differential contribution of c4 and hla-dq genes to systemic lupus erythematosus susceptibility

Hum Genet (1993) 91:579-584 human .. genet ics

�9 Springer-Verlag 1993

Differential contribution of C4 and HLA-DQ genes to systemic lupus erythematosus susceptibility

Dolores De Juan 1, Jos6 M. Martin-Villa l, Juan J. G6mez-Reino 2, Jos6 L. Vicario 3, Alfredo CorelV, Jorge Martlnez-Laso J, Djamal Benmammar 1, Antonio Arnaiz-Villena 1

1 Department of Immunology, Hospital "12 de Octubre", Madrid, Spain 2 Department of Rheumatology, Hospital "12 de Octubre", Madrid, Spain 3 Histocompatibility, Transfusion Center, C.A.M., Madrid, Spain

Received: 23 August 1992

Abstract. The particular histocompatability antigen (HLA) gene(s) that may confer systemic lupus erythematosus (SLE) susceptibility remains unknown. In the present study, 58 unrelated patients and 69 controls have been analyzed for their class I and class II serologic antigens, class II (DR and DQ) DNA restriction fragment length polymorphism, their deduced DQA1 and B1 exon 2 nucleotide sequences and their corresponding amino acid residues. By using the etiologic fraction (5) as an almost absolute measure of the strongest linkage disequilibrium of an HLA marker to the putative SLE susceptibility locus, it has been found that the strength of association of the HLA marker may be quantified as follows: DQAI*0501 (associated to DR3) or DQBI*0201 (associated to DR3) > non Asp 57 ~]DQ/Arg 52 c~DQ > DR3 > non Asp 57 ~DQ. Thus, molecular HLA DQ markers tend to be more accurate as susceptibility markers than the classical serologic markers (DR3). However, dominant or recessive non Asp 57 I]DQ susceptibility theories, as previously postulated for insulin-dependent diabetes mellitus, do not hold in our SLE nephritic population; indeed, three patients bear neither Arg 52 ~DQ nor Asp 57 [~DQ susceptibility factors. On the other hand, nonsusceptibility factors are included in our population in the A30B 18 CF 130- DR3DQ2(Dw25) haplotype and not in A1B8CS01- DR3DQ2(Dw24) ; this distinctive association has also been recorded in type I diabetes mellitus and may reflect the existence of common pathogenic HLA-linked factors for both diseases only in the A30B18CF10DR3DQ2- (Dw25) haplotype. Finally, the observed increase of deleted C4 genes (and not 'null' C4 proteins) in nephritic patients shows that C4 genes are disease markers, but probably without a pathogenic role.

Introduction

Several studies have shown that susceptibility to systemic lupus erythematosus (SLE) is partially controlled by poly-

Correspondence to: A. Arnaiz-Villena, Immunologfa, Hospital "12 de Octubre", E-28041 Madrid, Spain

morphic genes of the major histocompatability complex (MHC) system (Scherak et al. 1979; Howard et al. 1986; Hawkins et al. 1987). In a variety of ethnic groups, the associations with the histocompatibility antigens (HLA) have been related to the outcome and expression of the disease (Fronek et al. 1988). Most authors have found DR2 and DR3 significantly increased among Caucasian SLE patients. Moreover, most of those studies indicate that the A1B8CS01DR3DQ2 extended haplotype is strongly associated with the disease (Woodrow 1988).

Our previous results revealed that the BfF1 allele was increased in Spanish SLE patients with diffuse prolifera- tive glomerulonephritis (DPGN) (Martfn-Villa et al. 1986), suggesting that class III antigens play a role in determin- ing the clinical manifestations of the disease. Others have claimed that C4 null alleles are better susceptibility markers for SLE than class II antigens (Batchelor et al. 1987); however, the molecular basis of complete C4 deficiency in human individuals is complex and requires further study (Uring-Lambert et al. 1989).

Studies of HLA and disease associations carried out in different ethnic groups or clinical subgroups may help to clarify the relevance of these correlations (Schur et al. 1990). Moreover, HLA restriction fragment length polymorphism (RFLP) studies have been found to be useful in characterizing, at the DNA level, stronger and more pre- cise HLA and disease relationships, in particular with respect to SLE and HLA-DR alleles (Dunckley et al. 1986).

On the other hand, SLE presents a variety of clinical and serologic manifestations. DPGN is a severe form of renal disease secondary to a deposit of immune complexes. The course of the disease and prognosis of patients suffering from DPGN are different from those in patients with other types of renal disease. Therefore, it can be considered as a well-defined subset of renal disease and thus suitable for the definition of a possible HLA and SLE association.

We have therefore investigated the HLA class I, II and III genes, at both the serologic and DNA level, in a group of Spanish SLE patients, in order to pinpoint and charac- terize the SLE susceptibility genes within the HLA complex. We also wished to test whether the postulated sus-

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ceptibili ty hypothesis to other au to immune diseases, au- to immune insulin dependent diabetes melli tus ( IDDM), based on specific amino acid residues at certain positions of the H L A - D Q chains, applies to SLE, since both diseases are associated with the same extended haplotype in the Spanish population (G6mez-Reino et al. 1991; Vicario et al. 1992). The suggested differential H L A association with different SLE clinical forms has also been examined.

Materials and methods

Patients

Fifty-eight unrelated SLE patients, diagnosed according to the American College of Rheumatology criteria (Tran et al. 1982), were studied. Sixty-nine healthy unrelated individuals were used as controls.

Among the SLE patients, 36 with clinical evidence of renal disease had undergone kidney biopsy and were classified according to the World Health Organization criteria, slightly modified (Pirani and Pollak 1978), as suffering fiom DPGN. Patients with no clinical evidence of renal disease were not biopsied. In a previous un- published study of 20 SLE patients with no clinical evidence of renal disease, renal biopsy revealed only one with DPGN. Thus, patients with no evidence of renal disease were included in the non- DPGN group.

HLA class L H and HI typing

HLA class 1 (-A, -B, -C) and II (-DR, -DQ) typing was performed by a two-step microlymphocytotoxicity technique (Danilovs et al. 1980) on T and B lymphocytes, respectively, using IX and X In- ternational Histocompatibility Workshop HLA alloantisera. C4 and Bf typing was carried out on plasma samples by an electroim- munofixation technique, using goat anti-human C4 and Bf serum, respectively (Atlantic Antibodies, USA) (Rodrfguez de C6rboda et al. 1981; Regueiro and Arnaiz-Villena 1987), and specific C4A and C4B monoclonal antibodies when necessary, kindly donated by Dr. Schendel (O'Neill 1984).

DNA extraction, digestion, processing, and hybridization

the TaqI enzyme in both the SLE and control groups was carried out according to Schneider et al. (1986). C4B deleted genes were determined by comparing the intensities of the specific bands obtained (Schneider et al. 1986).

Statistical analysis

The distribution of serologic specificities and DNA allogenotopes in patients and controls was compared by calculating the chi square value with Yates' correction and Fisher's exact test (when appropriate). The relative risk (RR) and etiologic fraction (6) were calculated only in significant comparisons. P, 6, and RR values are shown in footnotes to the Tables. Comparisons of ~ values can de- termine which of the different HLA markers has the strongest association to SLE. ~is calculated as follows: (5=d-p/1-p, where d is the frequency of the antigen among the individuals with the disease, and p is the frequency of the antigen in the general population. This measure has advantages over RR values when the association is due to linkage disequilibrium between a genetic marker and the true genetic diseased marker, both being very close at the genomic level. Since the true susceptibility marker is unknown this type of analysis will tend to assign higher 6 values to the genetic marker placed closest to the true susceptibility markers or loci (Bengtsson and Thomson 1981; Thomson et al. 1983). In addition, 95% confi- dence intervals for ~ (12) , have been calculated as described (Thom- son et al. 1983).

Assignment of exon-2 DQA 1 and DQB- 1 sequences by DNA-RFLP analysis

DNA-RFLPs of the DQA1 and DQBI genes have been associated to Dw phenotypes (Bidwell 1988; Martfnez-Laso et al. 1989a, b), which in turn have been associated to exon-2 DQA 1 and DQB 1 sequences (Marsh and Bodmer 1989; Martfnez-Laso et al. 1991); therefore, it has been possible to associate DQA1 and DQBI allogenotopes with their corresponding exon-2-sequences, and there- after to record the presence or absence of Asp 57 in the ~3DQ chain and Arg 52 in the c~DQ chain. A total of 58 SLE patients (22 DPGN-positive, 36 DPGN-negative) and 69 control DNA were analyzed using this technique. In addition, the DNA of 108 control individuals was processed by both direct DNA sequence and this RFLP sequence assignment technique; there was a full coincidence of direct oligotyping DNA sequence assignment and indirect DNA-RFLP sequence assigmnent (Vicario et al. 1992).

DNA extraction, digestion with TaqI restriction endonuclease (Boehringer Mannheim), electrophoresis, transfer of the DNA fragments, hybridization, and washing were performed as previously described (Arnaiz-Villena et al. 1989, Vicario et al. 1990).

Results

Serologic HLA class H and class III antigens

HLA class H allogenotyping

A 517-bp Pstl fragment of the exon-specific DRB cDNA clone pRTVI (Bidwell and Jarred 1986), a 797-bp PstI fragment of the HLA-DQA cDNA clone pDCH1 (Auffray et al. 1982) and a 627- bp Avat coding region tYagment of the HLA-DQB cDNA clone pII-]3-1 (Larhamar et al. 1982) were used as probes in HLA class II allogenotyping. HLA class II DRB, DQA, and DQB allogenotopes were studied in both populations. The nomenclature used has been previously described (Bidwell 1988; Martfnez-Laso 1989a, b).

HLA class II1 allogenotyping

A 5.5-kb SalI/HindIIl fragment of the C4 cDNA clone pAT-A (Belt et al. 1984) kindly provided by R. D. Campbell (Oxford, UK) was used for C4 typing. In the case of 21-OH, a 490-bp PstI fragment of the 21-OH cDNA clone, pC21a, was used as a probe (White et al. 1986). Analysis of the RFLP patterns obtained with

HLA class I and H phenotypes. Serologic tissue typing shows (Table 1) a significant increase of the DR3 antigen ( P = 0 . 0 0 2 ) in the haplotype A 3 0 B 1 8 C F 1 3 0 D R 3 D Q 2 in the group of SLE patients with DPGN. Such an increase has not been found for the A 1 B 8 C S 0 1 D R 3 D Q 2 haplotype in either clinical subgroup (DPGN-posi t ive or DPGN- negative).

Bfphenotypes. A significantly higher frequency (P = 0.006) of the BfF1 allele was found in the DPGN-pos i t ive group (n = 22) than in the DPGN-nega t ive and control population (Table 1), confi rming our previously published data (Martin-Villa et al. 1986) and suggesting a possible deter- minant role of the A 3 0 B 18CF130DR3 DQ2 haplotype in the susceptibility for severe renal damage in our Spanish SLE patients.

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Table 1. Relevant associations of serologically determined HLA class I, II, and III antigens to SLE (%)

Table 2. Significant DRB1, DQA1, and DQB1 RFLP alleles and SLE associations (%)

HLA DPGN+ DPGN- Total Controls antigens (n = 22) (n = 36) (n = 58) (n = 69)

RFLP DPGN+ DPGN- Total Controls (n = 22) (n = 36) (n = 58) (n = 69)

B8 14 25 21 I0

B18 55 a 14 29 19 Cw5 50 17 29 26

DR2 18 39 31 28 DR3 68 b 25 41 29

A30BI8DR3 41 ~ 8 21 9

A1B8DR3 13 16 15 10 BfF1 41 d 14 24 11

C4AQ0 e 23 33 27 34

C4BQ0 e 50 36 41 29

C4AQ0 or BQ0 e 72 36 71 59

C4BQ0 BQ0 e 14 3 7 4

DPGN+ = DPGN-positive, DPGN- = DPGN-negative

a p = 0.003 DPGN+ vs controls; RR = 5.2, ~= 0.444 (_+ 0.004) P= 0.002 DPGN+ vs controls; RR= 5.2, ~= 0.549 (_+ 0.005)

c P=0.001 DPGN+ vs controls; RR=7.3, ~=0.352 (_+0.004) d p = 0.006 DPGN+ vs controls; RR = 5.3, ~= 0.337 (_+ 0.004) e For C4 allotypes comparisons, n =21 DPGN+; n=32 DPGN-;

n = 53 controls

C4 null phenotypes. The estimated C4AQ0 and BQ0 frequencies in diseased and control populat ion are listed in the Table 1. In contrast to other populat ions (Kemp et al. 1987; Goldestein et al. 1988), C4AQ0 is not increased in our diseased group (DPGN-posi t ive or DPGN-negat ive) .

HLA class H and class I l i allogenotyping

HLA class H allogenotyping. With respect to the DRB 1 allogenotypes, two different DR17 RFLP TaqI patterns, DR17.1 ( D R B I * 0 3 0 1 - D R B 3 * 0 1 0 1 ) and DR17.2 (DRBI*0301- DRB 3"0202) , were found. When comparing the DPGN- positive group versus the DPGN-negat ive or the control group, only a significant increase of the DR17.2 allele (DRB l * 0 3 0 1 - D R B 3 * 0 2 0 2 ) is observed (P = 0.003 and P = 0.0008, respectively). This allele is usual ly found associated with the B18 ant igen and is included in the extended haplotype A30B 18 CF 130DR3 DQ2, whereas the DR 17.1 (DRB 1 * 0301-DRB 3 * 0101) allele (not increased) is associated with B8 in the A 1 B 8 C S 0 1 D R 3 D Q 2 haplotype (Table 2).

Regarding the DQA1 and DQB 1 allogenotypes, D Q ~ 2 (DQAI*0501, associated to DR3) and DQI32a (DQB 1"0201, associated to DR3) alleles are also significantly increased in the DPGN-posi t ive group (P = 0.0004 versus DPGN- negative, and 0.00003 versus controls). This is probably because of their strong l inkage disequi l ibr ium with the DR17.2 (DRB l*0301-DRB 3"0202) antigen. The DQcOa (DQAI*0101) allele has a lower frequency in both the DPGN negative group and total SLE group when compared with healthy controls (P = 0.02) (Table 2).

DQA1 and DQB1 DNA and protein sequence assignment. With regard to D Q ~ I Asp 57-negative susceptibility, an

DR17

DR17.1 27 14 19 26

DR17.2 59 a 17 33 17 DQ~ la 18 8 b 12 c 30

DQ~ 2 82 d 30 50 29

DQI] 2a 82 d 30 50 29


a p = 0.003 DPGN+ vs DPGN-; RR = 7.2, S= 0.506 (+ 0.004) and P = 0.0008 DPGN+ vs controls; RR = 6.9, ~= 0.506 (+ 0.004)

b p = 0.02 DPGN- vs controls c p = 0.02 Total SLE vs controls a P--0.0004 DPGN+ vs DPGN-; RR= 11.0, S=0.743 (+0.004) and

P = 0.00003 DPGN+ vs controls; RR = 10.2, ~= 0.746 (+ 0.004)

Table 3. Comparison of the distribution of the Asp-57 [3DQ (protective) and Arg-52 c~DQ (susceptible) HLA haplotypes between patients and controls

Cases HLA" DQ a Total (m ([~)

DPGN+ b DPGN- SLE Controls (n =22) (n = 36) (n=58) (n =69)

1 S S 6 3 9 4 S S

2 S S 5 4 9 10 S P

3 S S 4 3 7 8 P S

4 P S 1 5 6 11 S P

5 S S 3 5 8 5 P P

6 P S 0 2 2 7 P S

7 S P 0 0 0 5 S P

8 P S 1 3 4 6 P P

9 P P 1 6 7 9 S P

10 P P 1 4 5 4 P P


a DQ~ chain S = Arg 52+ = susceptibility factor; DQa chain P = Arg 52- = protective factor; DQI] chain S = Asp 57- = susceptibility factor; DQI] chain P = Asp 57+ = protective factor b P=0.017 DPGN+ versus controls when the susceptible combinations (5 first cases) are considered; RR = 5.2, 6= 0.689 (+ 0.005)

increased frequency of Asp 57-negative I]DQ homozy- gote individuals has been found in our DPGN-pos i t ive population. No difference has been found when comparing the DPGN-negat ive or total SLE group with the control group (Table 3).

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Table 4. Frequency of C4 deleted genes in SLE patients and controls Total

DPGN+ (n=21)

DPGN- (n = 32)

SLE (it = 53)

Controls 0t=52)

C4 genes (%)

C4A del 19 16 17 15 C4B del 43 a 16 26 12 C4B short 67 72 70 71 C4B long 28 ~' 81 b 60 58

C4A del or C4B del 62 ~ 31 43 27

C4 null alleles caused by deletion (%)

C4AQ0 C4Adel 100 (4/ 4) C4BQ0 C4Bdel 90 (9/10) d C4AQ0 C4Adel

or C4BQ0 C4Bdel 93 (13/14) ~

50 (5/10) 38 (5/13)

43 (10/23)

64 (9/14)

61 (14/23)

62 (23/37)

47 (8/18)

40 (6/15)

42 (14/33)


~ P=0.007 DPGN+ vs controls; RR=5.8, 6=0.352 (-+0.004) t, P=0.04 DPGN+ vs controls and DPGN- vs controls ~ P=0.01 DPGN+ vs controls d P=0.01 DPGN+ vs controls and DPGN+ vs DPGN- " P=0.001 DPGN+ vs controls

For DQ[31 Asp 57-negative/DQ~ Arg 52-positive susceptibility, a high frequency of Asp 57-negative 13DQ/ Arg 52-positive o~DQ individuals has been found in our DPGN-positive population (DPGN-positive patients, n= 22, 86%; controls, n=69 , 55%, a= 0.70, RR=5.17. How- ever, three patients are placed under the central line in Table 3. They bear homozygous protective DQAI and DQB 1 genes and should not be diseased. In itself, this ex- ception invalidates the application of this susceptibility theory to SLE, as was previously demonstrated in diabetes. Again, no differences have been found when comparing the DPGN-negative or total SLE group with the control group (Table 3).

C4 genes. The analysis of the RFLPs shows (Table 4) that C4B but not C4A deleted genes have a higher frequency in the group of patients with DPGN when compared with the control group (P = 0.007). It is probably a reflection of the higher percentage of the A30B 18CF130DR3DQ2 extended haplotype, which carries a C4BQ0 deletion. More- over, the presence of C4 deleted genes (either C4 or C4B) is significantly raised (P = 0.01) among individuals with renal damage (DPGN-positive). Concerning the C4B gene sizes, C4B long is significantly increased (P = 0.04) in the DPGN-negative group, whereas it is significantly decreased (P=0.04) in the DPGN-positive group, when each is compared with the control group (Table 4).

Discuss ion

Serologic results show a significant increase of the haplotype A30B18CF130DR3DQ2 in the SLE group with DPGN; this is in concordance with our previous results (G6mez-Reino et al. 1991). This haplotype is character-

istic of type I diabetes in our population (Vicario et al. 1992) in contrast to the increased frequency of the extended haplotype A1B8CS01DR3DQ2 in other northern Caucasian diabetic populations (Morales et a1.1991). The former haplotype bears the BfF1 allele whose possible role in renal damage in SLE has been discussed elsewhere (Martfn-Villa et al. 1986). In addition, RFLP analysis has allowed us to subdivide further the HLA class lI specificities defined by serology, The significant increase of DR17.2 (DRBI*0301-DRB3*0202), the DR3 split present in the extended haplotype A30B18CFIDR3DQ2 in the DPGN-positive group, strongly suggests that the DR3 augmentation observed is attributable to the increase of this extended haplotype. In contrast to other reports, DR15 (the DR2 subtype most commonly found in our population) is not associated with either SLE or any of its clinical groups. Several studies have blamed the absence of concordant results of DR associations between different studies on technical serologic typing difficulties of SLE patients. This might be mainly a result of the presence of anti-lymphocyte antibodies in sera (Terasaki et al. 1970) and low levels of circulating B lymphocytes, often with poor viability (Stastny 1978). The good correlation (80%-100%) found between serologic assignment and RFLP typing in the present work (data not shown) ex- cludes the possibility of our having missed relevant antigens for analysis.

Moreover, the results obtained suggest that the differences in the non-coding region of HLA-B8DR3 (DR- B 1 * 0301-DRB 3"0101 ) and HLA-B 18DR3 (DRB 1 * 030 I- DRB3*0202) may be of importance for susceptibility to severe renal disease in patients of our ethnic origin, as has previously been described for other diseases (Segurado et al. 1991). However, DQo~2 (DQAI*0501) and DQ[32a (DQB 1 "0201 ) alleles, in linkage disequilibrium with both

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DR17 subtypes, are significantly increased in the DPGN- positive group, and appear to be related to this clinical group with the highest strength (8 = 0.74) (Table 2).

On the other hand, the analysis of C4 data further sup- ports the involvement of the A30B 18CF130DR3DQ2 extended haplotype in SLE nephritis susceptibility. It has been suggested that the C4A protein is critical in solubi- lization and elimination of immune complexes (IC) that lead to renal damage (Gatenby et al. 1990). However, we have found a similar C4A or C4B 'null' frequency in patients with a severe IC-mediated disease (DPGN) and in our control population. Notwithstanding, a significantly higher percentage of C4B deleted genes is found in the DPGN-positive group when compared with DPGN-negative patients and with controls. Although C4B has been involved in the clearance of IC, it has been shown to be less effective than the C4A isotype in this function. Thus, the increment of C4B deleted genes in the DPGN-positive group could merely reflect the higher frequency of the A30B18CF130DR3DQ2 haplotype in which that deletion is usually included. However, it is evident (Table 4) that although 'null' alleles are not increased in SLE with renal involvement, the percentage of the C4A or B 'null' phenotypes caused by deleted C4 genes (and not to lack of expression) is significantly higher in patients with renal disease (more than 90%) than in controls (about 50%) (Table 4). This does not favor the theory that involves C4 protein deficiency in the pathogeny of SLE, because of the lack of efficiency in IC clearance; rather, it suggests that deleted C4 genes are markers of neighboring susceptibility factors. In contrast, C4B long is usually associated with C4B 'pseudogenes'. Most of the non-deleted C4BQ0 alleles are explained by a gene conversion phenomenon at the C4B locus, resulting in homoduplicated expression at the C4A locus (Braun et al. 1990). It could be speculated that an excess of C4B long genes in DPGN-negative patients gives a double C4A gene dosage, thereby protecting against severe renal lesion in our SLE patients; the C4B long decrease in the DPGN-positive group could be un- derstood as being secondary to the C4B deleted increment.

A study by Fronek et al. (1990) shows that HLA- DR2 DQI alleles, especially the rare allele DQI31.AZH (DQBI*0502), confers a higher relative risk (RR = 14) of nephritis in Caucasian SLE patients. Other DQB 1 sequences, all related to DQ1 subtypes, are also increased in this clinical group. DQ~2a (DQBI*0201), increased in our DPGN-positive patients, does not share relevant amino acids (polymorphic sites) at any of the three hyper- variable regions of the DQB 1 second exon sequence, either with the DQ[31.1 allele (DQBI*0501) and DQI31.2 (DQB 1"0602) or with DQ~I .AZH (DQBI*0502), wide- ly represented in the nephritis SLE Caucasian group. Moreover, DQ0da (DQAI*0101), in linkage with DRI, is significantly decreased in the total SLE Spanish group (Table 2). These differences could arise because of the different ethnic origin of the groups studies. The DR2- DQI31 .AZH (DQB 1"0502) haplotype has not been found in the Spanish population. Moreover, different criteria were followed for the selection of patients, since our patients were classified according to renal biopsy findings

and clinical grounds whereas, in other studies, nephritis is defined only on clinical grounds. Thus, in SLE patients of Spanish descent, the extended haplotype A30B18- CF130DR3DQ2 appears as a susceptibility marker for DPGN. However, since the 6 value for the DQI32a allele (DQBI*0201, associated to DR3) is different from 1 (6= 0.74), the genetic contribution of other genes or their products needs further definition. Furthermore, differential environmental factors cannot be disregarded.

On the other hand, a hypothesis to explain susceptibility to autoimmune IDDM has recently been put forward (Khalil et al. 1990). It suggests that disease susceptibility is controlled by the presence of the amino acid arginine at residue 52 of the D Q a chain, and the absence of aspartate at residue 57 of the DQI3 chain. Since we have been able to assign exon-2 sequences based on the RFLP patterns obtained (see Materials and methods), we decided to test this hypothesis in another autoimmune disease, viz., SLE. Interestingly, both diseases are linked to the same extended haplotype in the Spanish population (i.e., A30B18- CF130DR3DQ2) in contrast to the A1B8CS01DR3DQ2 extended haplotype found in other Northern European populations. As shown in Table 3, this model does not ap- ply to our SLE patients. Although there is a significant increment of 'susceptible individuals' (belonging to the first five ~-[3 DQ heterodimer combinations) in the DPGN- positive group, RR and 8 values (Table 3) are smaller than those found with DQc~2 (DQAI*0501) and DQ~2a (DQB 1"0201) RFLPs. Finally, even with the present molecular methodology, an accurate assignment of the diseased locus is unlikely to be achieved, unless the pathogenic HLA mechanisms that lead to SLE are elucidated.

Acknowledgements. This work was supported by the Fondo de In- vestigaciones Sanitarias de la Seguridad Social, Spain.

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differential contribution of c4 and hla-dq genes to systemic lupus erythematosus susceptibility

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