DQA1 restriction fragment length polymorphisms and insulin-dependent diabetes mellitus: A Bg1II fragment labels a subset of B8,DR3 haplotypes uniquely associated with insulin-dependent diabetes mellitus

~A1 t Length Polymorphisms and Insulin-Dependent Diabetes Mellitus: A BgllI Fragment Labels a Subset of B8,DR3 Haplotypes Uniquely Associated with Ins~in-Dependent Diabetes Mellitus C. Carrier, N. Mollen, F, Gins~rg-Fellner, W. C. Rothman, and P. Rubinstein

ABSTRACT: Class II restriction fragment length polymorphism studies of 38 pedigrees with multiple cases of insulin-dependent diabetes mellitus revealed the existence of a DQAl-related polymorphism that distinguishes two kinds of HLA-B8,DR3 haplotypes. One of these, characterized b) the presence of DQA1-Bglll 7.20 kb. was present in all 14 examples inherited by patients and in 6 of the 12 B8,DR3 haplotypes not so inherited. The apparently complete association between the presence of this fragment and the "'affected" status of B8,DR3 haplotypes (p = 0.004, was confirmed in a separate group of 26 simplex pedigrees selectedfor the presence of this haplotype in the respective probands (combined p < 0.000l ~. ABBREVIATIONS IDDM insulin-dependent diabetes mellitus IEF isoelectric focusing LCL lymphoblastoid cell line RFLP restriction fragment length polymorphism

RR 10WS

relative risk Tenth International Histocompatibility Testing Workshop

INTRODUCTION It is now well established that DR4 haplotypes are heterogeneous in their association with insulin-dependent diabetes mellitus (IDDM) [ 1 - 5 ] : the linked D Q w 3 . 2 (now DQw8), but not DQw3.1 (DQw7), alleles identify high relative

From The Fred H. Allen Laboratory of lmmunogenetics. The Lindsley F. Kimball ResearchInstitute ~f the New York Blood Center. New York (C.C.."N.M.: M.W.: P.R,). Special Diagnostics. The Neu York Blood Program, New York (W.C.R.L and the Division of Pediatric Endocrinology, Mount Sinai School of Medicine of the City University of New York. New York (F.G.-F.). Address reprint requests to P. Rubinstein. M.D.. The Fred H. Allen Laboratory of lmmunogenems~ The Lindsley F. Kimball Research Institute. The New York Blood Center. 3t0 East 67th Street. New York. NY 10021. ReceivedApril 19 1989: revisedJuly 5, 1989.

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Human Immunology26. 344-352 (1989 ((~ AmericanSocietyfor Histocompatibilityand Immunogenetics.1989

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risk (RR) DR4 haplotypes [6]. Although evidence of a significant additive effect of selected variants of the DR4B 1 chain gene has been reported recently [7], this high RR suggests that DQB1 may be "the" susceptibility locus in DR4 haplotypes. While still not formally proven, this hypothesis is strongly supported by the negative association between IDDM and a DQB1 chain gene specifying aspartic acid in position 57 [8,9], while IDDM-associated and neutral DQ alleles have other, less charged, amino acids there. In contrast, evidence of a similar major IDDM-associated heterogeneity in risk has not been encountered thus far in the other major group of IDDMassociated haplotypes, those carrying DR3. These haplotypes are heterogeneous with regard to the DQA2 locus, to several class III loci, and to the second DRB chain gene (DRBIII), which encodes the DRw52 variants [ 10-17]. DRw52a is in linkage disequilibrium with the B8,DR3 and DRw52b with the "non-B8," DR3 haplotypes. These DR3 haplotypes have been felt to portend different relative risks (RR), based on serologic, cellologic, or molecular (protein or DNA) differences at one or more of these loci. DRw52a- and DRw52b-related differences in RR, however, appear to be stratified geographically: in some European populations B8,DR3,DRw52a may have higher risks [2], while B18,DR3,DRw52b may be more strongly associated with IDDM elsewhere [18,19]. Our family data as well as that of others, however, indicate that the haplotype relative risks (HRR) [20] for B8- and B18-carrying DR3 haplotypes are similar. DR3 haplotypes carrying neither of these alleles, though also IDDM-associated, may have lower HRRs ([2]; Rubinstein et al., unpublished). In this report, we present evidence that two subtypes of the common B8,DR3,DQw2 haplotypes are disclosed by a DQA1 restriction fragment length polymorphism (RFLP). This heterogeneity is associated with a major difference in IDDM association, such that one of the DQA1 variants appears to be invariably present in B8,DR3,DQw2 IDDM patients.

MATERIALS AND M E T H O D S Families. The pedigrees studied were selected from among 299 New York families of probands with IDDM typed for HLA and other genetic markers in this laboratory since 1978. A subset of 38 multiplex families has been used to prepare B-lymphoblastoid cell lines (B-LCLs) by Epstein-Barr-virus transformation of their B lymphocytes. All of these 38 families were initially tested for class lI RFLPs with six restriction enzymes (BamHI, BglII, EcoRI, HindIII, Pstl, and TaqI) (BRL Gaithersburg, MD) and three cDNA probes, designated by the 10th International Histocompatibility Testing Workshop (10WS) as DR/3, DQ~, and DO_33 (see below). Twenty-six additional, simplex families selected for having probands with the B8,DR3 haplotype were tested for DQA1 RFLPs by probing genomic DNA from their peripheral blood mononuclear cells. All the families were typed for the serologic variants of the HLA class I A, B, and C loci, the class II DR and DQ loci, the class III Bf locus, and for GLO allotypes using standard methods and antisera checked against our WS-typed panel. The isoelectric-point variants of the DR/3, DQ~, and DP/~ chains expressed by each of the haplotypes of 34 of the multiplex families were al~o determined by the previously described [21] high-resolution one-dimensional isoelectrofocusing technique. The allelic notations assigned to the products of the class II loci follow the currently recommended nomenclature [16] whenever possible. Further splits have been defined in this laboratory, however, and these are designated as proposed in our original descriptions [11,21]. In each family, the haplotypes present in at least one patient

346

P. Rubinstein et al. were labeled "affected" and those not present in any of the patients were consi& ered "unaffected" whether inherited by normal sibs or not [20].

Probes. The probes were selected from those included in the 10WS, they were, respectively, the 786-bp DR 3 [22], the 1200-bp DQ3 [23], and the 1600-bp DQ~ [24] cDNAs. The probes were labeled with 32p-dCTP using the oligopriming method [25].

DNA analysis. Genomic DNA was extracted from each of the cell lines according to the protocols of the 10WS and RFLPs determined exactly as described in ref. 10.

Computer assignment of RFLPs to haplotypes. The analysis of the segregation of each restriction fragment in all families was performed with RFrAP; a set of computer programs described by Rothman et al. [26].

Statistical analysis. The significance of the differences in the frequency of bands was estimated from 2 × 2 contingency tables using Fisher's exact test. RESULTS Analysis of the Segregation and IDDM Association of the DQA1 Restriction Fragments Generated by BglII Digestion The HLA haplotypes in these studies (N = 252) were classified by DR type, DR/3 isoelectric focusing and DQ3 variants, Dw specificity, and by the presence or absence of the class I alleles HLA-B8 (in DR3 haplotypes) and HLA-B14 (in DR1 haplotypes). Overall, the DQol cDNA probe hybridized with BgtII fragments of 12.50, 8.35, 7.20, 7.00, 6.40, 3.80, and 2.05 kb with the following correlations: 1. The 12.5-kb fragment was found in single examples each of DR1, DR3, DRw6, DR7, and DRw8 and m two examples each of DR4 and DR5 haptotypes. 2. The 8.35-kb fragment was present in some DQw2 haplotypes ~DR3 and DR7 I. These two fragments did not show significant associations, however, with either HLA alleles or IDDM alleles. 3. The 7.00-, 6.40-, and 2.05-kb fragments were found in all members of most o~ the families; thus, their segregation was generally uninformative. 4. The 3.80-kb fragment was present in most DQw2 haptotypes (DR3- and DR5-DQ~2.1) and in some DQw3 (DR5-DQw7) and DQw4 haplotypes (DRw8-DQ34.2). With the exception of two DR3 haplotypes that did not carry this fragment, its distribution was identical to the DQA1 fragments EcoRI ll.00 kb, HindIII 5.80 kb, and PstI 4.30 kb Idata not shown}; TaqI 4.77 kb also displayed a very similar distribution, except for its absence from DRw8-DQ34.2 haplotypes (data not shown}. All of these fragments were found in high linkage disequilibrium with the DRw52 specificity and were shared by DR3, DR5, and DRw8. 5. The BglII 7.20-kb DQA1 fragment (Figure 1) was detected in all examples of B14,DR1,Dw20,DQw5 and DRw13-Dw18 and in one example of the DRwl 3-Dw19 haplotype, in some of the DQw2 haplotypes also encoding B8, and in single examples of DR5-DQ32.1, DR7-DQ/~2.2, and DR4-DQw8

DQA1 RFLP and IDDM in B8,DR3 Haplotypes

347

OLI

a/b

c/d

b/c

a/d

a/c

a/d

8.35 K b - - • 7.2 K b ~

•

7.0 Kb 6.4

Kb-- ~

....

....

"~ ......

FIGURE 1 Southern blot of BglII-digested DNA from all members of family OLI hybridized to the DQa probe. The figure includes the pedigree and shows the segregation of the 7.20-kb fragment with the HLA "c" haplotype. The molecular sizes of the observed fragments are depicted at the left; the dot (e) shows the position of the 7.20-kb fragment.

haplotypes (Table 1). BgllI 7.20 kb was present in all 14 examples of B8,DR3,DQ~2.1 "affected" and in only 6 of the 12 "unaffected" haplotypes from the 38 multiplex families studied (p = 0.004). Additionally, 26 simplex families with at least one affected B8,DR3 haplotype each, were tested, and all these affected B8,DR3 haplotypes also had the 7.20-kb fragment (p = 0.0003). The overall association (Table 2) has a p < 0.0001. DISCUSSION The high sensitivity of the RFLP analysis of HLA polymorphisms has increased the probability of identifying the specific gene or genes that mediate the association between HLA and diseases such as I D D M [ 14,15,27-39]. The effectiveness of this analysis is limited by the difficulties in assigning a given restriction fragment (RF) to an individual haplotype, a limitation that is largely overcome by the study of its segregation in families [f0,26]. This study can be automated: the RFrAP computer programs [26] allow the identification of RF in linkage disequilibrium with specific alleles of closely linked genes. Using different restriction enzymes and HLA class II e D N A probes on 32 multiplex diabetic families, we have reevaluated [10] the RFLPs reported to recognize high-risk variants of the serologically defined IDDM-associated haplotypes [ 14,15,27-29,31,32,34,

348

P. Rubinstein et al. TABLE 1

T h e d i s t r i b u t i o n o f D Q A 1 - B g l I I 7.20 k b a m o n g 148 h a p l o t y p e s f r o m m u l t i p l e x families DQA1-BgIII 7.20 kb

B8-DR3-DQfl2.1 ~ B 14,DR1,Dw20,DQw5 DRwl 3-Dw18-DQw6 DR1,Dwl,DQw5 DR2, (DQw5 or DQw6) (non B8)-DR3-DQ/~2.1 ~ B8-DR3-DQw 1 DR4-DQw7 DR4-DQw8 DR4-DQ/33.4" DR5-DQw7 DR5-DQ32.1 ~ DRw 13-Dw 19-DQw6 DRw 14-Dw9-DQ34.3 ~ DR7-DQw9 DR7-DQ/32.2 ~ DRw8-DQB4.2 ~ DRw8-DQ,B4.3" DRw9-DQw8 Total

*

-

'['oral

20 6 3 0 0 0 0 0 1 0 0 1 1 0 0 i 0 0 0 33

6 0 1 11 14 15 1 12 26 2 11 0 4 l l 4 3 2 l 115

26 6 i I1 i~ i5 I 12 27 : Ii ! L "; 3 2 i 1,48

-Locally designatedallelicnotation;not in the officialHLA nomenclature[16]; see refs. I1 and 21

35,39]. I n that w o r k [10], w e s h o w e d that m o s t o f the significantly I D D M a s s o c i a t e d R F L P s o w e t h e i r association to t h e i r linkage d i s e q u i l i b r i a with e i t h e r D Q w 7 o r D Q w 8 . This agrees with the c o n s e n s u s that I D D M - s u s c e p t i b l e and - r e s i s t a n t D R 4 h a p l o t y p e s are largely d e f i n e d by their r e s p e c t i v e D Q B 1 variants. T h e D Q B 1 g e n e in D R 3 h a p t o t y p e s , h o w e v e r , d o e s not e x h i b i t similar variability and thus m a y n o t b e r e s p o n s i b l e for the p o s s i b l e h e t e r o g e n e i t i e s o f these h a p l o t y p e s w i t h r e s p e c t t o I D D M RRs. F o r e x a m p l e , the two b e s t - d e f i n e d D R 3 c a r r y i n g h a p l o t y p e s , B 8 , D R 3 and B 1 8 , D R 3 , differ consistently in t h e i r Bf, C 4 A , C 4 B , D R B I I I , and D Q A 2 alleles, and s o m e o f these have b e e n s u s p e c t e d o f m e d i a t i n g t h e p o s s i b l e h e t e r o g e n e i t y [ 1 0 - 1 5 , 1 7 ] . T h e two h a p l o t y p e s d i s p l a y n o r t h - s o u t h f r e q u e n c y g r a d i e n t s in E u r o p e , l e a d i n g to stratification and p o s s i b l y

TABLE 2

D Q A 1 - B g l I I 7.20 k b in B 8 , D R 3 affected and unaffected haplotypes DQA1-BgIII 7.20 kb +

DR3(B8)-DQ~2.1

Affected Unaffected Total

40 6 46

-

Total

0 40 6 12 6 52 Fisher's exact test p .( 0.0001

DQA1 RFLP and IDDM in B8,DR3 Haplotypes

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accounting for the already quoted findings of a stronger IDDM association with B8,DR3 in norhtern and B 18,DR3 in southern European countries. A different variation of DR3-bearing haplotypes has been disclosed in this study by the RF designated DQA1-BgIII 7.20 kb. This fragment, present on a subset of B8,DR3,DQw2 haplotypes, is absent from all B18,DR3 and other DRw52b haplotypes tested thus far. Among the carriers of B8,DR3 haplotypes, the presence of IDDM is, thus far, completely associated with the 7.20-kb fragment. As shown in Table 2, all 40 independent examples of the haplotype in patients and only 6 of the 12 B8,DR3 "unaffected" haplotypes are positive (Fisher's exact test p < 0.0001). This fragment produces weak hybridization signals with the DQc~ probe, which may account for its having escaped earlier recognition of RFLP studies of IDDM patients. Preliminary data from 10WS display a distribution of DQA 1-BglII 7.20 kb similar to that encountered here among class II haplotypes, although there is no mention of the B8,DR3 "split." In addition, DQA1-KpnI 8.40 kb was found to be identically distributed among 10WS cells. This DQA1 polymorphism, which is apparently not associated with IDDM among non-DR3 haplotypes, also divides DR specificities other than DR3. Most notably, among the 17 DR1 haplotypes included in this study, all six HLAB14,DR1,DQw5,Dw20, and no others, specified DQA1-BgIII 7.20 kb. The data indicate, therefore, that there is a subset of B8,DR3 haplotypes uniquely associated with IDDM which is characterized by the presence of the DQA 1-BglII 7.20-kb fragment. The frequencies of the two subsets of this haplotype in different populations may be different, which might contribute to the different risks for B8,DR3 measured in those populations. Studies designed to test for this possiblity are currently under way. The complete association between this "new" DQA1 marker and IDDM susceptibility in B8,DR3 haplotypes, if confirmed in such studies of independent samples, would support the suggestion that DQA1 may be "the" susceptibility locus of this haplotype. Owerbach et al. have also found evidence of DQA1 involvement in this susceptibility [40]. The only other very good marker of susceptibility (DQw8 in DR4 haplotypes) also concerns the DQ region but involves the DQB1 locus. Since, however, the RR of DR4,DQw8 haplotypes varies with their specific DR-Dw alleles [7], DQB1 could well be merely a marker for the susceptibility gene of DR4 haplotypes. It is thus possible that both DQ markers portend the presence of the same susceptibility factor in linkage disequilibrium with different markers in the different IDDM-associated haplotypes. This hypothesis is compatible with a plausible mechanism of expression of the genetic susceptibility: facilitating the formation of mixed-isotype DQ dimers. Such DQAI(DQw2)/DQBI(DQw8) dimers (and perhaps others) could contribute the necessary context for the required presentation of nominal antigens and autoantigens to T lymphocytes. Since multiple viruses [41-44] and several autoantigens [45,46] may participate in the triggering and/or maintenance of pancreatic/~-cell autoimmunity, the number and types of different "contexts" provided by a given genotype are both proposed to be important determinants of its influence on the susceptibility to IDDM. ACKNOWLEDGMENTS A preliminary version of this work was presented at the meeting of the American Society for Histocompatibility and Immunogenetics in San Francisco, November 1988. We thank S. Rodriguez de Cordoba and J. Rey-Campos for much constructive discussion,T. Huima for her photographic work, and V. Moore for help in the preparation of this manuscript. This work was supported in part by grant DK 19631-13 from the National Institutes of Health.

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