- Email: [email protected]
HLA polymorphism in type 1 diabetes Tunisians
Annales de Génétique 45 (2002) 45–50 www.elsevier.com/locate/angen
Original Article
HLA polymorphism in type 1 diabetes Tunisians H. Abid Kamoun a, S. Hmida b,*, H. Kaabi b, A. Abid c, H. Slimane Houissa a, M. Maamar b, N. Mojaat b, L. Ben Hamed b, A. Dridi b, M. Kamoun Zribi a, K. Nagati c, A. Haddad a, K. Boukef b b
a National Blood Transfusion Center, Tunis, Tunisia Service de Médecine Interne B Hôpital Charles Nicolle, Tunis, Tunisia c National Institute of Nutrition, Tunis, Tunisia
Received 16 November 2001; accepted 23 January 2002
Abstract Several studies of the association between HLA and type 1 diabetes have been carried out revealing differences between ethnic groups. Our study, as part of the studies that should be performed about this association in the rest of the word, aims at elucidating the HLA DRB1, DQB1 polymorphism in Tunisian type 1 diabetes. This study includes 43 unrelated type 1 diabetes patients, and their mean age at onset is less than 15 years. Analysis of the frequency of alleles and haplotypes in these subjects, compared to a reference group (n = 101) led to the following results. 1) The Tunisian insulin-dependent diabetics present similarities as well as differences with other ethnic groups (Caucasians, North Africans). 2) The haplotype DRB1*04 DQ*0302 and DRB1*03 DQB1*0201 is positively associated to type 1 diabetes. 3) The heterozygotic genotype DRB1*04 DQB1*0302 / DRB1*03 DQB1*0201 is strongly associated to type 1 diabetes. 4) The haplotypes DRB1*01501 DQB1*0602 and DRB1*11 DQB1*0301 proved to be protective. In addition, the study of the subtypes DRB1*04 showed that alleles DRB1*0405 predispose to type 1 diabetes, whereas the allele DRB1*0403, which is in linkage disequilibrium with the DQB1*0402 in the Tunisian population, has a protective effect. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Type 1 diabetes mellitus; HLA Cl II; HLA–DRB1, DQB1; DNA typing
1. Introduction Insulin-dependent diabetes mellitus (IDDM) or diabetes type 1 results from an auto-immune selective and irreversible destruction of β-cells of Langerhans [8,26]. The etiology is still a mystery but is known that it involves genetic and environmental factors. Genetically, the occurrence of diabetes mellitus depends on several genes and mainly on the one situated at the level of the major histo-compatibility complex. Among the different genes of the HLA system, those which belong to the class II molecules (DR, DQ, DP) play a major role in the presentation of the peptide which is the initial stage in the immune response [22,25]. Studies carried out during the last 10 years revealed that 90–95% of
* Corresponding author. Fax: 216 1562 957. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 0 0 3 - 3 9 9 5 ( 0 2 ) 0 1 1 0 4 - 8
Caucasian insulin-dependent diabetics belonged to the group HLA DR3 and/or DR4 against 40–50% from the reference group. A higher risk was observed in heterozygotes DR3/DR4 suggesting that predisposition should result from two rather than one genetic factors. Yet, studies of different populations revealed that the frequencies of the different HLA antigens and their associations with diseases varied with the ethnic and geographic origins of the populations [23,24,25,28,31]. DR2 is protective against IDDM patients, DR3 and DR4 are, on the other hand, associated to IDDM but this association is not constant [14,25]. The association is for example negative with DR3 (Japanese) and with DR4 (Chinese), but it is positive with DR9 (Chinese, Japanese, Blacks) and with DR7 (Blacks). That is why this study aims at completing and enriching the data already published on Tunisian type 1 diabetes by adopting a molecular approach [13].
46
Original article / Annales de Génétique 45 (2002) 45–50
2. Patients and method
hybrids by emission of light at 477 nm on conventional X-ray film [4].
2.1. Subjects 2.4. PCR–RFLP Forty-three type 1 diabetes patients were studied. Their mean age at onset was 10.0 ± 4 years, and the sex ratio (males/females) was 22:21. Diabetes mellitus was defined using the National Diabetes Data Group recommendations. Type 1 diabetes is easy to recognize when it occurs in children and adolescents. The control population consisted of 101 healthy Tunisian individuals randomly selected among volunteer blood donors [11]. Blood donor samples were collected from a blood center in Tunis and the diabetic patients were recruited throughout the country. 2.2. Methods 2.2.1. Blood sample Fifteen milliliters of fresh peripheral blood (5% EDTA) were collected per patient. 2.2.2. Preparation of genomic DNA Genomic DNA was extracted by the salting out procedure [19] with some modification as previously described by Gao et al. [10]. 2.2.3. Typing method HLA was typed by: * PCR–SSO = DRB; * PCR–RFLP = DQB; and * PCR–Reverse dot = Subtypes DRB1*04. 2.3. DNA typing by the polymerase chain reaction and hybridization sequence specific oligonucleotide probes (PCR–SSO) DNA samples were amplified with PCR. The primers for the amplification of DRB1, DRB3, DRB4 and DRB5 were: DRB = AMP-A = CCC ACA AGC CGT TTC TTG DRB = AMP-B = CCG CTG CAC TGT GAA GCT CT The annealing temperature was 55 °C. The SSO probes were 3'-end-labeled enzymatically by DNA deoxynucleotidyl transferase (Tdt, Boerhinger Manheim Ginbh, FRG) with one digoxigenin molecule, digoxigenin dioxy-uridine-triphosphate (DIG-11-dd UTP). The probe hybridization was performed by the tetramethyl ammonium chloride (TMAC) technique following the 11th international histo-compatibility workshop protocol [30]. The reagent was sheep Fab antigoxigenin IgG fragment conjugated to an alternative substrate of alkaline phosphatase producing chemiluminescence after reaction with the (3-2 spiroadamontane) 4 methoxy-4 (3 phosphatoxy) phenyl-1, 2 diox-etane (AMPPD) allowing the detection of
Genomic DNA (500 ng) previously was denatured for 5 mn at 94 °C, in a 100 µl reaction mixture containing: 10 mM Tris PH 8.3–50 mM KCl; 1.5 mM Mg Cl2, 0.1% gelatine; 200 µM dNTP; 2.5 units of Taq polymerase (eurobio); and 50 p moles of each primer. The two primers used were [18]: GH 29 = 5′GAG CTG CAG GTA GTT GTGTT GCA CAC3′ GH 28 b = CTA ATATATT GAG TTA TAAA GAC GCC CGTG ACG GCCCGGC CT GTGCTACT TCACCA ACG Amplification was accomplished by 30 cycles of incubation at 94 °C for 1 min, at 60 °C for 1 min and at 72 °C for 2 min. After amplification, a 10 µl aliquot of PCR reaction mixture was digested by the addition of 8 µl H2O D, 2 µl of the appropriate digested buffer and five units of restrictions endonucleases for 3 h. Three restrictions endonucleases were used: Hae III, MSPI, and Bsa HI (Sigma R 2760–R 4506–R 5628). The electrophoresis was performed on a 12% acrylamide gel. The restriction fragments were visualized by argent nitrate staining. Correlation between the fragments detected with three allele-specific restriction endonuclease and HLA DQB1 alleles is given in Table 1. 2.5. PCR–reverse dot blot hybridization In order to determine the subtypes DRB1*04, all the DR4-positive samples detected by PCR–SSO were investigated by PCR–reverse dot blot hybridization [10]. 2.6. Statistical analysis Allelic frequencies were calculated by direct counting. Delta value (∆) and haplotype frequencies were calculated according to the formula from Mattiuz et al. [17]. The significance of the linkage disequilibria between alleles was tested using chi-square analysis [12]. The odds ratio was calculated according to Woolfs formula and, by convention, expressed as a relative risk (RR). Hal-danes modification of the formula was used when one element of the equation was zero [27]. The level of significance was assessed by the χ2 test in comparisons between different groups and was set to 0.05.
Original article / Annales de Génétique 45 (2002) 45–50
47
Table 1 Correlation alleles DQB1/restriction fragments Hae III DQB1* 0501 0502 05031 05032 0504 0601 0602 0603 0604 0605 0201 0301 0302 03031 03032 0401 0402
2 2 1
+
+ +
Msp I 1 3 2
1 2 7
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + +
1 2 4
9 4
+ + + + + + + + + +
+ + + +
2 2 4
1 7 8
+ + + + + + + + + + + + + + + + +
Bsa HI 1 1 8
1 0 6
2 3 6
+ +
+ + + + + + + + + +
+ + + + +
+
+ + + +
+ + + + +
3. Results 3.1. Distribution of the DRB1, DQB1 alleles The distribution of DRB1 and DQB1 alleles among the healthy controls indicated that our population was in HardyWeinberg equilibrium. The well-known positive association of DR3 and DR4 with type 1 diabetes in the different ethnic origin population is confirmed in this study (Table 2). The HLA DRB1*03 allele was present in 37.2% of patients compared with only 24.3% in controls (P < 0.025). The prevalence of the HLA DRB1*04 allele was 40.7% among the patients compared with only 11.8% among controls (P < 2.9 10–8). Results obtained in this study confirm the protective role of DRB1*11 and DRB1*1501 with a relative risk of 0.217 and 0.01, respectively (P = 0.007 and 0.006).
1 4 7
1 2 8
1 0 7
8 8
+ + + + +
+ + + +
+ + + + +
+ + + + +
+ + + + + +
+ + + + + +
+ + +
The distribution of the DQB1 alleles (Table 3) reveals that the DQB1*0201 was significantly associated in type 1 diabetes patients (RR = 1.79, P = 0.025) and the DQB1*0302 allele was a strong risk factor, whatever the associated allele (RR = 8.9, P = 5.3 10–11). However, the results obtained in this study confirm the protective role of the DQB1*0501, DQB1*0602 and DQB1*0301 allele. 3.2. Haplotypes DRB1 DQB1 The haplotypes which present significant associations are shown in Table 4. Only eight positive associations were found in the group of Tunisian diabetics against 23 in the reference group. The haplotypes DRB1*04 DQB1*0302, and DRB1*03 DQB1*0201 were relatively frequent in our series. There is
Table 2 Distribution of DRB1 alleles among type 1 diabetic patients and control subjects Controls
Type 1 diabetes
DRB1*
2n
%
2n
%
01 1501 1601 03 04 11 12 13 14 07 08 09 10
14 21 7 49 24 29 1 19 6 25 3 1 3
6.9 10.4 3.5 24.3 11.8 14.3 0.5 9.4 2.9 12.8 1.5 0.5 1.5
2 1 1 32 35 3 0 6 0 6 0 0 0
2.3 1.1 1.1 37.2 40.7 3.5 0 7 0 7 0 0 0
RR: Relative risk. Only statistically significant RR are presented.
6 6
RR
P
0.101
0.006
1.84 5.13 0.217
0.025 2.9 E-08 0.007
48
Original article / Annales de Génétique 45 (2002) 45–50
Table 3 Distribution of DQB1 alleles among type I diabetic patients and control subjects Controls
Type 1 diabetes
DQB1*
2n
%
2n
%
RR
P
0501 0502 0503 0601 0602 0603 0604 0201 0301 0302 0303 0304
18 10 6 2 20 5 4 66 41 12 4 14
8.9 4.9 2.9 1 10 2.4 1.9 32.6 20.2 5.9 1.9 6.9
2 1 0 0 1 1 4 40 4 31 0 2
2.3 1.1 0 0 1.1 1.1 4.6 46.5 4.6 36 0 2.3
0.24
0.004
0.14
0.009
1.79 0.193 8.9
0.0258 0.0008 5.3 E-11
RR: Relative risk. Only statistically significant RR are presented.
3.3. Risks associated with the genotypes DRB1
a positive association with type 1 diabetes with a relative risk of 1.639 and 1.637, respectively (P = 3.1 × 10–7 and 1.96 × 10–5).
To evaluate the risk associated with the different combinations of DRB1, we considered three DRB1 alleles: DRB1*03, DRB1*04 and DRB1*X (X is different from DR3 and DR4) (Table 5).
On the other hand the haplotypes DRB1*1501 DQB1*0602 and DRB1*11 DQB1*0301 are protective (RR = 0.055, P = 0.0199 and RR = 0.145, P = 0.03, respectively).
Table 4 Study of haplotype frequency in diabetics in comparison with a reference group Controls
Diabetes type 1
Haplotypes
n
%
n
%
DRB1*01/DQB1*0501 DRB1*1501/DQB1*0602 DRB1*1601/DQB1*0502 DRB1*03 / DQB1*0201 DRB1*04/DQB1*0302 DRB1*11/DQB1*0301 DRB1*13/DQB1*0603 DRB1*07/DQB1*0201
13 17 6 28 16 23 5 18
2.4 8.8 3.5 21.3 11.6 11.6 4.5 10.1
2 1 1 31 27 3 5 5
1.2 0.53 0.63 30.7 17.7 1.87 2.82 4.7
RR
P
0.055
0.0199
1.637 1.639 0.145
1.96 E-05 3.1 E-07 0.0323
RR
P
7.6 7.4 0.1 9.8
0.00002 0.001 0.00002 0.00002
RR: Relative risk. Only statistically significant RR are presented. Table 5 The DRB1 genotypes of type 1 diabetes patients Controls Genotypes DRB1*03/X DRB1*04/X DRB1*03/DRB1*03 DRB1*03/DRB1*04 DRB1*04/DRB1*04 Non DR3/Non DR4 DRB1*03 and/or DRB1*04
Diabetes type 1 n
31 12 6 6 3 43 58
RR: Relative risk. Only statistically significant RR are presented.
% 30.7 11.9 5.9 5.9 3.0 42.6 57.4
n 8 5 5 14 8 3 40
% 18.60 11.62 11.62 32.55 18.6 6.97 93.0
Original article / Annales de Génétique 45 (2002) 45–50
49
Table 6 The DRB1*04 Subtypes DR4 positive subjects Diabetes type 1 Alleles DRB1*0401 DRB1*0402 DRB1*0403 DRB1*0404 DRB1*0405 DRB1*0406 DRB1*0407
n 1 12 2 0 20 0 0
Controls %
2.8 34.2 5.7 0 57.1 0 0
n 3 6 9 1 2 0 3
% 12.5 25 37.5 4.1 8.3 0 12.5
RR
P
0.1
0.002
14.705
0.0001
RR: Relative risk. Only statistically significant RR are presented.
Ninety-three percent of the patients were DRB1*03 and/or DRB1*04 compared with only 57.4% of the healthy controls (P = 2.76 10–5). Also, 32.5% of the patients were DRB1*03/*04 compared with only 5.9% of healthy controls (RR = 7.6, P = 0.00002). The relative risk conferred by the homozygosity of HLA DRB1*04 (RR = 7.4 and P = 0.001) was nearly the same as that of DRB1*03/*04 (RR = 7.6, P = 2 10–5). 3.4. DR4 subtypes associated with type 1 diabetes The above results of DRB1*04 typing were obtained using PCR–reverse dot (Table 6). Our result revealed that the DRB1*0405 allele is strongly associated with the disease (RR = 10.8, P = 0.0012) whereas DRB1*0403 had a significant protective effect (RR = 0.079, P = 0.007).
4. Discussion More difficulties still remain in the clinical classification of diabetes mellitus because of the existence of confused forms of the disease such as maturity-onset diabetes of the young and non-insulin-requiring slow type 1 diabetes of auto immune origin [5,8,16,20]. That is the justification of our choice of a population with a diabetes onset age less then 15 years for this study. The study of HLA polymorphism concerned 43 type 1 diabetes Tunisians, and was carried out to determine the predisposing and the protective alleles to type 1 diabetes. The results found have confirmed the major role played by the haplotype DRB1*04 DQB1*0302 and DRB1*03 DQB1*0201 and were in accordance with those found with other ethnics origins [3,5,7,9,21,27,29]. The protective effect of the DRB1*1501 DQB1*0602 has been shown in our population (RR = 0.055). This result was close to those reported in most Caucasian and Black populations. Furthermore the same effect is shown with DRB1*11 DQB1*0301 in our series and also in French, Greek and Japanese populations [1,3,5,29].
The relative risk conferred by the genotype HLA DRB1*04/*04 remains below that of HLA DRB1*03/*04 heterozygotes in our population and in all ethnic groups. It was reported [6] that the risk associated with DRB1*04/*04 homozygoty was usually lower than of DRB1*03/*03, but in our study, the DRB1*03/*03 homozygoty was not associated with type 1 diabetes. However, it is interesting to note that in several populations (such as the Basque, Turkish, Moroccan and Argentinean populations) the DRB1*03/*03 individuals had a similar or even higher risk than DRB1*03/*04 heterozygotes [6]. Among the DRB1*04 subtypes, susceptibility to type 1 diabetes, the DRB1*0405 allele was closely associated with the disease in this population. However, in another Tunisian study concerning 19 type 1 diabetes, the two alleles DRB1*0402 and DRB1*0405 were reported to be associated with type 1 diabetes [15]. What consolidates our result is what was found in the Algerian population. Indeed the DRB1*0405 allele is associated in a statistically significant way with type 1 diabetes, and this proves once again that the Algerian and Tunisian population belong to the same ethnic group. Alleles DRB1*0402 and DRB1*0405 proved to be neutral in Moroccan and Egyptian populations, respectively [6]. In the French population, however, susceptibility to diabetes is observed in individuals carrying DRB1*0402,*0405 and *0401 [2]. Susceptibility conferred by DQB1*0405 has been seen in the majority of the studied ethnic groups [5,7,21]. It can be seen from our study that the DRB1*0403 allele, which is in linkage disequilibrium with DQB1*0402, confers a protective effect with type 1 diabetes. This protective effect was found in several Caucasians group type 1 diabetes. In French and Norwegian populations DRB1*0403 is a rare allele, and its frequency was lower in patients than in controls but the difference was not statistically significant. It must be emphasized that the protection conferred by DRB1*0403 and *0406 is strong enough to overcome susceptibility conferred by DQA1*0301 DQB1*0302.
50
Original article / Annales de Génétique 45 (2002) 45–50
In conclusion, Tunisian type 1 diabetes patients do share a series of traits with diabetics of other origins but also have specific characteristics. These variations between ethic groups have been previously referred to and are well explained by differences in DR–DQ linkage disequilibria or DQ transcomplementation.
References [1] P. Alberto, G. Roberto, M. Rocio, L. Zuheir, H. Aweleh, A. Erlich, et al., HLA DQB1*0602 is associated with dominant protection from Diabetes. Even among Islet cell antibody, positive first degree relatives of patients with IDDM, Diabetes 44 (1995) 608–613. [2] A. Bart Vander, W. Chritine Van, S. Frans, H. Harry, V. Christina, G. Frans, Jocelyne F., the Belgian Diabetes Registry, DRB1*0403 protects against IDDM in Caucasians with the high-risk heterozygous DQB1*0201 genotype, Diabetes 44 (1995) 527–530. [3] C. Bortard, S. Caillot-Zucman, J. Tinsit, Insulin depend diabetes and human leucocyte antigens, Diabetes Metabolism 23 (1997) 22–28. [4] I. Bronstein, C. John, K. Voyta, Improved chemilumines once detection of alkaline phosphatase, Biofeedback 92 (1990) 160–160. [5] S. Caillat Zucman, H.J. Garchon, J. Tinsit, R. Assan, Ch. Boitard, S.I. Djilali, P. Bougneres, J.F. Bach, Age-dependant HLA genetic heterogeneity of type 1 insulin dependent diabetes mellitus, J. Clin. Invest. 90 (1992) 2242–2250. [6] S. Caillat Zucman, I. Djilali Saiah, J. Timsit, et al., Insulindependent diabetes mellitus (IDDM): 12th International Histocompatibility Work Shop Study, in: P.I. Terasaki (Ed.), Histocompatibility Work shop and Conference, Vol. 1, Oxford University Press, Oxford, 1992, pp. 389–397. [7] S. Djoulah, I. Khalil, J.P. Beressi, S. Ben Hamamouche, K. Bessaoud, I. Deschampls, L. Degos, J. Hors, HLA DRB1*0405 haplotype is most strongly associated with IDDM in Algeria, Eur. J. Immunogenet. 23 (1992) 127–127. [8] G.S. Eisenbarth, Type 1 Diabetes mellitus. A chronic auto-immune disease, N. Engl. J. Med. 314 (1986) 1360–1368. [9] H.A. Erlich, A. Zidler, J. Chang, S. Shaw, L.J. Raffel, W. Klitz, Y. Beshkow, et al., HLA Cl II alleles and susceptibility and resistance to insulin-dependent. Diabetes mellitus in Mexican, American families, Nat. Genet. 3 (1993) 358–364. [10] X. Gao, M.A. Fernandez Vina, W. Shumway, P. Stastny, DNA typing for Cl II HLA antigens with allele specific or group specific. Amplification typing for subsets of HLA DR4, Hum. Immunol. 27 (1990) 40–50. [11] S. Hmida, A. Goutier, A. Dridi, F. Quillivic, B. Genetet, K. Boukef, G. Semana, HLA Cl II gene polymorphism in Tunisian, Tissue Antigens 45 (1995) 63–68. [12] T. Imanishi, T. Akaza, A. Kimira, K. Tokunaga, T. Gojobori, Estimation of allele and haplotype frequencies for HLA and complement loci, in: K. Tsuji, M. Aizawa, T. Sasazuki (Eds.), HLA 1991, proceedings of the Eleventh International Histocompatibility Workshop and Conference, Vol. 1, Oxford University Press, Oxford, 1992, pp. 76–79. [13] F. Jenhani, R. Bardi, K. Ayed, S. Chammakhi, Extended HLA haplotypes in multiplex family with insulin-dependent. Diabetes mellitus in Tunisian population. HLA serological typing and RFLP analysis, Disease Markers 8 (1990) 327–331. [14] D. Jenkins, C. Mijovic, J. Fletcher, K.H. Jacobs, A.R. Brad Well, A.H. Barnett, Identification of susceptibility loci for type I (insulindependent) diabetes by trans-racial gene mapping, Diabetologia 33 (1990) 387–395.
[15] Y. Jul, X.F. Gu, R. Bardi, R. Krishnamoorty, D. Charron, A sample non radioactive method of DNA typing for subsets of HLA DR4. Prevalence Data on HLA-DR4 subsets in three diabetic population groups, Hum. Immunol. 33 (1991) 244–251. [16] J. Karjalaine, P. Salmela, J. Ilonen, H. Marja Suecel, M. Krip, A comparison of childhood and adulte type 1 diabetes mellitus, N. Engl. J. Med. 320 (1989) 881–886. [17] P.L. Mattuz, D. Ihde, A. Pizza, R. Cepellin, W.F. Bodmer, New approaches to the population genetics and aggregation analysis of the HLA system, in: P.I. Terasaki (Ed.), Histocompatibility Workshop and Conference, Oxford University Press, Oxford, 1992, pp. 76–79. [18] B. Mercier, C. Ferec, F. Dufosse, J.J. Huart, Improvement in HLA DQ B typing by PCR-RFLP. Introduction of a constant restriction site in one of the primers for digestion for control, Tissue Antigens 40 (1992) 86–89. [19] S.A. Miller, D.D. Dykes, H.F.A. Polesky, Sample salting out procedure for extracting DNA from human nucleated cells, Nucleic Acid Res. 16 (1988) 1215–1215. [20] National Diabetes Data Group, Classification and diagnostic of diabetes mellitus and other categories of glucose intolerance, Diabetes 29 (1979) 1039–1057. [21] S. Nejeutsev, H. Reijonen, B. Adojaan, L. Kovalchuk, A. Sochnevs, E.I. Schwartz, K.H. Akerblan, J. Ilonem, The effect of HLA-B allele on the IDDM risk defined by DRB1*04 subtypes and DQB1*0302, Diabetes 46 (1997) 1888–1892. [22] G.T. Nepom, H. Erlich, M.C.H. Cl, II molecules and auto immunity, Ann. Immunol. 9 (1991) 493–526. [23] P. Platz, B.K. Jakobsen, N. Morling, L.P. Ryder, A. Svejgaard, M. Thomsen, et al., HLA D and DR antigens in genetic analysis of insulin-dependant diabetes mellitus, Diabetologia 21 (1981) 108–115. [24] K.S. Ronningen, A. Surkland, T. Iwe, F. Vatdal, E. Thoshy, Distribution of HLA DR B1 alleles and DQ B1 genotypes among Norwegian patients with insulin-dependent diabetes mellitus, Tissue Antigens 37 (1991) 105–111. [25] M.J. Sheely, HLA and insulin-dependent diabetes mellitus, Diabetes 41 (1992) 123–129. [26] A. Sveijgaard, P. Platz, L.P. Ryder, Insulin-dependent diabetes mellitus, in: P.L. Terasaki (Ed.), Joint Report, UCLA, Los Angeles, 1980, pp. 638–656. [27] J.L. Twari, P.I. Terasaki, The Data and Statistical Methods, HLA and Disease Association, Springer Verlag, New York, 1985. [28] G. Thomson, W.P. Robinson, M.K. Kuhner, S. Joe, M.J. MacDonald, J.L. Gottschall, et al., Genetic heterogeneity, mode of inheritance, and risk estimate for a joint study of caucasiens with insulindependent diabetes mellitus, Am. J. Hum. Genet. 43 (1988) 799–816. [29] J.A. Todd, J.I. Bell, H.O. McDevitt, HLA DQ ß gene contribute to susceptibility and resistance insulin-dependent diabetes mellitus, Nature 329 (1987) 599–604. [30] P. Stasny, A. Kimura, DNA typing analysis of the DR4 group, in: K. Tsuji, M. Aizawa, T. Sasazuki (Eds.), HLA 1991, Proceedings of the Eleventh International Histocompatibility Workshop and Conference, Vol. 1, Oxford University Press, Oxford, 1992, pp. 465–470. [31] C.M. Vadheim, A. Zidler, J.I. Ratter, L. Langba, I.A. Schulman, M.R. Spenner, et al., Different HLA haplotypes in Mexicans with IDDM, Diabetes Care 12 (1989) 477–500.
Original Article
HLA polymorphism in type 1 diabetes Tunisians H. Abid Kamoun a, S. Hmida b,*, H. Kaabi b, A. Abid c, H. Slimane Houissa a, M. Maamar b, N. Mojaat b, L. Ben Hamed b, A. Dridi b, M. Kamoun Zribi a, K. Nagati c, A. Haddad a, K. Boukef b b
a National Blood Transfusion Center, Tunis, Tunisia Service de Médecine Interne B Hôpital Charles Nicolle, Tunis, Tunisia c National Institute of Nutrition, Tunis, Tunisia
Received 16 November 2001; accepted 23 January 2002
Abstract Several studies of the association between HLA and type 1 diabetes have been carried out revealing differences between ethnic groups. Our study, as part of the studies that should be performed about this association in the rest of the word, aims at elucidating the HLA DRB1, DQB1 polymorphism in Tunisian type 1 diabetes. This study includes 43 unrelated type 1 diabetes patients, and their mean age at onset is less than 15 years. Analysis of the frequency of alleles and haplotypes in these subjects, compared to a reference group (n = 101) led to the following results. 1) The Tunisian insulin-dependent diabetics present similarities as well as differences with other ethnic groups (Caucasians, North Africans). 2) The haplotype DRB1*04 DQ*0302 and DRB1*03 DQB1*0201 is positively associated to type 1 diabetes. 3) The heterozygotic genotype DRB1*04 DQB1*0302 / DRB1*03 DQB1*0201 is strongly associated to type 1 diabetes. 4) The haplotypes DRB1*01501 DQB1*0602 and DRB1*11 DQB1*0301 proved to be protective. In addition, the study of the subtypes DRB1*04 showed that alleles DRB1*0405 predispose to type 1 diabetes, whereas the allele DRB1*0403, which is in linkage disequilibrium with the DQB1*0402 in the Tunisian population, has a protective effect. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Type 1 diabetes mellitus; HLA Cl II; HLA–DRB1, DQB1; DNA typing
1. Introduction Insulin-dependent diabetes mellitus (IDDM) or diabetes type 1 results from an auto-immune selective and irreversible destruction of β-cells of Langerhans [8,26]. The etiology is still a mystery but is known that it involves genetic and environmental factors. Genetically, the occurrence of diabetes mellitus depends on several genes and mainly on the one situated at the level of the major histo-compatibility complex. Among the different genes of the HLA system, those which belong to the class II molecules (DR, DQ, DP) play a major role in the presentation of the peptide which is the initial stage in the immune response [22,25]. Studies carried out during the last 10 years revealed that 90–95% of
* Corresponding author. Fax: 216 1562 957. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 0 0 3 - 3 9 9 5 ( 0 2 ) 0 1 1 0 4 - 8
Caucasian insulin-dependent diabetics belonged to the group HLA DR3 and/or DR4 against 40–50% from the reference group. A higher risk was observed in heterozygotes DR3/DR4 suggesting that predisposition should result from two rather than one genetic factors. Yet, studies of different populations revealed that the frequencies of the different HLA antigens and their associations with diseases varied with the ethnic and geographic origins of the populations [23,24,25,28,31]. DR2 is protective against IDDM patients, DR3 and DR4 are, on the other hand, associated to IDDM but this association is not constant [14,25]. The association is for example negative with DR3 (Japanese) and with DR4 (Chinese), but it is positive with DR9 (Chinese, Japanese, Blacks) and with DR7 (Blacks). That is why this study aims at completing and enriching the data already published on Tunisian type 1 diabetes by adopting a molecular approach [13].
46
Original article / Annales de Génétique 45 (2002) 45–50
2. Patients and method
hybrids by emission of light at 477 nm on conventional X-ray film [4].
2.1. Subjects 2.4. PCR–RFLP Forty-three type 1 diabetes patients were studied. Their mean age at onset was 10.0 ± 4 years, and the sex ratio (males/females) was 22:21. Diabetes mellitus was defined using the National Diabetes Data Group recommendations. Type 1 diabetes is easy to recognize when it occurs in children and adolescents. The control population consisted of 101 healthy Tunisian individuals randomly selected among volunteer blood donors [11]. Blood donor samples were collected from a blood center in Tunis and the diabetic patients were recruited throughout the country. 2.2. Methods 2.2.1. Blood sample Fifteen milliliters of fresh peripheral blood (5% EDTA) were collected per patient. 2.2.2. Preparation of genomic DNA Genomic DNA was extracted by the salting out procedure [19] with some modification as previously described by Gao et al. [10]. 2.2.3. Typing method HLA was typed by: * PCR–SSO = DRB; * PCR–RFLP = DQB; and * PCR–Reverse dot = Subtypes DRB1*04. 2.3. DNA typing by the polymerase chain reaction and hybridization sequence specific oligonucleotide probes (PCR–SSO) DNA samples were amplified with PCR. The primers for the amplification of DRB1, DRB3, DRB4 and DRB5 were: DRB = AMP-A = CCC ACA AGC CGT TTC TTG DRB = AMP-B = CCG CTG CAC TGT GAA GCT CT The annealing temperature was 55 °C. The SSO probes were 3'-end-labeled enzymatically by DNA deoxynucleotidyl transferase (Tdt, Boerhinger Manheim Ginbh, FRG) with one digoxigenin molecule, digoxigenin dioxy-uridine-triphosphate (DIG-11-dd UTP). The probe hybridization was performed by the tetramethyl ammonium chloride (TMAC) technique following the 11th international histo-compatibility workshop protocol [30]. The reagent was sheep Fab antigoxigenin IgG fragment conjugated to an alternative substrate of alkaline phosphatase producing chemiluminescence after reaction with the (3-2 spiroadamontane) 4 methoxy-4 (3 phosphatoxy) phenyl-1, 2 diox-etane (AMPPD) allowing the detection of
Genomic DNA (500 ng) previously was denatured for 5 mn at 94 °C, in a 100 µl reaction mixture containing: 10 mM Tris PH 8.3–50 mM KCl; 1.5 mM Mg Cl2, 0.1% gelatine; 200 µM dNTP; 2.5 units of Taq polymerase (eurobio); and 50 p moles of each primer. The two primers used were [18]: GH 29 = 5′GAG CTG CAG GTA GTT GTGTT GCA CAC3′ GH 28 b = CTA ATATATT GAG TTA TAAA GAC GCC CGTG ACG GCCCGGC CT GTGCTACT TCACCA ACG Amplification was accomplished by 30 cycles of incubation at 94 °C for 1 min, at 60 °C for 1 min and at 72 °C for 2 min. After amplification, a 10 µl aliquot of PCR reaction mixture was digested by the addition of 8 µl H2O D, 2 µl of the appropriate digested buffer and five units of restrictions endonucleases for 3 h. Three restrictions endonucleases were used: Hae III, MSPI, and Bsa HI (Sigma R 2760–R 4506–R 5628). The electrophoresis was performed on a 12% acrylamide gel. The restriction fragments were visualized by argent nitrate staining. Correlation between the fragments detected with three allele-specific restriction endonuclease and HLA DQB1 alleles is given in Table 1. 2.5. PCR–reverse dot blot hybridization In order to determine the subtypes DRB1*04, all the DR4-positive samples detected by PCR–SSO were investigated by PCR–reverse dot blot hybridization [10]. 2.6. Statistical analysis Allelic frequencies were calculated by direct counting. Delta value (∆) and haplotype frequencies were calculated according to the formula from Mattiuz et al. [17]. The significance of the linkage disequilibria between alleles was tested using chi-square analysis [12]. The odds ratio was calculated according to Woolfs formula and, by convention, expressed as a relative risk (RR). Hal-danes modification of the formula was used when one element of the equation was zero [27]. The level of significance was assessed by the χ2 test in comparisons between different groups and was set to 0.05.
Original article / Annales de Génétique 45 (2002) 45–50
47
Table 1 Correlation alleles DQB1/restriction fragments Hae III DQB1* 0501 0502 05031 05032 0504 0601 0602 0603 0604 0605 0201 0301 0302 03031 03032 0401 0402
2 2 1
+
+ +
Msp I 1 3 2
1 2 7
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + +
1 2 4
9 4
+ + + + + + + + + +
+ + + +
2 2 4
1 7 8
+ + + + + + + + + + + + + + + + +
Bsa HI 1 1 8
1 0 6
2 3 6
+ +
+ + + + + + + + + +
+ + + + +
+
+ + + +
+ + + + +
3. Results 3.1. Distribution of the DRB1, DQB1 alleles The distribution of DRB1 and DQB1 alleles among the healthy controls indicated that our population was in HardyWeinberg equilibrium. The well-known positive association of DR3 and DR4 with type 1 diabetes in the different ethnic origin population is confirmed in this study (Table 2). The HLA DRB1*03 allele was present in 37.2% of patients compared with only 24.3% in controls (P < 0.025). The prevalence of the HLA DRB1*04 allele was 40.7% among the patients compared with only 11.8% among controls (P < 2.9 10–8). Results obtained in this study confirm the protective role of DRB1*11 and DRB1*1501 with a relative risk of 0.217 and 0.01, respectively (P = 0.007 and 0.006).
1 4 7
1 2 8
1 0 7
8 8
+ + + + +
+ + + +
+ + + + +
+ + + + +
+ + + + + +
+ + + + + +
+ + +
The distribution of the DQB1 alleles (Table 3) reveals that the DQB1*0201 was significantly associated in type 1 diabetes patients (RR = 1.79, P = 0.025) and the DQB1*0302 allele was a strong risk factor, whatever the associated allele (RR = 8.9, P = 5.3 10–11). However, the results obtained in this study confirm the protective role of the DQB1*0501, DQB1*0602 and DQB1*0301 allele. 3.2. Haplotypes DRB1 DQB1 The haplotypes which present significant associations are shown in Table 4. Only eight positive associations were found in the group of Tunisian diabetics against 23 in the reference group. The haplotypes DRB1*04 DQB1*0302, and DRB1*03 DQB1*0201 were relatively frequent in our series. There is
Table 2 Distribution of DRB1 alleles among type 1 diabetic patients and control subjects Controls
Type 1 diabetes
DRB1*
2n
%
2n
%
01 1501 1601 03 04 11 12 13 14 07 08 09 10
14 21 7 49 24 29 1 19 6 25 3 1 3
6.9 10.4 3.5 24.3 11.8 14.3 0.5 9.4 2.9 12.8 1.5 0.5 1.5
2 1 1 32 35 3 0 6 0 6 0 0 0
2.3 1.1 1.1 37.2 40.7 3.5 0 7 0 7 0 0 0
RR: Relative risk. Only statistically significant RR are presented.
6 6
RR
P
0.101
0.006
1.84 5.13 0.217
0.025 2.9 E-08 0.007
48
Original article / Annales de Génétique 45 (2002) 45–50
Table 3 Distribution of DQB1 alleles among type I diabetic patients and control subjects Controls
Type 1 diabetes
DQB1*
2n
%
2n
%
RR
P
0501 0502 0503 0601 0602 0603 0604 0201 0301 0302 0303 0304
18 10 6 2 20 5 4 66 41 12 4 14
8.9 4.9 2.9 1 10 2.4 1.9 32.6 20.2 5.9 1.9 6.9
2 1 0 0 1 1 4 40 4 31 0 2
2.3 1.1 0 0 1.1 1.1 4.6 46.5 4.6 36 0 2.3
0.24
0.004
0.14
0.009
1.79 0.193 8.9
0.0258 0.0008 5.3 E-11
RR: Relative risk. Only statistically significant RR are presented.
3.3. Risks associated with the genotypes DRB1
a positive association with type 1 diabetes with a relative risk of 1.639 and 1.637, respectively (P = 3.1 × 10–7 and 1.96 × 10–5).
To evaluate the risk associated with the different combinations of DRB1, we considered three DRB1 alleles: DRB1*03, DRB1*04 and DRB1*X (X is different from DR3 and DR4) (Table 5).
On the other hand the haplotypes DRB1*1501 DQB1*0602 and DRB1*11 DQB1*0301 are protective (RR = 0.055, P = 0.0199 and RR = 0.145, P = 0.03, respectively).
Table 4 Study of haplotype frequency in diabetics in comparison with a reference group Controls
Diabetes type 1
Haplotypes
n
%
n
%
DRB1*01/DQB1*0501 DRB1*1501/DQB1*0602 DRB1*1601/DQB1*0502 DRB1*03 / DQB1*0201 DRB1*04/DQB1*0302 DRB1*11/DQB1*0301 DRB1*13/DQB1*0603 DRB1*07/DQB1*0201
13 17 6 28 16 23 5 18
2.4 8.8 3.5 21.3 11.6 11.6 4.5 10.1
2 1 1 31 27 3 5 5
1.2 0.53 0.63 30.7 17.7 1.87 2.82 4.7
RR
P
0.055
0.0199
1.637 1.639 0.145
1.96 E-05 3.1 E-07 0.0323
RR
P
7.6 7.4 0.1 9.8
0.00002 0.001 0.00002 0.00002
RR: Relative risk. Only statistically significant RR are presented. Table 5 The DRB1 genotypes of type 1 diabetes patients Controls Genotypes DRB1*03/X DRB1*04/X DRB1*03/DRB1*03 DRB1*03/DRB1*04 DRB1*04/DRB1*04 Non DR3/Non DR4 DRB1*03 and/or DRB1*04
Diabetes type 1 n
31 12 6 6 3 43 58
RR: Relative risk. Only statistically significant RR are presented.
% 30.7 11.9 5.9 5.9 3.0 42.6 57.4
n 8 5 5 14 8 3 40
% 18.60 11.62 11.62 32.55 18.6 6.97 93.0
Original article / Annales de Génétique 45 (2002) 45–50
49
Table 6 The DRB1*04 Subtypes DR4 positive subjects Diabetes type 1 Alleles DRB1*0401 DRB1*0402 DRB1*0403 DRB1*0404 DRB1*0405 DRB1*0406 DRB1*0407
n 1 12 2 0 20 0 0
Controls %
2.8 34.2 5.7 0 57.1 0 0
n 3 6 9 1 2 0 3
% 12.5 25 37.5 4.1 8.3 0 12.5
RR
P
0.1
0.002
14.705
0.0001
RR: Relative risk. Only statistically significant RR are presented.
Ninety-three percent of the patients were DRB1*03 and/or DRB1*04 compared with only 57.4% of the healthy controls (P = 2.76 10–5). Also, 32.5% of the patients were DRB1*03/*04 compared with only 5.9% of healthy controls (RR = 7.6, P = 0.00002). The relative risk conferred by the homozygosity of HLA DRB1*04 (RR = 7.4 and P = 0.001) was nearly the same as that of DRB1*03/*04 (RR = 7.6, P = 2 10–5). 3.4. DR4 subtypes associated with type 1 diabetes The above results of DRB1*04 typing were obtained using PCR–reverse dot (Table 6). Our result revealed that the DRB1*0405 allele is strongly associated with the disease (RR = 10.8, P = 0.0012) whereas DRB1*0403 had a significant protective effect (RR = 0.079, P = 0.007).
4. Discussion More difficulties still remain in the clinical classification of diabetes mellitus because of the existence of confused forms of the disease such as maturity-onset diabetes of the young and non-insulin-requiring slow type 1 diabetes of auto immune origin [5,8,16,20]. That is the justification of our choice of a population with a diabetes onset age less then 15 years for this study. The study of HLA polymorphism concerned 43 type 1 diabetes Tunisians, and was carried out to determine the predisposing and the protective alleles to type 1 diabetes. The results found have confirmed the major role played by the haplotype DRB1*04 DQB1*0302 and DRB1*03 DQB1*0201 and were in accordance with those found with other ethnics origins [3,5,7,9,21,27,29]. The protective effect of the DRB1*1501 DQB1*0602 has been shown in our population (RR = 0.055). This result was close to those reported in most Caucasian and Black populations. Furthermore the same effect is shown with DRB1*11 DQB1*0301 in our series and also in French, Greek and Japanese populations [1,3,5,29].
The relative risk conferred by the genotype HLA DRB1*04/*04 remains below that of HLA DRB1*03/*04 heterozygotes in our population and in all ethnic groups. It was reported [6] that the risk associated with DRB1*04/*04 homozygoty was usually lower than of DRB1*03/*03, but in our study, the DRB1*03/*03 homozygoty was not associated with type 1 diabetes. However, it is interesting to note that in several populations (such as the Basque, Turkish, Moroccan and Argentinean populations) the DRB1*03/*03 individuals had a similar or even higher risk than DRB1*03/*04 heterozygotes [6]. Among the DRB1*04 subtypes, susceptibility to type 1 diabetes, the DRB1*0405 allele was closely associated with the disease in this population. However, in another Tunisian study concerning 19 type 1 diabetes, the two alleles DRB1*0402 and DRB1*0405 were reported to be associated with type 1 diabetes [15]. What consolidates our result is what was found in the Algerian population. Indeed the DRB1*0405 allele is associated in a statistically significant way with type 1 diabetes, and this proves once again that the Algerian and Tunisian population belong to the same ethnic group. Alleles DRB1*0402 and DRB1*0405 proved to be neutral in Moroccan and Egyptian populations, respectively [6]. In the French population, however, susceptibility to diabetes is observed in individuals carrying DRB1*0402,*0405 and *0401 [2]. Susceptibility conferred by DQB1*0405 has been seen in the majority of the studied ethnic groups [5,7,21]. It can be seen from our study that the DRB1*0403 allele, which is in linkage disequilibrium with DQB1*0402, confers a protective effect with type 1 diabetes. This protective effect was found in several Caucasians group type 1 diabetes. In French and Norwegian populations DRB1*0403 is a rare allele, and its frequency was lower in patients than in controls but the difference was not statistically significant. It must be emphasized that the protection conferred by DRB1*0403 and *0406 is strong enough to overcome susceptibility conferred by DQA1*0301 DQB1*0302.
50
Original article / Annales de Génétique 45 (2002) 45–50
In conclusion, Tunisian type 1 diabetes patients do share a series of traits with diabetics of other origins but also have specific characteristics. These variations between ethic groups have been previously referred to and are well explained by differences in DR–DQ linkage disequilibria or DQ transcomplementation.
References [1] P. Alberto, G. Roberto, M. Rocio, L. Zuheir, H. Aweleh, A. Erlich, et al., HLA DQB1*0602 is associated with dominant protection from Diabetes. Even among Islet cell antibody, positive first degree relatives of patients with IDDM, Diabetes 44 (1995) 608–613. [2] A. Bart Vander, W. Chritine Van, S. Frans, H. Harry, V. Christina, G. Frans, Jocelyne F., the Belgian Diabetes Registry, DRB1*0403 protects against IDDM in Caucasians with the high-risk heterozygous DQB1*0201 genotype, Diabetes 44 (1995) 527–530. [3] C. Bortard, S. Caillot-Zucman, J. Tinsit, Insulin depend diabetes and human leucocyte antigens, Diabetes Metabolism 23 (1997) 22–28. [4] I. Bronstein, C. John, K. Voyta, Improved chemilumines once detection of alkaline phosphatase, Biofeedback 92 (1990) 160–160. [5] S. Caillat Zucman, H.J. Garchon, J. Tinsit, R. Assan, Ch. Boitard, S.I. Djilali, P. Bougneres, J.F. Bach, Age-dependant HLA genetic heterogeneity of type 1 insulin dependent diabetes mellitus, J. Clin. Invest. 90 (1992) 2242–2250. [6] S. Caillat Zucman, I. Djilali Saiah, J. Timsit, et al., Insulindependent diabetes mellitus (IDDM): 12th International Histocompatibility Work Shop Study, in: P.I. Terasaki (Ed.), Histocompatibility Work shop and Conference, Vol. 1, Oxford University Press, Oxford, 1992, pp. 389–397. [7] S. Djoulah, I. Khalil, J.P. Beressi, S. Ben Hamamouche, K. Bessaoud, I. Deschampls, L. Degos, J. Hors, HLA DRB1*0405 haplotype is most strongly associated with IDDM in Algeria, Eur. J. Immunogenet. 23 (1992) 127–127. [8] G.S. Eisenbarth, Type 1 Diabetes mellitus. A chronic auto-immune disease, N. Engl. J. Med. 314 (1986) 1360–1368. [9] H.A. Erlich, A. Zidler, J. Chang, S. Shaw, L.J. Raffel, W. Klitz, Y. Beshkow, et al., HLA Cl II alleles and susceptibility and resistance to insulin-dependent. Diabetes mellitus in Mexican, American families, Nat. Genet. 3 (1993) 358–364. [10] X. Gao, M.A. Fernandez Vina, W. Shumway, P. Stastny, DNA typing for Cl II HLA antigens with allele specific or group specific. Amplification typing for subsets of HLA DR4, Hum. Immunol. 27 (1990) 40–50. [11] S. Hmida, A. Goutier, A. Dridi, F. Quillivic, B. Genetet, K. Boukef, G. Semana, HLA Cl II gene polymorphism in Tunisian, Tissue Antigens 45 (1995) 63–68. [12] T. Imanishi, T. Akaza, A. Kimira, K. Tokunaga, T. Gojobori, Estimation of allele and haplotype frequencies for HLA and complement loci, in: K. Tsuji, M. Aizawa, T. Sasazuki (Eds.), HLA 1991, proceedings of the Eleventh International Histocompatibility Workshop and Conference, Vol. 1, Oxford University Press, Oxford, 1992, pp. 76–79. [13] F. Jenhani, R. Bardi, K. Ayed, S. Chammakhi, Extended HLA haplotypes in multiplex family with insulin-dependent. Diabetes mellitus in Tunisian population. HLA serological typing and RFLP analysis, Disease Markers 8 (1990) 327–331. [14] D. Jenkins, C. Mijovic, J. Fletcher, K.H. Jacobs, A.R. Brad Well, A.H. Barnett, Identification of susceptibility loci for type I (insulindependent) diabetes by trans-racial gene mapping, Diabetologia 33 (1990) 387–395.
[15] Y. Jul, X.F. Gu, R. Bardi, R. Krishnamoorty, D. Charron, A sample non radioactive method of DNA typing for subsets of HLA DR4. Prevalence Data on HLA-DR4 subsets in three diabetic population groups, Hum. Immunol. 33 (1991) 244–251. [16] J. Karjalaine, P. Salmela, J. Ilonen, H. Marja Suecel, M. Krip, A comparison of childhood and adulte type 1 diabetes mellitus, N. Engl. J. Med. 320 (1989) 881–886. [17] P.L. Mattuz, D. Ihde, A. Pizza, R. Cepellin, W.F. Bodmer, New approaches to the population genetics and aggregation analysis of the HLA system, in: P.I. Terasaki (Ed.), Histocompatibility Workshop and Conference, Oxford University Press, Oxford, 1992, pp. 76–79. [18] B. Mercier, C. Ferec, F. Dufosse, J.J. Huart, Improvement in HLA DQ B typing by PCR-RFLP. Introduction of a constant restriction site in one of the primers for digestion for control, Tissue Antigens 40 (1992) 86–89. [19] S.A. Miller, D.D. Dykes, H.F.A. Polesky, Sample salting out procedure for extracting DNA from human nucleated cells, Nucleic Acid Res. 16 (1988) 1215–1215. [20] National Diabetes Data Group, Classification and diagnostic of diabetes mellitus and other categories of glucose intolerance, Diabetes 29 (1979) 1039–1057. [21] S. Nejeutsev, H. Reijonen, B. Adojaan, L. Kovalchuk, A. Sochnevs, E.I. Schwartz, K.H. Akerblan, J. Ilonem, The effect of HLA-B allele on the IDDM risk defined by DRB1*04 subtypes and DQB1*0302, Diabetes 46 (1997) 1888–1892. [22] G.T. Nepom, H. Erlich, M.C.H. Cl, II molecules and auto immunity, Ann. Immunol. 9 (1991) 493–526. [23] P. Platz, B.K. Jakobsen, N. Morling, L.P. Ryder, A. Svejgaard, M. Thomsen, et al., HLA D and DR antigens in genetic analysis of insulin-dependant diabetes mellitus, Diabetologia 21 (1981) 108–115. [24] K.S. Ronningen, A. Surkland, T. Iwe, F. Vatdal, E. Thoshy, Distribution of HLA DR B1 alleles and DQ B1 genotypes among Norwegian patients with insulin-dependent diabetes mellitus, Tissue Antigens 37 (1991) 105–111. [25] M.J. Sheely, HLA and insulin-dependent diabetes mellitus, Diabetes 41 (1992) 123–129. [26] A. Sveijgaard, P. Platz, L.P. Ryder, Insulin-dependent diabetes mellitus, in: P.L. Terasaki (Ed.), Joint Report, UCLA, Los Angeles, 1980, pp. 638–656. [27] J.L. Twari, P.I. Terasaki, The Data and Statistical Methods, HLA and Disease Association, Springer Verlag, New York, 1985. [28] G. Thomson, W.P. Robinson, M.K. Kuhner, S. Joe, M.J. MacDonald, J.L. Gottschall, et al., Genetic heterogeneity, mode of inheritance, and risk estimate for a joint study of caucasiens with insulindependent diabetes mellitus, Am. J. Hum. Genet. 43 (1988) 799–816. [29] J.A. Todd, J.I. Bell, H.O. McDevitt, HLA DQ ß gene contribute to susceptibility and resistance insulin-dependent diabetes mellitus, Nature 329 (1987) 599–604. [30] P. Stasny, A. Kimura, DNA typing analysis of the DR4 group, in: K. Tsuji, M. Aizawa, T. Sasazuki (Eds.), HLA 1991, Proceedings of the Eleventh International Histocompatibility Workshop and Conference, Vol. 1, Oxford University Press, Oxford, 1992, pp. 465–470. [31] C.M. Vadheim, A. Zidler, J.I. Ratter, L. Langba, I.A. Schulman, M.R. Spenner, et al., Different HLA haplotypes in Mexicans with IDDM, Diabetes Care 12 (1989) 477–500.