HLA-DQ RFLP variants of five HLA-DQw2-bearing major histocompatability complex extended haplotypes

We have analyzed genomic DNA in a large number of independent examples of five HLA-DQw2-bearing extended haplotypes for their associated subtypes by restriction fragment length polymorphism (RFLP) using DRB, DQA, and DQB probes after Taq I and Pst I digestion and Southern blotting. In addition to three previously described HLA-DQw2 subtypes, DQw2a, DQw2b, and DQw2c, we observed a fourth subtype, HLA-DQw2d, characterized by 5.8 kilobase (kb) DRB/Taq I, 2.4, 2.3, and 1.8 kb DQB/Taq I, and 8.0 and 2.3 kb DQA/Pst I fragments. All 22 independent examples of the extended haplotype [HLA-B8,SCO1,DR3] carried DQw2a and all 11 independent examples of [HLA-B18,F1C30,DR3] carried DQw2b. In addition, all independent examples (21 and 4, respectively) of two DR7-carrying extended haplotypes, [HLA-B44,FC31,DR7] and [HLA-Bw47,FC91,0,DR7], carried DQw2c and all independent examples of [HLA-Bw57,SC61,DR7] carried DQw2d. Our results show that the DNA in the DR/DQ region of extended haplotypes is relatively fixed and that different DQw2 subtypes characterize different DQw2-bearing extended haplotypes.     

Recognition of class II molecules by human T cells

The HLA-D region of individuals with the DRw11, w52, DQw3 haplotype encodes multiple molecular products of three distinct subregions, DR, DP, and DQ. Since each molecule can carry multiple stimulatory epitopes, the repertoire of allogeneic T-cell responses to determinants of this haplotype can be quite large. In the present experiments, alloreactive cloned T-cell lines recognized six distinct epitopes associated with DRw11, DRw52, DQw3 haplotypes. Panel studies established that three epitopes were DRwll-like and three were DRw52-like. Blocking with monoclonal antibodies showed that two DRw11-like epitopes were carried by DR-subregion products and one DRwll-like epitope was carried by DQ-subregion molecules. DRw52-like epitopes were detected on separate DR subregion-encoded molecules. One of them carried both DRwl1-and DRw52-like epitopes, the other carried two of the DRw52-like epitopes. These epitopes, which represent functional units that trigger T-cell responses, can be detected at the present time only with the methods used in this report. Conventional allogeneic T-cell responses represent the summation of responses to multiple epitopes encoded by different D-subregion genes.     

Allelic forms of the alpha- and beta-chain genes encoding DQw1-positive heterodimers

On chromosome 6, in the HLA region, the DQ subregion is located immediately centromeric to the DR subregion. Even though only three serological specificities to date have been officially recognized (DQwl, DQw2, and DQw3), it seems likely that the phenotypical polymorphism expressed by DQ molecules is much more complex. There are reasons to believe that fixed alpha-beta combinations exist, each of them associated with a different DR allele. DQw1 is a determinant present on DQ molecules that are found associated with DRI-, DR2-, and DRw6-positive haplotypes. By restriction fragment length polymorphism analysis, we recognized three allelic DQ-alpha and three allelic DQ-beta patterns associated with DQw1 . In addition, one of these alpha/beta pairs associated with DR1, two with DR2, and a fourth with DRw6. We have obtained evidence using nucleotide sequencing that there are as many allelic forms of DQ-alpha and DQ-beta genes as there are different molecular DQ-alpha and DQ-beta patterns. The DQ-alpha and DQ-beta chains of DQwl-positive molecules each are encoded by at least three distinctly different allelic genes, and particular alpha/beta gene combinations are associated with the same DR alleles as their corresponding molecular alpha/beta pairs.     

A novel association of DQ alpha and DQ beta genes in the DRw10 haplotype. Determination of a DQw1 specificity by the DQ beta-chain

The association of the class II genes of the DRw10 haplotype from a cell line, NASC, initiated from a member of a well characterized family, was analyzed by sequencing cDNA clones corresponding to DR beta I, DQ alpha, and DQ beta genes. An identical haplotype was also identified in the Raji cell line. In addition to typing as DRw10 and DQw1 with HLA typing sera both, the NASC and Raji cell lines were shown to react strongly with the monoclonal antibodies 109d6 (specific for DRw10 beta 1 and DRw53 beta 2 gene products) and Genox 3.5.3 (specific for DQw1) and exhibited the restriction fragment length polymorphism indicative of a DRw10, DQw1 haplotype. The DR beta 1 gene corresponding to the DRw10 specificity was found to have a first domain sequence different from all other DR beta I genes. Sequence analysis of the 3'-untranslated region of this DR beta-chain gene showed a significant divergence from the 3' untranslated region of the DRw53 family of haplotypes and a lesser divergence from that of the DRw52 and DR1/DR2 families. The sequence of the DQ beta genes corresponding to the DQw1 specificity in the DRw10 haplotype was found to be identical to the DQ beta gene from a DR1, DQw1 haplotype. Surprisingly, however, the DQ alpha gene did not resemble other DQw1-like DQ alpha genes, but was identical in sequence to the DQ alpha gene found in DR4 haplotypes. The novel association of DQ alpha and DQ beta genes in the DRw10 haplotype revealed in these studies may result from a double recombinational event. More consequentially, these studies strongly suggest that the DQw1 specificity recognized by Genox 3.5.3 is determined by the DQ beta chain and is not affected by the DQ alpha-chain.     

Recombination sites in the HLA class II region are haplotype dependent

We have analyzed DNA sequence polymorphisms of DQ alpha and DQ beta chains from three haplotypes from the DRw52 family: DR5 DQw1 (FPA, GM3106), DRw6 DQw1 (CB6B, 10w9060), and DRw6 DQw3 (AMALA, 10w9064). The results indicate that the DR5 DQw1 and DRw6 DQw1 haplotypes have arisen by recombination between the DR beta 1 and DQ alpha loci. This contrasts with our previous analysis of DR4 DQ"Wa", DR3 DQ"Wa", and DR7 DQw3 haplotypes, all of which appear to have arisen by virtue of recombination between DQ alpha and DQ beta. Thus, there appear to be at least two different sites where recombination has occurred within the DR and DQ subregions. These differing patterns of recombination were interpreted in the context of the three major family groups of class II haplotypes, the DRw53, DRw52, and DR1/2 haplotype families. The data indicate that haplotypes from these family groups tend to undergo recombination at different locations. We propose that these differences in site of recombination are a reflection of differences in the molecular organization of the haplotypes belonging to each family group.     

HLA-DQ system and insulin-dependent diabetes mellitus in Japanese: does it contribute to the development of IDDM as it does in Caucasians?

Fifty-six unrelated Japanese patients with insulin-dependent diabetes mellitus (IDDM) were HLA-typed, and restriction fragment length polymorphism (RFLP) analysis was performed after enzyme digestion with Bam HI and Taq I by using both DR and DQ probes. As previously reported, increased frequencies of Bw54, Cw1, DR4, and DRw53, which are in strong linkage disequilibrium in the Japanese population and make the characteristic Japanese haplotype, were confirmed. DQw4, a new allele of the DQ system recognized by the monoclonal antibody HU-46 and in linkage disequilibrium with this haplotype, presented the highest IDDM association. The RFLP analysis also showed the strongest correlation to IDDM when the DQ probe was applied. These results indicate that HLA-DQ might play the most important role in the development of IDDM in Japanese as well as in Caucasians. The correlation of DQ amino acid sequences strongly associated with IDDM in Japanese are discussed in this study, and contrasting results were found when such sequences were compared with those of Caucasians.Abbreviations used in this paper IDDM insulin-dependent diabetes mellitus - RFLP restriction fragment length polymorphism - Asp aspartic acid - Asp-57 aspartic acid at the 57th residue of the DQ chain - non-Asp-57 nonaspartic acid at the 57th residue of the DQ chain - R.R. relative risk of Woolf and Haldane     

The narcolepsy-associatedDRw15, DQw6, Dw2 haplotype has no uniqueHLA-DQA or -DQB restriction fragments and does not extend to theHLA-DP subregion

Almost all patients with cataplectic narcolepsy are DR2-positive. It has been suggested that thenon-DR2 allele/haplotype might not be neutral with respect to disease susceptibility. It has also been reported thatTaq IDQA andBam HI,Eco RI,Eco RV, andPst IDQB restriction fragments might differentiate between narcoleptic and healthy DR2-positive individuals. In the present study,HLA class II gene polymorphisms were investigated by restriction fragment length polymorphism (RFLP) analysis in 47 Swedish patients with cataplectic narcolepsy, 100 random controls, and DR2-associated homozygous cell lines. All patients hadTaq IDRBDQA-DQB patterns corresponding to theDRw15,DQw6, Dw2 haplotype. The non-DR2 haplotype was found to be neutral. This genotyped group of patients allows firm rejection of a recessive mode of inheritance and supports a dominant or additive model. NoDQA orDQB RFLPs were found that could differentiate between DR2-positive narcoleptics, DRw15,DQw6,Dw2-positive controls, orDw2-homozygous cell lines. No significantMsp IHLA-DP association was found. No linkage disequilibrium was observed between theDRw15,DQw6,Dw2 haplotype and alleles of theDP subregion in patients or controls. Thus, theHLA-D region-associated narcolepsy susceptibility gene may be located telomeric to theHLA-DP subregion. No RFLPs have been observed that can locate the narcolepsy susceptibility gene closer to theDQ than to theDR subregion.     

The “Sardinian”HLA-A30,B18,DR3,DQw2 haplotype constantly lacks the21-OHA andC4B genes. Is it an ancestral haplotype without duplication?

TheC4 and21-OH loci of the class III HLA have been studied by specific DNA probes and the restriction enzymeTaq I in 24 unrelated Sardinian individuals selected from completely HLA-typed families. All 24 individuals had theHLA extended haplotypeA30,Cw5,B18, BfF1,DR3,DRw52,DQw2, named “Sardinian” in the present paper because of its frquency of 15% in the Sardinian population. Eighteen of these were homozygous for the entire haplotype, and six were heterozygous at theA locus and blank (or homozygous) at all the other loci. In all completely homozygous cells and in four heterozygous cells at theA locus, the restriction fragments of the21-OHA (3.2 kb) andC4B (5.8 kb or 5.4 kb) genes were absent, and the fragments of theC4A (7.0 kb) and21-OHB (3.7 kb) genes were present. It is suggested that the “Sardinian” haplotype is an ancestral haplotype without duplication of theC4 and21-OH genes, practically always identical in its structure, also in unrelated individuals. The diversity of this haplotype in the class III region (about 30 kb less) may be at least partially responsible for its misalignment with most haplotypes, which have duplicatedC4 and21-OH genes, and therefore also for its decreased probability to recombine. This can help explain its high stability and frequency in the Sardinian population. The same conclusion can be suggested for the Caucasian extended haplotypeA1,B8,DR3 that always seems to lack theC4A and21-OHA genes.     

Molecular analysis of the HLA class II genes in two DRw6-related haplotypes, DRw13 DQw1 and DRw14 DQw3

We have compared the sequence polymorphism of HLA class II genes of two distinct DRw6 haplotypes. cDNA libraries were constructed from two lymphoblastoid cell lines: CB6B (10w9060) which types as DRw13 DQw1, and AMALA (10w9064) which types as DRw14 DQw3. Multiple sequence differences were found at the DR beta I, DQ alpha, and DQ beta loci when these two haplotypes were compared. The DR beta I allele found in the DRw14 DQw3 haplotype appears to have diverged primarily as a result of a gene conversion event with a DR1 allele acting as donor. In contrast, the DRw13 DQw1 haplotype appears to have arisen by means of a recombination event between the DR and DQ subregions. Thus, multiple genetic mechanisms, including point mutation, gene conversion, and recombination, have generated diversity among DRw6 haplotypes.     

Polymorphism of the HLA-D region in American blacks. A DR3 haplotype generated by recombination

The polymorphism of HLA class II molecules in man is particularly evident when comparisons between population groups are made. This study describes a DR3 haplotype commonly present in the American black population. Unlike the Northern European population in which almost all DR3 individuals are DQw2, approximately 50% of DR3-positive American blacks express a serologically undefined DQ allelic product. DNA restriction fragment analysis with the use of several unrelated individuals and an informative family has allowed us to identify unique DQ alpha- and beta-fragments associated with the DR3, DQw- haplotype. Based on fragment size, the DQ alpha genes of the DR3, DQw- and DRw8, DQw- haplotypes are similar as are the DQ beta genes of DR3, DQw-; DRw8, DQw-; and DR4, DQw- haplotypes. In addition, a DX beta gene polymorphism has been identified which is associated with some DR3 haplotypes including the American black DR3, DQw- haplotype. cDNA sequence analysis has revealed a DQw2-like alpha gene and a DQ beta gene which is similar to that previously described for a DR4, DQw- haplotype. It is postulated that recombination between DQ alpha and DQ beta genes and between the DQ and DX subregions has generated the various DR3 haplotypes and has played an important role in creating diversity in the HLA-D region.     

Characterization of specific HLA-DQ alpha allospecificities by genomic, biochemical, and serologic analysis

Bgl II restriction endonuclease digestion of genomic DNA from lymphoblastoid cell lines homozygous for HLA DR and DQ serological specificities, followed by hybridization with a DQ alpha cDNA probe, identified a genomic polymorphism characterized by two reciprocal patterns, one associated with DR 3, 5 and 8 and the other with DR 1, 2, 4, 7, and 9. The former pattern corresponded precisely to the reactivity of monoclonal antibody SFR20-DQ alpha 5, shown by Western blotting to react with isolated alpha-chains, but not with beta-chains. Additional variants of the DQ alpha genes were identified by using a locus-specific oligonucleotide probe for the DQ alpha gene, indicating differences among the DQ alpha 5-negative set of alleles. This analysis defines a set of DQ alpha allelic markers that are distinct from the well-established DQ serologic specificities DQw1, 2, 3 or "blank." Although most DQ alpha 5+ cells carry the DRw52 specificity associated with the DR beta 2 gene, analysis of DQ alpha polymorphisms on DR5, DQw1; DR8, DQw1; and DRw13, DQw1 cells verified that this DQ alpha family of alleles was not invariably linked to the DR beta 2 locus.     

Molecular characterization of a subtype of DQw1 recognized by hapten-specific T cells

Previous studies of HLA-restricted antigen recognition by cloned T cells have frequently demonstrated reactivity that did not correlate precisely with the expression of serologically defined HLA specificities. To further explore such discrepancies, we utilized monoclonal antibody (MoAb) blocking, partial NH₂-terminal amino acid sequencing, and Southern blot hybridization techniques to analyze the fine specificity of four autologous trinitrophenyl-specific T cell lines restricted to DR2-linked epitopes. MoAb blocking studies demonstrated that two of these lines recognized determinants on DR molecules while the other two recognized determinants on the same molecule that expresses the DQw1 determinant. However, these latter two lines appeared to recognize a DQw1-related determinant found primarily in association with DR2, but not the other DQw1-associated DR alleles, DR1 and DRw6. To ascertain whether these lines were defining a functional split of DQw1, we performed partial NH₂-terminal amino acid sequencing of the molecules precipitated with a DQw1-specific MoAb (Genox 3.53) from different stimulator lines. The results showed that these T cell lines recognized a subtype of DQw1 that is in linkage disequilibrium with DR2. Moreover, we identified characteristic restriction fragment length polymorphisms with a DQ -specific cDNA that correlated with stimulatory capacity for the DQw1-restricted lines. These results demonstrate that: (1) DQ molecules may provide restriction determinants that are incorrectly assigned to DR molecules on stimulator panel analyses; (2) cloned antigen-specific T cell lines recognize polymorphic regions of class II molecules not distinguished by either conventional typing antisera or xenogeneic MoAb; and (3) the DQw1 epitope(s) is located on a heterogeneous group of DQ molecules that differ from each other in the primary sequence of their chains.Abbreviations used in this paper ATCC American type culture collection; cpm, counts per minute - DNA deoxyribonucleic acid - EBV Epstein-Barr virus - FCS fetal calf serum - MoAb monoclonal antibody - PBMC peripheral blood mononuclear cells - % RAgS percent relative antigen stimulation - RFLP restriction fragment length polymorphism - SDS sodium dodecyl sulfate - S-RPMI supplemented-RPMI - TCL T-cell line - TNP trinitrophenyl     

Polymorphisms of DQ β genes in HLA-DR4 haplotypes from healthy and diabetic individuals

The restriction fragment length polymorphism (RFLP) of DQ was assessed in a panel of control and insulin-dependent diabetes (IDD) patients who were serologically typed as HLA-DR4 homozygotes or HLA-DR3, DR4 heterozygotes. Digestions of genomic DNA with Barn HI, Bg1 II, Pst I, Xba I, and Hind III revealed a total of 15 RFLPs in the panel of 71 HLA-DR4 chromosomes. These RFLPs were organized into six allelic groups on the basis of segregation analysis in families. Complete RFLP haplotypes for the 5 restriction enzymes could be constructed for 42 of the HLA-DR4 chromosomes. This analysis revealed 18 RFLP haplotypes of DQ associated with the DR4 chromosomes tested. Two of these haplotypes, designated DQ3.DR4.a and DQ3.DR4.b, accounted for over 50 % of the DR4 chromosomes analyzed. These two haplotypes were antithetical for the RFLPs detected by all five enzymes, indicating that they represent very distinct forms of DQ . The remaining 16 haplotypes were infrequent or unique and were closely related to either a DQ3.DR4.a or DQ3.DR4.b. Two of the RFLPs detected, a 5.8 kb Bg1 II fragment and a 10.5 kb Barn HI fragment, had increased frequencies in disease-associated chromosomes. However, none of the RFLPs we detected exhibited a statistically significant increase in IDD or control populations. In contrast, the DQ3.DR4.b DQ haplotype was significantly decreased in IDD-associated DR4 chromosomes. (P=0.04). These results suggest that the DQ3.DR4.b DQ allele may be protective for the development of IDD.     

Cloning and analysis of HLA class I cDNA encoding a new HLA-C specificity Cx52

HLA-C loci frequently have an unclassifiable blank (CwBL) specificity. It is unclear whether HLA-C specificities associated with the haplotypes of A24 Bw52 CwBL DR2 DQw1 and Aw33 B44 CwBL DRw13 DQw1 in Japanese (tentatively named Cx52 and Cx44, respectively) really exist. Southern hybridization experiments revealed that restriction enzyme-cleaved genomic DNA from AKIBA, consanguineous HLA homozygote, two other homozygotes with the former haplotype, and three homozygous cells with the latter haplotype hybridized strongly with an HLA-C-specific probe. We have screened the cDNA library constructed from AKIBA to isolate cDNA clones encoding the putative Cx52 antigen, and picked up 103 cDNA clones with HLA-class I DNA probes as possible candidates. By restriction enzyme mapping and Southern hybridization of selected clones, we identified three isotypes of cDNA clones, pA01, pB55, and pC68, which appeared to encode A24, Bw52, and Cx52, respectively. The nucleotide sequence of pC68 showed higher homology with exons of the HLA-C gene than with those of the HLA-A and HLA-B genes, especially in exons 6–8 which include the HLA-C-specific region. Comparison of amino acid sequences showed more than 86% homology among Cw1, Cw2, Cw3, and new pC68-encoded Cx52 proteins. These results support the notion that the inability to define C antigens serologically in this Cx52 haplotype is not due to a HLA-C gene deletion or mutation, but to the absence of typing sera.     

Linkage disequilibrium of HLA-SB1 with the HLA-A1, B8, DR3, SCO1 and of HLA-SB4 with the HLA-A26, Bw38, Dw10, DR4, SC21 extended haplotypes

Homozygous typing cells from 13 normal HLA-A1, B8, Dw3, DR3 and five normal HLA-A26, Bw38, Dw10, DR4 individuals were typed for the following markers: HLA-SB, MB, MT; complement proteins BF, C2, C4A, C4B; and GLO. Ninety-one percent of A1, B8, Dw3, DR3 homozygous individuals (HI) tested were homozygous for BF ^* S, C2 ^* C, C4A ^* QO, and C4B ^*1 (SCO1 complotype), which indicates that the SCO1 complotype is in linkage disequilibrium with the A1, B8, DR3 haplotype in randomly selected normal populations. Sixty-seven percent of HLA-A1, B8, Dw3, DR3, SCO1 positive HI also expressed SB1; since the frequency of SB 1 in random Caucasian populations is 11.2%, this finding indicates that SB1 is in linkage disequilibrium with the A1, B8, DR3, SCO1 extended haplotype. All HI with the A26, Bw38, Dw10, DR4 haplotype were homozygous for both SC21 and SB4, suggesting that SC21 and SB4 should be included in the A26, Bw38, Dw10, DR4 extended haplotype. On the other hand, neither of the GLO markers were found in association with either haplotype. The results of this study indicate that HLA-SB is included in some extended haplotypes and may be important in these markers for diseases such as insulin-dependent diabetes mellitus. This study also demonstrated an apparent influence of HLA-SB on primary mixed lymphocyte culture (MLC) responses. The mean relative response of primary MLCs between individuals matched for HLA-A, B, D, DR, MB and MT but not SB was 40% of that for the MLCs with mismatched HLA-D, significantly higher than the MLCs matched for all HLA and complotypes.     

A cellular and functional split in the DRw8 haplotype is due to a single amino acid replacement (DR β ser 57- asp 57 )

The single DR beta chain gene of the DRw8 haplotype has been suggested to carry both the DRw8 and the DRw52 epitopes. Cellular typing has shown that the DRw8 haplotype can be split into three subtypes, Dw8.1. Dw8.2, and Dw8.3, presumably due to a polymorphism in the DRw8 chain. Furthermore, Dw8.1 and Dw8.2 cells present influenza virus antigen to different T-cell clones. In the present study, DRw8/Dw8.2 chain cDNA was cloned and characterized. A comparison of this sequence with a partial DRw8/Dw8.1 chain gene suggested that the DRw8 split is due to a single amino acid replacement of ser ₅₇ -asp ₅₇ caused by three nucleotide substitutions in the same codon. In most DR haplotypes, two expressed DR beta chain genes exist. Comparing the nucleotide sequence of the single beta gene in the DRw8 haplotype to those of other DR beta genes revealed that the DRw8 beta gene sequence is most closely related to the DRBI genes of the DR3, 5, and w6 haplotypes. However, the comparisons also showed that it was not possible from sequence similarities to divide the DR beta genes into two or more distinct allelic series.     

Quantitative variation of C4 variant proteins associated with many MHC haplotypes

C4 protein variants were analyzed in 64 individuals, of which 51 were either homozygous or heterozygous for an extended major histocompatibility complex (MHC) haplotype (a fixed combination of MHC alleles). The relative amount of each C4 variant was measured by densitometric scanning of stained immunofixed electrophoretic patterns of neuraminidase- and carboxypeptidase-treated samples. The relative concentrations of C4 variants on any haplotype were stable and inherited in families. In five of the eight extended haplotypes investigated, the amount of one of the C4 variants relative to others in the same pattern was increased:[HLA-B8, SC01, DR3] and[HLA-B7, SC31, DR2] produced an approximately doubled amount of C4B1;[HLA-B18, S042, DR2] an increased amount of C4B2; and[HLA-B44, SC30, DR4] a double amount of C4A3. The extended haplotype[HLA-Bw57, SC61, DR7] gave rise to two to three times as much C4B1 as C4A6. In the extended haplotypes[HLA-B44, FC31, DR7] and[HLA-Bw62, SC33, DR4], the results did not clearly indicate differences in expression of the C4 isotypes. DNA analysis possibly supported an actual gene duplication only for the haplotype[HLA-B7, SC31, DR2]. The results suggest that, in addition to variation in the number of structural genes, other MHC-linked mechanisms may be involved in the regulation of the relative amounts of C4A or C4B protein specified by any haplotype.     

The genomic structure of two ancestral haplotypes carrying C4A duplications

Two major histocompatibility complex (MHC) ancestral haplotypes (AH) HLA A24, Bw52, C2C, BfS, C4A3 + 2, C4BQO, DRw15, DQw6 (52.1) and HLA A24, Cw7, B7, C2C, BfS, C4A3 + 3, C4B1, DR1, DQw5 (7.2), which occur with the haplotype frequencies of approximately 10% and 4% respectively in the Japanese population, carry duplicated C4A alleles by C4 allotyping. Southern blot analysis with Taq I indicated that the 52.1 AH has two C4 genes defined by 7.0 kilobase (kb) and 6.0 kb C4 hybridizing fragments but both encode C4A allotypes, being C4A3 and C4A2 respectively. The 7.2 AH carries two C4A3 and one C4B1 alleles and restriction lenght polymorphism (RFLP) analysis with Taq I showed that 6.0 kb and 7.0 kb fragments are in the proportion of 2:1. By pulsed field gel electrophoresis (PFGE) analysis, the lengths of the Pvul fragments carrying C4 and Cyp21 genes were approximately 390 kb for 52.1 and 440 kb to 7.2. The results indicate that the RFLP markers do not correlate with C4 isotype (A or B) or allotype and that the C4 gene copy number is a function of the number of genomic blocks containing C4 and Cyp21.     

The susceptibility to insulin-dependent diabetes mellitus bis associated with C4 allotypes independently of the association with HLA-DQ alleles in HLA-DR3, 4 heterozygotes

In the genetically homogeneous Danish population, 27 HLA-DR3,4 heterozygous patients with insulin-dependent diabetes mellitus (IDDM) and 19 DR3,4 heterozygous controls without family history of IDDM were investigated for HLA-region markers and Gm and Km immunoglobulin allotypes. The aim was to define susceptibility factors for IDDM development other than HLA-DR using a number of techniques: lymphocytotoxicity (HLA-DR and DQ antigens), cellular methods (Dw and DP typing), restriction fragment length polymorphism (DQ alleles), electrophoresis and immunofixation (BF and C4 allotypes), and passive hemagglutination inhibition (Gm and Km immunoglobulin allotypes). The complement allotype C4A3 and the HLA-DQw8 (DQw3.2) antigen were found in all of the patients, whereas this was the case for only 8 of the 19 controls (P=6 x 10^–6): five lacked C4A3, five others lacked DQw8, and one of the controls lacked both of these factors. Fourteen of the patients had the complement allotype C4B3 versus three of the controls (P=0.01). Previously reported family studies suggest that these alleles are part of the following haplotype: B15, BFS, C4A3, C4B3, DR4, Dw4, DQw8, and these factors were found together in ten of the patients versus one of the controls (P=0.01). The markers usually associated with DR3 did not show significant differences between IDDM patients and controls, and the non-HLA markers studied showed no significant deviation from what was expected. In addition to the susceptibility factor DQw8, the study suggests the existence of susceptibility genes for IDDM near the complement C4 genes on DR4-carrying haplotypes. Since recent works have shown that the structural gene for the monokine tumor necrosis factor alpha (TNF-) is located between the HLA-B and C4 loci and that TNF- might be of importance in IDDM pathogenesis, the hypothesis is put forward that the C4-associated IDDM susceptibility reflects linkage dis-equilibrium between the C4 gene and a gene controlling TNF- production. The high relative risk for IDDM in HLA-DR3,4 heterozygotes might be explained by the combined action of IDDM-specific susceptibility genes on DR4 haplotypes and DR3-linked susceptibility genes associated with predisposition to autoimmunity.     

DNA sequences near a meiotic recombinational breakpoint within the human HLA-DQ region

The molecular organization of HLA-DQ regions derived from DR7, DQw2, and DR4, DQw3 parental haplotypes and DR7, DQw3, a presumed recombinant haplotype, have been studied to define the sequences between DQA1 and DQB1 which may have been involved in this recombinational event. The breakpoint was localized in the intergenic region near the 3 end of the DQB1 gene by restriction mapping. DNA sequences in the immediate vicinity of the breakpoint in DR7, DQw2 (parental), and DR7, DQw3 (recombinant) haplotypes revealed the presence of (CA)₂₂ repeats, minisatellite-related sequences and GC-rich sequences. The intergenic regions varied considerably depending on the haplotype and contained several additional types of repetitive sequences including Alu and LINE repeats. Some of these sequences are related to sequences previously suggested to be involved in meiotic or somatic recombination. In particular, (CA)_n repeats, which can adopt the Z-DNA conformation, have previously been shown to promote recombination in several systems. Address correspondence and offprint requests to: J. Strominger.