu, South India.

Seventy-four randomly sampled Iyers, a Brahmin population of Tamil Nadu and preachers and followers of the Advaita philosophy, living in Madurai, were studied for their HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, C4A, C4B, and BF polymorphisms and compared with other populations. HLA alleles A1, A11.1, A24, A33, B35, B44, B51, B52, B57, Cw4, Cw6, Cw7, DR4, DR7, DR8, DR10, DR11, DR15, and DQ1 and C4A*3, C4A*4, C4A*6, C4A*Q0, C4B*1, and BF*S were represented in 15% of the samples studied. HLA alleles A25, A69, Cw3, Cw8, B45, B14, B39, B18, B50, and B56 were not identified. Various populations of Tamil Nadu were compared, but the Iyers of Madurai formed a separate cluster with Sourashtrans of Madurai and major group 4 (various Brahmin populations of Tamil Nadu); hill tribes (Irulas, Malayalis, and Badagas) and caste groups in the plains (Kallars and Nadars) formed distinct clusters. Comparison of the Iyers with other Indian and world populations revealed that Iyers form a distinct branch of the Indo-European and Central Asian tree. The Bhargavas of Lucknow, another Brahmin caste group from Uttar Pradesh, did not cluster with the Iyers but clustered with Central Asian populations. The Punjabis of Delhi clustered with European and Middle Eastern populations. Studies on two-locus haplotypes of Iyers revealed unique haplotypes in them (A26-B8, A33-B44, A33-Cw7, A1-B57, B8-DR3, B44-DR7, DR7-DQ2, C4A*32-C4B*Q0, and C4A*6-C4B*2), most of which were not identified in the Bhargavas of Lucknow and the Punjabis of Delhi. Thus it is possible that various Brahmin populations of India differ in their origin, migration, and settlement, although all of them adopted Hinduism in ancient times. A comparison of haplotypes in Iyers with the world population reveals a sharing of haplotypes with Southeast Asian populations. This implies that the ancestors of the Iyers of Madurai, who originated in the Eurasian steppes or Central Asia, might have migrated to India through Southeast Asia, thus developing the prevalent haplotypes en route.

Red cell enzymes and serum protein polymorphisms in three population groups of south India.

Blood and serum samples from random individuals of three populations in south India, the first being an endogamous group from the Nilgiri hills (Tamil Nadu), the second from the Shevroy hills (Tamil Nadu), and the third from a semi-urban area of Tamil Nadu, were screened for ESD, GLO1 and Hp polymorphisms. The allelic frequencies for these markers have been estimated. High GLO1*1 (0.379) frequency was observed in the tribal Malayalis, in contrast with other Indian population groups.

Prevalence of sickle cells in Irula, Kurumba, Paniya & Mullukurumba tribes of Nilgiris (Tamil Nadu, India).

A total of 1377 tribals, comprising Irulas (536), Paniyas (196), Kurumbas (87), Mullukrurumbas (156) and Soligas (402), living in the Nilgiris, Tamil Nadu, India were studied for sickle cell trait between 1981-85. Patients attending various tribal clinics at Arayure, Kozhikarai, Kothagiri and Biligiri Rengan hills for various ailments were screened at random by solubility test and by acetate paper electrophoresis, if required. HbAS carrier frequency was 30-37.8 per cent in all the tribals studied except Kurumbas (19.5%). The frequency of carriers were more (37.8%) on the western part of Nilgiris (Nedungode, Kappala and adjoining regions) than the eastern part (30%). Further, the prevalence of carriers was higher (47-49%) in the 10-19 yr age group amongst Paniyas and Mullukurumbas living in the western part of Nilgiris. An episodic, epidemic of malaria so rampant during the early part of this century in the western parts of Nilgiris might have eliminated many children with HbAA and hence the higher frequency of HbAS in this particular age group.

Association of pulmonary tuberculosis and HLA in south India.

In 204 patients with smear-positive pulmonary tuberculosis HLA-A10, B8 and DR2 were more frequently found than in 404 control subjects (p = 0.01); the greatest attributable risk (0.29) was associated with HLA-DR2. The radiographic extent of disease was also associated with HLA-DR2 (p = 0.0001). In 152 patients with smear-negative pulmonary tuberculosis, the frequencies of HLA-A10 and B8, but not DR2, were greater in the control subjects (p = 0.001 and 0.01 respectively). HLA-DR2 may be involved in the pathogenesis of advanced pulmonary tuberculosis. Study of endogamous, genetically disparate populations (caste) revealed other HLA associations (A3, B12 and DR4) unique to them, suggesting that genes linked with the HLA complex might also be significant in the pathogenesis of tuberculosis.

Spectrum of immune reactivity to mycobacterial (BCG) antigens in healthy hospital contacts in south India.

In an effort to study the immunological responses to antigens of tubercle bacilli, 49 tuberculin positive and 41 tuberculin negative hospital contacts aged 20-29 years (staff nurses and students working in Government Rajaji Hospital, Madurai, South India) were studied for serum antibodies (IgG, IgM and IgA classes) to BCG by ELISA and diameter of induration to PPD by Mantoux procedures. The two immunological parameters were correlated in regression analysis. The results have revealed higher anti-BCG serum antibody levels in hospital contacts than in non-contacts, significantly higher antibodies in tuberculin negative hospital contacts than in tuberculin positive hospital contacts, an inverse correlation of tuberculin reactivity and antibodies and a bimodal decline (regression) of antibodies against the increase in skin test induration. This study has thus suggested the existence of an immunological spectrum in hospital contacts from south India; persons at one pole of the spectrum were tuberculin negative and possessed significantly elevated antibody levels and those at the other pole of the spectrum were tuberculin positive and possessed low antibody levels. Thus the spectrum of immune reactivity may be due to an inherent susceptibility/resistance of an individual to Mycobacterium tuberculosis.

HLA Bw57 and DR7 association with psoriasis vulgaris in south India.

Eighty-three south Indian patients with psoriasis vulgaris were studied for HLA antigen frequencies and compared with 77 controls studied simultaneously. HLA Bw57, a split of B17 was found elevated in the patients. The two sexes differed in their age-at-onset curves: females had a preponderance to early onset of the disease, while the males had late onset. Among these patients, major group 3, a Western Brachycephal Armenoid group, revealed the highest risk for B17 & Bw57 but not major group 2, a Mediterranean one.