Extensive Study of DRB, DQA, and DQB Gene Polymorphism in 23 DR2-Positive, Insulin-Dependent Diabetes Mellitus Patients Dominique Zeliszewski, Jean-Marie Tiercy, Christian Boitard, Xue-Fan Gu, Michel Loche, Rajagopal Krishnamoorthy, Nancy Simonney, Jacques Elion, Jean-Franqois Bach, Bernard Mach, and Ghislaine Sterkers ABSTRACT: To gain insight into the HLA subregions involved in protection against insulin-dependent diabetes mellitus (IDDM) we investigated the polymorphism of HLA-DR and -DQ genes in 23 DR2 IDDM patients. Results show the following. (1) Fourteen patients (61%) possess the DRB1, DRB5, and DQB1 alleles found in DRw16/DQw5 healthy people. These data contrast with the 5% of DRw16 normally found in DR2 populations and are in agreement with former observations supporting that the DRw16 haplotype is not protective. (2) Nine DR2 patients, i.e., 39% versus 95% in published DR2 controls, possess the DRB alleles found in DRw15 unaffected people. Among them, six patients have also DQA1 and DQB1 alleles identical to those found in DRw15/ DQw6 healthy individuals. These data confirm that the DRwlS/DQw6 haplotype is protective but indicate that
ABBREVIATIONS IDDM insulin-dependent diabetes mellitus PCR polymerase chain reaction RFLP restriction fragment length polymorphism
none of the DR or DQ alleles, alone or in association, confers an absolute protection. (3) Our most striking results concern the very high frequency of recombinant haplotypes among the DRwl5 patients: 3 of 9. In these three patients recombinations led to the elimination of both DQB1 and DQA1 alleles usually associated with DRwl5. This strongly suggests that the occurrence of IDDM in these DRwl5 patients is due to the absence of the usual DQ product and thus reinforces the assumption that DQ rather than DR region is involved in the protection conferred by the DRwl 5/DQw6 haplotype. Finally, analysis of the non-DRwl5 haplotypes in heterozygous patients showed that IDDM can occur in the absence of any DQafl heterodimer of susceptibility. Human Immunology 33, 1 4 0 - I 4 7 (I992)
PBMC Asp Arg
peripheral blood mononuclear cells aspartic acid arginine
INTRODUCTION Insulin-dependent diabetes mellitus (IDDM) in an autoimmune disease whose precise etiology is still unFrom the Immunology Laboratory 1NSERM, H6pital Robert Debre (D.Z.; X.-F.G.: R.K.; N.S.,'J.E.: G.S.), Paris, France; Transplantation Immunology Unit, H6pital Cantonal Universitaire, and Department of Genetics and Microbiology, C.M.U. (].-M.T.; M.L.; B.M.), Geneva, Switzerland," and INSERM, HSpital Necker (C.B.; J.-F.B.), Paris, France. Address reprint requests to D. Zeliszewski, Immunology Laboratory, INSERM CJF 90-15, HSpital Robert Debre, 48 bd Serurier, 75019 Paris, France. Received September 14, 1991; acceptedNovember27, 1991.
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known. Several genes are likely to contribute to I D D M development. A m o n g them, at least one gene is located within the major histocompatibility complex (MHC)class II region [1, 2]. The definition of HLA molecules associated with I D D M development has been initially approached by determining the frequency of H L A - D R and D Q serologically defined specificities among patients as compared to control populations. First, a positive association mainly with H L A - D R 3 / D Q w 2 and D R 4 / D Q w 3 specificities [ 1 - 3 ] and a negative association mainly with D R 2 / D Q w l specificities [1, 4] have Human Immunology33, 140-147 (1992) © AmericanSocietyfor Histocompatibilityand Immunogenetics,1992
I n v o l v e m e n t o f H L A - D Q in I D D M P r o t e c t i o n
141
TABLE 1 D R 2 / D Q w l haplotypes usually found in healthy populations Atlelic polymorphism of DR and D Q loci Serological specificities
Serological subtypes
Cellular typing
DRB 1
DRB 5
DQA 1
DQB 1
DR2/DQwl
D R w l 5/DQw6 D R w l 5/DQw6 D R w l 6/DQw5
Dw2 Dwl2 Dw21 (AZH)
1501 1502 1601
0101 0102 0201
0102 0103 0102
0602 0601 0502
The DRA gene is not indicated because it is monomorphic. For details on the new nomenclature see ref 15.
been described. Later, IDDM associations were more precisely defined by using genomic rather than serological markers [5-10]. Indeed, restriction fragment length polymorphism (RFLP) and more recently oligonucleotide typing techniques allowed a precise definition of HLA polymorphism that could not be achieved by serological methods. Thus, IDDM susceptibility and IDDM protection were assigned to DQB subregion. This was reinforced by statistical analysis of DQBchain-deduced sequences with an apparent correlation with amino acid at position 57 for both protection and susceptibility [ 11, 12]. An exclusive role of DQB genes could, however, be an oversimplified conclusion since the same susceptible DQB1 allele is found in susceptible DR3 and neutral DR7 haplotypes as well. Furthermore, several reports have suggested that DQA and DRB might also be implicated in susceptibility [13, 14]. In this study we focused on the HLA-DR2/DQwl haplotype which is the most strongly protective against IDDM in view of its extremely low frequency in patients (less than 0.5%) as compared to controls ( 1 8 % 25%) [1, 4, 11]. At least three haplotypes within DR2/ D Q w l have been unequivocally identified by RFLP and oligotyping methods. For simplicity they are recalled in Table 1. In Caucasians, two main haplotypes have been described: one contains DRA, DRBl*1501, DRB5*0101, DQAI*0102, and DQBI*0602 alleles. These genes code for DRw15 and DQw6 products that are subdivisions of DR2 and DQwl, respectively [15]. The second haplotype contains DRA, DRBl*1601, DRB5*0201, DQAI*0102, and DQBI*0502 alleles. These genes code for DRw16 and DQw5 products that are also subdivisions of DR2 and DQwl, respectively (Table 1). Due to quasi absolute linkage disequilibrium between DR and D Q genes, other DRB, DQA, and DQB allelic combinations than those cited above are exceptional. Protection against IDDM has been correlated with the expression of DRw15/DQw6 but not DRw16/
DQw5 encoding haplotype, mainly from studies of DQB allelic polymorphism [9-12]. Indeed, DQBI*0602 allele was absent in most cohorts of IDDM patients. This led to the conclusion that this allele confers protection against diabetes. Yet in these studies, the polymorphism of other HLA-class II genes (DRB1, DRB5, or DQA1) linked to DQBI*0602 in healthy populations was not always investigated in DR2 patients. Thus, a clear mapping of protective genes to DQ region remains to be clearly established. This can be hopefully addressed by studying "recombinant" haplotypes, i.e., haplotypes presenting a disruption of the usual DR-DQ linkage disequilibrium. In this regard, in a previous study, we found by DQB RFLP analysis that the two DRw15 IDDM patients studied had a DQB gene different from that of healthy DRw15 individuals [ 16]. This strongly suggested a contrario that at least the HLA-DQB region was involved in protection. However, further analysis of DRB, DQA, and DQB polymorphism, on a larger panel of DRw15 IDDM patients, was required to definitely determine the respective contribution of each of these HLA class II genes in this protection. In the present study we collected the largest panel of DR2 IDDM patients studied so far, from several hundreds of serologically typed patients, and their DRB, DQA, and DQB allelic polymorphism was characterized by RFLP or oligonucleotide typing after specific D N A amplifications. METHODS Patients. Twenty-three
DR2 IDDM patients were selected from 358 HLA-phenotyped patients from Necker and R. Debre hospitals (Paris, France). These patients were characterized as presenting IDDM following World Health Organization criteria. Autoimmune diabetes was inferred from the presence of islet cell antibodies (above 5 Juvenile Diabetes Foundation units) in 57% of studied patients. Islet cell antibody determi-
142
TABLE 2
Name
D. Zeliszewski et al.
Oligonucleotide probes used in this study
Reference
Alleles identified
D11
23
D37 (DR) AV86 F67 V86 D57 A49 $57 V38 I37 E45 A57 D37 (DQ) V27 V57 DQAI.1 DQA1.2
25 25 25 25 20 20 20 20 20 20 20 20 20 20 24 24
DRB5*0101, 0201 DRB5*0102, 0202 DRB5*0101 DRB5*0201, 0202 DRBl*1601 DRBl*1501 DQB 1"0602, 0603 DQBI*0501 DQB 1"0502, 0504 DQBI*0501, 0502, 0503, 0504 DQBI*0201 DQBI*0301 DQBI*0302 DQBI*0601 DQB 1"0603, 0604 DQBI*0604, 0605, 0501 DQAI*0101 DQAI*0102, 0103, 0501
Amino acid position
Washing temperature (°C)
9-14
50
35-4O 83-88 64-69 85-89
50 54 50 50 58
55-60 47-52
55- 6O 35-40 34-39 42-48 54-59 34-39 25-30 55-60 32-37 32-37
nation was performed in reference to an international standard as part of four international workshop standardization programs. The prevalence of islet cell antibodies in DR2 patients studied reflected the overall sensitivity of the test in an age-matched population of IDDM patients considered independently of HLA phenotype [17]. Patients' peripheral blood mononuclear cells (PBMC) were isolated from blood samples by FicollHipaque gradient centrifugation. HLA serological typing was performed according to a standard procedure in a microlymphocytotoxicity assay. After serological typing residual PBMC were frozen until further use for DNA analysis.
DNA amplifications." T cells from residual PBMC were expanded in macrotiter plates with interleukin 2 (10 IU/ml) during 10 days, after initial stimulation by 1/zg/ ml of phytohemagglutinin A. By this method sufficient amounts of cells could be available from most patients for preparation of DNA. DNA was extracted by standard techniques. DRB1, DRB5, DQA1, or DQB1 first domain exons were selectively amplified from 1/zg genomic DNA by polymerase chain reaction (PCR) using the thermostable Taq-Polymerase, as described elsewhere [18-20].
Oligoprobes: Oligoprobes were synthesized by the /3cyanoethyl amidite method on a Pharmacia gene assembler. Their purification and labeling are described elsewhere [21, 22]. Oligoprobes used in this study are
56 56
58 54 58 64 54
52 56
58 58
described in Table 2, except those used for DRB 1 typing of non-DR2 haplotypes. These latter probes are extensively described elsewhere [23].
Oligotyping: Oligotyping was performed as described elsewhere [20, 22, 23]. Briefly, small aliquots (5%10%) of amplified DNA were denatured in 0.4N NaOH for 10 min at 20°C, neutralized with 1 vol of ammonium acetate 2M, and spotted on a Nylon membrane (Nytran, Schleicher and SchOll, New Hampshire) using the Minifold II slot-blot apparatus (Schleicher and SchiJll). Membranes were prehybridized (30 rain at 50°C) and hybridized (3 h at 50°C) in 5 × SSC, 20mM Na-phosphate, 10x Denhardt's, 5% SDS, and 100/zg/ ml denatured herring DNA. The hybridization mixture contained 0 . 5 - 1 × 106 cpm/ml of labeled oligoprobe. Blots were washed first 1 h at 50°C in 3 × SSC, 70 mM Na-phosphate (pH7), 10x Denhardt's, 5% SDS, and then 1 h in 1% SDS, 1 x SSC at different temperatures indicated in Table 2. Autoradiographs (3-12 h) were performed using x-OMAT XAR Kodak films (Rochester, NY) and CAWO SE4 intensifying screens.
DQA PCR-RFLP typing: DQA typing was performed as described elsewhere [24]. Briefly, after specific DQA gene amplification, three aliquots (8 ILl) were digested with three restriction endonucleases: Fok I, Dde I, or Rsa I. Each product of digestion was then subjected to 8% polyacrylamide gel electrophoresis (100 V, 25 mA) for about 3 h. After migration gels were incubated in ethidium bromide for 10 rain, washed, and photo-
Involvement of HLA-DQ in IDDM Protection
TABLE 3
143
Oligotyping analysis of DRB and DQB alleles in DR2 haplotypes from the 23 IDDM patients DQB 1 oligotyping:
Patient
DRB5
identification
Serological
number
typing
Dll
(DR2)
oligotyping: Probes (specificity) D37(DRw15)
probes (specificity)
AV86(DRw16)
D57(0602Y
$57(0502)
1
2/3
+b
+
_
+
3
2/4
+
+
-
+
_
4
2/3
+
+
-
5
2/3
+
+
-
+
-
6
2/w13
+
+
-
-
-
7
2/9
+
+
-
+
-
8
2/4
+
+
-
+
-
9
2/w12
+
+
-
+
-
10
214
+
+
-
11
2/7
+
-
+
-
+
12
2/w6
+
-
+
-
+
13
2/4
+
-
+
-
+
14
2/3
+
-
+
-
+
15
2/-
+
-
+
ND
ND
-
-
16
2/3
+
-
+
-
+
17
2./1
+
-
+
ND
ND
18
2/3
+
-
+
ND
ND
19
2,/1
+
-
+
-
+
20
2/-
+
-
+
-
+
21
2/'4
+
-
+
-
+
22
214
+
-
+
-
+
23
2/'3
+
-
+
-
+
24
2/3
+
-
+
-
+
T h e D Q B I * 0 6 0 2 allele was identified by the p r o b e D 5 7 but also by a negative hybridization with p r o b e V 2 7 (not shown). b Results are expressed as + or - according to hybridizations or not with the probes indicated.
graphed under UV light on polaroi'd films. Each migration pattern after digestion with the three enzymes corresponds to one D Q A allele. But for identifications of DQAI*0101 and DQAI*0102 alleles, one additional step is necessary, i.e., oligotyping. For this, slot-blots, hybridizations, and washing were performed as described above. DQA 1*0101 - and DQA 1*0102-specific probes are described in Table 2. RESULTS
Selection of patients: This study was restricted to wellcharacterized IDDM patients initially typed as DR2 by serology, and subsequently confirmed to be DR2 by oligotyping. All D N A samples were characterized by HLA-DR generic oligotyping [23], which included the use of oligoprobe D 11 specific for all DR2 alleles (Table 3, third column). DRB1 and DRB5 alleles characterization." The DR2 subtype of the 23 DR2 patients was identified by oligotyping of DRB5 polymorphism with two oligoprobes, D37
and AV86, recognizing DRB5*0101 and DRB5*0201/ 0202, respectively (see Table 2). Table 3 shows results concerning only the DR2 haplotype of the patients. As shown in Table 3, 9 of 23 IDDM patients were D37positive (fourth column) and are therefore identified as DRw15. The remaining 14 patients were AV86-positive (fifth column) and are therefore referred to as DRw16 patients. In most of these DR2 IDDM patients allelic polymorphism at locus DRB 1 was subsequently determined after locus-specific amplification [25] and hybridization with probe V86, which is specific for DRBI*1501, and probe F67, which is specific for DRB1*1601 (see Table 2). As expected from the strong DRB1-DRB5 linkage disequilibrium existing in healthy donors [25], all DRB5*0101 patients (i.e., DRw15) exhibited the DRB1*1501 allele (Table 4, second column), and all DRB5*0201 patients (i.e., DRw16) exhibited the DRB 1" 1601 allele (not shown). The DRB1 oligotyping of their non-DR2 haplotype was in agreement with that expected from serological typing (Table 4, and not shown).
144
D. Zeliszewski et al.
TABLE 4
DRB1, DQB1, and DQA1 Detailed typing of both haplotypes of the 9 DRw15 patients DQB1
Patient identification number
DRB 1 typing
DQB 1 oligotyping
Presence of D Q B Asp57
4 6 10 3 1 5 7 8 9
1501/0301 1501/1302 1501/04-1501/04-1501/0301 1501/0301 1501/0901 1501/04-I501/1201
-a/0201 0301/0604 0503/0302 0602/0302 0602/0201 0602/0201 0602/0303 0602/0302 0602/0301
A/NA A/NA A/NA A/NA A/NA A/A A/NA A/A
-/NA ~
DQA 1 typing ND 0601/0102 0401/0301 0102/0301 0102/0501 0102/0501 ND 0102/0301 0102/0501
No hybridization with any probe tested. b Results are indicated as A for an Asp at position 57 or NA for absence of Asp at position 57 on the corresponding t3 chains.
DQBI characterization: DQB 1 alleles were then characterized by oligotyping using 10 oligoprobes (see Table 2) as described [20]. As shown in Table 3 for the DR2 haplotype, all of the DRw16 IDDM patients tested exhibited the DQBI*0502 allele as identified with the oligoprobe $57, and as expected from the DR-DQ linkage disequilibrium usually found in DRw16 individuals. The DQBI*0602 allele, which is exclusively found in DRw15/DQw6 Caucasian individuals, is defined by a positive hybridization signal with probe D57 (0602 + 0603) and a negative hybridization signal with probe V27 (0603 + 0604). The probe D57 recognizes the aspartic acid (Asp) at position 57, which has been postulated to correlate with IDDM protection [11]. Based on the very strong DR-DQ linkage disequilibrium, our group of patients shown to be DRw15 should present the DQBI*0602 allele. This was indeed the case for 6 of 9 patients (see Table 3, sixth column). Moreover, two of these six patients are homozygous for the presence of an Asp at position 57 (see Table 4). But surprisingly, the remaining three DRw15 patients did not hybridize with the DQBl*0602-specific probe (patients 4, 6, and 10), suggesting a disruption of the usual DR-DQ disequilibrium. Table 4 shows the DQB 1 typing of both haplotypes for the 9 DRwl5 patients. In each case the DQB1 allele encoded by the DRwl5 haplotype is indicated in first position. Patient 4 has a hybridization pattern compatible with DQBI*0201 homozygosity. Patient 6 has a DQB 1"0301 allele (corresponding to DQw7), and a familial segregation analysis clearly showed that this allele is on the DRwl 5 haplotype, while the second haplotype
encodes for DRwl3 and DQw6 (not shown). Finally, in patient 10 the DRwl5 haplotype presents the DQBI*0503 allele usually linked to DRBI*1401 (corresponding to DRwl4). In all 9 heterozygous DRwl5 patients, the non-DR2 haplotype was conform to the expected DR-DQ associations. In summary the three DRwl5 IDDM patients that did not show the expected linkage disequilibrium with DQBl*0602 exhibited, respectively, the alleles DQBI*0201, 0301, 0503. It is of interest that two of these three alleles do contain an Asp at position 57 (see Table 4).
DQA1 characterization." DQA genes have been proposed to be also involved in IDDM susceptibility and resistance [13, 26]. It must be pointed out that no DR2 IDDM patient was included in these two reports. Thus, we characterized DQA1 genes from our DRwl5 patients by using a technique of RFLP analysis after specific DNA amplification by PCR [24]. This allows a rapid DQA 1 typing with only three restriction enzymes and two oligoprobes. DQA1 gene characterization of the six DQBl*0602-positive patients shows that all of them are DQAl*0102-positive, as are DRw15/DQw6 healthy individuals (see Table 4, fifth column). Concerning the three DQBl*0602-negative patients, patients 6 and 10 are both DQAl*0102-negative. As noted above, concerning patient 6, a familial segregation analysis allowed to conclude that alleles DQBI*0301 and DQAI*0601 encoding DQw7 are present on the DRw15 haplotype. It is interesting to note that the second haplotype (DRw13) presents a DQAI*0102 allele, linked as usual to DQBI*0604. Unfortunately, DQA 1 typing of patient 4 could not be performed because an insufficient amount of DNA was available. Finally, here again the non-DR2 haplotype of the 9 heterozygous DRw15 patients corresponds to DQA1DQB 1 associations that can be found in healthy populations [15, and unpublished observations]. If we consider both haplotypes, the DQAI*0102 allele usually found in DRw15 individuals is present in at least 6 of our DRw15 patients, including one of the three patients having recombined haplotypes. DISCUSSION We characterized the polymorphism of DRB1, DRB5, DQA1, and DQB1 genes in 23 DR2 IDDM patients. First, results presented herein show that 14 of 23 DR2 patients express DRB 1, DRB5, and DQB1 genes identical to those found in DRwl6/DQw5 healthy probands. Since the DQA1 locus is located between DR
Involvement of HLA-DQ in IDDM Protection
and DQB1 loci, we can anticipate that the DRw16 patients also express a DQA1 allele identical to that of healthy DRwl6 individuals. No recombination was found in our 14 DRwl6/DQw5 patients. In contrast, we found a very large proportion of recombinant haplotypes in the 9 remaining DR2 patients. In fact, 3 of 9, and 4 of 10 if we include former observations reported elsewhere [ 16], have recombinant haplotypes. They express DRB1 and DRB5 alleles similar if not identical to those found in DRwl5 healthy people but they do not express the DQB 1 allele usually present in DRwl5/DQw6 healthy individuals. This strongly suggests that elimination of the DQw6 product has restored the susceptibility to the disease. To our knowledge the three recombinant haplotypes reported herein have never been described in healthy people. We believe that, rather than being characteristic of IDDM patients, these haplotypes have not been found in unaffected people because of their exceptionally rare frequency. One unusual DR2 haplotype has already been described by others [27] and it is distinct from the new DR2 haplotypes we describe here. Because the DQAI*0102 allele is present in at least one of the DQBl*0602-negative patients (if we consider both haplotypes), this suggests a contrario that the DQB subregion is more closely associated with resistance to the disease than the DQA subregion. This is reinforced by the fact that DRwl 5 and DRwl6 individuals share the same DQA allele. Among the 23 DR2 IDDM patients studied, the frequencies of usual DRB, DQA1, and DQB1 alleles are 39%, 91%, and 26%, respectively. The latter frequency correlates with that found by Todd et al. [ 11 ]. HLA-associated protection against IDDM is correlated with the presence of Asp at position 57 on the DQ,8 chain. The fact that less than 5 % of diabetic individuals are heterozygous non-Asp/Asp (NA/A) versus 46% in controls has suggested [12] that protection observed for Asp behaved in a dominantlike manner. However, it must be pointed out that 8 of our 9 DRwl 5 patients are Asp57-positive on the DQ/3 chains, including two of the patients who have an unusual DQB1 allele (see Table 4). Moreover, we found two Caucasian patients with an Asp57 on both DQ]3 chains, and thus two "protective" DQB1 alleles. This pattern A/A is very rare among Caucasian diabetics [28] but is found among Asiatic patients [29, 30]. Thus, our data reinforce the idea that the protection conferred by Asp57 is not dominant and not absolute. More recently Khalil et al. [26] have implicated the DQA locus in both susceptibility and resistance to IDDM. In addition to DQfi-Asp57, they suggested that DQ~ chains which have an arginine (Arg) at position 52 are susceptible (S) while all Arg52-negative DQa chains
145
TABLE 5
Presence of putative susceptible or protective DQ~ or 3 chains among the 9 DRwl 5 patients
Patients
DQ~ ~
DQ/3 b
4 6 10 3
-' S/P S/S P/S P/S P/S P/S P/S
-/S P/S P/S P/S P/S P/S P/P P/S P/P
1
5 7 8 9
Arg52-positive oe chains are susceptible (S) and Arg52-negative c~ chains are protective (P) according to Khalil et aL [26]. h Asp57-negative/3 chai*~s are susceptible (S) and Asp57-positive/3 chains are protective (P) according to Todd et al. [11]. '
ND
are protective (P). According to their model, the development of diabetes depends on the presence at the cell surface of at least one heterodimer DQo43 "SS," this heterodimer being possibly created by transcomplementation. Table 5 shows the presence of S or P DQc~ and DQB chains in the 9 DRwl 5 patients, according to the classifications of Khalil et al. [26] and Todd et al. [11]. Two of our diabetic patients are DQfl P/P and most probably DQce P/S (see Table 5, patients 9 and 7). These patients can only express PS or PP DQ heterodimers and thus constitute exceptions to the proposed rule. The possibility that patients could develop IDDM while expressing two protective DQ products confirms that the inheritance and genetic feature of IDDM is complex. The DQc~/3 heterodimer in DRwl5/DQw6 haplotype does not confer an absolute protection even though it might correlate with a huge incidence of resistance to IDDM. Altogether these results indicate that resistance to IDDM cannot be fully explained by DQ polymorphism. The mechanism(s) by which some HLA products can confer a resistance to IDDM are unknown. The polymorphism of class II molecules has a major influence on the T-cell repertoire. Indeed, it is involved in both positive and negative selections o f T cells expressing certain T-cell receptors during thymic education. Thus, one possible mechanism that would account for the involvement of DQ genes in resistance against IDDM would be that the DQ molecule from the DRw15/DQw6 haplotype would lead to a clonal deletion of lymphocytes bearing a T-cell receptor directed against a putative islet autoantigen. This hypothesis is compatible with studies
146
p e r f o r m e d in nonobese diabetic mice suggesting that expression of the protective I-E class II product leads to a clonal deletion of specific autoreactive T-cell precursors [31]. H o w e v e r , since we found that the D Q product encoded for by the D R w 1 5 / D Q w 6 haplotype does not confer an absolute protection, we must postulate that in some individuals autoreactive clones would escape this clonal deletion possibly due to other genetic factors. An alternative although not exclusive hypothesis could be that the D Q region is involved in the control of suppressive mechanisms on T-cell clones directed against the putative autoantigen in the periphery. H e r e again such protection might be, in some situations, o v e r c o m e depending on polymorphic genes not linked with M H C . In conclusion, the detection of recombinant haplotypes in 3 out of 9 DRw15 I D D M patients provides further evidence that the D Q B subregion is strongly protective against I D D M . But this protection is not absolute and cannot be explained only by D Q gene polymorphism. ACKNOWLEDGMENTS
We thank B. Battistolo, A. Morrisson, and P. Roux-Chabbey for their excellent technical assistance, M. Seman for helpful comments on the manuscript, and I. Rivenez for typing the manuscript. This work was partly supported by an EMBO fellowship and by a "contrat de recherches cliniques de l'assistance publique. H6pitaux de Paris."
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D. Zeliszewski et al.
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19. Saiki RK, Gelfand DH, Stoffels S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487, 1988. 20. Morel C, Zwahlen F, Jeannet M, Mach B, Tiercy J-M: Complete analysis of HLA-DQB1 polymorphism and DR-DQ linkage disequilibrium by oligonucleotide typing. Hum Immunol 29:64, 1990.
Involvement of HLA-DQ in IDDM Protection
2 I. Angelini G, DePreval C, Gorski J, Mach B: High resolution analysis of the human HLA-DR polymorphism by hybridization with sequence-specific oligonucleotide probes. Proc Natl Acad Sci USA 83:4489, 1986. 22. Tiercy J-M, Gorski J, Jeannet M, Mach B: Identification and distribution of three serologically undetected alleles of HLA-DR by oligonucleotide. DNA typing analysis. Proc Natl Acad Sci USA 85:198, 1988. 23. Mach B, Tiercy J-M: Genotypic typing of H L A class II: from the bench to the bedside. Hum Immunol 30:278, 1991. 24. Ju LY, Gu XF, Larger E, Krishnamoorthy R, Charron D: Application of silver staining to the rapid typing of the polymorphism of HLA-DQ alleles by enzymatic amplification and allele specific restriction fragment length polymorphism. Electrophoresis 12:270, 1991. 25. Tiercy J-M, Jeannet M, Mach B: Oligonucleotide typing analysis for the linkage disequilibrium between the polymorphic DRB1 and DRB5 loci in DR2 haplotypes. Tissue Antigens 37:161, 1991. 26. Khalil I, d'Auriol L, Gobet M, Morin L, Lepage V, Deschamps I, Park MS, Degos L, Galibert F, Hors J: A combination of HLA-DQ3 Asp57-negative and HLA-DQ~
147
Arg 52 confers susceptibility to insulin-dependent diabetes mellitus. J Clin Invest 85:1315, 1990. 27. Erlich HA, Griffith RL, Bugawan TL, Ziegler R, Alper C, Eisenbarth G: Implication of specific DQBI alleles in genetic susceptibility and resistance by identification of IDDM siblings with novel HLA-DQB1 allele and unusual DR2 and DR1 haplotypes. Diabetes 40:478, 1991. 28. Ronnigen KS, Iwe T, Halstensen TS, Spurkland A, Thorsby E: The amino acid at position 57 of the HLADQ3 chain and susceptibility to develop insulin-dependent diabetes mellitus. Hum Immunol 26:215, 1989. 29. Bao MZ, Wang JX, Dorman JS, Trucco M: HLA-DQ3 non-Asp57 allele and incidence of diabetes in China and the USA. Lancet ii:8661:497, 1989. 30. Yamagata K, Nakajima H, Hanafusa T, Noguchi T, Miyazaki A, Miyagaura J, Sada M, Amemiya H, Tanaka T, Kono N, Tarui S: Aspartic acid at position 57 of DQ,8 chain does not protect against type I (insulin-dependent) diabetes mellitus in Japanese subjects. Diabetologia 32:762, 1989. 31. Reich E-P, Sherwin RS, Kanagawa O, Janeway Jr CA: An explanation for the protective effect of the MHC class I1 I-E molecule in routine diabetes. Nature 341:326, 1989,
ABBREVIATIONS IDDM insulin-dependent diabetes mellitus PCR polymerase chain reaction RFLP restriction fragment length polymorphism
none of the DR or DQ alleles, alone or in association, confers an absolute protection. (3) Our most striking results concern the very high frequency of recombinant haplotypes among the DRwl5 patients: 3 of 9. In these three patients recombinations led to the elimination of both DQB1 and DQA1 alleles usually associated with DRwl5. This strongly suggests that the occurrence of IDDM in these DRwl5 patients is due to the absence of the usual DQ product and thus reinforces the assumption that DQ rather than DR region is involved in the protection conferred by the DRwl 5/DQw6 haplotype. Finally, analysis of the non-DRwl5 haplotypes in heterozygous patients showed that IDDM can occur in the absence of any DQafl heterodimer of susceptibility. Human Immunology 33, 1 4 0 - I 4 7 (I992)
PBMC Asp Arg
peripheral blood mononuclear cells aspartic acid arginine
INTRODUCTION Insulin-dependent diabetes mellitus (IDDM) in an autoimmune disease whose precise etiology is still unFrom the Immunology Laboratory 1NSERM, H6pital Robert Debre (D.Z.; X.-F.G.: R.K.; N.S.,'J.E.: G.S.), Paris, France; Transplantation Immunology Unit, H6pital Cantonal Universitaire, and Department of Genetics and Microbiology, C.M.U. (].-M.T.; M.L.; B.M.), Geneva, Switzerland," and INSERM, HSpital Necker (C.B.; J.-F.B.), Paris, France. Address reprint requests to D. Zeliszewski, Immunology Laboratory, INSERM CJF 90-15, HSpital Robert Debre, 48 bd Serurier, 75019 Paris, France. Received September 14, 1991; acceptedNovember27, 1991.
140 0198-8859/92/$5.00
known. Several genes are likely to contribute to I D D M development. A m o n g them, at least one gene is located within the major histocompatibility complex (MHC)class II region [1, 2]. The definition of HLA molecules associated with I D D M development has been initially approached by determining the frequency of H L A - D R and D Q serologically defined specificities among patients as compared to control populations. First, a positive association mainly with H L A - D R 3 / D Q w 2 and D R 4 / D Q w 3 specificities [ 1 - 3 ] and a negative association mainly with D R 2 / D Q w l specificities [1, 4] have Human Immunology33, 140-147 (1992) © AmericanSocietyfor Histocompatibilityand Immunogenetics,1992
I n v o l v e m e n t o f H L A - D Q in I D D M P r o t e c t i o n
141
TABLE 1 D R 2 / D Q w l haplotypes usually found in healthy populations Atlelic polymorphism of DR and D Q loci Serological specificities
Serological subtypes
Cellular typing
DRB 1
DRB 5
DQA 1
DQB 1
DR2/DQwl
D R w l 5/DQw6 D R w l 5/DQw6 D R w l 6/DQw5
Dw2 Dwl2 Dw21 (AZH)
1501 1502 1601
0101 0102 0201
0102 0103 0102
0602 0601 0502
The DRA gene is not indicated because it is monomorphic. For details on the new nomenclature see ref 15.
been described. Later, IDDM associations were more precisely defined by using genomic rather than serological markers [5-10]. Indeed, restriction fragment length polymorphism (RFLP) and more recently oligonucleotide typing techniques allowed a precise definition of HLA polymorphism that could not be achieved by serological methods. Thus, IDDM susceptibility and IDDM protection were assigned to DQB subregion. This was reinforced by statistical analysis of DQBchain-deduced sequences with an apparent correlation with amino acid at position 57 for both protection and susceptibility [ 11, 12]. An exclusive role of DQB genes could, however, be an oversimplified conclusion since the same susceptible DQB1 allele is found in susceptible DR3 and neutral DR7 haplotypes as well. Furthermore, several reports have suggested that DQA and DRB might also be implicated in susceptibility [13, 14]. In this study we focused on the HLA-DR2/DQwl haplotype which is the most strongly protective against IDDM in view of its extremely low frequency in patients (less than 0.5%) as compared to controls ( 1 8 % 25%) [1, 4, 11]. At least three haplotypes within DR2/ D Q w l have been unequivocally identified by RFLP and oligotyping methods. For simplicity they are recalled in Table 1. In Caucasians, two main haplotypes have been described: one contains DRA, DRBl*1501, DRB5*0101, DQAI*0102, and DQBI*0602 alleles. These genes code for DRw15 and DQw6 products that are subdivisions of DR2 and DQwl, respectively [15]. The second haplotype contains DRA, DRBl*1601, DRB5*0201, DQAI*0102, and DQBI*0502 alleles. These genes code for DRw16 and DQw5 products that are also subdivisions of DR2 and DQwl, respectively (Table 1). Due to quasi absolute linkage disequilibrium between DR and D Q genes, other DRB, DQA, and DQB allelic combinations than those cited above are exceptional. Protection against IDDM has been correlated with the expression of DRw15/DQw6 but not DRw16/
DQw5 encoding haplotype, mainly from studies of DQB allelic polymorphism [9-12]. Indeed, DQBI*0602 allele was absent in most cohorts of IDDM patients. This led to the conclusion that this allele confers protection against diabetes. Yet in these studies, the polymorphism of other HLA-class II genes (DRB1, DRB5, or DQA1) linked to DQBI*0602 in healthy populations was not always investigated in DR2 patients. Thus, a clear mapping of protective genes to DQ region remains to be clearly established. This can be hopefully addressed by studying "recombinant" haplotypes, i.e., haplotypes presenting a disruption of the usual DR-DQ linkage disequilibrium. In this regard, in a previous study, we found by DQB RFLP analysis that the two DRw15 IDDM patients studied had a DQB gene different from that of healthy DRw15 individuals [ 16]. This strongly suggested a contrario that at least the HLA-DQB region was involved in protection. However, further analysis of DRB, DQA, and DQB polymorphism, on a larger panel of DRw15 IDDM patients, was required to definitely determine the respective contribution of each of these HLA class II genes in this protection. In the present study we collected the largest panel of DR2 IDDM patients studied so far, from several hundreds of serologically typed patients, and their DRB, DQA, and DQB allelic polymorphism was characterized by RFLP or oligonucleotide typing after specific D N A amplifications. METHODS Patients. Twenty-three
DR2 IDDM patients were selected from 358 HLA-phenotyped patients from Necker and R. Debre hospitals (Paris, France). These patients were characterized as presenting IDDM following World Health Organization criteria. Autoimmune diabetes was inferred from the presence of islet cell antibodies (above 5 Juvenile Diabetes Foundation units) in 57% of studied patients. Islet cell antibody determi-
142
TABLE 2
Name
D. Zeliszewski et al.
Oligonucleotide probes used in this study
Reference
Alleles identified
D11
23
D37 (DR) AV86 F67 V86 D57 A49 $57 V38 I37 E45 A57 D37 (DQ) V27 V57 DQAI.1 DQA1.2
25 25 25 25 20 20 20 20 20 20 20 20 20 20 24 24
DRB5*0101, 0201 DRB5*0102, 0202 DRB5*0101 DRB5*0201, 0202 DRBl*1601 DRBl*1501 DQB 1"0602, 0603 DQBI*0501 DQB 1"0502, 0504 DQBI*0501, 0502, 0503, 0504 DQBI*0201 DQBI*0301 DQBI*0302 DQBI*0601 DQB 1"0603, 0604 DQBI*0604, 0605, 0501 DQAI*0101 DQAI*0102, 0103, 0501
Amino acid position
Washing temperature (°C)
9-14
50
35-4O 83-88 64-69 85-89
50 54 50 50 58
55-60 47-52
55- 6O 35-40 34-39 42-48 54-59 34-39 25-30 55-60 32-37 32-37
nation was performed in reference to an international standard as part of four international workshop standardization programs. The prevalence of islet cell antibodies in DR2 patients studied reflected the overall sensitivity of the test in an age-matched population of IDDM patients considered independently of HLA phenotype [17]. Patients' peripheral blood mononuclear cells (PBMC) were isolated from blood samples by FicollHipaque gradient centrifugation. HLA serological typing was performed according to a standard procedure in a microlymphocytotoxicity assay. After serological typing residual PBMC were frozen until further use for DNA analysis.
DNA amplifications." T cells from residual PBMC were expanded in macrotiter plates with interleukin 2 (10 IU/ml) during 10 days, after initial stimulation by 1/zg/ ml of phytohemagglutinin A. By this method sufficient amounts of cells could be available from most patients for preparation of DNA. DNA was extracted by standard techniques. DRB1, DRB5, DQA1, or DQB1 first domain exons were selectively amplified from 1/zg genomic DNA by polymerase chain reaction (PCR) using the thermostable Taq-Polymerase, as described elsewhere [18-20].
Oligoprobes: Oligoprobes were synthesized by the /3cyanoethyl amidite method on a Pharmacia gene assembler. Their purification and labeling are described elsewhere [21, 22]. Oligoprobes used in this study are
56 56
58 54 58 64 54
52 56
58 58
described in Table 2, except those used for DRB 1 typing of non-DR2 haplotypes. These latter probes are extensively described elsewhere [23].
Oligotyping: Oligotyping was performed as described elsewhere [20, 22, 23]. Briefly, small aliquots (5%10%) of amplified DNA were denatured in 0.4N NaOH for 10 min at 20°C, neutralized with 1 vol of ammonium acetate 2M, and spotted on a Nylon membrane (Nytran, Schleicher and SchOll, New Hampshire) using the Minifold II slot-blot apparatus (Schleicher and SchiJll). Membranes were prehybridized (30 rain at 50°C) and hybridized (3 h at 50°C) in 5 × SSC, 20mM Na-phosphate, 10x Denhardt's, 5% SDS, and 100/zg/ ml denatured herring DNA. The hybridization mixture contained 0 . 5 - 1 × 106 cpm/ml of labeled oligoprobe. Blots were washed first 1 h at 50°C in 3 × SSC, 70 mM Na-phosphate (pH7), 10x Denhardt's, 5% SDS, and then 1 h in 1% SDS, 1 x SSC at different temperatures indicated in Table 2. Autoradiographs (3-12 h) were performed using x-OMAT XAR Kodak films (Rochester, NY) and CAWO SE4 intensifying screens.
DQA PCR-RFLP typing: DQA typing was performed as described elsewhere [24]. Briefly, after specific DQA gene amplification, three aliquots (8 ILl) were digested with three restriction endonucleases: Fok I, Dde I, or Rsa I. Each product of digestion was then subjected to 8% polyacrylamide gel electrophoresis (100 V, 25 mA) for about 3 h. After migration gels were incubated in ethidium bromide for 10 rain, washed, and photo-
Involvement of HLA-DQ in IDDM Protection
TABLE 3
143
Oligotyping analysis of DRB and DQB alleles in DR2 haplotypes from the 23 IDDM patients DQB 1 oligotyping:
Patient
DRB5
identification
Serological
number
typing
Dll
(DR2)
oligotyping: Probes (specificity) D37(DRw15)
probes (specificity)
AV86(DRw16)
D57(0602Y
$57(0502)
1
2/3
+b
+
_
+
3
2/4
+
+
-
+
_
4
2/3
+
+
-
5
2/3
+
+
-
+
-
6
2/w13
+
+
-
-
-
7
2/9
+
+
-
+
-
8
2/4
+
+
-
+
-
9
2/w12
+
+
-
+
-
10
214
+
+
-
11
2/7
+
-
+
-
+
12
2/w6
+
-
+
-
+
13
2/4
+
-
+
-
+
14
2/3
+
-
+
-
+
15
2/-
+
-
+
ND
ND
-
-
16
2/3
+
-
+
-
+
17
2./1
+
-
+
ND
ND
18
2/3
+
-
+
ND
ND
19
2,/1
+
-
+
-
+
20
2/-
+
-
+
-
+
21
2/'4
+
-
+
-
+
22
214
+
-
+
-
+
23
2/'3
+
-
+
-
+
24
2/3
+
-
+
-
+
T h e D Q B I * 0 6 0 2 allele was identified by the p r o b e D 5 7 but also by a negative hybridization with p r o b e V 2 7 (not shown). b Results are expressed as + or - according to hybridizations or not with the probes indicated.
graphed under UV light on polaroi'd films. Each migration pattern after digestion with the three enzymes corresponds to one D Q A allele. But for identifications of DQAI*0101 and DQAI*0102 alleles, one additional step is necessary, i.e., oligotyping. For this, slot-blots, hybridizations, and washing were performed as described above. DQA 1*0101 - and DQA 1*0102-specific probes are described in Table 2. RESULTS
Selection of patients: This study was restricted to wellcharacterized IDDM patients initially typed as DR2 by serology, and subsequently confirmed to be DR2 by oligotyping. All D N A samples were characterized by HLA-DR generic oligotyping [23], which included the use of oligoprobe D 11 specific for all DR2 alleles (Table 3, third column). DRB1 and DRB5 alleles characterization." The DR2 subtype of the 23 DR2 patients was identified by oligotyping of DRB5 polymorphism with two oligoprobes, D37
and AV86, recognizing DRB5*0101 and DRB5*0201/ 0202, respectively (see Table 2). Table 3 shows results concerning only the DR2 haplotype of the patients. As shown in Table 3, 9 of 23 IDDM patients were D37positive (fourth column) and are therefore identified as DRw15. The remaining 14 patients were AV86-positive (fifth column) and are therefore referred to as DRw16 patients. In most of these DR2 IDDM patients allelic polymorphism at locus DRB 1 was subsequently determined after locus-specific amplification [25] and hybridization with probe V86, which is specific for DRBI*1501, and probe F67, which is specific for DRB1*1601 (see Table 2). As expected from the strong DRB1-DRB5 linkage disequilibrium existing in healthy donors [25], all DRB5*0101 patients (i.e., DRw15) exhibited the DRB1*1501 allele (Table 4, second column), and all DRB5*0201 patients (i.e., DRw16) exhibited the DRB 1" 1601 allele (not shown). The DRB1 oligotyping of their non-DR2 haplotype was in agreement with that expected from serological typing (Table 4, and not shown).
144
D. Zeliszewski et al.
TABLE 4
DRB1, DQB1, and DQA1 Detailed typing of both haplotypes of the 9 DRw15 patients DQB1
Patient identification number
DRB 1 typing
DQB 1 oligotyping
Presence of D Q B Asp57
4 6 10 3 1 5 7 8 9
1501/0301 1501/1302 1501/04-1501/04-1501/0301 1501/0301 1501/0901 1501/04-I501/1201
-a/0201 0301/0604 0503/0302 0602/0302 0602/0201 0602/0201 0602/0303 0602/0302 0602/0301
A/NA A/NA A/NA A/NA A/NA A/A A/NA A/A
-/NA ~
DQA 1 typing ND 0601/0102 0401/0301 0102/0301 0102/0501 0102/0501 ND 0102/0301 0102/0501
No hybridization with any probe tested. b Results are indicated as A for an Asp at position 57 or NA for absence of Asp at position 57 on the corresponding t3 chains.
DQBI characterization: DQB 1 alleles were then characterized by oligotyping using 10 oligoprobes (see Table 2) as described [20]. As shown in Table 3 for the DR2 haplotype, all of the DRw16 IDDM patients tested exhibited the DQBI*0502 allele as identified with the oligoprobe $57, and as expected from the DR-DQ linkage disequilibrium usually found in DRw16 individuals. The DQBI*0602 allele, which is exclusively found in DRw15/DQw6 Caucasian individuals, is defined by a positive hybridization signal with probe D57 (0602 + 0603) and a negative hybridization signal with probe V27 (0603 + 0604). The probe D57 recognizes the aspartic acid (Asp) at position 57, which has been postulated to correlate with IDDM protection [11]. Based on the very strong DR-DQ linkage disequilibrium, our group of patients shown to be DRw15 should present the DQBI*0602 allele. This was indeed the case for 6 of 9 patients (see Table 3, sixth column). Moreover, two of these six patients are homozygous for the presence of an Asp at position 57 (see Table 4). But surprisingly, the remaining three DRw15 patients did not hybridize with the DQBl*0602-specific probe (patients 4, 6, and 10), suggesting a disruption of the usual DR-DQ disequilibrium. Table 4 shows the DQB 1 typing of both haplotypes for the 9 DRwl5 patients. In each case the DQB1 allele encoded by the DRwl5 haplotype is indicated in first position. Patient 4 has a hybridization pattern compatible with DQBI*0201 homozygosity. Patient 6 has a DQB 1"0301 allele (corresponding to DQw7), and a familial segregation analysis clearly showed that this allele is on the DRwl 5 haplotype, while the second haplotype
encodes for DRwl3 and DQw6 (not shown). Finally, in patient 10 the DRwl5 haplotype presents the DQBI*0503 allele usually linked to DRBI*1401 (corresponding to DRwl4). In all 9 heterozygous DRwl5 patients, the non-DR2 haplotype was conform to the expected DR-DQ associations. In summary the three DRwl5 IDDM patients that did not show the expected linkage disequilibrium with DQBl*0602 exhibited, respectively, the alleles DQBI*0201, 0301, 0503. It is of interest that two of these three alleles do contain an Asp at position 57 (see Table 4).
DQA1 characterization." DQA genes have been proposed to be also involved in IDDM susceptibility and resistance [13, 26]. It must be pointed out that no DR2 IDDM patient was included in these two reports. Thus, we characterized DQA1 genes from our DRwl5 patients by using a technique of RFLP analysis after specific DNA amplification by PCR [24]. This allows a rapid DQA 1 typing with only three restriction enzymes and two oligoprobes. DQA1 gene characterization of the six DQBl*0602-positive patients shows that all of them are DQAl*0102-positive, as are DRw15/DQw6 healthy individuals (see Table 4, fifth column). Concerning the three DQBl*0602-negative patients, patients 6 and 10 are both DQAl*0102-negative. As noted above, concerning patient 6, a familial segregation analysis allowed to conclude that alleles DQBI*0301 and DQAI*0601 encoding DQw7 are present on the DRw15 haplotype. It is interesting to note that the second haplotype (DRw13) presents a DQAI*0102 allele, linked as usual to DQBI*0604. Unfortunately, DQA 1 typing of patient 4 could not be performed because an insufficient amount of DNA was available. Finally, here again the non-DR2 haplotype of the 9 heterozygous DRw15 patients corresponds to DQA1DQB 1 associations that can be found in healthy populations [15, and unpublished observations]. If we consider both haplotypes, the DQAI*0102 allele usually found in DRw15 individuals is present in at least 6 of our DRw15 patients, including one of the three patients having recombined haplotypes. DISCUSSION We characterized the polymorphism of DRB1, DRB5, DQA1, and DQB1 genes in 23 DR2 IDDM patients. First, results presented herein show that 14 of 23 DR2 patients express DRB 1, DRB5, and DQB1 genes identical to those found in DRwl6/DQw5 healthy probands. Since the DQA1 locus is located between DR
Involvement of HLA-DQ in IDDM Protection
and DQB1 loci, we can anticipate that the DRw16 patients also express a DQA1 allele identical to that of healthy DRwl6 individuals. No recombination was found in our 14 DRwl6/DQw5 patients. In contrast, we found a very large proportion of recombinant haplotypes in the 9 remaining DR2 patients. In fact, 3 of 9, and 4 of 10 if we include former observations reported elsewhere [ 16], have recombinant haplotypes. They express DRB1 and DRB5 alleles similar if not identical to those found in DRwl5 healthy people but they do not express the DQB 1 allele usually present in DRwl5/DQw6 healthy individuals. This strongly suggests that elimination of the DQw6 product has restored the susceptibility to the disease. To our knowledge the three recombinant haplotypes reported herein have never been described in healthy people. We believe that, rather than being characteristic of IDDM patients, these haplotypes have not been found in unaffected people because of their exceptionally rare frequency. One unusual DR2 haplotype has already been described by others [27] and it is distinct from the new DR2 haplotypes we describe here. Because the DQAI*0102 allele is present in at least one of the DQBl*0602-negative patients (if we consider both haplotypes), this suggests a contrario that the DQB subregion is more closely associated with resistance to the disease than the DQA subregion. This is reinforced by the fact that DRwl 5 and DRwl6 individuals share the same DQA allele. Among the 23 DR2 IDDM patients studied, the frequencies of usual DRB, DQA1, and DQB1 alleles are 39%, 91%, and 26%, respectively. The latter frequency correlates with that found by Todd et al. [ 11 ]. HLA-associated protection against IDDM is correlated with the presence of Asp at position 57 on the DQ,8 chain. The fact that less than 5 % of diabetic individuals are heterozygous non-Asp/Asp (NA/A) versus 46% in controls has suggested [12] that protection observed for Asp behaved in a dominantlike manner. However, it must be pointed out that 8 of our 9 DRwl 5 patients are Asp57-positive on the DQ/3 chains, including two of the patients who have an unusual DQB1 allele (see Table 4). Moreover, we found two Caucasian patients with an Asp57 on both DQ]3 chains, and thus two "protective" DQB1 alleles. This pattern A/A is very rare among Caucasian diabetics [28] but is found among Asiatic patients [29, 30]. Thus, our data reinforce the idea that the protection conferred by Asp57 is not dominant and not absolute. More recently Khalil et al. [26] have implicated the DQA locus in both susceptibility and resistance to IDDM. In addition to DQfi-Asp57, they suggested that DQ~ chains which have an arginine (Arg) at position 52 are susceptible (S) while all Arg52-negative DQa chains
145
TABLE 5
Presence of putative susceptible or protective DQ~ or 3 chains among the 9 DRwl 5 patients
Patients
DQ~ ~
DQ/3 b
4 6 10 3
-' S/P S/S P/S P/S P/S P/S P/S
-/S P/S P/S P/S P/S P/S P/P P/S P/P
1
5 7 8 9
Arg52-positive oe chains are susceptible (S) and Arg52-negative c~ chains are protective (P) according to Khalil et aL [26]. h Asp57-negative/3 chai*~s are susceptible (S) and Asp57-positive/3 chains are protective (P) according to Todd et al. [11]. '
ND
are protective (P). According to their model, the development of diabetes depends on the presence at the cell surface of at least one heterodimer DQo43 "SS," this heterodimer being possibly created by transcomplementation. Table 5 shows the presence of S or P DQc~ and DQB chains in the 9 DRwl 5 patients, according to the classifications of Khalil et al. [26] and Todd et al. [11]. Two of our diabetic patients are DQfl P/P and most probably DQce P/S (see Table 5, patients 9 and 7). These patients can only express PS or PP DQ heterodimers and thus constitute exceptions to the proposed rule. The possibility that patients could develop IDDM while expressing two protective DQ products confirms that the inheritance and genetic feature of IDDM is complex. The DQc~/3 heterodimer in DRwl5/DQw6 haplotype does not confer an absolute protection even though it might correlate with a huge incidence of resistance to IDDM. Altogether these results indicate that resistance to IDDM cannot be fully explained by DQ polymorphism. The mechanism(s) by which some HLA products can confer a resistance to IDDM are unknown. The polymorphism of class II molecules has a major influence on the T-cell repertoire. Indeed, it is involved in both positive and negative selections o f T cells expressing certain T-cell receptors during thymic education. Thus, one possible mechanism that would account for the involvement of DQ genes in resistance against IDDM would be that the DQ molecule from the DRw15/DQw6 haplotype would lead to a clonal deletion of lymphocytes bearing a T-cell receptor directed against a putative islet autoantigen. This hypothesis is compatible with studies
146
p e r f o r m e d in nonobese diabetic mice suggesting that expression of the protective I-E class II product leads to a clonal deletion of specific autoreactive T-cell precursors [31]. H o w e v e r , since we found that the D Q product encoded for by the D R w 1 5 / D Q w 6 haplotype does not confer an absolute protection, we must postulate that in some individuals autoreactive clones would escape this clonal deletion possibly due to other genetic factors. An alternative although not exclusive hypothesis could be that the D Q region is involved in the control of suppressive mechanisms on T-cell clones directed against the putative autoantigen in the periphery. H e r e again such protection might be, in some situations, o v e r c o m e depending on polymorphic genes not linked with M H C . In conclusion, the detection of recombinant haplotypes in 3 out of 9 DRw15 I D D M patients provides further evidence that the D Q B subregion is strongly protective against I D D M . But this protection is not absolute and cannot be explained only by D Q gene polymorphism. ACKNOWLEDGMENTS
We thank B. Battistolo, A. Morrisson, and P. Roux-Chabbey for their excellent technical assistance, M. Seman for helpful comments on the manuscript, and I. Rivenez for typing the manuscript. This work was partly supported by an EMBO fellowship and by a "contrat de recherches cliniques de l'assistance publique. H6pitaux de Paris."
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