DR2+ haplotypes in insulin-dependent diabetes: analysis of DNA restriction fragment length polymorphisms

Insulin-dependent diabetes (IDD) is strongly associated with certain HLA class II (Ia) antigens. The frequency of DR2 is significantly reduced in IDD; among DR2+ patients, the frequency of the subtype specificity Dw2 defined with homozygous typing cells (HTCs) is significantly reduced compared to DR2+ controls, and the specificity LD-MN2, which we have defined using primed lymphocyte typing reagents, is significantly increased. We have studied DNA restriction fragment length polymorphisms (RFLP) of DR2-LD-MN2+ individuals and homozygous typing cells carrying specificities antigenically related to LD-MN2. Using a number of different restriction enzymes, a characteristic pattern of fragments could be defined for DR2-LD-MN2 using both DQ beta and DR beta cDNA probes. This pattern was shared with some but not all of the antigenically related HTCs, and was distinct from that of DR2-Dw2. The RFLP pattern of DR2-LD-MN2 obtained with the DQ beta probe is identical, except for one band, to that of DR1-Dw1, suggesting that at least some part of the DQ region is identical in these two haplotypes. These results indicate that analysis of RFLP patterns can be used to help identify the genetic regions and, eventually, genes most important in the association of HLA and IDD.     

A study of HLA-DR2 associated HLA-Dw/LD specificities

HLA-Dw2 and Dw12 are both associated with HLA-DR2; however, these specificities account for only 86% (161/188) of the DR2 + haplotypes in our North American Caucasian panel. In an attempt to identify new DR2 associated antigenic clusters, we have generated four primed lymphocyte (LD) typing (PLT) reagents in haploidentical familial combinations against DR2 + Dw blank haplotypes. These reagents were positively restimulated by 11 of 16 DR2 + Dw blank cells tested, with good discrimination from Dw2 and Dw12 + cells, thus identifying a new antigenic cluster provisionally termed LD-MN2. We have compared the LD-MN2 specificity with the specificity LD-5a defined by two DR2 + HTCs, BAS and REM, (Layrisse, Caracas) which have been included in the pre-1984 Workshop Cluster DB9. Although none of our DR2 + cells gave typing responses to these two HTCs defining LD-5a, PLT studies did indicate an interrelationship between these specificities and with the specificity tb24 defined with the HTC, FJO (Betuel). The LD-5a HTCs, four LD-5a heterozygous cells, and two additional HTCs (WJR-Hansen, Seattle and FJO/tb24-Betuel, Lyon) significantly restimulated the anti-MN2 PLT reagents, though usually not as strongly as the MN2+ cells. MN2+ cells primed against the LD-5a HTCs were restimulated by only the LD-5a + cells. Dw2 + cells primed against FJO were restimulated by some, but not all MN2 + cells. These results suggest that MN2, tb24, and LD-5a share some determinants, not shared with most cells which type as Dw2 and Dw12, though differing by other stimulatory determinants. These studies emphasize the necessity of studying new antigenic clusters by both PLT and HTC methodologies as well as testing different ethnic groups.     

Techniques used to define human MHC antigens: restriction fragment length polymorphisms.

Polymorphisms within the HLA-DRB1, -DRB3, -DQB1 and -DQ A1 genes are detectable using restriction fragment length polymorphism (RFLP) analysis. DNA is isolated from EDTA-treated blood or from spleen or lymph nodes. The DNA is digested to completion with the restriction endonuclease TaqI and resolved using agarose gel electrophoresis. The DNA after denaturation is then transferred to a nylon membrane (Southern blotting) and hybridised with radiolabelled cDNA probes: HLA-DR beta pRTV1, HLA-DQ beta pII-beta-1 and HLA-DQ alpha pDCH1. After autoradiography the membrane is dehybridised prior to rehybridisation. This system is very useful in those situations where serological assignment is difficult due to poor quality or low numbers of circulating B cells and where there is a lack of reliable antisera for certain specificities. The RFLP techniques can also define subtypes of DR and DQ serological specificities. However, certain alleles have the same RFLP. In some instances by identifying the DQ allele the DR allele can be determined by association due to linkage disequilibrium (e.g., DRw17-Dw25-DQw2 and DRw13-Dw25-DQw6). In other instances (e.g., DR1 and DRBr), the problem can be resolved using serology. In addition the RFLP system cannot be applied prospectively to the cadaver donor situation because of time restrictions. Thus the RFLP system complements existing serological techniques. However, it can be very useful as a quality control for the serological methods especially in the assessment of the quality of antisera and in the determination of discrepancies between centres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Southern hybridization analysis of DNA polymorphism in the HLA-D region

Restriction fragment-length polymorphisms (RFLP) were systematically analyzed by Southern hybridization with restriction endonuclease-digested genomic DNA from 28 HLA-homozygous B cell lines with Dw1-Dw19 specificity using the DR beta and DQ beta chain cDNAs as probes. These probes detected polymorphic fragments unique to each HLA-DR specificity. Furthermore, the DQ beta chain probes permitted us to distinguish between different Dw specificities with an identical DR type much more efficiently than with the DR beta chain probe. Distribution analysis of restriction fragments hybridizing to DR beta in relation to the DR and DQ specificities showed several sets of them forming ten clusters, some of which correlate with DRw53, DQw1, and DR alleles. This DNA typing technique allows the direct definition of HLA types at the gene level and provides a powerful tool for isolating genes controlling HLA-associated diseases.     

HLA-DR and DQ DNA genotyping in multiple sclerosis patients in Northern Ireland.

DNA from multiple sclerosis (MS) patients and healthy control individuals from the Northern Ireland population, was assessed by restriction fragment length polymorphism (RFLP) analysis to look for disease-associated polymorphisms. HLA-DR and -DQ allogenotyping was performed using a single enzyme (TaqI)/Multiple probe system. The TaqI/DR beta RFLPs correlate well with serologically defined specificities and in addition detect further subtypes of these associated with DQ or Dw specificities. The results confirm an association of MS with DR beta 15 and show a decreased frequency of DR beta 4 in the patients. An increased frequency of DR beta 17 in patients negative for DR beta 15 was also found. There is a decrease in the frequency of the TaqI/DQ beta 3 (TA10 + ve) RFLP and the TaqI/DQ alpha 2LL genotype in the MS patients, these not being associated with DR beta 15. In addition RFLP analysis of the DPA1 and DPB1 genes reveals an MspI/DP beta 3.0-kb fragment which occurs at a higher frequency in the patients than in the controls.     

Allogenotypes defined by short DQ alpha and DQ beta cDNA probes correlate with and define splits of HLA-DQ serological specificities

HLA-DR and -DQ serotyped cell lines and peripheral blood leucocytes were analysed by Southern blot allogenotyping. Using a short DQ beta cDNA probe, a DQ beta allelic series was defined by restriction fragment length polymorphism (RFLP) with the restriction endonuclease TaqI. This DQ beta allelic series correlates with, and defines splits of, the HLA-DQ serological specificities DQw1 (DQ beta 1a and DQ beta 1b RFLPs), DQw2 (DQ beta 2a and DQ beta 2b RFLPs) and DQw3 (DQ beta 3a and DQ beta 3b RFLPs). By sequential use of a short DQ alpha cDNA probe a second, DQ alpha allelic series is defined by RFLP. This series correlates to a lesser extent than DQ beta RFLPs with the HLA-DQ serological specificities. Thus, two DQ alpha RFLPs correlate with a single DQ serotype (DQ alpha 1a and DQ alpha 1c with DQw1), but three DQ alpha RFLPs correlate with more than one DQ serotype (DQ alpha 1b with DQw1 and DQw3; DQ alpha 2 with DQw2 and DQw3; DQ alpha 3 with DQw2 and DQw3). Individual DQ beta and DQ alpha RFLP subtypes appear to correlate with single, or associated HLA-DR specificities. Specific combinations of DQ beta with DQ alpha RFLPs also correlate with HLA-Dw splits of DR2 and DRw6. A system for HLA-DNA typing is described, which uses RFLP patterns generated by sequential hybridization of TaqI-digested DNAs with short DR beta, DQ beta and DQ alpha cDNA probes. The DQ beta and DQ alpha probes not only identify the DQ allele, but because of linkage disequilibrium with DR, help to assign the DR allele, which may not always be identified with a DR beta probe alone.     

HLA-DR typing using restriction fragment length polymorphism (RFLP) with one enzyme and two probes

A panel of 43 homozygous and 36 heterozygous highly selected cells, representing the most common DR-specificities, were investigated with the DNA hybridization technique. By using a single restriction enzyme, TaqI, and two probes, DR beta and DQ alpha, it was possible to construct assignment criteria giving a reasonable definition of DR1, 3, 4, 5, 7 + w9, w8, w10 and w11. The criteria sometimes require that certain bands must not be present. Therefore, in certain genotypic combinations, the presence or absence of the particular specificity on one haplotype cannot be decided. This is a problem only for DR2 and DRw6, which for this reason cannot be assigned in about 1/3 to 1/4 of the cases. The association between RFLP assignment and serological assignment is not absolute, the correlation coefficients ranking from 0.62 to 1.0. In the case of false negative RFLP assignment, this may be due to genetic heterogeneity, as in the case of a DR2 individual who proved to be Dw12 and not Dw2 associated. It is often stated that interpretation of the RFLP pattern is particularly difficult in random or heterozygous individuals compared to proven homozygotes. This is not the case in the present study, where in fact correlation coefficients between RFLP and serologically determined DR specificities were higher in the heterozygotes (range 0.79-1.00).     

An overview of the restriction fragment length polymorphism of the HLA-D region: its application to individual D-, DR- typing by computerized analyses

HLA-D/DR alleles as defined by cellular and serological typing can also be identified by biochemical methods. The Southern blot technique provides an additional typing facility which can be applied to DNA obtained from any source of nucleated cells. The polymorphism revealed by Southern blot analyses, the so-called restriction fragment length polymorphism (RFLP), depends upon the restriction enzyme and cDNA probes used. To identify HLA-DR specificities a protocol was developed based on the use of the results of southern blot analyses with several restriction enzymes and cDNA probes within a panel of HLA-D/DR homozygous cells representing the DR1 to DRw8 alleles. First, hybridizations with the 3' untranslated sequence of the DR beta cDNA probe, after digestion of the DNA with PvuII (PvuII-DR beta 3') allows the selective identification of DR1, DR2 and DRw8; DR3, DR5 and DRw6 are found as one group as well as DR4 and DR7 as another. Second, TaqI-DQ alpha hybridization allows the splitting of DRw6-Dw18, DRw6-Dw19 and DRw6-Dw9 from the DR3, DR5 and DRw6 group. The other alleles DR3, DR4, DR5, DRw6-Dw16 and DR7 are revealed by dehybridization and rehybridization of the blot with a DR beta cDNA probe. This protocol was used to test whether in a panel of 30 randomly chosen individuals the HLA-DR typing could be performed. The results were highly concordant to the serotyping. Furthermore by adding the Pst-DR beta and TaqI-DQ alpha RFLPs, most of the MLC defined Dw specificities could also be identified. An overview of the specific fragments described here has been summarized in matrices which can be used as references for DNA-typing in computerized analyses.     

New HLA-D Alleles Associated with DRI and DR2

The present study describes two new HLA-D specificities : LD 13, associated with DR1, and LD14 associated with DR2. LD13 is defined by an HTC who is the bc offspring of an a: A25, B18, DR7, Dw7/b: A33, B14, DR1, Dx father, and of a c: A24, B14, Dr1, Dx/d: A26, B41, DR5, Dw5 mother. This HTC was included both as a responder and as a stimulator in our cross-reference studies of 8W HTCs. While failing to cluster with any other 8W HTC, it typed 2 of 64 panel members carrying a "blank" HLA-D, linked to DR1. To exclude the possibility that HTC-LD13 might be a split of Dwl, the entire family was tested with the Family Set of 8W HTCs. No typing responses to any 8W Dw1 HTCs were observed. Furthermore, checkerboard experiments between HTC-LD13 and 8WDw1 HTCs showed strong reciprocal stimulation. The LD13 specificity was only found in Ashkenazi Jews and may be in linkage disequilibrium with HLA-B14. LD14 is defined by three, SD different, HTCs deriving from the same family of Sicilian descent. The family was included in the 8th Workshop and each HTC was shown to have inherited DR2, MT1 from both parents. When tested as stimulators, on our HLA-D reference panel, these cells were clustered in a distinct group, LD14, associated with DR2. None of the 8W HTCs appeared to belong to this cluster. The antigen frequency of LD14 is 0.03.     

Extended HLA-DQw2 haplotypes: molecular analysis.

The HLA-DQw2 specificity, homogeneous in serology, is strongly associated to two HLA-DR specificities: DR3 and DR7. These alleles are found mainly on DQw2 bearing extended haplotypes with strong linkage disequilibrium. We describe, with BamHI, HindIII and RsaI, two restriction fragments length polymorphisms (RFLP) for the A gene of DQw2. These two subtypes correlated with the DR3 and DR7 specificities. Interestingly, by non-equilibrium pH gradient electrophoresis (NEPHGE), two DQ alpha chains were also found, respectively correlated with the same DR specificities. In addition, HincII polymorphism allowed us to distinguish several patterns of B genes for (DR7) DQw2 haplotypes but without any detectable association with another HLA marker. However, only one DQ beta chain was found by NEPHGE in the (DR7) DQw2 haplotype. Furthermore, HincII discriminated the B genes of the two extended haplotypes: (B8, DR3) DQw2 and (B18, DR3) DQw2. The same result was found by NEPHGE: two DQ beta chains were described, corresponding to the same extended haplotypes. The use of exon-specific DQB probes showed that the genomic polymorphism in DQw2 haplotypes is located, at least, at the 3' end of the gene. These data add new characteristics to the different DQw2 extended haplotypes.     

Segregation of HLA-DR, -DQ and -DP phenotypes and restriction fragment length polymorphic fragments in two recombinant families

Members of two families were typed for HLA-DR, -DQ and -DP specificities by means of sera and local PLT bulk reagents. One B:C and one DR:DP cross-over were identified in both families. The restriction fragment length polymorphism (RFLP) was analyzed by southern blotting and the use of DR beta, DR alpha, DQ beta, DQ alpha, DP beta and DP alpha probes. Previous observations concerning typical RFLP patterns for serological DR/DQ- and cellular DP-specificities were confirmed. With respect to recombinant haplotypes, RFLP was informative in both families. All polymorphic fragments-(DRB, DQA and B as well as DPA and B)-segregated together in the HLA-B:DR cross-overs. In the DR:DP recombinant children, the DR and DQ fragments were separated from DP fragments, demonstrating that DP-types as defined by PLT and RFLP map close together. No cross-hybridization between the segregating fragments was detected with the various probes.     

New HLA-DR2-related specificities detected in South African Blacks and individuals of mixed ancestry

This study describes a new HLA-DR2-related specificity DR2LUM (CT) present in South African Blacks and individuals of mixed ancestry (Cape Coloureds). It can be distinguished from the "classic" DR2 specificities. DRw15 and DRw16, using serological and Southern blot techniques. Although no HLA-Dw specificity could be assigned to the DR2LUM(CT) cells, borderline typing reactions with Dw2 HTCs were observed. Southern blot analysis using a DRB probe and the TaqI enzyme has shown that DR2LUM(CT) shared a 1.6 kb fragment with DRw15 and a 4.7 kb fragment with DR1 and DRw10, indicating sequence homology between DR2LUM(CT) and these alleles. In addition, another unusual HLA-DR2 haplotype was found. The DR antigen was typed serologically as DRw16 but showed a combination of restriction fragments which are associated with both the DRw15 and DRw16 specificities. This study demonstrates the value of investigating non-Caucasoid populations in further characterizing the polymorphisms of the HLA class II genes.     

RFLP analysis of the rhesus monkey MHC class II DR subregion.

Restriction fragment length polymorphism (RFLP) analysis was performed on a panel of 39 serologically typed DR homozygous monkeys. DNA was digested with the restriction enzyme TaqI and hybridizations were carried out with a human leukocyte antigen (HLA)-DR beta 3'UT-specific probe. In addition a panel of 18 monkeys was analyzed comprising experimental autoimmune encephalomyelitis (EAE) susceptible and nonsusceptible animals. The number of DRB/TaqI fragments detected for the various DR specificities varied from two to six, suggesting that the number of DRB genes per haplotype is not constant. RFLP typing allows that most serologically defined DR specificities can be subdivided. This knowledge was applied to define the DR specificities of the animals used for EAE experiments.     

HLA-DR-DQ haplotypes defined by restriction fragment analysis. Correlation to serology

Through the analysis of RFLP (restriction fragment length polymorphism) of the HLA-DR beta, -DQ alpha, and -DQ beta genes from 70 serologically well-characterized individuals, we have established unique HLA-DR-DQ RFLP haplotypes correlating to all of the DR1-w14 specificities. The RFLP of DR beta, DQ alpha, and DQ beta genes is very high using the restriction enzyme TaqI and 21 DR-DQ RFLP haplotypes were defined with this restriction enzyme. Our analysis confirms the strong linkage disequilibrium between alleles in the DR and DQ loci. DR beta RFLP indicates a common ancestor for the DR alleles within either of the supertypic DRw52 and DRw53 specificities. The DQ beta gene shows a high degree of RFLP, and the RFLP alleles partly reflect the serologic DQw1-w3 specificities. The results presented here also demonstrate the heterogeneity of DRw6 (DRw13 and DRw14) associated haplotypes, and the DRw13 related Dw18 and Dw19 specificities were found to have distinct DR-DQ haplotypes. The DQw1 positive haplotypes DR1, 2, w10, w13, and w14 are related with regard to DQ alpha and DQ beta RFLPs and the DRw52 positive haplotypes DR3, w11, and w12, as well as the DRw53 positive haplotypes DR4, 7, and w9, are related with regard to DR beta and DQ alpha RFLPs. These findings indicate that polymorphic sequences around the DQ alpha gene are associated with DR beta and DQ beta polymorphism, which suggests a location of the DQ alpha gene between DR beta and DQ beta.     

Characterization of class II alpha genes and DLA-D region allelic associations in the dog

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the alpha genes of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (BamHI, EcoRI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I and Bgl II), separated by agarose gel electrophoresis and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabelled HLA cDNA probes corresponding to DQ, DP, DZ and DR alpha genes. Clear evidence was obtained for the canine homologues of DQ and DR alpha genes with simple bi- or tri-allelic polymorphism respectively. Evidence for a single, nonpolymorphic DP alpha gene was also obtained. However, the presence of a DZ alpha gene could not be clearly demonstrated in canine genomic DNA. This report extends our previous RFLP analysis documenting polymorphism of DLA class II beta genes in the same panel of homozygous typing cell dogs, and provides the basis for DLA-D genotyping at a population level. This study also characterizes the RFLP-defined preferential allelic associations across the DLA-D region in nine different homozygous typing cell specificities.     

Subtypes of HLA-DQ and -DR defined by DQB1 and DRB1 RFLPs: allele frequencies in the general population and in insulin-dependent diabetes (IDDM) and multiple sclerosis patients

We have used the HLA-DQB1 gene as a Southern hybridization probe with TaqI-digested genomic DNA in a study of 600 haplotypes from unrelated individuals and have characterized HLA-DQB1 RFLP patterns associated with the DR specificities DR1-DRw10 and DN1. For six of the specificities (DR2, 4, w6, 7, w8 and 9), we have also identified subtypes (multiple DQB1 band patterns), In a previous study (Cox Pt al. 1988), we identified RPLPs and subtypes with a DRB1 probe. Using the present results from DQB1 RFLPs to supplement those from DRB1 RFLPs, it was possible to discriminate among all the DR specificities with the exception of a minority of DR7 and DR9 subtypes. A comparison of DQB1 and DRB1 subtypes in the same subjects showed strong linkage disequilibrium for subtypes of some but not all DR specificities. We have also determined the allele frequencies of the DQBl subtypes in controls and in patients with insulin-dependent diabetes mellitus (IDDM) or multiple sclerosis (MS). A consideration of subtypes in patients and controls indicated that for most DR specificities, neither IDDM nor MS was more strongly associated with any of the DQBl subtypes than with the serologically defined DR antigens. The exveptions were the DQBl patterns corresponding to the DQw3.2 subtype of DR4 and the rarer subtype of DR2, which were found in higher frequency in IDDM patients, as has been previously reported.     

Analysis of HLA-DRB and -DQB gene RFLPs in DR7 homozygous cell lines: associations with Dw11, Dw17 and DB1

The DR7-associated Dw specificities, Dw11, Dw17 and DB1 were investigated with regard to DRB- and DQB-gene polymorphism, as revealed by RFLP analysis using the restriction enzyme TaqI. In the 22 DR7 homozygous cell lines investigated, each of these Dw specificities was found to correlate to one specific RFLP defined DR-DQ haplotype. In addition, a clear linkage disequilibrium to a specific HLA-B locus allele for each Dw specificity was noted, indicating that the Dw subtypes of DR7 often are associated with a conserved HLA-B-DR-DQ haplotype. Only one genetically homozygous cell line, PLH, deviated from these correlations. This cell line, notably derived from an individual with a deletion of the 21-hydroxylase B-gene (21-OHB), caries the HLA haplotype Bw47, DR7, DQw2, DB1, but displayed a DRB RFLP otherwise found in association with Dw17.     

Unusual HLA-DR,DQ haplotypes found in South African families of black, Asian Indian, and mixed ancestral origin.

Four non-Caucasoid families with the unusual HLA-DR,DQ haplotypes DRw17,DQw7; DR9,DQw2; DR4,DQw2; and DR4,DQw5 were studied. All four haplotypes showed identical serological patterns to those seen with the equivalent Caucasoid antigens, but no HLA-Dw specificities could be assigned. TaqI restriction fragment length polymorphism (RFLP) patterns observed using DRB, DQB, and DQA probes showed that the DRw17,DQw7 haplotype may have originated from a homologous crossover between a DRw17,DQw2 haplotype and a haplotype with DQw7. The results obtained for the DR9,DQw2 and DR4,DQw2 haplotypes suggest that these could have resulted from recombination events with an ancestral "black" DQw2 haplotype. From the RFLP data, it is difficult to postulate the origin of the DR4,DQw5 haplotype being from a single recombination event.     

Polymorphism of HLA-DR2,DQwl haplotypes in Asian Indians

We examined the polymorphism of DR2,DQw1 haplotypes in Epstein-Barr virus-transformed B-lymphoblastoid cell lines (HTCs) and unrelated (32 Canadian Caucasians and 24 Asian Indians) individuals by restriction fragment length polymorphism (RFLP) and oligonucleotide typing. The data demonstrate that three subtypes of DR2,DQw1 haplotypes, DRw15(B1.1501).DQw6a(A1.0102,B1.0602),DRw15(B1.1502). DQw6b(A1.0103,B1.0601), DRw16(B1.1601).DQw5(A1.0102,B1.0502) are present in HTCs and Canadian Caucasians. Of these, DRw15(B1.1501).DQw6a (A1.0102,B1.0602) haplotype was present in majority (81.3%) of Caucasians. Among Asian Indians, this haplotype was present only in one DR2,DQw1-positive individual. In addition, three new haplotypes representing different combinations of DRB1, DQA1 and DQB1 genes were demonstrable in Asian Indians. These new haplotypes are DRw15(B1.1501).DQw6b(A1.0103,B1.0601),DRw15(B1.1501). DQw5(A1.0102,B1.0502), and DRw15(B1.1501).DQw6c(A1.0102, B1.0601). The most frequent haplotypes among Asian Indians were DRw15(B1.1502).DQw6b(A1.0103,B1.0601) and DRw15(B1.1501). DQw6b(A1.0103,B1.0601). The distribution of subtypes of DR2,DQw1 haplotypes in Asian Indians was significantly different from that in Canadian Caucasians. The results in the present study have important implications for HLA and for HLA-disease associations.     

EXTENDED HLA-DQw2 HAPLOTYPES: MOLECULAR ANALYSIS

The HLA-DQw2 specificity, homogeneous in serology, is strongly associated to two HLA-DR specificities: DR3 and DR7. These alleles are found mainly on DQw2 bearing extended haplotypes with strong linkage disequilibrium. We describe, with BamHI, HindIII and RsaI, two restriction fragments length polymorphisms (RFLP) for the A gene of DQw2. These two subtypes correlated with the DR3 and DR7 specificities. Interestingly, by non-equilibrium pH gradient electrophoresis (NEPHGE), two DQα chains were also found, respectively correlated with the same DR specificities. In addition, Hindi polymorphism allowed us to distinguish several patterns of B genes for (DR7) DQw2 haplotypes but without any detectable association with another HLA marker. However, only one DQ(3 chain was found by NEPHGE in the (DR7) DQw2 haplotype. Furthermore, MncII discriminated the B genes of the two extended haplotypes: (B8, DR3) DQw2 and (B18, DR3) DQw2. The same result was found by NEPHGE: two DQβ chains were described, corresponding to the same extended haplotypes. The use of exon-specific DQB probes showed that the genomic polymorphism in DOw2 haplotypes is located, at least, at the 3’end of the gene. These data add new characteristics to the different DQw2 extended haplotypes.