Haplotypic variation of the transporter associated with antigen processing (TAP) genes and their extension of HLA class II region haplotypes

Stable cell surface presentation of HLA class I molecules requires active transport of antigenic peptides across the endoplasmic reticulum by products of two genes, TAP1 and TAP2, which map in the major histocompatibility complex class II region. Alleles of each gene are derived from a combination of variable sitesaat each locus. In this study, TAP1 and TAP2 alleles were identified in homozygous typing cell (HTC) lines, allowing resolution of specific haplotypes in conjunction with the highly polymorphic HLA class II region haplotypes. Three alleles at each TAP locus were found from which eight haplotypes could be assigned. Determination of TAP1 and TAP2 alleles in cell lines homozygous at DR, DQ, and DP created eight additional haplotypes beyond the number observed with these class II genes alone. Complete analysis of DR, DQ, TAP, and DP genotypes in 66 HTCs resulted in the following groups: 1) 46 homozygotes; 2) nine homozygous at DR, DQ, and TAP, but heterozygous at DP; 3) four homozygous at DR, DQ, and DP, but heterozygous at one or both TAP genes; 4) four homozygous at DR and DQ, but heterozygous at TAP and DP; and 5) three complex genotypes heterozygous at DP, TAP, and at least one of DQA1, DQB1, or DRB1 loci. TAP1 and TAP2 genes map in an area of frequent recombination. TAP alleles were determined in five DQB1, DPB1 recombinant individuals, three of which were informative. Recombination was found between DQB1 and the TAP loci in two individuals and between TAP and DPB1 in the other individual.     

Characterization of the MHC class II region in cattle. The number of DQ genes varies between haplotypes

The organization of the major histocompatibility complex (MHC) class II region in cattle was investigated by Southern blot analysis using human probes corresponding to DO, DP, DQ, and DR genes. Exon-specific probes were also employed to facilitate the assessment of the number of different bovine class II genes. The results indicated the presence of single DO and DR genes, at least three DR genes, while the number of DQ genes was found to vary between MHC haplotypes. Four DQ haplotypes, DQ ^{1 1} to DQ ^{2 4}, possessed a single DQ and a single DQ gene whereas both these genes were duplicated in eight other haplotypes, DQ ^{3 5} to DQ ^{9 12}. No firm evidence for the presence of bovine DP genes was obtained. The same human probes were also used to investigate the genetic polymorphism of bovine class II genes. DQ DQ , DR DR , and DO restriction fragment length polymorphisms (RFLPs) were resolved and in particular the DQ restriction fragment patterns were highly polymorphic. Comparison of the present result with the current knowledge of the class II region in other mammalian species suggested that the DO, DP, DQ, DR, and DZ subdivision of the class II region was established already in the ancestor of mammals. The DP genes appear to be the least conserved class II genes among mammalian species and may have been lost in cattle. The degree of polymorphism of different class II genes, as revealed by RFLP analyses, shows striking similarities between species.     

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.     

Genetic organization of the ovine MHC class II region

To study the class II genes of the major histocompatibility region of the sheep genome, human HLA class II genes corresponding to the known subregions in man (DR, DQ, DP, DO, and DZ) were used for Southern hybridization analysis of sheep DNA and to probe a sheep genomic library. Hybridizing bands were noted for all probes except DP. DQ and and DR appear to be present as multicopy genes, while DR-, DZ-, , and DO -like genes appear to be single copy. All bands detected with the DP probe were also detectable with other chain probes. From eight -bacteriophage clones of a sheep genomic library nine distinct class II genes were identified. These genes were characterized by differential hybridization analysis and restriction mapping. Two genes were DR -like, three DQ-like and four DQ -like. The extensive cross-hybridization observed with chain probes was not seen with chain probes. The results of this study suggest that the major histocompatibility complex class II region of the sheep has a similar genetic organization to that of man, with the provisional exception of the DP subregion.Abbreviation used in this paper OLA ovine major histocompatibility complex     

A study of the HLA-D region in patients with classic Kaposi's sarcoma

The HLA-D region in nine Sardinian patients with classic Kaposi's sarcoma was studied with two restriction enzymes, Eco RI and Eco RV, and two cDNA probes, DR and DQ. A total of 41 polymorphic restriction fragments were identified. One, an 11.5 kb Eco RV DQ fragment, was present in three of the patients but in none of the controls; a second, an 8.0 kb Eco RV DR fragment, was present in six patients and all the controls. No single fragment was identified which was significantly over or under-represented in either group.     

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.     

The major histocompatibility complex of tassel-eared squirrels

The complexity and polymorphism of sequences related to the class I and class II genes of mammalian major histocompatibility complexes (MHCs) were investigated in the tassel-eared squirrel subspecies Sciurus aberti kaibabensis or Kaibab squirrel. Kaibab squirrels are geographically isolated on the Kaibab plateau north of the Grand Canyon in Arizona. Genomic DNA from 22 individuals was digested with Eco RI and Barn HI, electrophoresed, blotted, and hybridized with a panel of human class I and class II probes. Sequences homologous to DR, DR , DQ, and DQ probes were observed. A single, nonpolymorphic DR-related sequence and multiple, polymorphic DQ-related sequences were observed. Hybridization with DR and DQ probes revealed multiple, polymorphic sequences with such specificity that no bands were observed to hybridize with both probes. The level of polymorphism of sequences exceeded that observed with sequences. Further, three Eco RI bands apparently included at least parts of both and sequences. Hybridization of genomic blots with the HLA-B7 class I probe revealed a number of bands comparable in size range and number to other mammalian species. However, only a minor percentage of bands were observed to segregate. The inheritance of these five families of sequences appeared to be neither concordant nor random in the sample population. Based on prior conclusions in other species, these class I and class II sequences are presumed to map to the Kabib MHC, TLSA. Although DQ- and DQ -related sequences were concordantly inherited, segregating sequences in the other families could not be assigned to identifiable, segregating haplotypes. These observations suggest that the present-day TSLA haplotypes have been derived from a limited number of progenitor haplotypes through repeated, intra-TSLA recombination.     

Sequence analysis of the grey mouse lemur (<Emphasis Type="Italic">Microcebus murinus</Emphasis>) <Emphasis Type="Italic">MHC class II DQ</Emphasis> and <Emphasis Type="Italic">DR</Emphasis> region

We here report the genomic organisation of the grey mouse lemur (Microcebus murinus) MHC class II DQ and DR region based on BAC clone analysis. The sequenced Mimu-MHC haplotype spans 343 kb and encompasses the genes TAP2, DOB, DQB, DQA, DRB, DRA, BTNL2 and a further BTNL gene. The DQ and DR genes of this haplotype are not duplicated. Mimu-DOB is not transcribed and represents a pseudogene due to deletions and premature stop codons. Analysis of BAC clone DNA, a cDNA sample and eight genomic DNA samples suggests that Mimu-DRB, Mimu-DQA and Mimu-DQB are highly polymorphic with the majority of peptide-binding residues being affected by polymorphisms. In contrast, Mimu-DRA is moderately polymorphic, and the variable amino acid positions are not part of the peptide-binding region. Phylogenetic analysis of Mimu-DQA and Mimu-DQB and other primate DQA and DQB genes indicates that duplication of DQA and DQB loci occurred in Anthropoidea after the split from Strepsirrhini.     

Southern blot analysis of HLA-DP gene polymorphisms in Caucasoid rheumatoid arthritis (RA) patients and controls

Despite extensive analysis of the incidence ofHLA-DR andHLA-DQ allele frequencies in defined autoimmune disease groups, there is very little information available onHLA-DP allele frequencies. This is largely becauseHLA-DP typing has until recently been restricted to primed lymphocyte typing (PLT). However, allelic polymorphism of theHLA-DP subregion can now be studied by Southern blot analysis or genotyping withDPA1 andDPB1 probes. By direct counting of allele-specific DNA fragments, we have analyzed the frequencies of five majorDP genotypes (DPw1, DPw2, DPw3/6, DPw4, andDPw5), in a large number of Caucasoid rheumatoid arthritis (RA) patients (n=74), and controls (n=91). The predicted frequency ofDP alleles in both patient and control groups was comparable to PLT-determinedDP allele frequencies in normal Caucasoids. However, the gene frequency ofDPw4 was increased in the RA patients, with 51% of the patients studied scoring asDPw4, 4 homozygotes. With the exception of one possible combination (DPw5 andDRw6) in the controls, no significant linkage disequilibrium was detected betweenDP andDR alleles in either patient or control groups. Thus the prevalence ofDPw4 in the RA patients is independent of any disease association with theDR loci, and may represent a new class II association with RA.     

Complement C4 and heat shock protein 70 (HSP70) genotypes and type I diabetes mellitus

Type I diabetes is strongly associated with the major histocompatibility complex (MHC) class II region (DR and DQ loci), and to a lesser extent the class III region (complement C4 loci). Restriction fragment length polymorphism analysis was employed to investigate the C4 and heat shock protein 70 (HSP70) loci of 176 patients with type I diabetes and 92 healthy controls. In the patient population there was an excess of deletions of the C4A locus (48.5% vs 22.1%, P < 0.0005). The HSP70 probe in conjunction with the restriction endonuclease Pst I detects two alleles of 9 or 8.5 kilobases (kb). The 8.5 kb allele was significantly increased in the patient group compared to healthy controls (0.569 vs 0.353, respectively, P < 0.0005). Furthermore, a C4A deletion nearly always occurred with the 8.5 kb HSP70 allele, suggesting that it may be a marker of the HLA-A1, B8, C4A deletion, DR3 extended haplotype.     

A SINE insertion provides information on the divergence of the HLA-DQA1 and HLA-DQA2 genes

The class II region of the human major histocompatibility complex (MHC) contains a cluster of highly polymorphic genes organized into at least three subloci (DR, DQ, and DP), each encoding a subset of surface antigens participating in the modulation of the immune response. Genetic diversity in this system is brought about by two major mechanisms, hypermutation and trans-species evolution. The DQ subregion contains a pair of closely related A genes, HLA-DQA1 and HLA-DQA2, whose phylogenetic relationship is uncertain, although their generation by duplication of an ancestral A gene before or after speciation can be implied. We report here the presence of a member of the Alu repetitive family immediately 5 to the HLA-DQA1 gene. The sequence of this element indicates that it may have integrated by transposition at the time of divergence of hominoids from Old World monkeys. HLA-DQA2 carries an empty integration target site in place of the Alu, thereby suggesting that the insertion of Alu near HLA-DQA1 was preceded by the separation of the two genes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M32372.     

Dissection of HLA class II haplotypes in HLA-DR4 homozygous individuals

In order to identify better markers for HLA-DR4-associated autoimmune disorders, we have studied the complexity of the HLA class II region in DR4-positive cells at the DNA level and compared the DNA polymorphism with that defined by serology, mixed lymphocyte culture (MLC) reactivity, and protein chemistry. At the DNA level, HLA-DR4 can be characterized by a homogeneous pattern of bands hybridizing to HLA class II cDNA probes. Besides, subtypes can be defined within DR4 using HLA-DR , -DQ , and -DQ cDNA probes in Southern blot analysis. Three subtypes are found using the DR cDNA probe. One of these subtypes correlates with the cellularly defined Dw15 specificity, another with the serologically defined LB4 and LB14 specificities. None of the restriction fragment length polymorphism (RFLP) patterns coincide with the MLC-defined DR4 subtypes Dw4, DW10, Dw13, and Dw14 separately. Variation of two fragments hybridizing to the DQ cDNA probe obtained after either Pvu II or Taq I digestion yields three subtypes. Pvu II- and Eco RI-digested DR4 DNA give rise to three DQ detectable subtypes. Correlation between these subtypes, isoelectric point variation of DQ molecules, and the DQ-related allelic system TA10/2B3 are demonstrated. Some of the patterns obtained with DQ and DQ cDNA probes display heterozygosity in the DQ region, as demonstrated by family segregation. No correlation was observed between DQ and the cellularly defined Dw determinants. A new polymorphism has been obtained with the DQ probe, probably due to DX polymorphism. DR RFLP divides the LB 14 supertypic specificity into two new subtypes. A combination of the four different techniques applied to a panel of 16 DR4 homozygous cell lines reveals at least nine different haplotypes in DR4. These newly defined haplotypes may be of help in further studies concerning the relationship of micropolymorphism with several diseases.     

HLA-D gene studies in relation to immune responsiveness to a grass allergen Lol p III

The grass pollen allergen Lol p III (M _r11 000) is a well-characterized antigen that has been found useful in immunogenetic studies of human immune responsiveness. Since immune responsiveness to this allergen is associated with HLA-DR3, we investigated whether there was any sequence in the HLA-D region that would render a person susceptible [antibody (Ab)-positive] to the allergen. By sequence-specific oligonucleotide (SSO) slot-blot and sequence analyses of polymerase chain reaction (PCR)-amplified genomic DNA from Lol p III responder and nonresponder subjects, Ab responsiveness was found to be strongly associated with the sequence Glu-Tyr-Ser-Thr-Ser (EYSTS), present in the first polymorphic regions of DRI polypeptide chains of DR3, DR11 (split of DR5), and DRw6. Of the 41 grass-allergic subjects investigated, 19 had the EYSTS sequence, of whom 18 (95%) were Lol p III immunoglobulin G (IgG) Ab responder; among the 22 EYSTS^- subjects, ten were Lol p III responders (P=0.0001, relative risk=21.6). No such association was found with any polymorphic sequences in othe DR chains,or in DQI and DQI chains. These findings suggest that the EYSTS sequence is important in the presentation of an epitope of Lol p III; other sequence(s) may be involved in the presentation of othe epitope(s). To our knowledge, this is the first demonstration of a strong association between a specific HLA sequence and immune responsiveness to a well-defined antigen. The paper shows that presence of the EYSTS sequence classifies subjects as Lol p III responders in 18/19 cases.     

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.     

First domain encoding sequence mediates human class II beta-chain gene cross-hybridization

HLA class II molecules are surface heterodimers which are essential in the initiation of immune responses. The amount of polymorphism expressed by the different class II molecules is largely dependent on the polymorphic structure of their beta chains. Cross-hybridization between, class II beta genes frequently hampered restriction fragment length polymorphism analysis of donor genomic DNA. In this report we show that the cross-hybridization between human class II beta genes is mediated by a region of high homology, rich in C and G residues, between the first domain encoding sequences of DP, DQ, and DR genes. The removal of the DNA segment containing this region from the fragments used as labeled probes against the corresponding fragments of the genes at other loci or against endonuclease digested genomic DNA completely eliminated or drastically reduced the cross-hybridization. Also, the RFLP patterns generated with the shortened probes were more informative and much simpler to interpret than were these generated with probes made from the original genes.     

Ancestral and recombinant 16-locus HLA haplotypes in the Hutterites

 Prior studies in the Schmiedeleut Hutterites of South Dakota have demonstrated associations between human leukocyte antigen (HLA) haplotype matching and fetal loss (Ober et al. 1992) and mate preferences (Ober et al. 1997), as well as deficiencies of homozygotes for HLA haplotypes (Kostyu et al. 1993). These studies were based on the serologically-defined five-locus HLA-A, -C, -B, -DR, -DQ haplotype. To further elucidate the effects of specific major histocompatibility (MHC) loci or regions on fetal loss and mate choice, we genotyped a sample of Hutterites for 14 MHC loci by DNA or biochemical methods. Typing for additional loci in the HLA-A to HLA-DPB1 region increased the number of recognized Hutterite MHC haplotypes to 67, and further localized the site of cross-over in 9 of 15 recombinant haplotypes. Hutterite MHC haplotype sequences are similar to those observed in outbred Caucasians, suggesting that the influence of HLA haplotypes on fetal loss and mating structure may be general. Received: 1 May 1998 / Revised: 2 December 1998     

Exon-specific oligonucleotide probes localize HLA-DQ β allelic polymorphisms

The HLA genetic region consists of a large multigene complex which includes a number of highly homologous and genes encoding class II polypeptides, clustered in three major loci, DP, DQ, and DR. Analysis of genomic polymorphisms at each of these loci is of considerable interest due to the role of particular structural polymorphisms in immune function, but this analysis has been hampered by difficulty in distinguishing between such highly homologous loci. We have identified locus-specific and exon-specific class II gene sequences in order to produce synthetic oligonucleotide probes which hybridize specifically to DQ genes. Two such oligonucleotide probes are described which are specific for the ₁ and ₂exons of DQ (DC) which identify DQ genes in digests of cellular DNA and which can be used to characterize restriction sites flanking the two oligonucleotide-specific regions. By sequentially hybridizing these probes in modified Southern analyses, we have been able to generate a tentative restriction map of a newly identified DQ allele from digests of total genomic DNA. This oligonucleotide mapping technique discriminates between two HLA-DQw3 ⁺ alleles, DQ3.1 and DQ3.2, permitting the recognition of structural polymorphisms with DQ which are highly associated with type I diabetes mellitus.     

Linkage relationships in the bovine MHC region. High recombination frequency between class II subregions

Class II genes of the bovine major histocompatibility complex (MHC) have been investigated by Southern blot analysis using human DNA probes. Previous studies revealed the presence of bovine DO , DQ , DQ , DR and DR genes, and restriction fragment length polymorphisms for each of these genes were documented. In the present study, the presence of three additional class II genes, designated DZ , DY , and DY , are reported. DZ was assumed to correspond to the human DZ gene while the other two were designated DY because their relationship to human class II genes could not be firmly established. The linkage relationships among bovine class II genes and two additional loci, TCP1B and C4, were investigated by family segregation analysis and analysis of linkage disequilibrium. The results clearly indicated that all these loci belong to the same linkage group. This linkage group is divided into two subregions separated by a fairly high recombination frequency. One region includes the C4, DQ , DQ , DR and DR loci and the other one is composed of the DO DY , DY , and TCPIB loci. No recombinant was observed within any of these subregions and there was a strong or fairly strong linkage disequilibrium between loci within groups. In contrast, as many as five recombinants among three different families were detected in the interval between these subregions giving a recombination frequency estimate of 0.17 ± 0.07. The fairly high recombination frequency observed between class 11 genes in cattle is strikingly different from the corresponding recombination estimates in man and mouse. The finding implies either a much larger molecular distance between some of the bovine class II genes or alternatively the presence of a recombinational hot spot in the bovine class II region.     

The location ofC2, C4, andBF relative toHLA-B andHLA-D

The loci forHLA-A, B, C, D, andDR are known to be closely linked to the structural loci for the complement components C2, BF, and the duplicated loci for C4, C4A and C4B. Conflicting evidence has been presented for the order of these genes. However, new techniques have made possible identification of markers in theHLA-D andC4 region for nearly all identified haplotypes. In our population we have confirmed fiveHLA-B-D crossovers and in each case informative allotypes of C2, BF, or C4A and C4B segregated withHLA-D orDR suggesting that the loci for these proteins lie close toHLA-D andDR. These findings may be of importance for resolving problems encountered in the assignment ofHLA-D alleles.     

An extended HLA-DQ-DR haplotype rather than DRB1 alone contributes to RA predisposition

 In the present study, we tested our hypothesis on the role of a DQ-DR haplotype in rheumatoid arthritis (RA) predisposition. Using two groups of patients and controls, one from The Netherlands and one from Switzerland, we found that DQA1*0301-homozygous and DQA1*0301//DQA1*0101/04-heterozygous individuals are highly predisposed to RA in both populations, while DQA1*0101/04-homozygous are not. The DQA1*0301-DRB1*0403/06/07 and DQA1*0301-DRB1*0901 haplotypes are not associated with RA by themselves but strongly increase the risk of developing disease in DQA1*0301- and DQA1*0101/04-heterozygous. DRB1 alleles carrying the motif DERAA in their third hypervariable region, i.e., *0103, *0402, *1102, *1103, *1301, and *1302, provide a long-lasting protection against RA in DQA1*0101/04- but not in DQA1*0301-positive individuals. These data show that considering both DQ and DR gives a better distinction between patients and controls than the shared epitope hypothesis. Received: 5 March 1998 / Revised: 21 April