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HLA class II-associated predisposition to insulin-dependent diabetes mellitus in a Polish population
HLA Class II-Associated Predisposition to Insulin-Dependent Diabetes Mellitus in a Polish Population M. Krokowski, J. Bodalski, A. Bratek, Ch. Boitard, and S. Caillat-Zucman ABSTRACT: Susceptibility and resistance to insulindependent diabetes mellitus (IDDM) are strongly associated with alleles of HLA class II DR and DQ genes. We have studied HLA DRB1, DQA1, DQB1 allele and haplotype distribution in 152 IDDM children and 103 unrelated healthy individuals from the region of Lodz in central Poland by the polymerase chain reaction and hybridisation with allele-specific oligonucleotide probes. The DRB1*04 allele showed the strongest association with IDDM in the Polish population (OR 5 3.87). The DRB1*03 allele was also associated with predisposition to the disease (OR 5 3.25), particularly in DR3/4 heterozygous individuals (OR 5 14.47). Among DR4 subtypes, DRB1*0401 was the most frequent both in patients and controls, whereas DRB1*0403 was rarely observed in patients and conferred a significant protection from IDDM. The DRB1*04-DQA1*0301-DQB1*0302 hap-
lotype conferred the highest risk to develop IDDM. The presence of DRB1*0401 on this haplotype reinforced the disease risk whereas DRB1*0403 had a dominant protective effect even in the presence of the predisposing DQB1*0302 allele (OR 5 0.24). The DRB1*1501DQA1*0102-DQB1*0602 haplotype conferred a dominant protective effect (OR 5 0.04). The different behaviour of the DRB1*04-DQB1*0302 haplotypes in conferring IDDM risk confirms that DRB1 by itself is strongly associated with IDDM independently from DQB1, with DRB1*0401 being a high frequency/ moderate risk allele, and DRB1*0403 a high frequency/ low risk allele in the Polish population. Human Immunology 59, 451– 455 (1998). © American Society for Histocompatibility and Immunogenetics, 1998. Published by Elsevier Science Inc.
ABBREVIATIONS HLA human leukocyte antigens IDDM insulin-dependent diabetes mellitus
PCR SSOP
INTRODUCTION Susceptibility to insulin-dependent diabetes mellitus (IDDM) is determined by a combination of environmental and genetic factors. In human, two main genomic regions, the HLA region on chromosome 6p21 (IDDM1) and the insulin gene region on chromosome 11p15 (IDDM2), show both linkage and association with IDDM [1, 2]. In addition, several other putative IDDM
From the Institute of Pediatrics, University School of Medicine, Lodz, Poland (M.K., J.B., A.B.), and the Laboratory of Immunology and INSERM U25, Hopital Necker, Paris, France (C.B., S.C.-Z.). Address reprint requests to: Marcin Krokowski, MD, PhD, Laboratory of Immunopathology, Institute of Pediatrics, Medical University of Lodz, Sporna 36/50, 91-738 Lodz, Poland; Tel: (4842) 562979; Fax: (4842) 561874; E-Mail: [email protected]. Received December 15, 1997; revised March 10, 1998; accepted March 24, 1998. Human Immunology 59, 451– 455 (1998) © American Society for Histocompatibility and Immunogenetics, 1998 Published by Elsevier Science Inc.
polymerase chain reaction sequence specific oligonucleotide probes
susceptibility loci have been identified in wide whole genome screening studies, some of them still needing to be confirmed [3, 4]. The contribution of HLA to the genetic risk of IDDM is estimated at approximately 50% [3]. However, population studies have shown that HLA associations may vary depending on the geographic and ethnic origin [5]. In Caucasians, predisposition to IDDM is mostly associated with the DRB1*03-DQB1*0201 and/or DRB1*04-DQB1*0302 haplotypes, whereas the DRB1*15-DQB1*0602 haplotype confers a strong protection from the disease [1, 6]. Although the DQ locus is usually considered as primarily associated with disease susceptibility, some DR alleles, in particular DR4 subtypes have been shown to greatly influence the risk to develop IDDM. So, the net effect on predisposition to or 0198-8859/98/$19.00 PII S0198-8859(98)00036-6
452
protection from IDDM results from the combination of DQ and DR alleles. This study is the first report concerning the distribution of HLA class II alleles among IDDM patients in a Polish population. PATIENTS AND METHODS Patients The population studied consisted of 152 IDDM children from the region of Lodz in central Poland. Mean age at IDDM onset was 9.5 years (range 0.4 –17.5 years). In 49.3% of cases, IDDM occurred before the age of 10 years. The control population consisted of 103 unrelated healthy blood donors from the same region of Lodz. HLA Typing Genomic DNA was extracted from peripheral blood cells and the second exon of the DRB1, DQA1 and DQB1 genes was amplified by the polymerase chain reaction (PCR) using specific flanking primers. Alleles were identified following hybridisation with sequence specific oligonucleotide probes (SSOP) according to the XIIth International Histocompatibility Workshop protocols [7]. A total of 25 probes was used for DRB1 generic typing, 18 for DRB1*04 subtyping, 18 for DQA1, and 24 for DQB1 typing. Statistical Analysis Allele and phenotype frequencies were determined at each locus. Odds ratios (OR) were calculated according to the Woolf’s formula with Haldane’s modification when necessary. The p value was defined by chi-square analysis using a 2 3 2 contingency table or Fisher’s exact test when appropriate, and corrected (pc) for the number of comparisons. The level of significance was set to 0.05. RESULTS The DRB1, DQA1 and DQB1 allele frequencies are shown in Tables 1 and 2. DRB1 Locus The DRB1*04 allele showed the strongest association with IDDM in the Polish population (38.8% in IDDM patients versus 14.1% in controls, OR 5 3.87, pc , 0.0001). The DRB1*03 allele frequency was also significantly increased in IDDM patients compared to controls (OR 5 3.25). DRB1*03/03 or 04/04 homozygosity was not more frequently observed than expected. DRB1*03 was neutral in the absence of DRB1*04 on the other chromosome (19.7% of patients vs 16.5% of controls). By contrast, the DRB1*03/04 heterozygous combination was strongly associated with IDDM predisposition
M. Krokowski et al.
TABLE 1 DRB1 allele frequencies among IDDM patients (152) and healthy controls (103)
DRB1 01 15 16 03 04 07 08 11 12 13 14
IDDM n 5 304 (%)
Controls n 5 206 (%)
33 (10.9) 4 (1.3) 14 (4.6) 85 (28.0) 118 (38.8) 20 (6.5) 9 (3.0) 6 (2.0) 2 (0.6) 13 (4.3) 0 (0)
35 (17.0) 30 (14.6) 6 (2.8) 22 (10.7) 29 (14.1) 29 (14.1) 4 (1.9) 22 (10.7) 3 (1.5) 19 (9.2) 7 (3.4)
pc
OR [95% CI]
,0.001
0.08 [0.03–0.23]
,0.001 ,0.0001
3.25 [1.95–5.40] 3.87 [2.46–6.11]
,0.001
0.17 [0.07–0.42]
n—number of alleles OR is only indicated when the corrected p value is less than 0.05.
(30.3% in IDDM vs 2.9% in control subjects, OR 5 14.47; p , 0.0001). Altogether, the vast majority of patients (92.1%) expressed the DRB1*03 and/or DRB1*04 phenotype. The DRB1*15 allele conferred a strong protection TABLE 2 Distribution of DQA1 and DQB1 alleles among IDDM patients and healthy controls
DQA1 0101 0102 0103 0201 0301 0401 0501 0601 DQB1 02 0301 0302 0303 0402 0501 0502 05031 0601 0602 0603 0604 0605
IDDM n 5 304 (%)
Controls n 5 206 (%)
33 (10.8) 23 (7.6) 8 (2.6) 20 (6.6) 118 (38.8) 9 (3.0) 93 (30.6) 0 (0)
42 (20.4) 40 (19.4) 11 (5.3) 29 (14.1) 29 (14.1) 2 (1.0) 51 (24.7) 2 (1.0)
105 (34.5) 19 (6.3) 106 (34.9) 1 (0.3) 9 (3.0) 33 (10.9) 14 (4.6) 0 1 (0.3) 2 (0.6) 6 (2.0) 8 (2.6) 0
46 (22.3) 41 (19.9) 18 (8.7) 6 (2.9) 2 (1.0) 35 (17.0) 6 (2.9) 7 (3.4) 2 (1.0) 28 (13.6) 9 (4.4) 5 (2.4) 1 (0.5)
pc
OR [95% CI]
0.03 ,0.001
0.48 [0.29–0.78] 0.34 [0.20–0.59]
,0.0001
3.87 [2.46–6.11]
,0.001 ,0.001
0.27 [0.15–0.48] 5.59 [3.26–9.58]
,0.001
0.04 [0.01–0.18]
n—number of alleles; OR is only indicated when the corrected p value is less than 0.05.
Insulin-Dependent Diabetes Mellitus
453
TABLE 3 Distribution of DRB1*04-DQA1-DQB1 haplotypes among IDDM patients and healthy controls
DRB1
DQA1
DQB1
DR4-positive IDDM n 5 118 (%)
0401 0401 0402 0403 0403 0403 0407 0407 0417 0417
0301 0301 0301 0301 0301 0301 0301 0301 0301 0301
0301 0302 0302 0301 0302 02 0301 0302 0302 02
4 (3.4) 83 (70.3) 9 (7.6) 1 (0.9) 10 (8.5) 0 6 (5.1) 1 (0.8) 3 (2.5) 1 (0.9)
DR4-positive controls n 5 29 (%) 8 (27.6) 7 (24.2) 2 (6.9) 0 8 (27.6) 1 (3.4) 2 (6.9) 0 1 (3.4) 0
p
OR [95% CI]
0.0003 ,0.0001
0.09 [0.03–0.33] 7.45 [2.92–19.04]
,0.01
0.24 [0.09–0.69]
n—number of DRB1*04-DQA1-DQB1 haplotypes; OR is only indicated when the corrected p value is less than 0.05.
from IDDM (OR 5 0.08). The DRB1*11 frequency was also decreased in diabetics relative to controls (OR 5 0.17). DQA1 and DQB1 Loci The DQA1*0301 frequency was increased (OR 5 3.87) whereas the DQA1*0102 and DQA1*0101 frequencies were decreased (OR 5 0.34 and 0.48, respectively) in IDDM patients relative to controls. Among DQB1 alleles, only DQB1*0302 conferred a strong predisposing effect (OR 5 5.59). The DQB1*0602 and DQB1*0301 alleles were both associated with protection from the disease (OR 5 0.04 and 0.27, respectively). Haplotype Analysis Haplotypes were deduced from the known linkage disequilibria in Caucasians. The DRB1*04-DQA1*0301DQB1*0302 haplotype conferred the highest risk to develop IDDM (OR 5 5.6, p , 0.0001). However, it appeared that the different DR4 subtypes modulated this risk (Table 3). The presence of DRB1*0401 on this haplotype reinforced the disease risk (OR 5 7.45), whereas DRB1*0403 had an opposite protective effect even in the presence of the predisposing DQB1*0302 allele (OR 5 0.24). The DRB1*03-DQA1*0501-DQB1*0201 haplotype was associated with predisposition to IDDM (OR 5 3.25, p , 0.0001), particularly when combined to the DRB1*04-DQA1*0301-DQB1*0302 haplotype in heterozygous individuals (28.3% of IDDM patients compared to none of controls, OR 5 82.2, p , 0.0001). The DRB1*1501-DQA1*0102-DQB1*0602 haplotype was found in 0.7% of IDDM patients compared to 13.6% of controls, thus conferring a dominant protective effect (OR 5 0.04, p , 0.0001). The DRB1*0401DQA1*0301-DQB1*0301, DRB1*11-DQA1*0501-
DQB1*0301 and DRB1*0403-DQA1*0301-DQB1* 0302 haplotypes were also found at lower frequencies in IDDM patients than in controls (OR 5 0.09, p 5 0.0003; OR 5 0.17, p , 0.0001, and OR 5 0.24, p , 0.01, respectively). Age-Dependent Associations The distribution of the DRB1-DQB1 haplotypes and genotypes was analysed according to the age at IDDMonset (below or over 10 years). No significant differences were observed (data not shown). DISCUSSION This study is the first report on HLA association with IDDM in the Polish population. Altogether, IDDM is associated with the DRB1*04-DQA1*0301-DQB1*0302 and/or DRB1*03-DQA1*0501-DQB1*0201 haplotypes, like in most other Caucasian groups [1, 8]. The heterozygous combination confers a strong synergistic effect, as usually observed. By contrast, homozygosity for the DR3 haplotype does not increase the risk to develop the disease in the Polish group, at variance with other populations such as Basque, Turkish or Argentinian [7], where DRB1*03/03 individuals have a similar or even higher risk than DRB1*03/04 heterozygotes. Furthermore, the DR3 haplotype, in absence of DR4 as other haplotype, is not significantly associated with IDDM susceptibility, which distinguishes the Polish population from others like Belgian, Sardinian, or French [9 –11]. Like in most populations studied so far, the DRB1*1501-DQB1*0602 haplotype confers a strong protection from IDDM [6, 10, 12]. In our patient group, only two patients carried this haplotype, which was associated with the DRB1*0403-DQB1*0302 or DRB1*0407-DQB1*0302 on the other chromosome. Analysis of the DRB1*04-subtype distribution is par-
454
ticularly informative, since it has been shown to greatly vary depending on the population studied. A modifying effect of certain DRB1*04 subtypes on the susceptibility encoded by DQ alleles is demonstrated by the fact that not all DRB1*04-DQB1*0302 haplotypes have an IDDM-predisposing effect. Thus, DRB1*0401 and 0404 have been shown associated with IDDM in Norway [12], DRB1*0402 and 0405 in France [11, 13] and in Mexican Americans [14], DRB1*0404 in Mexican Mestizos [7], and DRB1*0401 and 0402 in Australia [15]. DRB1*0405 confers a strong risk in Sardinians, Spaniards, North Africans, Blacks and Japanese [16 –18]. Conversely, protection from the disease is provided by DRB1*0403 in Sardinians, Belgians and Chinese [18 – 21], DRB1*0404 in French [11, 13], DRB1*0403, 0408, and 0411 in Mexican-Americans [14], and DRB1*0406 in Orientals [20 –22]. In our Polish population, the DRB1*0401 subtype represents almost 74% of all DR4 alleles and confers a significant risk to IDDM when combined to DQB1*0302. The DRB1*0402 is very rarely observed and DRB1*0404 and 0405 are absent in both patient and control groups. DRB1*0403 is strongly protective, even in the presence of the susceptible DQB1*0302 allele. The different behaviour of the DRB1*04DQB1*0302 haplotypes in conferring IDDM risk provides further evidence that DRB1 by itself is strongly associated with IDDM independently from DQB1, and is in favor of a hierarchy of IDDM susceptibility among DR4 subtypes as previously proposed [23], with DRB1*0401 being a high frequency/moderate risk allele, and DRB1*0403 being a high frequency/low risk allele in the Polish population. Among the various DRBDQB allelic combinations, a single dose of protective DR or DQ allele, such as DRB1*0403, is sufficient to provide dominant protection. In this sense, screening for prediabetes only by DQ genotyping may be misleading since protective genes can influence the penetrance of the DQA/DQB susceptible genotypes. On the other hand, susceptibility seems to require a combination of DR and DQ alleles (interactive or additive effect).
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ACKNOWLEDGMENTS
This work has been supported by the following grants from INSERM U25 N° EO 001/94 and KBN N° 4 P05E 01810. We thank E. Audran, P. Przednowed, and I. Texier for their technical assistance, and Dr. D. Marciniak-Bielak for collaboration.
REFERENCES 1. Thomson G, Robinson WP, Kuhner MK, Joe S, MacDonald MJ, Gottschall JL, Barbosa J, Rich SS, Bertrams J, Baur MP, Partanen J, Tait BD, Schober E, Mayr WR,
10.
11.
Ludvigsson J, Lindblom B, Farid R, Thompson C, Deschamps I: Genetic heterogeneity, modes of inheritance, and risk estimates for a joint study of Caucasians with insulin-dependent diabetes mellitus. Am J Hum Genet 43:799 – 816, 1988. Bennett ST, Lucassen AM, Gough SC, Powell EE, Undlien DE, Pritchard LE, Merriman ME, Kawaguchi Y, Dronsfield MJ, Pociot F, Nerup J, Bouzekri N, CambonThomsen A, Ronningen KS, Barnett AH, Bain SC, Todd JA: Susceptibility to human type 1 diabetes at IDDM2 is determined by tandem repeat variation at the insulin gene minisatellite locus. Nat Genet 9:284 –292, 1995. Davies JL, Kawaguchi Y, Bennett ST, Copeman JB, Cordell HJ, Pritchard LE, Reed PW, Gough SC, Jenkins SC, Palmer SM, Balfour KM, Rowe BR, Farrall M, Barnett AH, Bain SC, Todd JA: A genome-wide search for human type 1 diabetes susceptibility genes. Nature 371:130 – 136, 1994. Owerbach D, Gabbay KH: The search for IDDM susceptibility genes: the next generation. Diabetes 45:544 –551, 1996. She JX: Susceptibility to type I diabetes: HLA-DQ and DR revisited. Immunol Today 17:323–329, 1996. Baisch JM, Weeks T, Giles R, Hoover M, Stastny P, Capra JD: Analysis of HLA-DQ genotypes and susceptibility in insulin-dependent diabetes mellitus. N Engl J Med 322:1836 –1841, 1990. Caillat-Zucman S, Djilali-Saiach I, Timsit J, Bonifacio E, Sepe V, Collins P, Bottazzo GF, Maclaren N, Delamaire M, Martin S, Yamamoto AM, McWeeney S, Valdes AM, Babron MC, Thorsby E, Clerget-Darpoux F, Thomson G, Bach JF and the participating centers. Insulin dependent diabetes mellitus (IDDM): 12th International Histocompatibility Workshop study. In D. Charron (ed): HLA. Genetic diversity of HLA. Functional and Medical implications. EDK, 389 –398, 1997. Sanjeevi CB, Hook P, Landin Olsson M, Kockum I, Dahlquist G, Lybrand TP, Lernmark A: DR4 subtypes and their molecular properties in a population-based study of Swedish childhood diabetes. Tissue Antigens 47:275–283, 1996. Buyse I, Sandkuyl LA, Zamani Ghabanbasani M, Gu XX, Bouillon R, Bex M, Dooms L, Emonds MP, Duhamel M, Marynen P, Cassiman JJ: Association of particular HLA class II alleles, haplotypes and genotypes with susceptibility to IDDM in the Belgian population. Diabetologia 37:808 – 817, 1994. Cucca F, Muntoni F, Lampis R, Frau F, Argiolas L, Silvetti M, Angius E, Cao A, De Virgiliis S, Congia M: Combinations of specific DRB1, DQA1, DQB1 haplotypes are associated with insulin-dependent diabetes mellitus in Sardinia. Hum Immunol 37:85–94, 1993. Caillat Zucman S, Garchon HJ, Timsit J, Assan R, Boitard C, Djilali S, Bougneres P, Bach JF: Age-dependent HLA genetic heterogeneity of type 1 insulin-dependent diabetes mellitus. J Clin Invest 90:2242–2250, 1992.
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12. Ronningen KS, Spurkland A, Iwe T, Vartdal F, Thorsby E: Distribution of HLA-DRB1, -DQA1 and -DQB1 alleles and DQA1-DQB1 genotypes among Norwegian patients with insulin-dependent diabetes mellitus. Tissue Antigens 37:105–111, 1991. 13. Harfouch Hammoud E, Timsit J, Boitard C, Bach JF, Caillat Zucman S: Contribution of DRB1*04 variants to predisposition to or protection from insulin dependent diabetes mellitus is independent of dq. J Autoimmun 9:411– 414, 1996. 14. Erlich HA, Zeidler A, Chang J, Shaw S, Raffel LJ, Klitz W, Beshkov Y, Costin G, Pressman S, Bugawan T, Rotte JI: HLA class II alleles and susceptibility and resistance to insulin dependent diabetes mellitus in Mexican–American families. Nat Genet 3:358 –364, 1993. 15. Tait BD, Drummond BP, Varney MD, Harrison LC: HLA-DRB1*0401 is associated with susceptibility to insulin-dependent diabetes mellitus independently of the DQB1 locus. Eur J Immunogenet 22:289 –297, 1995. 16. Ronningen K, S., Spurkland A, Tait B, D., Drummond B, Lopez-Larrea C, Baranda S, Menendez-Diaz MJ, CaillatZucman S, Beaurain G, Garchon HJ, Ilonen J, Reijonen H, Knip M, Boehm BO, Rosak C, Looliger C, Uhnl M, Ottenhoff T, Contu L, Carcassi C, Savi M, Zanelli P, Neri TM, Yamaguchi K, Kimura A, Dong RP, Chikuba N, Nagataki S, Gorodezky C, Debaz H, Robles C, Coimbra HB, Martinho A, Ruas MA, Sachs JA, Garcia-Pachedo JM, Biro A, Nikaein A, Dombrausky L, Gonwa T, Zmijewski C, Monos DS, Kamoun M, Layrisse Z, Magli MC, Balducci P, Thorsby E: HLA class II association in insulin dependent diabetes mellitus among Black, Caucasoid and Japanese. In Anonymous: HLA 1991. Oxford: Oxford University Press, 713–722, 1992. 17. Morales P, Martinez Laso J, Martin Villa JM, Corell A,
455
18.
19.
20.
21.
22.
23.
Vicario JL, Varela P, Perez Aciego P, Arnaiz Villena A: High frequency of the HLA-DRB1*0405-(Dw15)-DQw8 haplotype in Spaniards and its relationship to diabetes susceptibility. Hum Immunol 32:170 –175, 1991. Cucca F, Lampis R, Frau F, Macis D, Angius E, Masile P, Chessa M, Frongia P, Silvetti M, Cao A, Deviriliis S, Congia M: The distribution of DR4 haplotypes in Sardinia suggests a primary association of type 1 diabetes with DRB1 and DQB1 loci. Hum Immunol 43:301–308, 1995. Van der Auwera B, van Waeyenberge C, Schuit F, Heimberg H, Vandewalle C, Gorus F, Flament J: DRB1*0403 protects against IDDM in Caucasians with the high-risk heterozygous DQA1*0301-DQB1*0302/DQA1*0501DQB1*0201 genotype. Belgian Diabetes Registry. Diabetes 44:527–530, 1995. Huang HS, Peng JT, She JY, Zhang LP, Chao CC, Liu KH, She JX: HLA-encoded susceptibility to insulin-dependent diabetes mellitus is determined by DR and DQ genes as well as their linkage disequilibria in a Chinese population. Hum Immunol 44:210 –219, 1995. Penny MA, Jenkins D, Mijovic CH, Jacobs KH, Cavan DA, Yeung VT, Cockram CS, Hawkins BR, Fletcher JA, Barnett AH: Susceptibility to IDDM in a Chinese population. role of HLA class. Diabetes 41:914 –919, 1992. Awata T, Kuzuya T, Matsuda A, Iwamoto Y, Kanazawa Y: Genetic analysis of HLA class II alleles and susceptibility to type 1 (insulin-dependent) diabetes mellitus in Japanese subjects. Diabetologia 1992:35:419 – 424, 1992. Undlien DE, Friede T, Rammensee HG, Joner G, Dahl Jorgensen K, 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 46:143–149, 1997.
lotype conferred the highest risk to develop IDDM. The presence of DRB1*0401 on this haplotype reinforced the disease risk whereas DRB1*0403 had a dominant protective effect even in the presence of the predisposing DQB1*0302 allele (OR 5 0.24). The DRB1*1501DQA1*0102-DQB1*0602 haplotype conferred a dominant protective effect (OR 5 0.04). The different behaviour of the DRB1*04-DQB1*0302 haplotypes in conferring IDDM risk confirms that DRB1 by itself is strongly associated with IDDM independently from DQB1, with DRB1*0401 being a high frequency/ moderate risk allele, and DRB1*0403 a high frequency/ low risk allele in the Polish population. Human Immunology 59, 451– 455 (1998). © American Society for Histocompatibility and Immunogenetics, 1998. Published by Elsevier Science Inc.
ABBREVIATIONS HLA human leukocyte antigens IDDM insulin-dependent diabetes mellitus
PCR SSOP
INTRODUCTION Susceptibility to insulin-dependent diabetes mellitus (IDDM) is determined by a combination of environmental and genetic factors. In human, two main genomic regions, the HLA region on chromosome 6p21 (IDDM1) and the insulin gene region on chromosome 11p15 (IDDM2), show both linkage and association with IDDM [1, 2]. In addition, several other putative IDDM
From the Institute of Pediatrics, University School of Medicine, Lodz, Poland (M.K., J.B., A.B.), and the Laboratory of Immunology and INSERM U25, Hopital Necker, Paris, France (C.B., S.C.-Z.). Address reprint requests to: Marcin Krokowski, MD, PhD, Laboratory of Immunopathology, Institute of Pediatrics, Medical University of Lodz, Sporna 36/50, 91-738 Lodz, Poland; Tel: (4842) 562979; Fax: (4842) 561874; E-Mail: [email protected]. Received December 15, 1997; revised March 10, 1998; accepted March 24, 1998. Human Immunology 59, 451– 455 (1998) © American Society for Histocompatibility and Immunogenetics, 1998 Published by Elsevier Science Inc.
polymerase chain reaction sequence specific oligonucleotide probes
susceptibility loci have been identified in wide whole genome screening studies, some of them still needing to be confirmed [3, 4]. The contribution of HLA to the genetic risk of IDDM is estimated at approximately 50% [3]. However, population studies have shown that HLA associations may vary depending on the geographic and ethnic origin [5]. In Caucasians, predisposition to IDDM is mostly associated with the DRB1*03-DQB1*0201 and/or DRB1*04-DQB1*0302 haplotypes, whereas the DRB1*15-DQB1*0602 haplotype confers a strong protection from the disease [1, 6]. Although the DQ locus is usually considered as primarily associated with disease susceptibility, some DR alleles, in particular DR4 subtypes have been shown to greatly influence the risk to develop IDDM. So, the net effect on predisposition to or 0198-8859/98/$19.00 PII S0198-8859(98)00036-6
452
protection from IDDM results from the combination of DQ and DR alleles. This study is the first report concerning the distribution of HLA class II alleles among IDDM patients in a Polish population. PATIENTS AND METHODS Patients The population studied consisted of 152 IDDM children from the region of Lodz in central Poland. Mean age at IDDM onset was 9.5 years (range 0.4 –17.5 years). In 49.3% of cases, IDDM occurred before the age of 10 years. The control population consisted of 103 unrelated healthy blood donors from the same region of Lodz. HLA Typing Genomic DNA was extracted from peripheral blood cells and the second exon of the DRB1, DQA1 and DQB1 genes was amplified by the polymerase chain reaction (PCR) using specific flanking primers. Alleles were identified following hybridisation with sequence specific oligonucleotide probes (SSOP) according to the XIIth International Histocompatibility Workshop protocols [7]. A total of 25 probes was used for DRB1 generic typing, 18 for DRB1*04 subtyping, 18 for DQA1, and 24 for DQB1 typing. Statistical Analysis Allele and phenotype frequencies were determined at each locus. Odds ratios (OR) were calculated according to the Woolf’s formula with Haldane’s modification when necessary. The p value was defined by chi-square analysis using a 2 3 2 contingency table or Fisher’s exact test when appropriate, and corrected (pc) for the number of comparisons. The level of significance was set to 0.05. RESULTS The DRB1, DQA1 and DQB1 allele frequencies are shown in Tables 1 and 2. DRB1 Locus The DRB1*04 allele showed the strongest association with IDDM in the Polish population (38.8% in IDDM patients versus 14.1% in controls, OR 5 3.87, pc , 0.0001). The DRB1*03 allele frequency was also significantly increased in IDDM patients compared to controls (OR 5 3.25). DRB1*03/03 or 04/04 homozygosity was not more frequently observed than expected. DRB1*03 was neutral in the absence of DRB1*04 on the other chromosome (19.7% of patients vs 16.5% of controls). By contrast, the DRB1*03/04 heterozygous combination was strongly associated with IDDM predisposition
M. Krokowski et al.
TABLE 1 DRB1 allele frequencies among IDDM patients (152) and healthy controls (103)
DRB1 01 15 16 03 04 07 08 11 12 13 14
IDDM n 5 304 (%)
Controls n 5 206 (%)
33 (10.9) 4 (1.3) 14 (4.6) 85 (28.0) 118 (38.8) 20 (6.5) 9 (3.0) 6 (2.0) 2 (0.6) 13 (4.3) 0 (0)
35 (17.0) 30 (14.6) 6 (2.8) 22 (10.7) 29 (14.1) 29 (14.1) 4 (1.9) 22 (10.7) 3 (1.5) 19 (9.2) 7 (3.4)
pc
OR [95% CI]
,0.001
0.08 [0.03–0.23]
,0.001 ,0.0001
3.25 [1.95–5.40] 3.87 [2.46–6.11]
,0.001
0.17 [0.07–0.42]
n—number of alleles OR is only indicated when the corrected p value is less than 0.05.
(30.3% in IDDM vs 2.9% in control subjects, OR 5 14.47; p , 0.0001). Altogether, the vast majority of patients (92.1%) expressed the DRB1*03 and/or DRB1*04 phenotype. The DRB1*15 allele conferred a strong protection TABLE 2 Distribution of DQA1 and DQB1 alleles among IDDM patients and healthy controls
DQA1 0101 0102 0103 0201 0301 0401 0501 0601 DQB1 02 0301 0302 0303 0402 0501 0502 05031 0601 0602 0603 0604 0605
IDDM n 5 304 (%)
Controls n 5 206 (%)
33 (10.8) 23 (7.6) 8 (2.6) 20 (6.6) 118 (38.8) 9 (3.0) 93 (30.6) 0 (0)
42 (20.4) 40 (19.4) 11 (5.3) 29 (14.1) 29 (14.1) 2 (1.0) 51 (24.7) 2 (1.0)
105 (34.5) 19 (6.3) 106 (34.9) 1 (0.3) 9 (3.0) 33 (10.9) 14 (4.6) 0 1 (0.3) 2 (0.6) 6 (2.0) 8 (2.6) 0
46 (22.3) 41 (19.9) 18 (8.7) 6 (2.9) 2 (1.0) 35 (17.0) 6 (2.9) 7 (3.4) 2 (1.0) 28 (13.6) 9 (4.4) 5 (2.4) 1 (0.5)
pc
OR [95% CI]
0.03 ,0.001
0.48 [0.29–0.78] 0.34 [0.20–0.59]
,0.0001
3.87 [2.46–6.11]
,0.001 ,0.001
0.27 [0.15–0.48] 5.59 [3.26–9.58]
,0.001
0.04 [0.01–0.18]
n—number of alleles; OR is only indicated when the corrected p value is less than 0.05.
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453
TABLE 3 Distribution of DRB1*04-DQA1-DQB1 haplotypes among IDDM patients and healthy controls
DRB1
DQA1
DQB1
DR4-positive IDDM n 5 118 (%)
0401 0401 0402 0403 0403 0403 0407 0407 0417 0417
0301 0301 0301 0301 0301 0301 0301 0301 0301 0301
0301 0302 0302 0301 0302 02 0301 0302 0302 02
4 (3.4) 83 (70.3) 9 (7.6) 1 (0.9) 10 (8.5) 0 6 (5.1) 1 (0.8) 3 (2.5) 1 (0.9)
DR4-positive controls n 5 29 (%) 8 (27.6) 7 (24.2) 2 (6.9) 0 8 (27.6) 1 (3.4) 2 (6.9) 0 1 (3.4) 0
p
OR [95% CI]
0.0003 ,0.0001
0.09 [0.03–0.33] 7.45 [2.92–19.04]
,0.01
0.24 [0.09–0.69]
n—number of DRB1*04-DQA1-DQB1 haplotypes; OR is only indicated when the corrected p value is less than 0.05.
from IDDM (OR 5 0.08). The DRB1*11 frequency was also decreased in diabetics relative to controls (OR 5 0.17). DQA1 and DQB1 Loci The DQA1*0301 frequency was increased (OR 5 3.87) whereas the DQA1*0102 and DQA1*0101 frequencies were decreased (OR 5 0.34 and 0.48, respectively) in IDDM patients relative to controls. Among DQB1 alleles, only DQB1*0302 conferred a strong predisposing effect (OR 5 5.59). The DQB1*0602 and DQB1*0301 alleles were both associated with protection from the disease (OR 5 0.04 and 0.27, respectively). Haplotype Analysis Haplotypes were deduced from the known linkage disequilibria in Caucasians. The DRB1*04-DQA1*0301DQB1*0302 haplotype conferred the highest risk to develop IDDM (OR 5 5.6, p , 0.0001). However, it appeared that the different DR4 subtypes modulated this risk (Table 3). The presence of DRB1*0401 on this haplotype reinforced the disease risk (OR 5 7.45), whereas DRB1*0403 had an opposite protective effect even in the presence of the predisposing DQB1*0302 allele (OR 5 0.24). The DRB1*03-DQA1*0501-DQB1*0201 haplotype was associated with predisposition to IDDM (OR 5 3.25, p , 0.0001), particularly when combined to the DRB1*04-DQA1*0301-DQB1*0302 haplotype in heterozygous individuals (28.3% of IDDM patients compared to none of controls, OR 5 82.2, p , 0.0001). The DRB1*1501-DQA1*0102-DQB1*0602 haplotype was found in 0.7% of IDDM patients compared to 13.6% of controls, thus conferring a dominant protective effect (OR 5 0.04, p , 0.0001). The DRB1*0401DQA1*0301-DQB1*0301, DRB1*11-DQA1*0501-
DQB1*0301 and DRB1*0403-DQA1*0301-DQB1* 0302 haplotypes were also found at lower frequencies in IDDM patients than in controls (OR 5 0.09, p 5 0.0003; OR 5 0.17, p , 0.0001, and OR 5 0.24, p , 0.01, respectively). Age-Dependent Associations The distribution of the DRB1-DQB1 haplotypes and genotypes was analysed according to the age at IDDMonset (below or over 10 years). No significant differences were observed (data not shown). DISCUSSION This study is the first report on HLA association with IDDM in the Polish population. Altogether, IDDM is associated with the DRB1*04-DQA1*0301-DQB1*0302 and/or DRB1*03-DQA1*0501-DQB1*0201 haplotypes, like in most other Caucasian groups [1, 8]. The heterozygous combination confers a strong synergistic effect, as usually observed. By contrast, homozygosity for the DR3 haplotype does not increase the risk to develop the disease in the Polish group, at variance with other populations such as Basque, Turkish or Argentinian [7], where DRB1*03/03 individuals have a similar or even higher risk than DRB1*03/04 heterozygotes. Furthermore, the DR3 haplotype, in absence of DR4 as other haplotype, is not significantly associated with IDDM susceptibility, which distinguishes the Polish population from others like Belgian, Sardinian, or French [9 –11]. Like in most populations studied so far, the DRB1*1501-DQB1*0602 haplotype confers a strong protection from IDDM [6, 10, 12]. In our patient group, only two patients carried this haplotype, which was associated with the DRB1*0403-DQB1*0302 or DRB1*0407-DQB1*0302 on the other chromosome. Analysis of the DRB1*04-subtype distribution is par-
454
ticularly informative, since it has been shown to greatly vary depending on the population studied. A modifying effect of certain DRB1*04 subtypes on the susceptibility encoded by DQ alleles is demonstrated by the fact that not all DRB1*04-DQB1*0302 haplotypes have an IDDM-predisposing effect. Thus, DRB1*0401 and 0404 have been shown associated with IDDM in Norway [12], DRB1*0402 and 0405 in France [11, 13] and in Mexican Americans [14], DRB1*0404 in Mexican Mestizos [7], and DRB1*0401 and 0402 in Australia [15]. DRB1*0405 confers a strong risk in Sardinians, Spaniards, North Africans, Blacks and Japanese [16 –18]. Conversely, protection from the disease is provided by DRB1*0403 in Sardinians, Belgians and Chinese [18 – 21], DRB1*0404 in French [11, 13], DRB1*0403, 0408, and 0411 in Mexican-Americans [14], and DRB1*0406 in Orientals [20 –22]. In our Polish population, the DRB1*0401 subtype represents almost 74% of all DR4 alleles and confers a significant risk to IDDM when combined to DQB1*0302. The DRB1*0402 is very rarely observed and DRB1*0404 and 0405 are absent in both patient and control groups. DRB1*0403 is strongly protective, even in the presence of the susceptible DQB1*0302 allele. The different behaviour of the DRB1*04DQB1*0302 haplotypes in conferring IDDM risk provides further evidence that DRB1 by itself is strongly associated with IDDM independently from DQB1, and is in favor of a hierarchy of IDDM susceptibility among DR4 subtypes as previously proposed [23], with DRB1*0401 being a high frequency/moderate risk allele, and DRB1*0403 being a high frequency/low risk allele in the Polish population. Among the various DRBDQB allelic combinations, a single dose of protective DR or DQ allele, such as DRB1*0403, is sufficient to provide dominant protection. In this sense, screening for prediabetes only by DQ genotyping may be misleading since protective genes can influence the penetrance of the DQA/DQB susceptible genotypes. On the other hand, susceptibility seems to require a combination of DR and DQ alleles (interactive or additive effect).
M. Krokowski et al.
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ACKNOWLEDGMENTS
This work has been supported by the following grants from INSERM U25 N° EO 001/94 and KBN N° 4 P05E 01810. We thank E. Audran, P. Przednowed, and I. Texier for their technical assistance, and Dr. D. Marciniak-Bielak for collaboration.
REFERENCES 1. Thomson G, Robinson WP, Kuhner MK, Joe S, MacDonald MJ, Gottschall JL, Barbosa J, Rich SS, Bertrams J, Baur MP, Partanen J, Tait BD, Schober E, Mayr WR,
10.
11.
Ludvigsson J, Lindblom B, Farid R, Thompson C, Deschamps I: Genetic heterogeneity, modes of inheritance, and risk estimates for a joint study of Caucasians with insulin-dependent diabetes mellitus. Am J Hum Genet 43:799 – 816, 1988. Bennett ST, Lucassen AM, Gough SC, Powell EE, Undlien DE, Pritchard LE, Merriman ME, Kawaguchi Y, Dronsfield MJ, Pociot F, Nerup J, Bouzekri N, CambonThomsen A, Ronningen KS, Barnett AH, Bain SC, Todd JA: Susceptibility to human type 1 diabetes at IDDM2 is determined by tandem repeat variation at the insulin gene minisatellite locus. Nat Genet 9:284 –292, 1995. Davies JL, Kawaguchi Y, Bennett ST, Copeman JB, Cordell HJ, Pritchard LE, Reed PW, Gough SC, Jenkins SC, Palmer SM, Balfour KM, Rowe BR, Farrall M, Barnett AH, Bain SC, Todd JA: A genome-wide search for human type 1 diabetes susceptibility genes. Nature 371:130 – 136, 1994. Owerbach D, Gabbay KH: The search for IDDM susceptibility genes: the next generation. Diabetes 45:544 –551, 1996. She JX: Susceptibility to type I diabetes: HLA-DQ and DR revisited. Immunol Today 17:323–329, 1996. Baisch JM, Weeks T, Giles R, Hoover M, Stastny P, Capra JD: Analysis of HLA-DQ genotypes and susceptibility in insulin-dependent diabetes mellitus. N Engl J Med 322:1836 –1841, 1990. Caillat-Zucman S, Djilali-Saiach I, Timsit J, Bonifacio E, Sepe V, Collins P, Bottazzo GF, Maclaren N, Delamaire M, Martin S, Yamamoto AM, McWeeney S, Valdes AM, Babron MC, Thorsby E, Clerget-Darpoux F, Thomson G, Bach JF and the participating centers. Insulin dependent diabetes mellitus (IDDM): 12th International Histocompatibility Workshop study. In D. Charron (ed): HLA. Genetic diversity of HLA. Functional and Medical implications. EDK, 389 –398, 1997. Sanjeevi CB, Hook P, Landin Olsson M, Kockum I, Dahlquist G, Lybrand TP, Lernmark A: DR4 subtypes and their molecular properties in a population-based study of Swedish childhood diabetes. Tissue Antigens 47:275–283, 1996. Buyse I, Sandkuyl LA, Zamani Ghabanbasani M, Gu XX, Bouillon R, Bex M, Dooms L, Emonds MP, Duhamel M, Marynen P, Cassiman JJ: Association of particular HLA class II alleles, haplotypes and genotypes with susceptibility to IDDM in the Belgian population. Diabetologia 37:808 – 817, 1994. Cucca F, Muntoni F, Lampis R, Frau F, Argiolas L, Silvetti M, Angius E, Cao A, De Virgiliis S, Congia M: Combinations of specific DRB1, DQA1, DQB1 haplotypes are associated with insulin-dependent diabetes mellitus in Sardinia. Hum Immunol 37:85–94, 1993. Caillat Zucman S, Garchon HJ, Timsit J, Assan R, Boitard C, Djilali S, Bougneres P, Bach JF: Age-dependent HLA genetic heterogeneity of type 1 insulin-dependent diabetes mellitus. J Clin Invest 90:2242–2250, 1992.
Insulin-Dependent Diabetes Mellitus
12. Ronningen KS, Spurkland A, Iwe T, Vartdal F, Thorsby E: Distribution of HLA-DRB1, -DQA1 and -DQB1 alleles and DQA1-DQB1 genotypes among Norwegian patients with insulin-dependent diabetes mellitus. Tissue Antigens 37:105–111, 1991. 13. Harfouch Hammoud E, Timsit J, Boitard C, Bach JF, Caillat Zucman S: Contribution of DRB1*04 variants to predisposition to or protection from insulin dependent diabetes mellitus is independent of dq. J Autoimmun 9:411– 414, 1996. 14. Erlich HA, Zeidler A, Chang J, Shaw S, Raffel LJ, Klitz W, Beshkov Y, Costin G, Pressman S, Bugawan T, Rotte JI: HLA class II alleles and susceptibility and resistance to insulin dependent diabetes mellitus in Mexican–American families. Nat Genet 3:358 –364, 1993. 15. Tait BD, Drummond BP, Varney MD, Harrison LC: HLA-DRB1*0401 is associated with susceptibility to insulin-dependent diabetes mellitus independently of the DQB1 locus. Eur J Immunogenet 22:289 –297, 1995. 16. Ronningen K, S., Spurkland A, Tait B, D., Drummond B, Lopez-Larrea C, Baranda S, Menendez-Diaz MJ, CaillatZucman S, Beaurain G, Garchon HJ, Ilonen J, Reijonen H, Knip M, Boehm BO, Rosak C, Looliger C, Uhnl M, Ottenhoff T, Contu L, Carcassi C, Savi M, Zanelli P, Neri TM, Yamaguchi K, Kimura A, Dong RP, Chikuba N, Nagataki S, Gorodezky C, Debaz H, Robles C, Coimbra HB, Martinho A, Ruas MA, Sachs JA, Garcia-Pachedo JM, Biro A, Nikaein A, Dombrausky L, Gonwa T, Zmijewski C, Monos DS, Kamoun M, Layrisse Z, Magli MC, Balducci P, Thorsby E: HLA class II association in insulin dependent diabetes mellitus among Black, Caucasoid and Japanese. In Anonymous: HLA 1991. Oxford: Oxford University Press, 713–722, 1992. 17. Morales P, Martinez Laso J, Martin Villa JM, Corell A,
455
18.
19.
20.
21.
22.
23.
Vicario JL, Varela P, Perez Aciego P, Arnaiz Villena A: High frequency of the HLA-DRB1*0405-(Dw15)-DQw8 haplotype in Spaniards and its relationship to diabetes susceptibility. Hum Immunol 32:170 –175, 1991. Cucca F, Lampis R, Frau F, Macis D, Angius E, Masile P, Chessa M, Frongia P, Silvetti M, Cao A, Deviriliis S, Congia M: The distribution of DR4 haplotypes in Sardinia suggests a primary association of type 1 diabetes with DRB1 and DQB1 loci. Hum Immunol 43:301–308, 1995. Van der Auwera B, van Waeyenberge C, Schuit F, Heimberg H, Vandewalle C, Gorus F, Flament J: DRB1*0403 protects against IDDM in Caucasians with the high-risk heterozygous DQA1*0301-DQB1*0302/DQA1*0501DQB1*0201 genotype. Belgian Diabetes Registry. Diabetes 44:527–530, 1995. Huang HS, Peng JT, She JY, Zhang LP, Chao CC, Liu KH, She JX: HLA-encoded susceptibility to insulin-dependent diabetes mellitus is determined by DR and DQ genes as well as their linkage disequilibria in a Chinese population. Hum Immunol 44:210 –219, 1995. Penny MA, Jenkins D, Mijovic CH, Jacobs KH, Cavan DA, Yeung VT, Cockram CS, Hawkins BR, Fletcher JA, Barnett AH: Susceptibility to IDDM in a Chinese population. role of HLA class. Diabetes 41:914 –919, 1992. Awata T, Kuzuya T, Matsuda A, Iwamoto Y, Kanazawa Y: Genetic analysis of HLA class II alleles and susceptibility to type 1 (insulin-dependent) diabetes mellitus in Japanese subjects. Diabetologia 1992:35:419 – 424, 1992. Undlien DE, Friede T, Rammensee HG, Joner G, Dahl Jorgensen K, 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 46:143–149, 1997.