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HLA, complement C2, C4, properdin factor B and glyoxalase types in South Indian diabetics
Diabetes Research and Clinical Practice, 1 (1985) 41-47
Elsevier
41
DRC 00005
HLA, complement C2, C4, properdin factor B and glyoxalase types in South Indian diabetics* R.L. K i r k TM, P . R . R a n f o r d 1, S.W. S e r j e a n t s o n ~, A . R . T h o m p s o n 1, S.M. M u n i r a t h n a m C h e t t y 2, Lily J o h n 3, V. M o h a n * , A. R a m a c h a n d r a n 4, C. S n e h a l a t h a * a n d M. V i s w a n a t h a n * 1Human Biology Department. John Curtin School of Medical Research, Canberra, Australia, 2Medical College Hospital. Coimbatore, 3Christian Medical College. Vellore, and *Diabetes Research Centre, Madras, India
(Received 7 October 1984, accepted 10 December 1984)
Key words: Insulin-dependentdiabetes mellitus; Non-insulin-dependentdiabetes mellitus; South India; Chromosome 6 markers; HLA;
BF; C2; C4A; C4B
Summary A series of diabetic patients from 3 centres in South India have been tested for H L A A, HLA B, BF, C2, C4A, C4B and G L O types. For insulin-dependent diabetes mellitus (IDDM) patients there was a significant increase in H L A B8, of BF F and decrease of C4 A6. No significant variation in HLA, BF, C2 or G L O frequencies was found in non-insulin-dependent diabetes mellitus ( N I D D M ) patients, but there was a significant decrease in C4B 1 and an increase in C4B 2. The H L A and BF association in South Indian I D D M patients is very different from that reported previously in North India.
Introduction The prevalence of diabetes mellitus in India approximates 2% of adults aged 15 years and over [1], although the study under the auspices of the Indian Council for Medical Research showed there are marked differences between sub-populations within the country. For example, there is a higher prevalence among urban compared with rural groups
* This project was supported in part through N.I.H. Grant no. Rol AM 25446 to Professor Paul Zimmet. ** Address for correspondence: Dr. R.L. Kirk, Human Biology Department, John Curtin School of Medical Research, P.O. Box 334, Canberra 2601, Australia.
and among vegetarians the prevalence is twice that among non-vegetarians. Estimates of the relative prevalence of insulindependent diabetes (IDDM) and non-insulin-dependent diabetes ( N I D D M ) in Indians are more difficult to obtain. Omar [2] studying diabetes in South Africa considers that I D D M among Indians has a prevalence of 0.02% and such low prevalences have been reported in India by other workers [3-5]. I D D M among Indians also is claimed to be rare in Singapore [6] and in Fiji [7]. In addition to I D D M and N I D D M other clinical forms such as J-type diabetes and tropical pancreatic diabetes are present in parts of India and are characterized by insulin-dependence but resistance to ketosis [8].
0168-8227/85/$03.30 © 1985 ElsevierScience Publishers B.V. (Biomedical Division)
42 Recently attention has been given to the distribution of genetic markers in Indian diabetic patients. H a m m o n d and Asmal [9] presented data on HLA A and HLA B types for Indian diabetics in South Africa and Serjeantson et al. [10] carried out a similar survey in Fiji. Subsequently, Srikanta et al. [11] have studied HLA types in I D D M patients in Delhi and Kirk et al. [12] have added to this by showing a significant relationship with the properdin factor B type, BF SI in the Delhi I D D M patients. However, in a later study Kirk et al. [13] have shown that in South Indian I D D M the association is with BF F not BF S1. We have now been able to extend the series of patients examined and have also typed them for HLA A and HLA B, for complement C2, complement C4A and C4B and glyoxalase. The results presented here are contrasted with those for a series of non-insulin-dependent diabetics ( N I D D M ) and non-diabetic controls from the same geographical area.
Patients and Methods
Blood samples were obtained from a total of 96 IDDM and 73 N I D D M patients and 115 controls from 3 centres in South India (Madras, Vellore and Coimbatore). Patients were included in the study only if their place of birth was in 1 of the 4 southern states: Andhra Pradesh, Karnataka, Kerala and Tamil Nadu. Lymphocytes were separated on Ficoll-Isopaque columns and put into transport medium for shipment to Canberra, where HLA A and B testing by standard microcytotoxicity methods was completed on 32 IDDM, 49 N I D D M and 47 controls. At the time of separation of lymphocytes plasma and red cell aliquots were frozen and transported to Canberra in dry ice. BF typing was carried out by immunofixation using a specific anti-BF antiserum (Atlantic Antibodies) after agarose gel electrophoresis [14]. C2 typing was performed according to the method of Lachmann and Hobart [15] and C4A and C4B typing was done on desialized samples following the method of Awdeh and Alper [16]. Glyoxalase (GLO) typing was per-
TABLE I AGE D I S T R I B U T I O N O F D I A B E T I C PATIENTS SAMPLED IN SOUTH I N D I A
N1DDM (n = 73)
I D D M (n = 96)
Age of onset 0 6 I1 16 21 26 >30
%
Age of
%
onset 5 10 15 20 25 30
6.3 22.9 31.3 22.9 10.4 3.1 3.1
0 21 26 31 36 > 40
20 25 30 35 40
0 1.4 8.2 I1.0 16.4 63.0
formed by starch gel electrophoresis using the technique described by Ghosh [17]. I D D M patients were selected on the basis of age-of-onset less than 40 years, being ketosis prone and having a complete dependence on exogenous insulin. N I D D M patients were more heterogeneous with onset ranging from 21 years upwards (Table 1). All N I D D M patients were maintained by diet or oral drugs, though in some cases, exogenous insulin was required after the disease had persisted for more than 10 years. Fasting C-peptide values for a subset of these patients shows a clear distinction between those classified as I D D M (n = 40) and N I D D M (n = 42). The mean value for the former was 0.04 ± 0.003 pmol/ml whilst, for the latter, the mean was 0.36 q- 0.05 compared to 0.61 ± 0.04 for controls [18].
Results and Discussion
(1) H L A A and B The antigen frequencies for HLA A and H L A B together with the corresponding gene frequencies, estimated by maximum likelihood, are given in Table 2. The values for the control series are similar to those obtained by Wolf et al. [19] in a study of leprosy in South India. For I D D M patients, HLA AI and H L A B40 were significantly decreased and
43 TABLE 2 HLA A A N D HLA B A N T I G E N F R E Q U E N C I E S IN S O U T H I N D I A 1N I D D M A N D N I D D M D I A B E T I C PATIENTS A N D MATCHED CONTROLS
Controls (n = 47)
IDDM (n = 32)
NIDDM (n = 49)
n
%
n
%
n
%
14 16 2 18 13 0 0 6 2 2 4 6 2
29.8 34.0 4.3 38.3 27.7 0 0 12.8 4.3 4.3 8.5 12.8 4.3
3 6 6 17 12 1 2 5 3 0 1 3 I
9.4" 18.7 18.7" 53.1 37.5 3.1 6.2 15.6 9.4 0 3.1 9.4 3.1
18 20 3 16 17 0 0 3 4 I 5 4 0
36.7 40.8 6.1 32.7 34.7 0 0 6.1 8.2 2.0 10.2 8.2 0
17 4 2 0 0 5 13 4 4 4 2 14 2 2 13 I 4
36.2 8.5 4.3 0 0 10.6 27.7 8.5 8.5 8.5 4.3 29.8 4.3 4.3 27.7 2.1 8.5
12 7 9 1 1 2 4 3 I 3 0 6 0 0 2 I 1
37.5 21.9 28.1 b 3.1 3.1 6.2 12.5 9.4 3.1 9.4 0 18,7 0 0 6.2" 3.1 3. I
18 4 I 2 0 5 14 0 2 2 3 12 4 I 8 1 I
36.7 8.2 2,0 4.1 0 10.2 28.6 0 4.1 4.1 6.1 24.5 8.2 2.0 16.3 2.0 2.0
HLA A alltigell 1 2 3 9 11 19 25 26 28 29 30/31 32 33
HLA B anligetl 5 7 8 13 14 15 17 18 21 22 27 35 37 38 40 41 44 0.05 > P > 0.01. b 0.01 > P > 0.001.
HLA A3 and HLA B8 were significantly increased. However, there were no significant differences between antigen frequencies for N I D D M patients and controls. An increase in HLA B8 has been reported pre-
viously by Hammond and Asmal [9] for a small series (n = 23) of juvenile onset diabetics among Indians in South Africa who were categorized as 'Dravidian', that is, of South Indian origin. Hammond and Asmal, however, also reported a signifi-
44 TABLE 3 D I S T R I B U T I O N O F P R O P E R D I N F A C T O R B (BF) P H E N O T Y P E S A N D G E N E F R E Q U E N C I E S Phenotype nos.
% Gene frequencies
BF
no.
S
FS
F
FSI
SS1
BF*S
BF*F
BF*SI (0.7)
IDDM NIDDM Control
96 72 I 15
33 35 56
37 33 46
22 3 8
2 I I
2 0 4
54.7 71.5 70.4
43.2 a 27.8 27.4
2.1 0.7 2.2
0.01 > P > 0.001.
cant increase in HLA Bw52. We were not able to discriminate between Bw51 and Bw52 in our H L A B5 so it is possible that a similar increase in Bw52 might have been present in our own series. Neither did we observe the new antigenic type HLA B5IND which Hammond and Asmal found significantly increased in their 'Dravidian" diabetic series. The increase in HLA B8 among South Indian IDDM patients is in marked contrast to the absence of this association for IDDM patients in North India, as noted by H a m m o n d and Asmal [9] and also by Srikanta et al. [11]. These latter authors found a striking association with HLA Bw21, which was present in 35% of their patients compared to only 4% of controls. Compared with IDDM patients of European origin the increase in HLA B8 is the only common feature. The increases in HLA BI5 and BI8 which also are characteristic for IDDM in Europeans are not found among Indians, whether from the North or the South.
(2) Properdin.factor B Previously we have noted that the BF*F allele is strongly associated with I D D M in South India [13]. The present results extend and confirm this finding, as shown in Table 3: the BF*F gene frequency is 43% in I D D M compared to 27 and 28% respectively in controls and N I D D M . In persons of European origin there is a significant association with the rare factor BF F1 [20,21] which is in strong linkage disequilibrium with H L A B I8. In India not only is HLA B18 relatively infrequent, with a gene frequency approximating 2% [22,23] but BF F1 is absent. On the other hand BF SI, which is of slightly lower frequency than BF F1 in Europeans, is present in India and is very strongly associated with I D D M in North India with a relative risk of 7.3 [12]. However, BF SI is not increased in the present series of I D D M patients from South India.
(3) Complement C2 No previous reports on complement C2 types in
TABLE 4 D I S T R I B U T I O N O F C O M P L E M E N T C2 P H E N O T Y P E S A N D G E N E F R E Q U E N C I E S Phenotype nos.
% Gene frequencies
C2
no.
I- I
2- I
2-2
I-V
C2" I
C2"2
C2" V
IDDM NIDDM Control
96 72 114
87 58 102
7 I1 12
I 2 0
1 1 0
94.8 88.9 94.7
4.7 10.4 5.3
0.5 0.7 0
45 Indian populations are available. The results given in Table 4 reveal no difference in phenotypes between IDDM patients and controls in the present series. There is an increase in the C2"2 gene frequency among NIDDM patients (10.4% vs. 5.3%) but the difference is not significant at the P = 0.05 level. Although the C2"2 gene frequency for the Indian controls in the present series is similar to that for Europeans, it is of interest that the significant increase in C2"2 which has been reported in IDDM
patients in Australia, Europe and the U.S.A. by various workers [24,25] is not present among the South Indian IDDM patients. In Europeans, C2"2 is in linkage disequilibrium with the IDDM risk factor HLA BI5 [26-28] so that the absence of any increase of C2"2 in the present series is consistent with the lack of increase o f H L A BI5 in the IDDM patients.
(4) Complement C4A and C4B No previous reports on complement C4A and C4B
TABLE 5 C4 P H E N O T Y P E A N D G E N E F R E Q U E N C I E S IN SOUTH I N D I A IDDM n
NIDDM %
n
Control %
n
%
C4A
phenotype 2-2 2-3 2-6 3-3 3-4 3-5 3-6 4-4 4-6 6-6 0-0
1 1 1 70 9 1 1 1 0 1 2
1.1 1.1 1.1 79.7 10.3 1.1 1.1 1.1 0 1.1 2.3
0 1 0 46 8 0 8 3 1 I 0
0 1.5 0 67.7 11.7 0 11.7 4.4 1.5 1.5 0
1 0 I 66 6 1 14 3 2 4 1
1.0 0 1.0 66.7 6.1 1.0 14.2 3.0 2.0 4.0 1.0
Total
88
100.0
68
100.0
99
I00.0
1-1 1-2 1-3 1-5 1.1-I.1 a 2-2 3-3 0-0
64 13 1 2 0 2 1 5
72.7 14.8 1.1 2.3 0 2.3 1.1 5.7
38 I5 0 1 2 II 0 I
55.9 22.0 0 1.5 2.9 16.2 0 1.5
79 14 1 0 0 4 0 1
79.8 14.1 1.0 0 0 4.1 0 1.0
Total
88
100.0
68
100.0
99
100.0
C4A allele
IDDM %
NIDDM %
Control %
2 3 4 5 6 0
1.7 74.7 5.8 0.6 1.7 b 15.5
0.7 74.5 9.7 0 7.6 7.5
1.0 67.6 5.8 0.5 11.2 13.9
C4B allele
IDDM %
NIDDM %
Control %
l I.I 2 3 5 0
67.9 0 8.7 1.2 1.1 21.1
56.2 b 1.5 22.1 b 0 0.7 19.5
78.3 0 9.7 0.5 0 11.5
C4B phenoo'pe
a Two individuals gave a C4B pattern with bands slightly more anodal than the bands for C4B 1. This pattern has been designated C4B l.l. b 0.01 > P > 0.001.
46 typing have been published for populations in India. Table 5, therefore, provides valuable data for the normal controls as well as the I D D M and N I D D M series. A comparison of antigen frequencies reveals a significant decrease in C4A 6 among I D D M patients, but no significant decrease for C4B antigens. By contrast 2 C4B antigens are significantly associated with N I D D M , C4B 1 being decreased and C4B 2 increased in N I D D M patients. Since C4A 4 and C4B 2 are in linkage disequilibrium in Europeans [29] it is of interest that the C4A 4 antigen frequency is increased in our N I D D M patients compared to controls (17.6% vs. 11.1%) although the difference is not significant.
for a complete range of chromosome 6 markers. For Europeans such screening results in a sharp discrimination between I D D M and N I D D M patients. No consistent pattern of association with any of the markers on chromosome 6 have been demonstrated in the case of N I D D M . By contrast, series of I D D M patients reveal increases in several H L A A, B and D R antigens with BF S and BF F1, with C2 2 and with C4A Q0 and C4B 3. Because of linkage disequilibrium the pattern of increases in these various factors leads to an increase in some extended haplotypes, the most common of which in European I D D M patients are: (I) H L A B8, DR3; BF S; C2 1; C4A Q0; C4B 1 (2) H L A B15, DR4; BF S; C2 1; C4A 3; C4B 3 (3) H L A B18, DR3; BF F1; C2 1; C4A 3; C4B
(5) Gb, oxalase Ghosh [17] gives the GLO*I gene frequencies for Indians in the range of 14-30%, with the majority of values between 21 and 28 %. In the present series all the GLO*I values fall within this range and although the GLO*I frequencies, as shown in Table 6, for I D D M and N I D D M are increased above that in the controls, the differences are not significant. The absence of an association between one of the glyoxalase alleles and I D D M is in agreement with our failure to find a glyoxalase association in a large series of I D D M patients in Australia [30]. The same study reported a disturbance in the Hardy-Weinberg distribution of glyoxalase types among N I D D M patients, but there is no such effect in the present series of N I D D M patients in South India (X211) = 0.8 > P > 0.3). Few diabetic populations other than ones in Europe or of European origin have been screened
Q0 In the present series of I D D M patients in South India, we have not been able to identify these extended haplotypes, with the possible exception of no. (1). We were unable to type these samples for H L A DR. If the D R type is ignored only 9 of the I D D M patients carried H L A B8 and 7 of these could have been also BF S; C2 1; C4A Q0; C4B 1. However, for 5 of these the BF allele could have been BF*F, and because of the significant increase in BF*Fin the diabetic series this seems more likely. Moreover we cannot with certainty assign C4A Q0 to this haplotype. Family studies will be needed to determine the extended haplotypes without ambiguity. The present results show that the chromosome 6 markers clearly distinguish I D D M patients in South India from those in North India. The increase in H L A B21 and BF S1 present in the latter
TABLE 6 DISTRIBUTION OF GLYOXALASE PHENOTYPES AND GENE FREQUENCIES % Gene frequencies
Phenotype nos. GLO
no.
I-1
2-1
2-2
GLO*I
GLO*2
IDDM NIDDM Control
75 72 96
6 7 9
29 25 23
40 40 64
27.3 27.1 21.3
72.7 72.9 78.7
47 is not a feature of the South Indian series. Nor was there any example of the HLA BI8, BF F1 combination which is commonly found among European I D D M patients. Clearly, whatever the susceptibility factor is, it occurs in different haplotype combinations in different parts of the world and this differentiation is present even within India itself. References I Ahuja, M.M.S., Epidemiological studies on diabetes mellitus in India. In: M.M.S. Ahuja (ed.), Epidemiology of Diabetes hi Developing Countries, Interprint, New Delhi, 1979, pp. 29-38. 2 Omar, M.A.K., A Study of Diabetes Mellitus in Young Africans and Indians (Age of Onset under 35) in Natal (Thesis for Doctor of Medicine), University of Natal, Durban, 1982. 3 Patel, J.C. and Talwalker, N.G., Diabetes in the Tropics, Diabetes Association of India, Bombay, 1966. 4 Viswanathan, M., Mohammed, N. and Krishnamoorthy, M., Diabetes in the Young. A study of 166 cases. In: J.C. Patel and N.G. Talwalker (eds.), Diabetes in the Tropics, Diabetes Association of India, Bombay, 1966, pp. 277 281. 5 Sathe, R.V., The problem of diabetes mellitus in India, J. h, dian Med. Assoc.. 61: 12-16, 1973. 6 Tulloch, J.A., Diabetes Mellitus hi the Tropics, Livingstone, Edinburgh, 1962. 7 Cassidy, J., Diabetes in Fiji, N.Z. Med. J. 66: 167-172, 1967. 8 Viswanathan, M., Pancreatic diabetes in India: an overview. In: S. Podolsky, and M. Viswanathan, (eds.), Secondary Diabetes: Tire Spectrum of the Diabetic Syndromes, Raven Press, New York, 1980, pp. 105 116. 9 Hammond, M.G. and Asmal, A.C., HLA and insulin dependent diabetes in South African Indians, Tissue Antigens, 15: 244 248, 1980. 10 Serjeantson, S.W., Ryan, D.P., Ram, P. and Zimmet, P., HLA and non-insulin dependent diabetes in Fiji Indians, Med. J. Aust., 1:462 463, 1981. 11 Srikanta, N.K., Mehra, M.C., Vaidya, A.N., Malaviya, A.N. and Ahuja, M.M.S., HLA antigens in type 1 (insulin dependent) diabetes mellitus in north India, Metabolism, 30: 992 993, 1981. 12 Kirk, R.L., Ranford, P.R., Theophilus, J., Ahuja, M.M.S., Mehra, N.K. and Vaidya, M.C., The rare factor BfS1 of the properdin system strongly associated with insulin-dependent diabetes in north India, Tissue Antigens, 20: 303-304, 1982. 13 Kirk, R.L., Ranford, P.R., Viswanathan, M., Mohan, V., Ramachandran, A., Snehalatha, C., Munirathnam Chetty, S.M. and John, L., Another association between the properdin system (BF) and insulin-dependent diabetes in south India, Tissue antigens. 22:170 171, 1983. 14 Alper, C.A., Boenisch, T. and Watson, L., Genetic poly-
15
16
17 18
19
20
21
22
23
24
25
26
27
28
29
30
morphism in human glycine-rich beta-glycoprotein, J. Exp. Med.. 135: 68-80, 1972. Lachmann, P.J. and Hobart, M.J., Complement Technology. In: D.M. Weir (ed.), Handbook of Experimental Immunology, Blackwell Scientific Publications, Oxford, 1978, pp. 5A1 23. Awdeh, Z.L. and Alper, C.A., Inherited polymorphism of human C4 as revealed by desialyzation, lmmunobiology, 158: 35 41, 1980. Ghosh, A.K., Polymorphism of red cell glyoxalase I, Hum. Genet., 39: 91-95, 1977. Mohan, V., Snehalatha, C., Ramachandran, A., Jayashree, R. and Viswanathan, M., Pancreatic/~-cell function in tropical pancreatic diabetes, Metabolism, 32: 1091-1092, 1983. Wolf, E., Fine, P.E.M., Pritchard, J., Watson, B., Bradley, D.J., Festenstein, H., Chacko, C.J.G. and Stevens, A., HLA-A, B and C antigens in south Indian families with leprosy, Tissue Antigens, 15: 436-446, 1980. Raum, D., Awdeh, Z. and Alper, C.A., BF types and the mode of inheritance of insulin-dependent diabetes mellitus (IDDM), Immunogenetics, 12: 59-74, 1981. Kirk, R.L., Serjeantson, S.W., Theophilus, J., Zimmet, P., Whitehouse, S. and Court, J.M., Age relationship between insulin-dependent diabetes and rare alleles of properdin factor B, Lancet, ii, 537, 1979. Mittal, K.K., Naik, S., Sansonetti, N., Cowhered, R., Kumar, R. and Wong, D.M., The HLA antigens in Indian Hindus, Tissue Antigens, 20:223 226, 1982. Verma, I.C., Mathews, A.R. and Choudhary, S.S., Histocompatibility antigens (A, B and C loci) in indians in Delhi, Indian J. Med. Res., 78: 233-238, 1983. Kirk, R.L., Ranford, P.R., Court, J. and Zimmet, P., Distribution of complement C2 and C6 types in Australian cases of diabetes mellitus, Med. J. Aust., 2: 614-615, 1980. Raum, D., Sosenko, S.M., Gabbay, K.H., Stein, R. and AIper, C.A., Bf and C2 polymorphism in insulin dependent diabetes, J. Immunol., 124: 1537, 1980. Alper, C.A., Inherited structural polymorphism in human C2: evidence for genetic linkage between C2 and BF, J. Exp. Med., 144:1111 1115, 1976. Meo, T., Atkinson, J., Bernoco, M., Bernoco, D. and Cepellini, R., Mapping of HLA locus controlling C2 structural variants and linkage disequilibrium between C22 and Bwl5, Eur. J. Immunol., 6: 916-919, 1976. Dewald, G. and Rittner, C., Polymorphism of the second component of human complement (C2), Vox Sang., 37: 47 54, 1979. Awdeh, Z,L., Raum, D., Yunis, E.J. and Alper, C.A., Extended HLA/complement allele haplotypes: Evidence for T/t-like complex in man, Proc. Natl. Acad. Sci. U.S.A., 80: 259 263, 1983. Kirk, R.L., Theophilus, J., Whitehouse, S., Court, J. and Zimmet, P., Genetic susceptibility to diabetes mellitus: the distribution of properdin factor B (Bf) and glyoxalase (GLO) phenotypes, Diabetes, 28: 949-951, 1979.
Elsevier
41
DRC 00005
HLA, complement C2, C4, properdin factor B and glyoxalase types in South Indian diabetics* R.L. K i r k TM, P . R . R a n f o r d 1, S.W. S e r j e a n t s o n ~, A . R . T h o m p s o n 1, S.M. M u n i r a t h n a m C h e t t y 2, Lily J o h n 3, V. M o h a n * , A. R a m a c h a n d r a n 4, C. S n e h a l a t h a * a n d M. V i s w a n a t h a n * 1Human Biology Department. John Curtin School of Medical Research, Canberra, Australia, 2Medical College Hospital. Coimbatore, 3Christian Medical College. Vellore, and *Diabetes Research Centre, Madras, India
(Received 7 October 1984, accepted 10 December 1984)
Key words: Insulin-dependentdiabetes mellitus; Non-insulin-dependentdiabetes mellitus; South India; Chromosome 6 markers; HLA;
BF; C2; C4A; C4B
Summary A series of diabetic patients from 3 centres in South India have been tested for H L A A, HLA B, BF, C2, C4A, C4B and G L O types. For insulin-dependent diabetes mellitus (IDDM) patients there was a significant increase in H L A B8, of BF F and decrease of C4 A6. No significant variation in HLA, BF, C2 or G L O frequencies was found in non-insulin-dependent diabetes mellitus ( N I D D M ) patients, but there was a significant decrease in C4B 1 and an increase in C4B 2. The H L A and BF association in South Indian I D D M patients is very different from that reported previously in North India.
Introduction The prevalence of diabetes mellitus in India approximates 2% of adults aged 15 years and over [1], although the study under the auspices of the Indian Council for Medical Research showed there are marked differences between sub-populations within the country. For example, there is a higher prevalence among urban compared with rural groups
* This project was supported in part through N.I.H. Grant no. Rol AM 25446 to Professor Paul Zimmet. ** Address for correspondence: Dr. R.L. Kirk, Human Biology Department, John Curtin School of Medical Research, P.O. Box 334, Canberra 2601, Australia.
and among vegetarians the prevalence is twice that among non-vegetarians. Estimates of the relative prevalence of insulindependent diabetes (IDDM) and non-insulin-dependent diabetes ( N I D D M ) in Indians are more difficult to obtain. Omar [2] studying diabetes in South Africa considers that I D D M among Indians has a prevalence of 0.02% and such low prevalences have been reported in India by other workers [3-5]. I D D M among Indians also is claimed to be rare in Singapore [6] and in Fiji [7]. In addition to I D D M and N I D D M other clinical forms such as J-type diabetes and tropical pancreatic diabetes are present in parts of India and are characterized by insulin-dependence but resistance to ketosis [8].
0168-8227/85/$03.30 © 1985 ElsevierScience Publishers B.V. (Biomedical Division)
42 Recently attention has been given to the distribution of genetic markers in Indian diabetic patients. H a m m o n d and Asmal [9] presented data on HLA A and HLA B types for Indian diabetics in South Africa and Serjeantson et al. [10] carried out a similar survey in Fiji. Subsequently, Srikanta et al. [11] have studied HLA types in I D D M patients in Delhi and Kirk et al. [12] have added to this by showing a significant relationship with the properdin factor B type, BF SI in the Delhi I D D M patients. However, in a later study Kirk et al. [13] have shown that in South Indian I D D M the association is with BF F not BF S1. We have now been able to extend the series of patients examined and have also typed them for HLA A and HLA B, for complement C2, complement C4A and C4B and glyoxalase. The results presented here are contrasted with those for a series of non-insulin-dependent diabetics ( N I D D M ) and non-diabetic controls from the same geographical area.
Patients and Methods
Blood samples were obtained from a total of 96 IDDM and 73 N I D D M patients and 115 controls from 3 centres in South India (Madras, Vellore and Coimbatore). Patients were included in the study only if their place of birth was in 1 of the 4 southern states: Andhra Pradesh, Karnataka, Kerala and Tamil Nadu. Lymphocytes were separated on Ficoll-Isopaque columns and put into transport medium for shipment to Canberra, where HLA A and B testing by standard microcytotoxicity methods was completed on 32 IDDM, 49 N I D D M and 47 controls. At the time of separation of lymphocytes plasma and red cell aliquots were frozen and transported to Canberra in dry ice. BF typing was carried out by immunofixation using a specific anti-BF antiserum (Atlantic Antibodies) after agarose gel electrophoresis [14]. C2 typing was performed according to the method of Lachmann and Hobart [15] and C4A and C4B typing was done on desialized samples following the method of Awdeh and Alper [16]. Glyoxalase (GLO) typing was per-
TABLE I AGE D I S T R I B U T I O N O F D I A B E T I C PATIENTS SAMPLED IN SOUTH I N D I A
N1DDM (n = 73)
I D D M (n = 96)
Age of onset 0 6 I1 16 21 26 >30
%
Age of
%
onset 5 10 15 20 25 30
6.3 22.9 31.3 22.9 10.4 3.1 3.1
0 21 26 31 36 > 40
20 25 30 35 40
0 1.4 8.2 I1.0 16.4 63.0
formed by starch gel electrophoresis using the technique described by Ghosh [17]. I D D M patients were selected on the basis of age-of-onset less than 40 years, being ketosis prone and having a complete dependence on exogenous insulin. N I D D M patients were more heterogeneous with onset ranging from 21 years upwards (Table 1). All N I D D M patients were maintained by diet or oral drugs, though in some cases, exogenous insulin was required after the disease had persisted for more than 10 years. Fasting C-peptide values for a subset of these patients shows a clear distinction between those classified as I D D M (n = 40) and N I D D M (n = 42). The mean value for the former was 0.04 ± 0.003 pmol/ml whilst, for the latter, the mean was 0.36 q- 0.05 compared to 0.61 ± 0.04 for controls [18].
Results and Discussion
(1) H L A A and B The antigen frequencies for HLA A and H L A B together with the corresponding gene frequencies, estimated by maximum likelihood, are given in Table 2. The values for the control series are similar to those obtained by Wolf et al. [19] in a study of leprosy in South India. For I D D M patients, HLA AI and H L A B40 were significantly decreased and
43 TABLE 2 HLA A A N D HLA B A N T I G E N F R E Q U E N C I E S IN S O U T H I N D I A 1N I D D M A N D N I D D M D I A B E T I C PATIENTS A N D MATCHED CONTROLS
Controls (n = 47)
IDDM (n = 32)
NIDDM (n = 49)
n
%
n
%
n
%
14 16 2 18 13 0 0 6 2 2 4 6 2
29.8 34.0 4.3 38.3 27.7 0 0 12.8 4.3 4.3 8.5 12.8 4.3
3 6 6 17 12 1 2 5 3 0 1 3 I
9.4" 18.7 18.7" 53.1 37.5 3.1 6.2 15.6 9.4 0 3.1 9.4 3.1
18 20 3 16 17 0 0 3 4 I 5 4 0
36.7 40.8 6.1 32.7 34.7 0 0 6.1 8.2 2.0 10.2 8.2 0
17 4 2 0 0 5 13 4 4 4 2 14 2 2 13 I 4
36.2 8.5 4.3 0 0 10.6 27.7 8.5 8.5 8.5 4.3 29.8 4.3 4.3 27.7 2.1 8.5
12 7 9 1 1 2 4 3 I 3 0 6 0 0 2 I 1
37.5 21.9 28.1 b 3.1 3.1 6.2 12.5 9.4 3.1 9.4 0 18,7 0 0 6.2" 3.1 3. I
18 4 I 2 0 5 14 0 2 2 3 12 4 I 8 1 I
36.7 8.2 2,0 4.1 0 10.2 28.6 0 4.1 4.1 6.1 24.5 8.2 2.0 16.3 2.0 2.0
HLA A alltigell 1 2 3 9 11 19 25 26 28 29 30/31 32 33
HLA B anligetl 5 7 8 13 14 15 17 18 21 22 27 35 37 38 40 41 44 0.05 > P > 0.01. b 0.01 > P > 0.001.
HLA A3 and HLA B8 were significantly increased. However, there were no significant differences between antigen frequencies for N I D D M patients and controls. An increase in HLA B8 has been reported pre-
viously by Hammond and Asmal [9] for a small series (n = 23) of juvenile onset diabetics among Indians in South Africa who were categorized as 'Dravidian', that is, of South Indian origin. Hammond and Asmal, however, also reported a signifi-
44 TABLE 3 D I S T R I B U T I O N O F P R O P E R D I N F A C T O R B (BF) P H E N O T Y P E S A N D G E N E F R E Q U E N C I E S Phenotype nos.
% Gene frequencies
BF
no.
S
FS
F
FSI
SS1
BF*S
BF*F
BF*SI (0.7)
IDDM NIDDM Control
96 72 I 15
33 35 56
37 33 46
22 3 8
2 I I
2 0 4
54.7 71.5 70.4
43.2 a 27.8 27.4
2.1 0.7 2.2
0.01 > P > 0.001.
cant increase in HLA Bw52. We were not able to discriminate between Bw51 and Bw52 in our H L A B5 so it is possible that a similar increase in Bw52 might have been present in our own series. Neither did we observe the new antigenic type HLA B5IND which Hammond and Asmal found significantly increased in their 'Dravidian" diabetic series. The increase in HLA B8 among South Indian IDDM patients is in marked contrast to the absence of this association for IDDM patients in North India, as noted by H a m m o n d and Asmal [9] and also by Srikanta et al. [11]. These latter authors found a striking association with HLA Bw21, which was present in 35% of their patients compared to only 4% of controls. Compared with IDDM patients of European origin the increase in HLA B8 is the only common feature. The increases in HLA BI5 and BI8 which also are characteristic for IDDM in Europeans are not found among Indians, whether from the North or the South.
(2) Properdin.factor B Previously we have noted that the BF*F allele is strongly associated with I D D M in South India [13]. The present results extend and confirm this finding, as shown in Table 3: the BF*F gene frequency is 43% in I D D M compared to 27 and 28% respectively in controls and N I D D M . In persons of European origin there is a significant association with the rare factor BF F1 [20,21] which is in strong linkage disequilibrium with H L A B I8. In India not only is HLA B18 relatively infrequent, with a gene frequency approximating 2% [22,23] but BF F1 is absent. On the other hand BF SI, which is of slightly lower frequency than BF F1 in Europeans, is present in India and is very strongly associated with I D D M in North India with a relative risk of 7.3 [12]. However, BF SI is not increased in the present series of I D D M patients from South India.
(3) Complement C2 No previous reports on complement C2 types in
TABLE 4 D I S T R I B U T I O N O F C O M P L E M E N T C2 P H E N O T Y P E S A N D G E N E F R E Q U E N C I E S Phenotype nos.
% Gene frequencies
C2
no.
I- I
2- I
2-2
I-V
C2" I
C2"2
C2" V
IDDM NIDDM Control
96 72 114
87 58 102
7 I1 12
I 2 0
1 1 0
94.8 88.9 94.7
4.7 10.4 5.3
0.5 0.7 0
45 Indian populations are available. The results given in Table 4 reveal no difference in phenotypes between IDDM patients and controls in the present series. There is an increase in the C2"2 gene frequency among NIDDM patients (10.4% vs. 5.3%) but the difference is not significant at the P = 0.05 level. Although the C2"2 gene frequency for the Indian controls in the present series is similar to that for Europeans, it is of interest that the significant increase in C2"2 which has been reported in IDDM
patients in Australia, Europe and the U.S.A. by various workers [24,25] is not present among the South Indian IDDM patients. In Europeans, C2"2 is in linkage disequilibrium with the IDDM risk factor HLA BI5 [26-28] so that the absence of any increase of C2"2 in the present series is consistent with the lack of increase o f H L A BI5 in the IDDM patients.
(4) Complement C4A and C4B No previous reports on complement C4A and C4B
TABLE 5 C4 P H E N O T Y P E A N D G E N E F R E Q U E N C I E S IN SOUTH I N D I A IDDM n
NIDDM %
n
Control %
n
%
C4A
phenotype 2-2 2-3 2-6 3-3 3-4 3-5 3-6 4-4 4-6 6-6 0-0
1 1 1 70 9 1 1 1 0 1 2
1.1 1.1 1.1 79.7 10.3 1.1 1.1 1.1 0 1.1 2.3
0 1 0 46 8 0 8 3 1 I 0
0 1.5 0 67.7 11.7 0 11.7 4.4 1.5 1.5 0
1 0 I 66 6 1 14 3 2 4 1
1.0 0 1.0 66.7 6.1 1.0 14.2 3.0 2.0 4.0 1.0
Total
88
100.0
68
100.0
99
I00.0
1-1 1-2 1-3 1-5 1.1-I.1 a 2-2 3-3 0-0
64 13 1 2 0 2 1 5
72.7 14.8 1.1 2.3 0 2.3 1.1 5.7
38 I5 0 1 2 II 0 I
55.9 22.0 0 1.5 2.9 16.2 0 1.5
79 14 1 0 0 4 0 1
79.8 14.1 1.0 0 0 4.1 0 1.0
Total
88
100.0
68
100.0
99
100.0
C4A allele
IDDM %
NIDDM %
Control %
2 3 4 5 6 0
1.7 74.7 5.8 0.6 1.7 b 15.5
0.7 74.5 9.7 0 7.6 7.5
1.0 67.6 5.8 0.5 11.2 13.9
C4B allele
IDDM %
NIDDM %
Control %
l I.I 2 3 5 0
67.9 0 8.7 1.2 1.1 21.1
56.2 b 1.5 22.1 b 0 0.7 19.5
78.3 0 9.7 0.5 0 11.5
C4B phenoo'pe
a Two individuals gave a C4B pattern with bands slightly more anodal than the bands for C4B 1. This pattern has been designated C4B l.l. b 0.01 > P > 0.001.
46 typing have been published for populations in India. Table 5, therefore, provides valuable data for the normal controls as well as the I D D M and N I D D M series. A comparison of antigen frequencies reveals a significant decrease in C4A 6 among I D D M patients, but no significant decrease for C4B antigens. By contrast 2 C4B antigens are significantly associated with N I D D M , C4B 1 being decreased and C4B 2 increased in N I D D M patients. Since C4A 4 and C4B 2 are in linkage disequilibrium in Europeans [29] it is of interest that the C4A 4 antigen frequency is increased in our N I D D M patients compared to controls (17.6% vs. 11.1%) although the difference is not significant.
for a complete range of chromosome 6 markers. For Europeans such screening results in a sharp discrimination between I D D M and N I D D M patients. No consistent pattern of association with any of the markers on chromosome 6 have been demonstrated in the case of N I D D M . By contrast, series of I D D M patients reveal increases in several H L A A, B and D R antigens with BF S and BF F1, with C2 2 and with C4A Q0 and C4B 3. Because of linkage disequilibrium the pattern of increases in these various factors leads to an increase in some extended haplotypes, the most common of which in European I D D M patients are: (I) H L A B8, DR3; BF S; C2 1; C4A Q0; C4B 1 (2) H L A B15, DR4; BF S; C2 1; C4A 3; C4B 3 (3) H L A B18, DR3; BF F1; C2 1; C4A 3; C4B
(5) Gb, oxalase Ghosh [17] gives the GLO*I gene frequencies for Indians in the range of 14-30%, with the majority of values between 21 and 28 %. In the present series all the GLO*I values fall within this range and although the GLO*I frequencies, as shown in Table 6, for I D D M and N I D D M are increased above that in the controls, the differences are not significant. The absence of an association between one of the glyoxalase alleles and I D D M is in agreement with our failure to find a glyoxalase association in a large series of I D D M patients in Australia [30]. The same study reported a disturbance in the Hardy-Weinberg distribution of glyoxalase types among N I D D M patients, but there is no such effect in the present series of N I D D M patients in South India (X211) = 0.8 > P > 0.3). Few diabetic populations other than ones in Europe or of European origin have been screened
Q0 In the present series of I D D M patients in South India, we have not been able to identify these extended haplotypes, with the possible exception of no. (1). We were unable to type these samples for H L A DR. If the D R type is ignored only 9 of the I D D M patients carried H L A B8 and 7 of these could have been also BF S; C2 1; C4A Q0; C4B 1. However, for 5 of these the BF allele could have been BF*F, and because of the significant increase in BF*Fin the diabetic series this seems more likely. Moreover we cannot with certainty assign C4A Q0 to this haplotype. Family studies will be needed to determine the extended haplotypes without ambiguity. The present results show that the chromosome 6 markers clearly distinguish I D D M patients in South India from those in North India. The increase in H L A B21 and BF S1 present in the latter
TABLE 6 DISTRIBUTION OF GLYOXALASE PHENOTYPES AND GENE FREQUENCIES % Gene frequencies
Phenotype nos. GLO
no.
I-1
2-1
2-2
GLO*I
GLO*2
IDDM NIDDM Control
75 72 96
6 7 9
29 25 23
40 40 64
27.3 27.1 21.3
72.7 72.9 78.7
47 is not a feature of the South Indian series. Nor was there any example of the HLA BI8, BF F1 combination which is commonly found among European I D D M patients. Clearly, whatever the susceptibility factor is, it occurs in different haplotype combinations in different parts of the world and this differentiation is present even within India itself. References I Ahuja, M.M.S., Epidemiological studies on diabetes mellitus in India. In: M.M.S. Ahuja (ed.), Epidemiology of Diabetes hi Developing Countries, Interprint, New Delhi, 1979, pp. 29-38. 2 Omar, M.A.K., A Study of Diabetes Mellitus in Young Africans and Indians (Age of Onset under 35) in Natal (Thesis for Doctor of Medicine), University of Natal, Durban, 1982. 3 Patel, J.C. and Talwalker, N.G., Diabetes in the Tropics, Diabetes Association of India, Bombay, 1966. 4 Viswanathan, M., Mohammed, N. and Krishnamoorthy, M., Diabetes in the Young. A study of 166 cases. In: J.C. Patel and N.G. Talwalker (eds.), Diabetes in the Tropics, Diabetes Association of India, Bombay, 1966, pp. 277 281. 5 Sathe, R.V., The problem of diabetes mellitus in India, J. h, dian Med. Assoc.. 61: 12-16, 1973. 6 Tulloch, J.A., Diabetes Mellitus hi the Tropics, Livingstone, Edinburgh, 1962. 7 Cassidy, J., Diabetes in Fiji, N.Z. Med. J. 66: 167-172, 1967. 8 Viswanathan, M., Pancreatic diabetes in India: an overview. In: S. Podolsky, and M. Viswanathan, (eds.), Secondary Diabetes: Tire Spectrum of the Diabetic Syndromes, Raven Press, New York, 1980, pp. 105 116. 9 Hammond, M.G. and Asmal, A.C., HLA and insulin dependent diabetes in South African Indians, Tissue Antigens, 15: 244 248, 1980. 10 Serjeantson, S.W., Ryan, D.P., Ram, P. and Zimmet, P., HLA and non-insulin dependent diabetes in Fiji Indians, Med. J. Aust., 1:462 463, 1981. 11 Srikanta, N.K., Mehra, M.C., Vaidya, A.N., Malaviya, A.N. and Ahuja, M.M.S., HLA antigens in type 1 (insulin dependent) diabetes mellitus in north India, Metabolism, 30: 992 993, 1981. 12 Kirk, R.L., Ranford, P.R., Theophilus, J., Ahuja, M.M.S., Mehra, N.K. and Vaidya, M.C., The rare factor BfS1 of the properdin system strongly associated with insulin-dependent diabetes in north India, Tissue Antigens, 20: 303-304, 1982. 13 Kirk, R.L., Ranford, P.R., Viswanathan, M., Mohan, V., Ramachandran, A., Snehalatha, C., Munirathnam Chetty, S.M. and John, L., Another association between the properdin system (BF) and insulin-dependent diabetes in south India, Tissue antigens. 22:170 171, 1983. 14 Alper, C.A., Boenisch, T. and Watson, L., Genetic poly-
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