HLA-Dw clusters associated with DR4 in Israeli Jews and the definition of a new DR4 associated Dw subtype: Dw"SHA"

A homozygous typing cell (HTC), that identifies a newly defined HLA-Dw determinant Dw"SHA," is described. The donor of the HTC was a Yeminite Jew and an offspring of first cousin marriage. This cell is not included in the known DR4-associated specificity clusters Dw4,Dw10,Dw13,Dw14,Dw15, or the provisional cluster Dw"KT2." Dw"SHA" was shown to segregate with DR4 positive haplotypes in family analysis, and in a population study was present in three of 43 unrelated DR4 positive individuals. This new Dw determinant was detected in Israeli Jews of Yemenite origin bearing the haplotype HLA-Bw41,DR4,DQw3. This indicates that Bw41,DR4,DQw3,Dw"SHA" may represent a typical allele combination in Yemenite Jews. Among 43 DR4 haplotypes in Israeli Jews, Dw10 had the highest antigen frequency (41.8%), whereas in American Caucasoids and in Japanese, Dw4 and Dw15 were most frequent, 44% and 40.5%, respectively.     

Is predisposition to pemphigus vulgaris in Jewish patients mediated by HLA-Dw10 and DR4?

Twenty-one Israeli Jewish pemphigus vulgaris (PV) patients were studied for the HLA-D lymphocyte defined determinants and the serologically defined antigens of the HLA-A, B, and DR series. HLA-D typing revealed that Dw10 is significantly associated with PV: 86% of patients vs 18% of controls carried Dw10, and DR4 was present in 86% of patients as compared to 38% in the controls. The most striking observation was that all Dw10 positive patients were also positive for DR4, and no other patient carried DR4 alone. The relative risk for a Dw10-DR4 carrier to develop PV was estimated at 31.9, higher than that observed for Dw10 alone (RR 26.7) or DR4 alone (RR 9.6). Probably HLA-Dw10 predisposes for pemphigus vulgaris.     

The HLA system in the Korean population

The frequencies of HLA-A, B, C, DR, and DQ antigens, HLA-D (HTC-defined) haplotypes, and the HLA-linked genetic markers glyoxalase I (GLO), factor B (Bf), C2 and C4 were studied in 162 healthy unrelated Koreans. Antigens A2, A24, A26, B44, B51, Bw62, B35, Cw1, Cw3, DR2, DR4, DRw6, DR7, and DRw8 were observed at frequencies of 15% or greater, and GLO-2, BfS, C4A*3, C2C, C4A*4, C4B*1, and C4B*2 were also frequently observed. The antigens A23, A25, B18, Bw42, Bw47, and B21 were not observed at all. HLA-DR4 was the most common class II antigen and was associated with a series of HLA-D-defined haplotypes including Dw4, Dw10, Dw13, and Dw15. The HLA-DRw6, DR2,Dw8, and DRw8 haplotypes were also found frequently. DR2 haplotypes were either Dw2 or Dw12, while all DRw8 haplotypes tested corresponded to the DB7 or Dw "8.3" specificity that has been described in other Oriental populations. Significant linkage disequilibrium was found between the alleles A2,Cw1; A30,B13; A30,Cw6; A30,DR7; Cw1,Bw22; Cw5,B12; Cw6,B13; Cw6,DR7; B7,DR1; B12,Dw6; B12,DR7; B12,Dw7; B13,DR7, B17,DR3; Bw22,C4B*6; DRw6,BfF; and C4A*4,C4B*2. A comparison of gene frequencies and commonly observed haplotypes between Koreans, Chinese, Japanese, and Caucasians showed that while Koreans share several characteristics in common with other Oriental populations, there are allelic frequencies and haplotypes in Koreans that are distinct.     

HLA-DP in rheumatoid arthritis

Frequencies of HLA-DR, Dw and DPw specificities were compared between rheumatoid arthritis (RA) patients, Felty's syndrome (FS) patients and normal controls. It was confirmed that the frequency of DR4 was increased in RA patients (54% (n = 111) vs 23% (n = 272), relative risk (RR) = 3.98, P less than 0.001). Cellular typing showed a highly significant increase in HLA-Dw14 in the entire RA population (17% (n = 32) vs 2% (n = 242), RR = 11.90, P less than 0.001), and a tendency towards an increase of HLA-Dw14 in DR4+ RA patients compared to DR4+ controls (28% (n = 32) vs 11% (n = 47), RR = 3.29, P less than 0.05). Regarding DPw specificities, the only significance was for a negative association with DPw3 (13% vs 22% (n = 254), RR = 0.51, P less than 0.05), with an additional tendential decrease of DPw1 (11% vs 19%, RR = 0.53, not significant (NS]. The decrease of DPw3 was more marked in DR4- RA patients (RR = 0.33, P less than 0.05) than in DR4+ RA patients (RR = 0.69, NS). In FS patients, 96% of whom were DR4+, decreased DPw1 was very marked, whereas the frequency of DPw3 was unaltered compared to DR4+ normals. These alterations in frequencies were not caused by linkage disequilibria between HLA-DR and -DP alleles. Thus, taken together, these data suggest that, in the presence of the major DR4-associated "susceptibility" gene(s) for RA, DPw1 may have "protective" effects, whereas in the absence of DR4, the presence of DPw3 has significant "protective" activity.     

Extended HLA haplotypes in families with insulin-dependent diabetes mellitus in Northern Finland

Haplotypes including HLA, Bf and C4 loci were analyzed in a material comprising 55 families with diabetic children. One hundred and ten haplotypes found in IDDM patients were compared with 101 haplotypes present only in healthy family members. Two complotypes, BfSC4A3B3 and SC4A0B1 . were significantly more common (P <0.05) in the diabetic haplotypes, and these were in most cases found in haplotypic combinations with HLA-B15,Dw4,DR4 and HLA-B8.Dw3,DR3 genes, respectively. The B8/DR3 haplotype was better conserved, as 72% included the BfSC4A0B1 complotype as compared with only 35% of the B15/DR4 haplotypes with "high risk" C4A3B3 complement alleles (p <0.05). DR3 was found in 26% of the diabetic haplotypes and DR4 in 43%. DR4 associated with the Dw4 in 69% of cases and with D w14 in 26% of the diabetic haplotypes. Our results confirm that the two phenotypes found earlier to be associated with IDDM in Northern Finland, e.g. "B15, BfS, C4A3B3, Dw4, DR4" and "B8, BfS, C4A0B1, Dw3, DR3" are inherited as haplotypes.     

Analysis of DR and DQ gene products of the DR4 haplotype in patients with IDDM: Possible involvement of more than one locus

Fifteen DR4-bearing haplotypes from twelve patients with insulin-dependent diabetes mellitus (IDDM) were analyzed serologically, cellularly, and biochemically. The HLA-Dw composition of these DR4-positive haplotypes was Dw4 (46%), Dw14 (22%), and Dw10 (33%). The biochemical analysis by two-dimensional electrophoresis (2D-PAGE) of the DR beta chains showed that each Dw specificity is characterized by a specific DR4 beta chain that appears to be identical in normal and diabetic individuals. Analysis of DQ beta chains in the DR4-bearing haplotypes revealed that certain Dw specificities such as Dw4 are characterized by the presence of either the DQw7 (formerly DQw3.1) or DQw8 (formerly DQw3.2) alleles, which generate the Dw4.1 or Dw4.2 subtypes, respectively. Others such as Dw14 and Dw10 are characterized by the presence of the DQw8 allele. In our sample of 12 patients the Dw4.2 (Dw4, DR4βI-4 DQw8) and Dw10 (Dw10, DR4βI-1, DQw8) subtypes were predominant. It is concluded that individual DR β and DQ β gene products from the DR4-bearing haplotype of IDDM patients are identical to those of normal control subjects and that Dw14 as well as Dw10 are involved in disease susceptibility. We suggest that disease susceptibility may be influenced by more than one locus within the HLA-D region.     

HLA-D clusters associated with DR2 and the definition of HLA-D"AZH": a new DR2 related HLA-D specificity in Israel

In order to investigate the HLA-D clusters associated with DR2 in Israeli Jews, 40 DR2 positive unrelated individuals were studied with a panel of DR2 associated homozygous typing cells (HTC's) which detect the lymphocyte defined specificities HLA-Dw2, Dw12, Dw9 and D-WJR. The results confirmed the existence of two distinct HLA-D clusters associated with the same serologically defined DR2. Of 40 individuals 22.5% (9/40) were Dw2 and 50% (20/40) were Dw12 carriers. Yet, no HLA-D specificity could be assigned to the remaining 11 DR2 positive individuals. In the present study we have defined a unique DR2-associated Dw specificity, HLA-D"AZH". The donor of the HTC was of Moroccan origin and an offspring of a first cousin marriage. This cell was not typeable with the known DR2-associated homozygous typing cells nor with other HTC's which define the well established HLA-Dw1 to Dw11 specificities. It was shown to segregate with DR2 positive HLA haplotypes in family analysis and in a population study, typed out 7 of 11 unrelated DR2 positive, Dw blank individuals, thus identifying a unique and new HLA-D cluster provisionally designated D"AZH".     

Determinants not correlated with HLA-Dw,DR, or SB detected by primed lymphocyte typing

We have identified a new HLA-Dw cluster, defined by five HTCs: 8W309 from the Eighth International Workshop, MN-LS and Bin-40 obtained locally. THO (Hansen), and RZoo (Hsu). Although highly associated with DR4, LD40 appears to be distinct from Dw4 and Dw10 {Hum Immunol 4:249. 1982}. PLT studies on LD40 were performed using intrafamilial PLT prepared in haploidentical combinations in which both stimulator and responder carried DR4 on the second haplotype and priming was only against LD40 or associated determinants. These reagents were apparently LD40-specific, as they were restimulated by ali DR4-LD40-positive cells with good discrimination from DR4-positive, LD40-negative cells.Whereas priming in a HLA-Dw-incompatible. DR-compatible combination produces PLT reagents with reactivities associated with the incompatible Dw specificity, further analysis of the D region is simplified if there is Dw and DR matching in the priming combination. A second type of reagent was generated using intrafamilial PLT prepared in a family in which two LD40 haplotypes were segregating: responder and stimulator shared one haplotype, and both carried DR4-LD40 on the second haplotype but associated with different A, B, and C antigens. This reagent appeared to recognize determinant(s) associated with several different haplotypes: among the subcultures derived from this reagent, several were found in which positive restimulation did not correlate with any particular A, B, C, DR, Dw, or SB/PL3/D^β type.These results suggest that the PLT test may detect (a) shared or cross-reactive antigenic determinants of HLA-Dw/DR as presently defined and/or (b) determinants distinct from Dw and DR. Although some of the latter, as detected by subcultures, appear to correlate with SB specificities, other show no correlation with presently defined Dw, DR, or SB antigens.     

Role of the mixed lymphocyte culture (MLC) reaction in marrow donor selection: Matching for transplants from related haploidentical donors

Abstract: The utility of the MLC assay as a test of HLA-D region matching and predictor of acute graft-versus-host disease (GvHD) was evaluated in 157 patients receiving marrow grafts from HLA-A, B identical related haploidentical donors. All donors and recipients were tested by HLA-DR serology, by Dw phenotyping with homozygous typing cells (HTC) and by standard MLC. Ninety-nine of the donor-recipient pairs were mismatched for a serologically defined HLA-DR antigen while 109 pairs were mismatched for the HLA-D region by HTC typing. Donor anti-recipient relative responses (RR) in MLC, corresponding to the GvHD vector in marrow transplantation, ranged from -4% to 100%, with a median of 25%. A comparison of reactivity in MLC with presence or absence of matching by Dw phenotyping, however, showed a significant overlap in the distribution of RRs from HLA-Dw matched versus Dw mismatched pairs, suggesting that the MLC was not a reliable predictor of HLA-Dw matching. Using an optimally-defined cutoff of 3% RR, the MLC was correlated with risk of developing clinically significant grades II–IV acute GvHD (p = 0.03) but not with risk of developing severe grades III-IV GvHD (p = 0.18). In contrast, matching by Dw phenotype was a significant predictor of GvHD, with Dw-compatible transplant recipients less likely to develop either grades II-IV (p = 0.004) or III-IV (p = 0.036) GvHD than Dw-incompatible transplant recipients. Overall, these results underscore the difficulty in using the MLC to measure HLA-D region compatibility and predict the risk of severe graft-versus-host disease among patients receiving related haploidentical marrow grafts. HLA-D (HTC) typing results correlate primarily with DRB compatibility, and with the advent of DRB1 allele matching by sequence-specific oligonucleotide probes (SSOP) or by direct sequencing, the precision in donor matching achievable with these methods is far greater than with either HLA-D typing or direct MLC testing.     

HLA antigens and complotypes in insulin-dependent diabetes mellitus

One hundred and thirty-six Finnish patients with insulin-dependent (type I) diabetes mellitus were investigated for the HLA-A, B, D and DR antigens as well as the Bf and C4 allotypes. The statistically significant increase in the frequencies of HLA-A9, B8, B15, Dw3, Dw4, DR3, DR4, C4A0 and C4B3 was observed when compared with the healthy controls. About 79% of the patients had HLA-DR4, and 53% had HLA-DR3 antigens. A rare C4 allele C4B3 was found in 21% of the patients, whereas only in 2% among the controls (relative risk 16.35). The etiological fraction (EF) values indicated that HLA D/DR alleles were the best markers for IDDM, the observed EF for HLA-DR4 in diabetes was as high as 0.70. Examination of HLA, Bf and C4 phenotypes suggested that at least two supratypes "B15 BfS C4A3B3 D(R)4" and "B8 BfS C4A0B1 D(R)3" were markers for the susceptibility to type I diabetes, one third of our patients had either of these supratypes. The protective role of DR2 and Dw2 antigens was also confirmed: no HLA-Dw2 positive patients and only one with HLA-DR2 was found.     

Increased frequency of HLA-DPw2 in pauciarticular onset juvenile chronic arthritis

Thirty-six unrelated Danish patients with pauciarticular Juvenile Chronic Arthritis (PJCA) and 120 controls were typed for HLA-DPw1-w6 and the local specificity CDPHEI with bulk-expanded Primed Lymphocyte Typing (PLT) cells. The frequency of HLA-DPw2 was 52.8% in PJCA patients and 16.7% in controls (relative risk, RR = 4.5; P less than 0.001). The antigens HLA-Dw5 and/or Dw8 were present in 50% of the patients and in 21.3% of the controls (RR = 4.2; p less than 10(-3)). DPw2 was not associated (in linkage disequilibrium) with Dw5/w8 in patients or in controls, and the DP and D associations with PJCA were independent of each other. However, the combined presence of DPw2 and Dw5 and/or Dw8 gave a significantly higher risk of PJCA than each antigen alone indicating interaction of DP and DR gene products. PJCA is the first disease definitely found to be associated with a DP antigen.     

HLA-A, B, C, DR antigens, Bf, C4 and glyoxalase I (GLO) polymorphisms in French Basques with insulin-dependent diabetes mellitus (IDDM)

The Basques were previously shown to present a high frequency of HLA—B18 and Bf Fl. which are known to be associated with insulin dependent diabetes mellitus (IDDM). During the VIII International Histocompatibility Workshop, we studied HLA-A, B, C, DR; Bf, C4 and GLO.I polymorphisms in 51 unrelated French Basque IDDM patients and in 50 controls. Haplotypes were established by family studies in all controls and some patients. Two haplotypes were frequently found in the controls: HLA- A1, Bw57, Bf S, C4 FIS, DR7 and HLA- Aw30, Cw5, B18, Bf Fl, C4 Fs°, DR3. The first one was not found in the patients. All the components of the second haplotype had increased frequencies possibly as a consequence of linkage disequilibrium with HLA— DR3 : a highly significant association between IDDM and HLA-DR3 was observed (90.2% vs 24.0%, relative risk (RR) = 29.1. P < 10⁻¹¹). The HLA-DR4 frequency was slightly increased (37.3% vs 16.0%). and HLA—DR2 was not found. The silent allele C4 s ° was particularly associated with early diagnosed IDDM (86.7% in patients with age at onset under 20 years vs 57.1% in other patients, P < 0.02). The high relative risk for HLA-DR3/DR4 heterozygous vs that of individuals, possibly HLA-DR3 homozygous, supported the hypothesis that two HLA-DR linked genetic factors could be involved in the inheritance of IDDM susceptibility.     

HLA class III haplotypes in multicase rheumatoid arthritis families

The class III complement proteins (C2, BF, C4A, and C4B) were studied in 57 multicase rheumatoid arthritis (RA) families. When the gene frequencies for RA probands were compared to a normal control panel (162 haplotypes), a significantly higher frequency of the rare variant C4B*3 was observed (p less than 0.05). No significant differences were seen for the other C2, BF, C4A, or C4B alleles. The most common haplotype found in the probands was HLA-Cw5,B44,C2*C,BF*S,C4A*3,C4B*3,DR4, occurring with a frequency of 0.088. Haplotypes containing HLA-DR4 and Bw62 were found to carry either C4A*3,C4B*3; C4A*3,C4B*1; or C4A*4,C4B*2. When only haplotypes containing DR4 were compared between probands and controls, the frequency of the C4B*3-bearing haplotype remained higher in the probands. It is concluded that Bw62,C4A*3,C4B*3DR4 is a haplotype which is especially associated with RA. The low frequency in the RA population of this haplotype indicates that C4B*3 has a minor role in overall RA susceptibility.     

The new HLA-DRw6- and 8- associated HLA-Dw HAG specificity defined by homozygous typing cell 9W 1802

Intrafamilial primary mixed lymphocyte culture (MLC) typing established that an HLA-A, B, C homozygous, DP heterozygous donor HAG was homozygous for HLA-Dw and behaved as a homozygous typing cell (HTC). Both haplotypes of the HTC were HLA-DR identical, but could not be assigned a clear DR specificity, giving reactions with sera containing antibodies against DRw6, DRw8 and TA10. MLC checkerboard studies failed to assign the HTC HAG specificity to any established or provisional cluster, suggesting that it defined a new Dw specificity. Primed lymphocyte typing (PLT) clones derived from intra-familial priming against either HAG haplotype displayed heterogeneous reactivity patterns. One clone was restimulated only by family members and unrelated donors positive for Dw HAG. Other clones were restimulated by determinants associated with either Dw8 or Dw6. Blocking of stimulation with monoclonal antibodies against different class II molecules suggested that while stimulatory determinants associated with Dw HAG and Dw8 were classifiable as HLA-D related, those associated with Dw6 were of a DP-like nature.     

HLA-D region heterogeneity in a Nigerian population

HLA class II antigens were studied in a panel of 130 Nigerians. Complex patterns of associations were seen between HLA-Dw, -DR and -DQ specificities, differing widely from those reported for other populations. A number of Dw types were associated with the same DR antigen: Dw'1N' and Dw'BERN' with DR1, Dw2 and Dw'2N' with DR2, Dw5 and Dw'5N' (Dw5 + Dw'F5') with DRw11. It was also observed that a Dw type associated with more than one DR antigen: HLA-Dw3 was assigned to individuals who were DR3 negative and similarly Dw10, Dw13 and Dw14 to individuals negative for DR4. HLA-DRw8 and Dw8 were completely dissociated in Nigerians, and Dw8 did not show a preferential DR association. These results demonstrate that DR and DQ identity between HTC stimulator and responder cell is not necessarily a prerequisite for Dw to be assigned. Preliminary studies show that subtypes of HLA-Dw1 and Dw8 detected by HTC typing correlate with restriction fragment length polymorphisms (RFLPs) detected with a combination of Bgl II enzyme and DRA/DRB cDNA probes. HLA-DP antigen frequencies differed between Nigerians and British Caucasoids. The most common DP antigen in Nigerians was DPw1, compared with DPw4 in Caucasoids. HLA-DPw6 appeared to be absent or rare in both Nigerians and British Caucasoids. Only five out of 68 Nigerians tested were assigned two DP specificities. The association between HLA-DR3 and DPw1 reported in Caucasoid panels was absent in Nigerians.     

Italian extended HLA haplotypes in Congenital Adrenal Hyperplasia

In order to complete the data on human 21-Hydroxylase deficiency, we present a study on HLA markers in 35 Italian families (14 from Northern, eight from Central and 13 from Southern Italy) with one affected child. Three children from the issue of first cousin marriages were homozygous for the whole HLA haplotype. Extended haplotypes shared by unrelated patients were not found, and a total absence of the HLA Bw47 allele among the haplotypes carrying the disease as well as normal haplotypes was observed. The absence of A1 Cw7 B8 BfS C4AQ0 C4B1 DR3 extended haplotype was instead confirmed. Allele frequencies in the different clinical forms were analyzed: BfSO7 allele frequency was significantly increased on haplotypes of the salt-wasting form (p less than 0.01). We noticed two duplications (C4B1-2) of C4B genes, on haplotypes involved in the disease. Allele distribution in the regions studied showed that Bw22 (w55), Cw3 and DR2 were characteristic of Northern patients, while B15 was found in patients from Central Italy.     

HLA-Dw14 and HLA-DR3 haplotypes share a functional determinant recognized by a human alloreactive T-cell clone

In the process of studying the fine specificity of HLA class II molecules, we identified an alloreactive T-cell clone raised to a HLA-Dw14 homozygous cell line that was specifically stimulated by Dw14+ homozygous typing cells but negatively with cells expressing the HLA-Dw4,-Dw10, -Dw13, and -Dw15 subspecificities of DR4. Of interest, this clone was also equivalently activated by stimulation with all DR3 cells and cell lines tested. Negative responses were obtained using a panel of 87 non-DR3 and non-Dw14 cells, including cell lines of the Tenth Histocompatibility Workshop. A monoclonal antibody inhibition study revealed the relevant stimulating determinant to be on HLA-DR molecules in both Dw14- and DR3-positive cells. A comparison of the DR beta 1-chain-inferred amino acid sequences suggests that formation of a topologically equivalent stimulating determinant would involve the participation of two noncontiguous regions of the third diversity region of DR beta 1. The putative recognition conformation detected by the clone is most probably specified by the presence of a valine at position 86 and a nonnegatively charged residue at positions 70, 71, and 74, since these are the only residues where DR3 and Dw14 are distinguishable from all other HLA-DR types. These findings illustrate that the functional ability of class II molecules is not necessarily either illustrated or predicted by serologic typing or by simple considerations of amino acid sequence.     

The HLA system in Inupiat and central Yupik Alaskan eskimos

We have studied HLA antigen profiles of the Inupiaq and Yupik-speaking peoples, two of the four Eskimo linguistic groups residing in Alaska. A relatively restricted polymorphism of HLA-A and -B locus antigens was noted. Only 35% of A locus specificities and 37% of B locus specificities tested for were detected in each population. The most common A locus alleles were A2, A24, and A28; the most common B locus alleles were B51(5), B27, B35, Bw60(40), Bw61(40), and Bw62(15). The antigens Cw3 (75 and 69%) and DR4 (81 and 67%) were found in high frequency in both groups. HLA-DR1, DR2, and DR7 were detected infrequently, while DR3 was not detected at all. DR4 was frequently associated in both Inupiats and Yupiks with Dw4, a specificity that was thought to occur only in Caucasian populations. A statistically significant difference between Inupiats and Yupiks was found for polymorphism at the A locus, but no significant differences were found for polymorphisms at the B, C, D, or DR loci. Analysis of HLA linkage disequilibrium revealed the presence of several novel haplotypes not previously described in other populations, suggesting that the selective factors responsible for positive associations observed in these Native Alaskans were probably distinct.     

Association of HLA-Bw65 with two major complotypes

The association between the HLA-B14 subtypes Bw64 and Bw65 and complement allotypes (C2, Bf and C4) was investigated in both population and family studies. Bf, C4A and C4B allotyping was performed on 37 Bw64 and 35 Bw65 positive unrelated Welsh/English subjects. Sixteen HLA-Bw65 bearing haplotypes were characterized for HLA-ABC, DR and DQ antigens and complement allotypes, including C2. The findings of the population study suggested that the complement haplotype associated with Bw64 is BfS, C4A2, C4B2. The population and family studies revealed two major complement haplotypes associated with HLA-Bw65: (i) C2C, BfF, C4A3, C4A1 - often associated with HLA-A3, Cw8 and DRw13, and (ii) C2C, BfS, C4A2, C4B2 - often associated with HLA-Aw33, Cw8 and DR1 or with A28, Cw8 and DRw13. The HLA-Bw65 bearing haplotypes of three families carried a C4B2B1 duplication of the C4B locus. In these families three C4B gene products were identified in the Bw65 positive members using an anti-C4B monoclonal antibody. It is suggested that most, if not all, HLA-Bw65 bearing haplotypes may possess a C4B locus duplication.     

Five HLA-D clusters associated with HLA-DR4

In order to investigate the HLA-D clusters associated with DR4, 54 DR4-positive, Dw4- and Dw10-negative responders, together with selected Dw4- or Dw10-positive responders, were tested with 22 HTCs that define DR4-associated D specificities. The results are consistent with previous data defining four distinct D clusters--Dw4, Dw10, DB3, and DYT--and have identified a new cluster provisionally termed LD40. In addition, the DB3 cluster is complex and appears to give typing response patterns overlapping those of the KT2 cluster originally defined as being associated with DR4 in Japanese populations. Of 116 DR4-positive haplotypes tested, 44% typed as Dw4, 18% were LD40, 16% were Dw10, 9% were DB3, 3% were DYT, and 10% gave no typing response to the HTCs defining any of these clusters. These studies are informative not only in defining the DR4-associated D clusters and in supporting the concept that D and DR cannot be considered identical but also in emphasizing the complexity of the D region.