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HLA-DR and -DQ associations with insulin-dependent diabetes mellitus in a population of Turkey
HLA-DR and -DQ Associations with Insulin-Dependent Diabetes Mellitus in a Population of Turkey Gu¨her Saruhan-Direskeneli, F. Aytu¨l Uyar, Firdevs Bas˛, Hu¨lya Gu¨no¨z, Ru¨veyde Bundak, Nurc˛in Saka, and Feyza Darendeliler ABSTRACT: Genetic susceptibility to insulin-dependent diabetes mellitus (IDDM) has been shown to be associated with MHC in many studies. To extend this data with a population with relatively low IDDM incidence, MHC DRB, DQA, and DQB have been investigated by polymerase chain reaction and sequence specific oligonucleotide probe hybridization (PCR/SSO) in 178 IDDM patients from Turkey and compared to 248 healthy controls. Significant differences are detected between IDDM and control groups in the frequencies of DRB1*0402 DQA1*03 DQB1*0302 (28.1% vs. 5.2%, p ⬍ 0.0001, OR: 7.1) and DRB1*0301 DQA1*0501 DQB1*02 (57% vs. 18.1%, p ⬍ 0.0001, OR: 6.1). Among the negative associations, the most strong ones are with DRB1*1401 DQA1*0101 DQB1*0503 (0.6% vs. 8.9%, p ⬍ 0.0001, OR: 0.1), DRB1*1502 DQA1*0103 DQB1*0601 (1.1% vs. 7.7%, p ⫽ 0.0023, OR: 0.1), DRB1*1301 DQA1*0103 DQB1*0603 (0.6% vs. 6.9%, p ⫽ 0.0039, OR: 0.2) and DRB1*1101 DQA1*0501 ABBREVIATIONS IDDM insulin-dependent diabetes mellitus MHC major histocompatibility complex
INTRODUCTION Insulin-dependent diabetes mellitus (IDDM) is a chronic inflammatory disorder of insulin-producing -cells of From the Istanbul University Istanbul Medical Faculty Physiology Department (G.S.-D., F.A.U.), Istanbul University, Electro-Neurophysiology Research and Application Centre (G.S.-D.), Istanbul University Istanbul Medical Faculty Paediatrics Department (F.B., H.G., R.B., N.S., F.D.), Istanbul, Turkey. A part of this study has been contributed to the XII.International Histocompatibility Workshop. Address reprint requests to: Dr. Gu¨her Saruhan-Direskeneli, Istanbul U¨niversitesi Istanbul Tıp Faku¨ltesi, Fizyoloji AD 34390, C¸apa Istanbul, Turkey; Tel/Fax: ⫹(90) 212 533 94 68; E-Mail: gsaruhan@istanbul. edu.tr. Received July 13, 1999; accepted November 4, 1999. Human Immunology 61, 296 –302 (2000) © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc.
DQB1*0301 (3.9% vs. 12.1%, p ⬍ 0.0001, OR: 0.2). When the DRB, DQA or DQB genotypes of the susceptible alleles are compared, the most strong susceptibility marker of the disease is found to be DRB1*0301/*04 (31.4% vs. 2.8%, p ⬍ 0.0001, OR: 15.8) and among these, heterozygote genotype DRB1*0301/*0401 (4.5% vs. 0, p ⫽ 0.0008, OR: 24.8). These results confirm the positive associations with IDDM previously observed in other Caucasian populations and reveal many negative and strong associations which maybe underlining several characteristics that distinguish Turkish diabetics form other Caucasians. Human Immunology 61, 296 –302 (2000). © American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: HLA-DRB1; HLA-DQA1; HLA-DQB1; IDDM; Turkey
PCR/SSO
polymerase chain reaction/sequencespecific oligonucleotide
pancreatic islets. Genetic contribution to the development of the disease is implicated by the 15-fold higher risk of the first-degree relatives of the patients’ [1]. Mapping studies in IDDM has identified IDDM1 interval at chromosome 6p21 as the first major susceptibility marker, which also contains the MHC region [2]. However, the extensive polymorphism and the functional relevance of MHC genes to an immune response have resulted in many population-based association studies. HLA-DR3 and -DR4 were shown to be strongly associated with the disease in Caucasoids [3]. Many studies have shown associations not only of DRB1, but also of DQA1, DPB1, and DQB1 alleles with IDDM. The 0198-8859/00/$–see front matter PII S0198-8859(99)00182-2
HLA Class II Associations of IDDM in Turkey
highest risk for developing the disease has been associated with heterozygous DR3/DR4 phenotype, particularly in combination with DQA1*0501-DQB1*0201/ DQA1*0301-DQB1*0302 alleles [4 – 6]. Many different negative associations with IDDM were also reported, DRB1*1501 DQA1*0102 DQB1*0602 being the strongest [7–9]. As the alleles of class II haplotypes show strong linkage disequilibria in many populations studied, it has been difficult to identify the susceptibility locus to IDDM [10]. A frequently raised hypothesis suggested that the associated DRB1 alleles are conferring susceptibility, whereas the associated DQ alleles are protecting, and the protection is dominant over the susceptibility [7, 10]. In this regard the pattern of associations in distinct ethnic groups were examined to dissect the susceptibility to IDDM [11]. With this approach, IDDM patients and healthy controls from the population of Turkey are evaluated and comparisons of the distribution of the putative class II “haplotypes,” as well as genotypes are made in this study.
MATERIALS AND METHODS Patients and Controls A total of 178 unrelated patients with IDDM were investigated. Male/female ratio and mean age were 1.1 and 12.3, respectively. The mean age of IDDM diagnosis was 7.5. All patients as well as the controls were of Turkish descent with mixed ethnic origin, patients were selected randomly in Istanbul Medical Faculty referral Hospital at the Department of Pediatrics, Growth Development and Pediatric Endocrinology Unit. The control group consisted of 248 unrelated healthy individuals, which has been investigated before [12]. DNA Typing of DRB, DQA and DQB DNA was prepared from venous blood by using salting out method. The second exons of MHC DRB, DQA1 and DQB1 were amplified with polymerase chain reaction (PCR) using the protocols of the XII.International Histocompatibility Workshop (IHWS): 2DRBAMP-A for “generic” and 2DRBAMP-4, 2DRBAMP-2 or 2DRBAMP-3 for group specific typings were used with 2DRBAMP-B [13]. The primers 2DQAAMP-A and 2DQAAMP-B were used for DQA1 typing and 2DQBAMP-A/2DQBAMP-B primers were used for DQB1. Hybridizations were performed by tetramethylammoniumchloride (TMAC) technique by using biotinylated sequence specific probes, following the IHWS Protocol [13]. The results of hybridizations were evaluated for DRB1.
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Statistical Analysis The results were compared using 2 test and with Fisher’s exact test when appropriate. Odds ratios (OR) were estimated according to Woolf’s formula [14]. A p value of ⬍0.05 was accepted significant and the correction of Bonferonni for multiple tests was applied by multiplying p by the number of comparisons. Confidence intervals (CI) are given for 95%. Individual associations were compared following Svejgaard et al. [15]. RESULTS Phenotypic Associations As the phenotypic combinations of the DRB1, DQA1 and DQB1 alleles were in accordance with the known haplotype assignments and were confirmed in 24 IDDM families studied (data not shown), the phenotypic combinations of HLA class II alleles were deduced as putative “haplotypes” and these are compared in this study, although no formal family study has been done. The distribution among the children with IDDM has shown the positive association of DR3 and DR4: 87% of the patients are DR3 and/or DR4 positive compared to 40% of the controls. Significant differences are demonstrated between the “haplotypic” phenotype frequencies in IDDM and control groups (Table 1). The most strongly IDDM-associated phenotypic combination of alleles was DRB1*0301 DQA1*0501 DQB1*02 with an OR of 6.1 (102 vs. 45, p ⬍ 0.0001). This combination of alleles has been detected concomitantly throughout the group. The “haplotypes” with DRB1*04 were also significantly more frequent in IDDM patients than in controls (109 vs. 61, p ⬍ 0.0001, OR: 4.8). Among the DR4 “haplotypes” (Table 2), DRB1*0402 DQA1*03 DQB1*0302 was the most prominent marker of susceptibility to IDDM (OR: 7.1), followed by DRB1*0401 DQA1*03 DQB1*0302 and DRB1*0405 DQA*03 DQB1*0302. The negative associations with IDDM were diverse in our study (Table 1): DRB1*1401 DQA1*0101 DQB1*0503 has given the lowest OR with 0.06 (p ⬍ 0.0001). The other putative “haplotypes” conferring protection for IDDM were DRB1*1303 DQA1*0501 DQB1*0301, DRB1*1502 DQA1*0103 DQB1*0601, DRB1*1301 DQA1*0103 DQB1*0603, DRB1*1101 DQA1*0501 DQB1*0301, DRB1*0701 DQA1*0201 DQB1*02, DRB1*1104 DQA1*0501 DQB1*0301 and DRB1*1501 DQA1*0102 DQB1*0602. The negative association of DRB1*1303 with IDDM was the only one not reported before, but this association did not reach the significance level after correction. Associations in Non-DR3/Non-DR4 Patients Considering different associations shown previously in non-DR3/non-DR4 IDDM patients [16], we have com-
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TABLE 1 Phenotype frequencies of the DRB1-DQA1-DQB1 combinations in IDDM patients and controls IDDM
Controls
p
DRB1
DQA1
DQB1
n ⴝ 178
%
n ⴝ 248
%
*0101 *0102 *0301 *04 *04 *07 *0701 *08 *08 *09 *1001 *1101 *1102 *1103 *1104 *1201/2 *1301 *1302 *1302 *1303 *1401 *1402 *1501 *1502 *1502 *1601 *1601 *0803/5 *0701 *1404 *1101 *1301 Othersa
*0101 *0101 *0501 *03 *03 *02 *02 *0601 *0401 *03 *0101 *0501 *0501 *0501 *0501 *0501 *0103 *0102 *0102 *0501 *0101 *0101 *0102 *0103 *0102 *0102 *0103 *0401 *02 *0101 *0501 *0103
*0501 *0501 *0201 *0302 *0301 *0201 *0303 *0301 *0402 *0303 *0501 *0301 *0301 *0301 *0301 *0301 *0603 *0604 *0605 *0301 *0503 *0503 *0602 *0601 *0601 *0502 *0502 *0301 *0302 *0503 *0304 *0605
18 2 102 102 7 8 5 1 4 1 1 7 1 1 7 2 1 6 1 0 1 1 9 2 1 23 1 0 0 0 0 0 0
10 1.1 57 57 3.9 4.5 2.8 0.6 2.2 0.6 0.6 3.9 0.6 0.6 3.9 1.1 0.6 3.4 0.6
20 7 45 54 7 35 3 4 8 5 10 47 7 6 30 8 17 19 4 10 22 1 30 19 0 28 0 4 3 3 2 2 13
8.1 2.8 18 22 2.8 14.1 1.2 1.6 3.2 2 4 19 2.8 2.4 12.1 3.2 6.9 7.7 1.6 4 8.9 0.4 12.1 7.7
0.6 0.6 5.1 1.1 0.6 12.9 0.6
OR
95% CI
<0.0001 <0.0001
6.1 4.8
3.9–9.4 3.6–8.5
0.001
0.3
0.1–0.6
<0.0001
0.2
0.08–0.4
0.0029
0.3
0.1–0.7
0.0039
0.2
0.03–0.7
0.0063a <0.0001
0.06 0.06
0.003–1.1 0.007–0.4
0.016 0.0023
0.4 0.1
0.2–0.8 0.03–0.6
11.3 1.6 1.2 1.2 0.8 0.8 5.2
“Others” refer to single combinations detected in controls. OR:Odds ratio. a Refers to p not significant after correction.
pared the phenotype frequencies in 23 patients with 147 controls not bearing any DRB1*03 or DRB1*04 allele. Among these patients DRB1*0101 DQA1*0101 DQB1*0501 frequency was increased to 30%, whereas only 11% of the controls were positive for this phenotype (7 vs. 17, p ⫽ 0.002, OR: 3.3, CI: 1,2–9,3). HLA-DR vs. -DQ To determine the strongest association for alleles of associated combinations, we applied the analysis described by Svejgaard and Ryder [15]. The combined presence of associated DRB1, DQA1, and DQB1 alleles was also high in the Turkish population making the interpretation difficult. DRB1*0301 of the DR3 “haplotype” showed higher OR value for independent association, compared to the DQB1*02, but CI were overlapping. Among the DR4 “haplotypes,” comparisons of associated DRB1 with DQB1 alleles have shown that DRB1*0401-,
*0402- or *0405- DQB1*0302 combinations are probably interacting to confer higher susceptibility. Among all alleles of DRB1, DQA1, and DQB1, DRB1*0402 has given a higher OR (7.3) by its single presence, compared to “haplotypes” with DR4. At the comparisons among the negatively associated phenotypic combinations, the differences did not point out any single locus or any combination of the alleles to be negatively associated with the disease independently. The alleles of the negatively associated “haplotypes,” also carried with the positively associated ones, namely DQA1*0501 and DQB1*02, did not show any protective effect, implicating dominance of DRB1 allele susceptibility over a probable protection. DRB Genotypes If DRB1 genotypes are compared, the increase of DRB1*03/04 is very significant in IDDM group (31.5%
HLA Class II Associations of IDDM in Turkey
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TABLE 2 Putative DR4 “haplotype” distribution in IDDM and control groups DRB1
DQA
DQB1
IDDM
n *0401 *0402 *0403 *0404 *0405 *0407 *0408 *0401 *0402 *0403 *0404 *0405 *0407 *0408 *0407 *X *X *X
CONTROLS
178 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03
*0302 *0302 *0302 *0302 *0302 *0302 *0302 *0301 *0301 *0301 *0301 *0301 *0301 *0301 *0304 *02 *0304 *0301
%
22 50 8 6 19 5 5 1 1
12.4 28.1 4.5 3.4 10.7 2.8 2.8 0.6 0.6
1 1 1 2 1 2
0.6 0.6 0.6 1.1 0.6 1.1
1
0.6
248
p
OR
95% CI
0.0001 <0.0001
4.9 7.1
2.02–11.6 3.7–13.4
0.0023
3.6
1.5–8.4
%
7 13 19 10 8 1 2 3
2.8 5.2 7.7 4 3.2 0.4 0.8 1.2
1
0.4
1 2
0.4 0.8
2
0.8
1
0.4
In DR4 heterozygots the phenotypes are assigned in favor of most frequent combination. X refers to the unknown DR4 allele already included in DQB1*0302 combinations.
vs. 2.8%, OR: 15.8) (Table 3). Among these, the combined presence of DRB1*0401 (OR: 24.8), *0405 (21.7) or *0402 (OR: 21.0) with DRB1*0301 have been detected most frequently in the patients. This genotype was associated with the highest risk of 51.4, if compared with the individuals lacking both factors [15]. The stratification of DR4 alleles in this comparison has revealed that DRB1*0401, *0405 and *0402 alleles with
DRB1*0301 were associated with OR of 53.8, 45.9, and 32.8, respectively. In the IDDM group, we found 10.7% “homozygous” patients both in DRB1*03 and in DRB1*04, whereas in the controls only 0.8 % were “homozygous” for DRB1*03 and 2.4% for DRB1*04. These homozygous genotypes were strongly associated with IDDM (OR: 14.7 and 4.8, respectively). Considering any other allele
TABLE 3 Comparison of the genotypes in IDDM and control groups DRB1
*0301 *0301 *0301 *0301 *0301 *0301 *0301 *0301 *0301 *04 *04 *04 *0301 X *11 *11
DRB1
*04 *0401 *0402 *0403 *0404 *0405 *0407 *0408 *0301 *04 X *01 X X *13 *07
IDDM
CONT
N ⫽ 178
%
56 8 26 5 4 7 3 3 19 19 34 7 27 23 1 1
31.5 4.5 14.6 2.8 2.2 3.9 1.7 1.7 10.7 10.7 19.1 3.9 15.2 12.9 0.6 0.6
N ⴝ 248
p
OR
95% CI
<0.0001 0.0008 <0.0001
15.8 24.8 21.0
7.0–35.7 1.4–432.4 4.9–89.9
0.002
21.7
1.2–383.2
<0.0001 0.0005
14.7 4.8
3.4–64.0 1.9–12.3
0.038
5.0
1.0–24.5
<0.0001 0.0057 0.029
0.1 0.1 0.1
%
7
2.8
2 2 2
0.8 0.8 0.8
1
0.4
2 6 38 2 36 147 14 10
0.8 2.4 15.3 0.8 14.5 59.3 5.6 4.0
The most frequent genotypes in the “X/X” group are shown only, X being non-DR3/non-DR4.
0.06–0.2 0.01–0.7 0.20–1.1
300
than DR3 or DR4 as X, the DR4/X genotype did not show any association with IDDM, although DRB1*04/ *01 genotype only was also significantly more frequent in IDDM patients (3.9% vs. 0.8%, OR: 5.0) as reported before [17]. The genotype described as X/X has conferred a strong protection to IDDM (23 vs. 147, OR: 0.1, p ⬍ 0.0001), among them DRB1*11/*13 and DRB1*11/*07 significantly different (1 vs. 14 and 1 vs. 10, respectively). When the susceptible DQB1 (Non-Asp at 57.aa position) and DQA1 (Arg at 52.position) alleles were compared with the other “protective” alleles [18], the genotype with four susceptible DQ alleles has conferred an OR of 14.5, whereas the protective genotype with four protective DQ alleles has given an OR of 0.04. DISCUSSION The distributions of DRB1, DQA1 and DQB1 alleles in IDDM patients have revealed that positive associations are in accordance with the known associations in Caucasians and the putative susceptible “haplotypes” for IDDM are highly increased in patients’ group in Turkey. Among associated alleles, DRB1*0402, *0405, *0401 and DRB1*0301 and the phenotypic combinations of DQ alleles with these, have conferred the highest risk for developing IDDM. Many different negative associations have also been confirmed in our study. Our results extend the “haplotypic” disease-associations to the Turkish population [5, 6]. As none, but DRB1*0402, of the allelic associations of DRB1, DQA1, or DQB1 were stronger than the phenotypic combinations, only putative “haplotypes” are evaluated. The highly conserved DR3 haplotype has been shown to be most strongly associated with IDDM in Sardinian before [19]. Although DR3 also associated with IDDM in other populations, the association was not as strong as in this study, which can be traced back to the relatively lower frequency of this “haplotype” in Turkey (18.1% in controls). The risk conferred by DR3 in Turkish patients has been calculated as high as 60.7, when the homozygous genotype is compared with the non-DR3 genotype [6]. The lower incidence of IDDM in Turkey and the stronger association with DRB1*0301 may suggest a real impact of it on IDDM susceptibility. The strongest association with DRB1*0402 is reported relatively infrequently in other studies and can be related to the relatively higher frequency of *0402 among other DR4 alleles (22%) in the Turkish population compared to other Caucasians, whereas the association with *0405 compare well with the reports from Sardinia [20], Morocco [11], Mexican American [8], Yemenite Jews [21], Japan [22], and French IDDM patients of all age groups favorably [23]. These relatively
G. Saruhan-Direskeneli et al.
stronger associations are in accordance with many different populations around Mediterranean [20, 23–25]. Compared to northern countries [26 –29], although associated with IDDM, the frequency of DRB1*0401 is relatively lower in our controls (15% of DR4) and DRB1*0404 is not associated, implicating the different association patterns in accordance to north-south gradient of HLA distribution. Especially DRB1*0403, being the most frequent DR4 allele in this population and suggested to be dominantly protective over other susceptibility markers in other populations [30], and *0406 and *0408 did not reveal any association with IDDM. Many negatively associated haplotypes were reported before to be protective for IDDM in different populations [11, 22]. Interestingly, the most frequently reported negative association with DRB*1501 haplotype, is only one of many others and not the dominant protection marker in this population [31]. The possible lower incidence of IDDM in Turkey (2.5/100000, unpublished observation) can be attributed to many negative associations with IDDM compared to Northern countries. Although the associations with distinct DRB1*04 alleles are not shown to be independent of DQB1*0302, the comparison among the DQB1*0302 individuals have confirmed the predisposing effect of the respective DRB1 alleles, namely DRB1*0402, *0401 and *0405 (OR: 3.0, 1.8 and 1.3). The allelic diversity of DR4 subtypes may have contributed to the DR4-associated susceptibility even in the presence of DQB1*0302. The positive associations with IDDM are found similar to Sardinian [20], but the reported dominance of DQB1 protection is not supported, as the negative associations in our data are also with haplotypes carrying DRB1 alleles different than DR4, whereas we did not detect any negative association with DR4. The positive association of DR3/DR4 heterozygous combination supported the putative synergistic role of independent susceptibility alleles [6]. In comparisons of genotypes with susceptible and protective DRB1 alleles, no dominant protective effect could be detected: DR3 or DR4 with DR15 is found 4% in both groups, with no dominance of protective DRB1*15. In conclusion, this study confirms the positive associations of IDDM with DR3 and DR4 in a population from Turkey as shown before by serology [32]. The IDDM associated genotype HLA DR4/3 of the Caucasians, was also significantly increased in the Turkish IDDM patients. Although many different negative associations are demonstrated, the dominant protective effect of the negatively associated alleles and the haplotype of DRB1*1501 is not confirmed. As the major susceptibility locus, the results may implicate the importance of DRB1, but the alleles of DQA1 and DQB1 loci commonly born on the same haplotypes are also associated
HLA Class II Associations of IDDM in Turkey
with IDDM. Our results favor the hypothesis that both DRB1 and DQB1 influence IDDM susceptibility as well as protection.
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8.
ACKNOWLEDGMENTS
The skillful assistance of S¸ule Aydın is gratefully acknowledged. 9.
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Congia M: The distribution of DR4 haplotypes in Sardinia suggests a primary association of type 1 diabetes with DRB1 and DQB1 loci. Hum Immunol 43:301, 1995. Israel S, Kwon OJ, Weintrob N, Sprecher E, Bloch K, Assa S, Brautbar C, Vardi P: HLA class II immunogenetics of IDDM in Yemenite Jews. Hum Immunol 59:728, 1998. Yasunaga S, Kimura A, Hamaguchi K, Ronningen KS, Sasazuki T: Different contribution of HLA-DR and -DQ genes in susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM). Tissue Antigens 47:37, 1996. Caillat-Zucman S, Garchon HJ, Timsit J, Assan R, Boitard C, Djilali-Saiah I, Bougneres P, Bach JF: Age-dependent HLA genetic heterogeneity of type 1 insulin-dependent diabetes mellitus. J Clin Invest 90:2242, 1992. Djoulah S, Khalil I, Beressi JP, Benhamamaouch S, Bessaoud K, Deschamps I, Degos L, Hors J: The HLADRB1*0405 hapotype is most strongly associated with IDDM in Algerians. Eur J Immunogenet 19:381, 1992. Khalil I, Spyropoulou M, Mallet C, Loste MN, Douay C, Laperriere J, Bartzokas C, Lepage V, Charron D, Stavropoulos C: HLA class II polymorphism and IDDM susceptibility in the Greek population. Eur J Immunogenet 20:193, 1993. Noble JA, Valdes AM, Cook M, Klitz W, Thomson G, Erlich HA: The role of HLA class II genes in insulindependent diabetes mellitus: molecular analysis of 180
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Caucasian, multiplex families. Am J Hum Genet 59: 1134, 1996. Reijonen H, Nejentsev S, Tuokko J, Koskinen S, Tuomilehto-Wolf E, Åkerblom HK, Ilonen J and The Childhood Diabetes in Finland Study Group: HLA-DR4 subtype and -B alleles in DQB1*0302 positive haplotypes associated with IDDM. Europ J Immunogenet 24:357, 1997. Sanjeevi CB, Ho¨o¨k P, Landin-Olsson M, Kockum I, Dahlquist G, Lybrand TP, Lernmark Å: DR4 subtypes and their molecular properties in a population-based study of Swedish childhood diabetes. Tissue Antigens 47:275, 1996. Krokowski M, Bodalski J, Bratek A, Boitard C, CaillatZucman S: HLA class II-associated predisposition to insulin-dependent diabetes mellitus in a Polish population. Hum Immunol 59:451, 1998. Undlien DE, Friede T, Rammensee HG, Joner G, DahlJorgensen J, Sovik O, Akselsen HE, Knutsen I, Ronningen KS, Thorsby E: HLA-encoded genetic predisposition in IDDM: DR4 subtypes may be associated with different degrees of protection. Diabetes 46:143, 1997. Sheehy MJ: HLA and insulin-dependent diabetes a protective perspective. Diabetes 41:123, 1992. Ozsahin H, Haktan M, Ozbakir F, Aydin A, Yazici H: The type 1 diabetes and HLA-DR in Turkey. Diabete Metab 17:421, 1991.
INTRODUCTION Insulin-dependent diabetes mellitus (IDDM) is a chronic inflammatory disorder of insulin-producing -cells of From the Istanbul University Istanbul Medical Faculty Physiology Department (G.S.-D., F.A.U.), Istanbul University, Electro-Neurophysiology Research and Application Centre (G.S.-D.), Istanbul University Istanbul Medical Faculty Paediatrics Department (F.B., H.G., R.B., N.S., F.D.), Istanbul, Turkey. A part of this study has been contributed to the XII.International Histocompatibility Workshop. Address reprint requests to: Dr. Gu¨her Saruhan-Direskeneli, Istanbul U¨niversitesi Istanbul Tıp Faku¨ltesi, Fizyoloji AD 34390, C¸apa Istanbul, Turkey; Tel/Fax: ⫹(90) 212 533 94 68; E-Mail: gsaruhan@istanbul. edu.tr. Received July 13, 1999; accepted November 4, 1999. Human Immunology 61, 296 –302 (2000) © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc.
DQB1*0301 (3.9% vs. 12.1%, p ⬍ 0.0001, OR: 0.2). When the DRB, DQA or DQB genotypes of the susceptible alleles are compared, the most strong susceptibility marker of the disease is found to be DRB1*0301/*04 (31.4% vs. 2.8%, p ⬍ 0.0001, OR: 15.8) and among these, heterozygote genotype DRB1*0301/*0401 (4.5% vs. 0, p ⫽ 0.0008, OR: 24.8). These results confirm the positive associations with IDDM previously observed in other Caucasian populations and reveal many negative and strong associations which maybe underlining several characteristics that distinguish Turkish diabetics form other Caucasians. Human Immunology 61, 296 –302 (2000). © American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: HLA-DRB1; HLA-DQA1; HLA-DQB1; IDDM; Turkey
PCR/SSO
polymerase chain reaction/sequencespecific oligonucleotide
pancreatic islets. Genetic contribution to the development of the disease is implicated by the 15-fold higher risk of the first-degree relatives of the patients’ [1]. Mapping studies in IDDM has identified IDDM1 interval at chromosome 6p21 as the first major susceptibility marker, which also contains the MHC region [2]. However, the extensive polymorphism and the functional relevance of MHC genes to an immune response have resulted in many population-based association studies. HLA-DR3 and -DR4 were shown to be strongly associated with the disease in Caucasoids [3]. Many studies have shown associations not only of DRB1, but also of DQA1, DPB1, and DQB1 alleles with IDDM. The 0198-8859/00/$–see front matter PII S0198-8859(99)00182-2
HLA Class II Associations of IDDM in Turkey
highest risk for developing the disease has been associated with heterozygous DR3/DR4 phenotype, particularly in combination with DQA1*0501-DQB1*0201/ DQA1*0301-DQB1*0302 alleles [4 – 6]. Many different negative associations with IDDM were also reported, DRB1*1501 DQA1*0102 DQB1*0602 being the strongest [7–9]. As the alleles of class II haplotypes show strong linkage disequilibria in many populations studied, it has been difficult to identify the susceptibility locus to IDDM [10]. A frequently raised hypothesis suggested that the associated DRB1 alleles are conferring susceptibility, whereas the associated DQ alleles are protecting, and the protection is dominant over the susceptibility [7, 10]. In this regard the pattern of associations in distinct ethnic groups were examined to dissect the susceptibility to IDDM [11]. With this approach, IDDM patients and healthy controls from the population of Turkey are evaluated and comparisons of the distribution of the putative class II “haplotypes,” as well as genotypes are made in this study.
MATERIALS AND METHODS Patients and Controls A total of 178 unrelated patients with IDDM were investigated. Male/female ratio and mean age were 1.1 and 12.3, respectively. The mean age of IDDM diagnosis was 7.5. All patients as well as the controls were of Turkish descent with mixed ethnic origin, patients were selected randomly in Istanbul Medical Faculty referral Hospital at the Department of Pediatrics, Growth Development and Pediatric Endocrinology Unit. The control group consisted of 248 unrelated healthy individuals, which has been investigated before [12]. DNA Typing of DRB, DQA and DQB DNA was prepared from venous blood by using salting out method. The second exons of MHC DRB, DQA1 and DQB1 were amplified with polymerase chain reaction (PCR) using the protocols of the XII.International Histocompatibility Workshop (IHWS): 2DRBAMP-A for “generic” and 2DRBAMP-4, 2DRBAMP-2 or 2DRBAMP-3 for group specific typings were used with 2DRBAMP-B [13]. The primers 2DQAAMP-A and 2DQAAMP-B were used for DQA1 typing and 2DQBAMP-A/2DQBAMP-B primers were used for DQB1. Hybridizations were performed by tetramethylammoniumchloride (TMAC) technique by using biotinylated sequence specific probes, following the IHWS Protocol [13]. The results of hybridizations were evaluated for DRB1.
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Statistical Analysis The results were compared using 2 test and with Fisher’s exact test when appropriate. Odds ratios (OR) were estimated according to Woolf’s formula [14]. A p value of ⬍0.05 was accepted significant and the correction of Bonferonni for multiple tests was applied by multiplying p by the number of comparisons. Confidence intervals (CI) are given for 95%. Individual associations were compared following Svejgaard et al. [15]. RESULTS Phenotypic Associations As the phenotypic combinations of the DRB1, DQA1 and DQB1 alleles were in accordance with the known haplotype assignments and were confirmed in 24 IDDM families studied (data not shown), the phenotypic combinations of HLA class II alleles were deduced as putative “haplotypes” and these are compared in this study, although no formal family study has been done. The distribution among the children with IDDM has shown the positive association of DR3 and DR4: 87% of the patients are DR3 and/or DR4 positive compared to 40% of the controls. Significant differences are demonstrated between the “haplotypic” phenotype frequencies in IDDM and control groups (Table 1). The most strongly IDDM-associated phenotypic combination of alleles was DRB1*0301 DQA1*0501 DQB1*02 with an OR of 6.1 (102 vs. 45, p ⬍ 0.0001). This combination of alleles has been detected concomitantly throughout the group. The “haplotypes” with DRB1*04 were also significantly more frequent in IDDM patients than in controls (109 vs. 61, p ⬍ 0.0001, OR: 4.8). Among the DR4 “haplotypes” (Table 2), DRB1*0402 DQA1*03 DQB1*0302 was the most prominent marker of susceptibility to IDDM (OR: 7.1), followed by DRB1*0401 DQA1*03 DQB1*0302 and DRB1*0405 DQA*03 DQB1*0302. The negative associations with IDDM were diverse in our study (Table 1): DRB1*1401 DQA1*0101 DQB1*0503 has given the lowest OR with 0.06 (p ⬍ 0.0001). The other putative “haplotypes” conferring protection for IDDM were DRB1*1303 DQA1*0501 DQB1*0301, DRB1*1502 DQA1*0103 DQB1*0601, DRB1*1301 DQA1*0103 DQB1*0603, DRB1*1101 DQA1*0501 DQB1*0301, DRB1*0701 DQA1*0201 DQB1*02, DRB1*1104 DQA1*0501 DQB1*0301 and DRB1*1501 DQA1*0102 DQB1*0602. The negative association of DRB1*1303 with IDDM was the only one not reported before, but this association did not reach the significance level after correction. Associations in Non-DR3/Non-DR4 Patients Considering different associations shown previously in non-DR3/non-DR4 IDDM patients [16], we have com-
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TABLE 1 Phenotype frequencies of the DRB1-DQA1-DQB1 combinations in IDDM patients and controls IDDM
Controls
p
DRB1
DQA1
DQB1
n ⴝ 178
%
n ⴝ 248
%
*0101 *0102 *0301 *04 *04 *07 *0701 *08 *08 *09 *1001 *1101 *1102 *1103 *1104 *1201/2 *1301 *1302 *1302 *1303 *1401 *1402 *1501 *1502 *1502 *1601 *1601 *0803/5 *0701 *1404 *1101 *1301 Othersa
*0101 *0101 *0501 *03 *03 *02 *02 *0601 *0401 *03 *0101 *0501 *0501 *0501 *0501 *0501 *0103 *0102 *0102 *0501 *0101 *0101 *0102 *0103 *0102 *0102 *0103 *0401 *02 *0101 *0501 *0103
*0501 *0501 *0201 *0302 *0301 *0201 *0303 *0301 *0402 *0303 *0501 *0301 *0301 *0301 *0301 *0301 *0603 *0604 *0605 *0301 *0503 *0503 *0602 *0601 *0601 *0502 *0502 *0301 *0302 *0503 *0304 *0605
18 2 102 102 7 8 5 1 4 1 1 7 1 1 7 2 1 6 1 0 1 1 9 2 1 23 1 0 0 0 0 0 0
10 1.1 57 57 3.9 4.5 2.8 0.6 2.2 0.6 0.6 3.9 0.6 0.6 3.9 1.1 0.6 3.4 0.6
20 7 45 54 7 35 3 4 8 5 10 47 7 6 30 8 17 19 4 10 22 1 30 19 0 28 0 4 3 3 2 2 13
8.1 2.8 18 22 2.8 14.1 1.2 1.6 3.2 2 4 19 2.8 2.4 12.1 3.2 6.9 7.7 1.6 4 8.9 0.4 12.1 7.7
0.6 0.6 5.1 1.1 0.6 12.9 0.6
OR
95% CI
<0.0001 <0.0001
6.1 4.8
3.9–9.4 3.6–8.5
0.001
0.3
0.1–0.6
<0.0001
0.2
0.08–0.4
0.0029
0.3
0.1–0.7
0.0039
0.2
0.03–0.7
0.0063a <0.0001
0.06 0.06
0.003–1.1 0.007–0.4
0.016 0.0023
0.4 0.1
0.2–0.8 0.03–0.6
11.3 1.6 1.2 1.2 0.8 0.8 5.2
“Others” refer to single combinations detected in controls. OR:Odds ratio. a Refers to p not significant after correction.
pared the phenotype frequencies in 23 patients with 147 controls not bearing any DRB1*03 or DRB1*04 allele. Among these patients DRB1*0101 DQA1*0101 DQB1*0501 frequency was increased to 30%, whereas only 11% of the controls were positive for this phenotype (7 vs. 17, p ⫽ 0.002, OR: 3.3, CI: 1,2–9,3). HLA-DR vs. -DQ To determine the strongest association for alleles of associated combinations, we applied the analysis described by Svejgaard and Ryder [15]. The combined presence of associated DRB1, DQA1, and DQB1 alleles was also high in the Turkish population making the interpretation difficult. DRB1*0301 of the DR3 “haplotype” showed higher OR value for independent association, compared to the DQB1*02, but CI were overlapping. Among the DR4 “haplotypes,” comparisons of associated DRB1 with DQB1 alleles have shown that DRB1*0401-,
*0402- or *0405- DQB1*0302 combinations are probably interacting to confer higher susceptibility. Among all alleles of DRB1, DQA1, and DQB1, DRB1*0402 has given a higher OR (7.3) by its single presence, compared to “haplotypes” with DR4. At the comparisons among the negatively associated phenotypic combinations, the differences did not point out any single locus or any combination of the alleles to be negatively associated with the disease independently. The alleles of the negatively associated “haplotypes,” also carried with the positively associated ones, namely DQA1*0501 and DQB1*02, did not show any protective effect, implicating dominance of DRB1 allele susceptibility over a probable protection. DRB Genotypes If DRB1 genotypes are compared, the increase of DRB1*03/04 is very significant in IDDM group (31.5%
HLA Class II Associations of IDDM in Turkey
299
TABLE 2 Putative DR4 “haplotype” distribution in IDDM and control groups DRB1
DQA
DQB1
IDDM
n *0401 *0402 *0403 *0404 *0405 *0407 *0408 *0401 *0402 *0403 *0404 *0405 *0407 *0408 *0407 *X *X *X
CONTROLS
178 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03 *03
*0302 *0302 *0302 *0302 *0302 *0302 *0302 *0301 *0301 *0301 *0301 *0301 *0301 *0301 *0304 *02 *0304 *0301
%
22 50 8 6 19 5 5 1 1
12.4 28.1 4.5 3.4 10.7 2.8 2.8 0.6 0.6
1 1 1 2 1 2
0.6 0.6 0.6 1.1 0.6 1.1
1
0.6
248
p
OR
95% CI
0.0001 <0.0001
4.9 7.1
2.02–11.6 3.7–13.4
0.0023
3.6
1.5–8.4
%
7 13 19 10 8 1 2 3
2.8 5.2 7.7 4 3.2 0.4 0.8 1.2
1
0.4
1 2
0.4 0.8
2
0.8
1
0.4
In DR4 heterozygots the phenotypes are assigned in favor of most frequent combination. X refers to the unknown DR4 allele already included in DQB1*0302 combinations.
vs. 2.8%, OR: 15.8) (Table 3). Among these, the combined presence of DRB1*0401 (OR: 24.8), *0405 (21.7) or *0402 (OR: 21.0) with DRB1*0301 have been detected most frequently in the patients. This genotype was associated with the highest risk of 51.4, if compared with the individuals lacking both factors [15]. The stratification of DR4 alleles in this comparison has revealed that DRB1*0401, *0405 and *0402 alleles with
DRB1*0301 were associated with OR of 53.8, 45.9, and 32.8, respectively. In the IDDM group, we found 10.7% “homozygous” patients both in DRB1*03 and in DRB1*04, whereas in the controls only 0.8 % were “homozygous” for DRB1*03 and 2.4% for DRB1*04. These homozygous genotypes were strongly associated with IDDM (OR: 14.7 and 4.8, respectively). Considering any other allele
TABLE 3 Comparison of the genotypes in IDDM and control groups DRB1
*0301 *0301 *0301 *0301 *0301 *0301 *0301 *0301 *0301 *04 *04 *04 *0301 X *11 *11
DRB1
*04 *0401 *0402 *0403 *0404 *0405 *0407 *0408 *0301 *04 X *01 X X *13 *07
IDDM
CONT
N ⫽ 178
%
56 8 26 5 4 7 3 3 19 19 34 7 27 23 1 1
31.5 4.5 14.6 2.8 2.2 3.9 1.7 1.7 10.7 10.7 19.1 3.9 15.2 12.9 0.6 0.6
N ⴝ 248
p
OR
95% CI
<0.0001 0.0008 <0.0001
15.8 24.8 21.0
7.0–35.7 1.4–432.4 4.9–89.9
0.002
21.7
1.2–383.2
<0.0001 0.0005
14.7 4.8
3.4–64.0 1.9–12.3
0.038
5.0
1.0–24.5
<0.0001 0.0057 0.029
0.1 0.1 0.1
%
7
2.8
2 2 2
0.8 0.8 0.8
1
0.4
2 6 38 2 36 147 14 10
0.8 2.4 15.3 0.8 14.5 59.3 5.6 4.0
The most frequent genotypes in the “X/X” group are shown only, X being non-DR3/non-DR4.
0.06–0.2 0.01–0.7 0.20–1.1
300
than DR3 or DR4 as X, the DR4/X genotype did not show any association with IDDM, although DRB1*04/ *01 genotype only was also significantly more frequent in IDDM patients (3.9% vs. 0.8%, OR: 5.0) as reported before [17]. The genotype described as X/X has conferred a strong protection to IDDM (23 vs. 147, OR: 0.1, p ⬍ 0.0001), among them DRB1*11/*13 and DRB1*11/*07 significantly different (1 vs. 14 and 1 vs. 10, respectively). When the susceptible DQB1 (Non-Asp at 57.aa position) and DQA1 (Arg at 52.position) alleles were compared with the other “protective” alleles [18], the genotype with four susceptible DQ alleles has conferred an OR of 14.5, whereas the protective genotype with four protective DQ alleles has given an OR of 0.04. DISCUSSION The distributions of DRB1, DQA1 and DQB1 alleles in IDDM patients have revealed that positive associations are in accordance with the known associations in Caucasians and the putative susceptible “haplotypes” for IDDM are highly increased in patients’ group in Turkey. Among associated alleles, DRB1*0402, *0405, *0401 and DRB1*0301 and the phenotypic combinations of DQ alleles with these, have conferred the highest risk for developing IDDM. Many different negative associations have also been confirmed in our study. Our results extend the “haplotypic” disease-associations to the Turkish population [5, 6]. As none, but DRB1*0402, of the allelic associations of DRB1, DQA1, or DQB1 were stronger than the phenotypic combinations, only putative “haplotypes” are evaluated. The highly conserved DR3 haplotype has been shown to be most strongly associated with IDDM in Sardinian before [19]. Although DR3 also associated with IDDM in other populations, the association was not as strong as in this study, which can be traced back to the relatively lower frequency of this “haplotype” in Turkey (18.1% in controls). The risk conferred by DR3 in Turkish patients has been calculated as high as 60.7, when the homozygous genotype is compared with the non-DR3 genotype [6]. The lower incidence of IDDM in Turkey and the stronger association with DRB1*0301 may suggest a real impact of it on IDDM susceptibility. The strongest association with DRB1*0402 is reported relatively infrequently in other studies and can be related to the relatively higher frequency of *0402 among other DR4 alleles (22%) in the Turkish population compared to other Caucasians, whereas the association with *0405 compare well with the reports from Sardinia [20], Morocco [11], Mexican American [8], Yemenite Jews [21], Japan [22], and French IDDM patients of all age groups favorably [23]. These relatively
G. Saruhan-Direskeneli et al.
stronger associations are in accordance with many different populations around Mediterranean [20, 23–25]. Compared to northern countries [26 –29], although associated with IDDM, the frequency of DRB1*0401 is relatively lower in our controls (15% of DR4) and DRB1*0404 is not associated, implicating the different association patterns in accordance to north-south gradient of HLA distribution. Especially DRB1*0403, being the most frequent DR4 allele in this population and suggested to be dominantly protective over other susceptibility markers in other populations [30], and *0406 and *0408 did not reveal any association with IDDM. Many negatively associated haplotypes were reported before to be protective for IDDM in different populations [11, 22]. Interestingly, the most frequently reported negative association with DRB*1501 haplotype, is only one of many others and not the dominant protection marker in this population [31]. The possible lower incidence of IDDM in Turkey (2.5/100000, unpublished observation) can be attributed to many negative associations with IDDM compared to Northern countries. Although the associations with distinct DRB1*04 alleles are not shown to be independent of DQB1*0302, the comparison among the DQB1*0302 individuals have confirmed the predisposing effect of the respective DRB1 alleles, namely DRB1*0402, *0401 and *0405 (OR: 3.0, 1.8 and 1.3). The allelic diversity of DR4 subtypes may have contributed to the DR4-associated susceptibility even in the presence of DQB1*0302. The positive associations with IDDM are found similar to Sardinian [20], but the reported dominance of DQB1 protection is not supported, as the negative associations in our data are also with haplotypes carrying DRB1 alleles different than DR4, whereas we did not detect any negative association with DR4. The positive association of DR3/DR4 heterozygous combination supported the putative synergistic role of independent susceptibility alleles [6]. In comparisons of genotypes with susceptible and protective DRB1 alleles, no dominant protective effect could be detected: DR3 or DR4 with DR15 is found 4% in both groups, with no dominance of protective DRB1*15. In conclusion, this study confirms the positive associations of IDDM with DR3 and DR4 in a population from Turkey as shown before by serology [32]. The IDDM associated genotype HLA DR4/3 of the Caucasians, was also significantly increased in the Turkish IDDM patients. Although many different negative associations are demonstrated, the dominant protective effect of the negatively associated alleles and the haplotype of DRB1*1501 is not confirmed. As the major susceptibility locus, the results may implicate the importance of DRB1, but the alleles of DQA1 and DQB1 loci commonly born on the same haplotypes are also associated
HLA Class II Associations of IDDM in Turkey
with IDDM. Our results favor the hypothesis that both DRB1 and DQB1 influence IDDM susceptibility as well as protection.
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8.
ACKNOWLEDGMENTS
The skillful assistance of S¸ule Aydın is gratefully acknowledged. 9.
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