Primate DRB genes from the DR3 and DR8 haplotypes contain ERV9 LTR elements at identical positions

The HLA-DRB genes of the human major histocompatibility complex constitute a multigene family with a varying number of DRB genes in different haplotypes. To gain further knowledge concerning the evolutionary relationship, the complete nucleotide sequence was determined for a region spanning introns 4 and 5 of the three DRB genes (DRB1*0301, DRB2 and DRB3*0101) from a DR52 haplotype and the single DRB gene (DRB1*08021) in the DR8 haplotype. These analyses identified an endogenous retroviral long terminal repeat element (ERV9 LTR3), inserted at identical positions in intron 5 of the functional DRB genes in these two haplotypes. Comparison of the nucleotide sequence from introns 4 and 5 including the ERV9 LTR elements revealed a strong similarity between the three expressed DRB genes. The DRB3*0101 and DRB1*08021 genes were most similar in this comparison. These findings provide further evidence for a separate duplication in a primordial DR52 haplotype followed by a gene contraction event in the DR8 haplotype. A homologous element was found in a chimpanzee DRB gene from a DR52 haplotype. This represents the first characterized ERV9 LTR element in a nonhuman species. The corresponding introns of the DRB genes in the DR4 haplotype contain no ERV9 LTRs. In contrast, these genes have insertions of distinct Alu repeats, implying distinct evolutionary histories of DR52 and DR53 haplotypes, respectively. Phylogenetic analyses of DRB introns from DR52, DR53, and DR8 haplotypes showed a close relationship between the DRB2 and DRB4 genes. Thus, the ancestral DR haplotype that evolved to generate the DR52 and DR53 haplotypes most likely shared a primordial common DRB gene.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X82660–X82663     

Ancestral haplotypes reveal the role of the central MHC in the immunogenetics of IDDM

The major histocompatibility complex (MHC) contains multiple and diverse genes which may be relevant to the induction adn regulation of autoimmune responses in insulin dependent diabetes mellitus (IDDM). In addition to HLA class I and II, the possible candidates include TNF, C4, and several other poorly defined polymorphic genes in the central MHC region. This study describes two approaches which take advantage of the fact that the relevant genes are carried by highly conserved ancestral haplotypes such as 8.1 (HLA-B8, TNFS, C4AQO, C4B1, DR3, DQ2). First, three diabetogenic haplotypes (two Caucasoid and one Mongoloid) have been compared and it has been shown that all three share a rare allele of BAT3 as well as sharing DR3, DQ2. In 43 sequential patients with IDDM the cross product ration for BAT3S was 4.8 (p<0.01) and 6.9 for HLA-B8 plus BAT3S (p<0.001). Second, partial or recombinant ancestral haplotypes with either HLA class I (HLA-B8) or II (HLA-DR3, DQ2) alleles were identified. Third, using haplotypic polymorphisms such as the one in BAT3, we have shown that all the patients carrying recombinants of the 8.1 ancestral haplotype share the central region adjacent to HLA-B. These findings suggest that both HLA and non-HLA genes are involved in conferring susceptibility to IDDM, and that the region between HLA-B and BAT3 contains some of the relevant genes. By contrast, similar approaches suggest that protective genes map to the HLA class II region.     

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.     

Polymorphic analysis of the three MHC-linked HSP70 genes

Three genes encoding members of the M _r 70 000 heat shock protein family (HSP70) are known to lie in the class III region of the human major histocompatibility complex. IN order to determine whether these genes or their protein products exhibit any polymorphism the three genes have been specifically amplified from genomic DNA and sequenced. The HSP70-1 and HSP70-2 genes encode the major heat-inducible HSP70. A comparison of the nucleotide sequences of these genes from B8, SC01, DR3, B18, F1C30, DR3, and B7, SC30, DR2 haplotypes has revelad only very limited sequence variation which is not associated with any amino acid polymorphism. The HSP70-Hom gene encodes a protein that is highly related to HSP70-1, but which is not heat-inducible. Nucleotide sequence analysis of this gene from different haplotypes has revealed a Met Thr amino acid substitution at residue 493 in a number of the haplotypes tested. This variable amino acid lies in the proposed peptide-binding site of the HSP70-Hom protein. Address correspondence and offprint requests to: R. D. Campbell.     

Susceptibility to IDDM Is marked by MHC supratypes rather than individual alleles

107 patients with insulin-dependent diabetes mellitus (IDDM) were typed for HLA A, B, C, and DR antigens, and for complement C4A, C4B, and Bf alleles, and the results were compared with those of a combined reference group of 332 appropriately matched healthy subjects. Supratypes (allelic combinations) were identified from the phenotype of each group, and it was shown that the frequency of several supratypes is increased in patients with IDDM, in particular supratypes (A1 Cw7) B8 C4AQ0 C4B1 BfS DR3 (P = 0.0001), (A30 Cw-) B18 C4A3 C4BQ0 BfF1 DR3 (P = 0.0003), (A2 Cw3) B62 C4AR C4B2.9 BfS DR4 (P = 0.0002), and three other supratypes including DR4. It was also shown that increases in the frequency of individual alleles are secondary to increases in supratype frequency. Moreover, supratypes appeared to interact; the presence of two relevant supratypes being particularly important. The absolute risk of IDDM was approximately 0.5 in subjects who were homozygous for B18 C4A3 C4BQ0 BfF1 DR3. We concluded that genetic susceptibility is best recognized by MHC supratypes rather than isolated alleles, and that supratype combinations make the identification of even greater disease risk possible.     

Structure and evolution of the promoter regions of the DQA genes

HLA-DQ antigens are unique among the class II antigens in that their chains are highly polymorphic. In the present study, we characterized the general structure of the promoter regions of the DQA genes derived from different DR haplotypes and defined their nucleotide sequence polymorphisms. The promoter of each DQA1 allele contains three sequence motifs which are not present in non-DQA related class II genes: one identical to a tumor necrosis factor (TNF) response element, one similar to an NFB binding element, and one similar to a W motif. All DQA alleles lack TATA and CCAAT boxes in the proximal promoter region but carry other sequence elements characteristic of MHC class II genes, including S, X, X2, and Y boxes, and a pyrimidine-rich tract upstream of the X box. Nucleotide sequence polymorphisms among the various DQA1 alleles were noted within the promoter region, with some of the differences mapping within, or close to, regulatory elements that are important for the expression of MHC class II genes. All DQA1 alleles carry an unrearranged, full length, Alu-Sx related repeat immediately upstream of the proximal promoter region. This repeat was not present in the DQA2 (DXA) genes analyzed, confirming that DQ locus duplication probably occurred before integration of the Alu repeat into the primordial DQA1 locus, some 31–43 million years (myr) ago. The DQA2 promoter region is highly conserved between DR4 and DR3 haplotypes, with the degree of conservation exceeding that expected from the neutral mutation rate.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence data base and have been assigned the accession numbers M97 454-M97 464. Correspondence to: E. Morzycka-Wroblewska.     

Class III gene rearrangements in Thai/Chinese supratypes containing null or defective C4 alleles

Class III gene rearrangements have been examined in Thai/Chinese individuals with supratypes bearing defective or null C4 alleles. Genomic DNA from C4 null supratypes was probed with an almost full-length 21-OH probe following digestion with Taq I and Kpn I. The HLA-B17 C4A3 BQO BfS DR3 Thai/Chinese supratypes (which may be associated with insulin-dependent diabetes mellitus in Orientals) lacks a 3.2 kb Taq I and a 3.9 kb Kpn I fragment hybridizing with the 21-OH probe. Similar gene rearrangements are found in Caucasoid diabetogenic supratypes HLA-B18 C4A3 BQO BfF1 DR3 and HLA-B8 C4AQ0 BI BfS DR3. Interethnic comparisons suggest that class II and class III interactions may be important in disease susceptibility. By contrast, neither of two Thai/Chinese supratypes with C4AQ0 appear to have major class III gene rearrangements; disease association studies will determine the significance of C4deficiency per se. As in Caucasoids, the electrophoretically fast C4 allele, C4A6, in Orientals has been shown to correlate with a 12 kb Bg1 II fragment hybridizing with a C4 probe. It is likely that the HLA-B17 C4A6 BI BfS DR7 supratype marks a highly conserved MHC chromosomal segment.     

Evolutionary relationship between human major histocompatibility complexHLA-DR haplotypes

 HLA-DR haplotypes of the human major histocompatibility complex are organized in five different groups. They can be identified based on the serological specificity expressed by the polymorphic DRB1 locus and by the presence of a characteristic set of DRB genes. The nucleotide sequences of introns 4 and 5 of the two DRB genes (DRB1 ^* 01 and DRB6 ^* 01) from a DR1 haplotype and the three DRB genes (DRB1 ^* 15, DRB6 ^* 15, and DRB5 ^* 15), from a DR51 haplotype were determined. This study identified endogenous retroviral long terminal repeat elements (ERV9 LTR) located at identical positions in intron 5 of the DRB1 genes in both the DR1 and DR51 haplotypes. Phylogenetic analyses revealed a close evolutionary relationship between these two haplotypes. The DRB5 gene, unique for the DR51 haplotype, may have been lost by a recent gene deletion event creating the DR1 haplotype. A model for the evolution of the human DR haplotypes involving separate duplication and contraction events is presented. Received: 10 October 1995 / Revised: 22 November 1995     

Family studies of IgA deficiency

Seventeen immunoglobulin A (IgA)-deficient subjects and other members from 13 families were examined at HLA-A, B and DR, C4A, C4B, and Bf loci. Of the 29 independent haplotypes in the IgA-deficient: subjects, 22 included deletions, duplications, or defects at the C4 or 21-hydroxylase loci. It is suggested that there may be a gene regulating serum IgA concentrations in this same region of chromosome 6. Three main supratypes explain most of the previously reported tHLA associations with IgA deficiency. These are A1, Cw7, B8, C4AQ0, C4B1, BfS, DR3, Bw65(14), C4A2, C4B1/2, BfS, and Bw57(17), C4A6, C4B1, BfS. All three are proposed to carry a gene for IgA deficiency, while other supratypes carrying the same B allele generally do not. Other supratypes possibly associated with IgA deficiency were also identified. A survey of about 150 individuals with at least 1 of the 3 main supratypes revealed only 2 IgA-deficient subjects, and these were among the 20 that had 2 of these supratypes. This suggests the possibility of a recessive mode of inheritance, with penetrance determined by another factor which is not major histocompatibility complex-linked. All the supratypes found in this group of IgA-deficient subjects would then carry the putative recessive allele for IgA deficiency.     

An approach to the localization of the susceptibility genes for generalized myasthenia gravis by mapping recombinant ancestral haplotypes

The association of HLA A1, B8, and DR3 with generalized myasthenia gravis (GMG) ini Caucasoids is well established, but no particular gene has been implicated and there is still no adequate explanation in functional terms. In study we have taken advantage of sequential genomic markers between B8 and DR3 so as to map the location of susceptibility gene(s) on the A1, B8, DR3 (8.1) ancestral haplotype. By studying 51 patients, we have delineated a region between HLA B and TNF which is shared by 29/29 patients with B8 and DR3, 19/19 patients with B8 but not DR3 and 2/3 patients with DR3 but not B8. The potential importance of this region was confirmed by examining a similar disease induced by D-Penicillamine (D-PenMG) and associated with different HLA class II alleles (DR1 and/or DR7). Among these patients, 7/16 (44%) have B8, often with other markers of 8.1. These results implicate at least two categories of genes in determining susceptibility to MG; one located in the region between HLA B and TNF may be immunoregulatory, whereas the second, located in the class II region, may relate to the inducing factor (e. g., DR1 or DR7 in D-PenMG).     

Associations of MHC ancestral haplotypes with resistance/susceptibility to AIDS disease development

We tested the association of MHC ancestral haplotypes with rapid or slow progression to AIDS by comparing their frequencies in the French genetics of resistance/susceptibility to immunodeficiency virus cohort with that reported in a control French population. Seven ancestral haplotypes were identified in the genetics of resistance/susceptibility to immunodeficiency virus cohort with a frequency >1%. The 8.1 (odds ratio (OR) = 3, p = 0.006), 35.1 (OR = 5.7, p = 0.001), and 44.2 (OR = 3.4, p = 0.007) ancestral haplotypes were associated with rapid progression, whereas the 35.2 (OR = 3.6, p = 0.001), 44.1 (OR = 5.4, p < 10(-4)), and 57.1 (OR = 5.8, p < 10(-4)) ancestral haplotypes were associated with slow progression to AIDS. Although the frequency of each ancestral haplotype is low in the population, the OR were quite higher than those previously obtained for single HLA allele associations, with some p values as low as 10(-4). The analysis of the recombinant fragments of these haplotypes allowed the identification of the MHC regions in the 35.1, 35.2, and 44.2 haplotypes associated with rapid progression to AIDS and the MHC regions of the 44.1 and 57.1 haplotypes associated with slow progression to AIDS. Previous studies have identified single HLA alleles associated with disease progression. Our results on recombinant fragments confirm the direct role of HLA-B35 in rapid progression. Associations with HLA-A29 and -B57 might be due to linkage disequilibrium with other causative genes within the MHC region.     

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.     

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.     

Order of class III genes relative to HLA genes determined by the haplotype method

The B18 C4A3 C4BQ0 BfF1 DR3 haplotype was found to be ideal for determining the order of C4 and Bf relative to HLA-B and DR by the haplotype method. All the copies of this haplotype are assumed to be derived from a single ancestral haplotype. Sixteen of the twenty-six BfFl-containing haplotypes carried all of the alleles from this ancestral haplotype. Most of the other BfFl-containing haplotypes could be derived from the ancestral haplotype by a single crossover event for one of the two possible gene orders. This suggests that B18 C4A3 C4BQ0 BfFl DR3 is the sole source of the BfFl allele. The uncommon C4 type on B18 C4A3 C4BQ0 BfFl DR3 facilitates recognition of the BfFl-containing products of recombination between Bf and C4. One such recombinant haplotype was found which shows that the orientation of the class III genes is as follows: C4 is closest to HLA-B and Bf is closest to HLA-DR. This gene order is supported by all the earlier unequivocal results obtained using the haplotype method (Olaisen et al. 1983, Marshall et al. 1984a). Combining these results with the information on class III genes obtained from overlapping cosmid clones (Carroll et al. 1984) and earlier mapping studies (Robson and Lamm 1984) shows that HLA-B is telomeric to 21B. C4B, 21A, C4A, Bf and C2 then follow 21B in that order covering 120 kb. HLA-DR is located further toward the centromere.     

Rearrangement of 21-hydroxylase genes in disease-associated MHC supratypes

Human cDNA probes for 21-hydroxylase (21-OH) and for complement component C4 are used on restriction digests of the members of several families with interesting supratypes. The presence of two Taq I fragments of 3.7 kb and 3.2 kb in size with a 21-OH probe is confirmed in most individuals who show no evidence of C4 deletions or 21-OH deficiency. Most individuals also show a doublet of weakly hybridizing bands at approximately 2.5 kb, the smaller of which is part of the 21A gene. The arrangement of the 21-OH genes on disease-associated supratypes was examined, and it is shown that copies of the same supratype from unrelated individuals are usually identical. Evidence is provided for deletions of 21A on the B8, C$AQ0 C4B1, BfS, DR3 and B18, C4A3, C4BQ0, BjF1, DR3 supratypes and a duplication of 21A on the B14, C4A2, C4B1/B2, BfS supratype. Gene rearrangements may be relevant to diseases such as juvenile onset diabetes mellitus.Publication Q8549 of the Departments of Clinical Immunology, Royal Perth Hospital and The Queen Elizabeth II Medical Centre, Perth, Western Australia     

Genetic heterogeneity of insulin-dependent (type I) diabetes mellitus: evidence from a study of extended haplotypes

Two hundred subjects with insulin-dependent (type I) diabetes mellitus (IDDM) were typed for HLA-B, HLA-DR, and properdin factor B (Bf). HLA and Bf antigen and haplotype frequencies in subjects were compared with control frequencies derived from the 8th HLA Workshop. Frequencies of extended haplotypes (defined by B-Bf-DR alleles on a chromosome) were also contrasted with control frequencies. Significant positive associations between IDDM and HLA-B8, DR3, DR4, BfS, and BfF1 were confirmed, as were significant negative associations between IDDM and HLA-B7, DR2, DR5, DR7, and BfF. One haplotype (B7-BfS-DR2) exhibited significant negative association, while five haplotypes (B8-BfS-DR3, B8-BfS-DR4, B15-BfS-DR4, B18-BfF1-DR3, and B40-BfS-DR4) exhibited significant positive associations with IDDM. In this sample, 64% of all probands carried at least one of the high-risk haplotypes. In conclusion, the occurrence of five "high-risk" haplotypes associated with IDDM provides evidence for previously undocumented genetic heterogeneity and suggests that possibly more than two HLA-region genes may be involved in IDDM susceptibility.     

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.     

Polymorphisms of Mitochondrial ATPASE 8/6 Genes and Association with Milk Production Traits in Holstein Cows

The maternal effect has been widely proposed to affect the production traits in domestic animals. However, the sequence polymorphisms of mitochondrial DNA (mtDNA) and association with milk production traits in Holstein cows have remained unclear. In this study, we investigated the single nucleotide polymorphisms (SNPs) of mtDNA ATPase 8/6 genes and association with four milk production traits of interest in 303 Holstein cows. A total of 18 SNPs were detected among the 842 bp fragment of ATPase 8/6 genes, which determined six haplotypes of B. taurus (H1-H4) and B. indicus (H5-H6). The mixed model analysis revealed that there was significant association between haplotype and 305-day milk yield (MY). The highest MY was observed in haplotype H4. However, we did not detect statistically significant differences among haplotypes for the traits of milk fat (MF), milk protein (MP), and somatic cell count (SC). The overall haplotype diversity and nucleotide diversity of ATPase 8/6 genes were 0.563 ± 0.030 and 0.00609 ± 0.00043, respectively. The results suggested that mitochondrial ATPase 8/6 genes could be potentially used as molecular marker to genetically improve milk production in Holstein cows.     

Two distinct subtypes of theHLA-DRw12 haplotypes in the Japanese population detected by nucleotide sequence analysis and oligonucleotide genotyping

We determined the DNA sequence of the enzymatically amplified second exon of theDRB1 gene of theDRw12 haplotypes derived from three Japanese donors and found two distinct subtypes of theDRw12 haplotype. The two subtypes, designatedDRw12a andDrw12b, had single-base substitutions that predicted one amino acid change at residue number 67. The sequence of theDrw12a andDRw12b subtypes differed from those of the otherDR haplotypes, but in the first hypervariable region of theDRB1 gene the sequences were identical to those of theDRw8(Dw8.1) andDRw8(Dw8.3) haplotypes. TheDRw12a andDRw12b subtypes were detected in a wide range of Japanse donors by genotyping with sequence-specific oligonucleotide probes synthesized according to the DNA sequence of the two subtypes. Results of this study demonstrated that theDRw12 haplotypes in the Japanese population are genetically diverse, as many otherDR haplotypes are. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M27509, M27510, M27511.