Polymorphisms in the TNF gene cluster and MHC serotypes in the West of Scotland

 We examined the distribution of polymorphic elements within the tumor necrosis factor (TNF) gene cluster in 105 unrelated individuals and determined their relationship to class I and class II major histocompatibility complex (MHC) antigens, and to the highly polymorphic microsatellites TNFa and TNFb. The data demonstrate the contribution of elements within the TNF cluster to two extended haplotypes which are recognized to straddle the MHC. The A1.B8.DR3 haplotype appears to contain the TNF alleles TNFa2, TNFb3, LT.Nco-1.B ^* 1, and TNF-308.2, while the A3.B7.DR2 haplotype is associated with the TNF alleles TNFa11, TNFb6, TNFc1, LT.Nco-1.B ^* 2, LT.AspH1.1, TNF-308.1, and TNFe1. The presence of other extended associations which covered smaller parts of the MHC was also suggested. In most cases, the associations described here were in keeping with previously described extended haplotypes which dominate the structure of the MHC, but these did not always match completely. Taken together, these data suggest that the structure of the TNF locus is well integrated into the rest of the MHC but that important ethnic differences may exist. Received: 12 June 1996 / Revised: 27 September 1996     

Cw * 1701 defines a divergent African HLA-C allelic lineage

 The complete sequence of a new HLA-C allele, Cw ^* 1701, was determined from a South African Zulu individual. Unique features that distinguish Cw ^* 1701 from other HLA-C alleles include multiple point substitutions and an 18 nucleotide insertion in exon 5, which encodes the transmembrane domain. In a phylogenetic analysis of HLA-C sequences, Cw ^* 1701 forms a third, distinct allelic lineage. A comparison of the transmembrane domain of Cw ^* 1701 with other HLA-B and -C alleles reveals that duplications and deletions have been common in the evolution of these loci. A polymerase chain reaction based typing method was used to determine the distribution of this unusual allele in human populations. In contrast to the other two lineages of HLA-C alleles, the Cw ^* 17 lineage is found at high frequencies only in populations of African descent. In addition, the HLA-B/Cw ^* 17 haplotype diversity is higher in Africa. Received: 29 June 1996  / Revised: 20 December 1996     

A phylogenetic analysis of cattle DRB3 alleles with a deletion of codon 65

 One of the most common cattle major histocompatibility complex DRB3 alleles, ^* 0201, includes a deletion of codon 65 encoding one residue in the α-helical chain. The mutation is functionally interesting and is likely to influence peptide binding. Exon 2 of two additional del65 alleles, ^* 3301 and ^* 4101, have now been sequenced with the aim to investigate the evolutionary relationship of this allelic group. Despite a fairly large genetic distance between the three alleles (11–17 nucleotide substitutions causing 8–11 amino acid substitutions) we found clear indications of a common ancestry. The α-helical region was very similar or identical among the alleles whereas the β-strand region was quite divergent. The results indicated that interallelic recombination has contributed to the diversification of the del65 group. Deletion of codon 65 has also been found in a roe deer DRB1 allele and a cattle DQB3 allele. Sequence comparisons of the cattle and roe deer DRB del65 alleles refuted the possibility of a trans-species persistence of a del65 allelic lineage but the two species may share a short ancestral sequence motif including del65. In addition to del65, the cattle DQB3 allele did not show any striking sequence similarities to the DRB alleles. Received: 20 March 1997 / Revised: 17 June 1997     

H2-M polymorphism in mice susceptible to collagen-induced arthritis involves the peptide binding groove

 The ability to develop type II collagen (CII)-induced arthritis (CIA) in mice is associated with the major histocompatibility I-A gene and with as yet poorly defined regulatory molecules of the major histocompatibility complex (MHC) class II antigen processing and presentation pathway. H2-M molecules are thought to be involved in the loading of antigenic peptides into the MHC class II binding cleft. We sequenced H2-Ma, H2-Mb1, and H2-Mb2 genes from CIA-susceptible and -resistant mouse strains and identified four different Ma and Mb2 alleles and three different Mb1 alleles defined by polymorphic residues within the predicted peptide binding groove. Most CIA-resistant mouse strains share common Ma, Mb1, and Mb2 alleles. In contrast, H2-M alleles designated Ma-III, Ma-IV, Mb1-III, and Mb2-IV could be exclusively identified in the CIA-susceptible H2 ^r and H2 ^q haplotypes, suggesting that allelic H2-M molecules may modulate the composition of different CII peptides loaded onto MHC class II molecules, presumably presenting “arthritogenic” epitopes to T lymphocytes. Received: 8 December 1995 / Revised: 16 January 1996     

HLA-G gene polymorphism in a Japanese population

 Polymorphism of the HLA-G gene in a Japanese population was investigated employing polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis, PCR sequence-specific oligonucleotide (SSO) analysis, and DNA direct sequencing. Nucleotide sequence variations in exons 2, 3, and 4 of the HLA-G gene in 54 healthy Japanese individuals were examined. In addition, seven Japanese samples carrying common HLA haplotypes were analyzed. In total, nine single-base substitutions compared with the sequence of G ^* 01011 were identified: one in intron 1 (nucleotide position 970), one in exon 2 (the third base of codon 57: G → A), three in intron 2 (1264, 1276, and 1292), three in exon 3 (the third base of codon 93: C → T, the third base of codon 107: A → T, and the first base of codon 110: C → A), and one in intron 3 (2334). The substitution at codon 110 was non-synonymous and led to an amino acid substitution from leucine to isoleucine. The other three nucleotide substitutions in exons were synonymous. Through analysis of combinations of the exon 2, 3, and 4 nucleotide sequences we identified four alleles, which we provisionally designated GJ1, GJ2, GJ3, and GJ4. The allele frequencies were estimated to be 0.33, 0.16, 0.45, and 0.06, respectively. Nucleotide sequences of GJ1, GJ2, and GJ4 were identical to G ^* 01011, the clone 7.0E, and G ^* 01013, respectively. GJ3 was a newly observed allele and was officially designated G ^* 0104 by the WHO Nomenclature Committee in January 1996. Strong positive associations were observed between HLA-G alleles and HLA-A, -B, or -DRB1 alleles. Received: 15 February 1996 / Revised: 26 March 1996     

Diversity associated with the second expressed H L A - D R B locus in the human population

Although diversity within the HLA-DRB region is predominantly focused in the DRB1 gene, the second expressed DRB loci, DRB3, DRB4, and DRB5, also exhibit variation. Within DRB1 ^* 15 or DRB1 ^* 16 haplotypes, four new variants were identified: 1) two new DRB5 alleles, DRB5 ^* 0104 and DRB5 ^* 0204, 2) a haplotype carrying a DRB1 ^* 15 or ^* 16 allele without the usual accompanying DRB5 allele, and 3) a haplotype carrying a DRB5^* 0101 allele without a DRB1 ^* 15 or ^* 16 allele. The evolutionary origins of these haplotypes were postulated based on their associations with the DRB6 pseudogene. Within HLA haplotypes which carry DRB3, a new DRB3 ^* 0205 allele and one unusual DRB3 association were identified. Finally, two new null DRB4 alleles are described: DRB4 ^* 0201N, which exhibits a deletion in the second exon, and a second allele, DRB4 ^* null, which lacks the second exon completely. Gene conversion-like events and variation in the number of functional genes through reciprocal recombination and inactivation contribute to the diversity observed in the second expressed HLA-DRB loci. Received: 2 November 1996 / Revised: 23 December 1996     

Unique peptide binding characteristics of the disease-associated DQ(α1*0501, β1*0201) vs the non-disease-associated DQ(α1*0201, β1*0202) molecule

 To understand the dominant association of celiac disease (CD) with the presence of HLA-DQ(α1^*0501, β1^*0201), the peptide binding characteristics of this molecule were compared with that of the structurally similar, but non-CD-associated DQ(α1^*0201, β1^*0202) molecule. First, naturally processed peptides were acid-extracted from immuno-affinity-purified DQ molecules of both types. Both molecules contained the Ii-derived CLIP sequence and a particular fragment of the major histocompatibility complex (MHC) class I α chain. Use of truncated analogues of these two peptides in cell-free peptide binding assays indicated that identical peptide frames are used for binding to the two DQ2 molecules. Detailed substitution analysis of the MHC class I peptide revealed identical side chain requirements for the anchor residues at p6 and p7. At p1, p4, and p9, however, polar substitutions (such as N, Q, G, S, and T) were less well tolerated in the case of the DQ(α1^*0201, β1^*0202) molecule. The most striking difference between the two DQ molecules is the presence of an additional anchor residue at p3 for the DQ(α1^*0201, β1^*0202) molecule, whereas this residue was found not to be specifically involved in binding of peptides to DQ(α1^*0501, β1^*0201). Similar results were obtained applying substitution analysis of the CLIP sequence. Molecular modelling of the DQ2 proteins complexed with the MHC class I and CLIP peptide corresponds well with the binding data. The results suggest that both CLIP and the MHC class I peptide bind DQ(α1^*0501, β1^*0201) and DQ(α1^*0201, β1^*0202) in a DR-like fashion, following highly similar binding criteria. This detailed characterization of unique peptide binding properties of the CD-associated DQ(α1^*0501, β1^*0201) molecule should be helpful in the identification of CD-inducing epitopes. Received: 21 March 1997 / Revised: 28 May 1997     

Extended HLA-DPB1 polymorphism: an RNA approach for HLA-DPB1 typing

Most of the 119 human leukocyte antigen (HLA)-DPB1 alleles are defined by polymorphism in six hypervariable regions (HVRs) in exon 2 of the HLA-DPB1 gene. We investigated how DPB1 polymorphism is represented in the entire coding region. An RNA sequencing-based typing (SBT) approach was developed for the identification of HLA-DPB1 polymorphism from the 5′ untranslated region (UTR) through the 3′-UTR. B-cell lymphoblastoid cell lines, encoding 16 different DPB1 alleles, were studied. Results show additional HLA-DPB1 polymorphism in exons 1, 3, 4 and 5 and the 5′ and 3′-UTR. Four new HLA-DPB1 alleles were identified, DPB1*0502, DPB1*0602, DPB1*0802 and DPB1*0902, which have exon 2 sequences identical to other DPB1 alleles but differ in the extended region. The additional polymorphism represents two main polymorphic lineages in the DPB1 alleles. Among the HVRs in exon 2, only HVR F correlates with these two main lineages.     

Sequence-based association analysis of HLA class I and II alleles in Japanese supports conservation of common haplotypes

 Alleles of HLA-A, B, C, DRB1, DQB1, and DPB1 loci were fully determined in 117 healthy Japanese. A ^* 2402, A ^* 3303, A ^* 1101, A ^* 0201, B ^* 4403, B ^* 5201, Cw ^* 0102, Cw ^* 1403, Cw ^* 0304, Cw ^* 0702, Cw ^* 0801, and Cw ^* 1202 showed frequencies of over 10%. Multi-locus haplotype frequencies were estimated by the maximum likelihood method. Strength of association between C and B loci was comparable with that between DRB1 and DQB1 loci. Alleles unidentified by a serological method and having very similar nucleotide sequences (A2: A ^* 0201, A ^* 0206, A ^* 0207, B61: B ^* 4002, B ^* 4006) were carried by different haplotypes. Several frequent five-locus haplotypes were identified including A ^* 3303-Cw ^* 1403-B ^* 4403-DRB1 ^* 1302-DQB1 ^* 0604, and A ^* 2402-Cw ^* 1202-B ^* 5201-DRB1 ^* 1502-DQB1 ^* 0601. These sequence-based haplotypes corresponded to serology-based common haplotypes which have already been described in Japanese. These findings indicate that common HLA haplotypes consist of particular sets of HLA alleles and that these haplotypes have been conserved through recent human evolution. Received: 25 November 1996 / Revised: 20 January 1997     

Haplotypic polymorphisms of the TNFB gene

The NTFB genes from two major histocomptibility complex (MHC) ancestral haplotypes have been compared. The genes carried by the ancestral haplotypes 8.1 (A1,B8,BfS,C4AQ0, C4B1,DR3) and 57.1 (A1,B57, BfS,C4A6,C4B1,DR7) were cloned and sequenced to determine the degree of polymorphism. In this report we show that the r e spective TNF genes are allelic and have unique nucleotide sequences. The data demonstrate the presence of three nucleotide differences between the TNFB alleles of 8.1 and 57.1. Two of the differences occur in untranslated regions of the gene but the third nucleotide change results in amino acid differences in the mature TNFB protein. These polymorphisms may have implications with respect to differential regulation in disease-and nondisease-associated haplotypes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M55913.     

Generation of the HLA-B35, -B5, -B16, and B15 groups of alleles studied by intron 1 and 2 sequence analysis

 HLA-B is the most polymorphic of the major histocompatibility complex classical class I loci. This polymorphism is mainly in exons 2 and 3, which code for the molecule’s α1 and α2 domains and include the antigenic peptide binding site. Recent studies have indicated that not only exons but also the intron 2 region may be involved in the generation of certain HLA-B alleles such as B ^* 3906 and B ^* 1522. To study the degree of intron 2 participation and the mechanisms that generate polymorphism at the HLA-B locus, intron 1 and 2 sequences from the HLA-B35, -B5, -B16 and -B15 groups of alleles were obtained. A group-specific intronic polymorphism was found: namely, B ^* 5301 shows intron 1 and 2 sequences identical to those found in all B35 alleles studied. On the other hand, B ^* 5101 and B ^* 52012 show the same intron 1 and 2 sequences and their intron 1 is the same as that found in the B35 group. This suggests that B5 and B35 groups of alleles may have arisen from a common ancestor. All known B16 alleles show the same introns 1 and 2, with the exception of B ^* 39061 and B ^* 39062, and all B15 alleles also bear the same introns 1 and 2, with the exception of B ^* 1522. Variability at intron 1 is more restricted than at intron 2, and the use of intron 1 for HLA-B allele phylogenetic analysis is better for grouping alleles of a postulated common origin. In conclusion, there is a remarkable conservation of intronic sequences within related HLA-B alleles, which probably reflects a common origin and perhaps a selective force avoiding DNA changes. Intronic sequences are also potentially useful to design DNA typing strategies. Received: 11 March 1997 / Revised: 29 May 1997     

Common chimpanzees have greater diversity than humans at two of the three highly polymorphic MHC class I genes

 MHC class I polymorphism improves the defense of vertebrate species against viruses and other intracellular pathogens. To see how polymorphism at the same class I genes can evolve in different species we compared the MHC-A, MHC-B, and MHC-C loci of common chimpanzees and humans. Diversity in 23 Patr-A, 32 Patr-B, and 18 Patr-C alleles obtained from study of 48 chimpanzees was compared to diversity in 66 HLA-A, 149 HLA-B, and 41 HLA-C alleles obtained from a study of over 1 million humans. At each locus, alleles group hierarchically into families and then lineages. No alleles or families are shared by the two species, commonality being seen only at the lineage level. The overall nucleotide sequence diversity of MHC class I is estimated to be greater for modern chimpanzees than humans. Considering the numbers of lineages, families, and alleles, Patr-B and Patr-C have greater diversity than the HLA-B and HLA-C, respectively. In contrast, Patr-A has less polymorphism than HLA-A, due to the absence of A2 lineage alleles. The results are consistent with ancestral humans having passed through a narrower population bottleneck than chimpanzees, and with pathogen-mediated selection having favored either preservation of A2 lineage alleles on the human line and/or their extinction on the chimpanzee line. Received: 8 December 1999 / Accepted: 30 December 1999     

Germline TCR-A restriction of immunoglobulin E responses to allergen

 Immunoglobulin E responses to known environmental antigens (allergens) may serve as a general model to investigate germline genetic restriction of the immune response. We have previously shown genetic linkage between IgE responses to major allergens and the T-cell receptor (TCR) A/D locus, but not to TCR-B, implying that elements in TCR A/D restrict the ability to react to specific antigens. We now show, in two sets of subjects from the same population, a strong allelic association between a VA8.1 polymorphism (VA8.1 ^* 2) and reactivity to Der p II, a major antigenic component of the house dust mite Dermatophagoides pteronyssinus. Association was also seen between Der p II IgE titres and HLA-DRB1 ^* 1501 alleles. Reactivity to Der p II was confined to subjects who were positive for VA8.1 ^* 2 and HLA-DRB1 ^* 1501, demonstrating germline HLA-DR and TCR-A interaction in restricting the response to exogenous antigen. Received: 28 January 1997 / Revised: 28 February 1997     

Structure of tumor necrosis factor-alpha haploblocks in European populations

DNA variants in the tumor necrosis factor-α (TNF) and linked lymphotoxin-α genes, and specific alleles of the highly polymorphic human leukocyte antigen B (HLA-B) gene have been implicated in a plethora of immune and infectious diseases. However, the tight linkage disequilibrium characterizing the central region of the human major histocompatibility complex (MHC) containing these gene loci has made difficult the unequivocal interpretation of genetic association data. To alleviate these difficulties and facilitate the design of more focused follow-up studies, we investigated the structure and distribution of HLA-B-specific MHC haplotypes reconstructed in a European population from unphased genotypes at a set of 25 single nucleotide polymorphism sites spanning a 66-kilobase long region across TNF. Consistent with the published data, we found limited genetic diversity across the so-called TNF block, with the emergence of seven common MHC haplotypes, termed TNF block super-haplotypes. We also found that the ancestral haplotype 8.1 shares a TNF block haplotype with HLA-B*4402. HLA-B*5701, a known protective allele in HIV-1 pathogenesis, occurred in a unique TNF block haplotype.     

An approach to mapping haplotype-specific recombination sites in human MHC class III

Studies of the major histocompatibility complex (MHC) in mouse indicate that the recombination sites are not randomly distributed and their occurrence is haplotype-dependent. No data concerning haplotype-specific recombination sites in human are available due to the low number of informative families. To investigate haplotype-specific recombination sites in human MHC, we here describe an approach based on identification of recombinant haplotypes derived from one conserved haplotype at the population level. The recombination sites were mapped by comparing polymorphic markers between the recombinant and assumed original haplotypes. We tested this approach on the extended haplotype HLA A3; B47; Bf ^* F; C4A ^* 1; C4B ^* Q0; DR7, which is most suitable for this analysis. First, it carries a number of rare markers, and second, the haplotype, albeit rare in the general population, is frequent in patients with 21-hydroxylase (21OH) defect. We observed recombinants derived from this haplotype in patients with 21OH defect. All these haplotypes had the centromeric part (from Bf to DR) identical to the original haplotype, but they differed in HLA A and B. We therefore assumed that they underwent recombinations in the segment that separates the Bf and HLA B genes. Polymorphic markers indicated that all break points mapped to two segments near the TNF locus. This approach makes possible the mapping of preferential recombination sites in different haplotypes.     

Trans-species origin of Mhc-DRB polymorphism in the chimpanzee

Trans-specific evolution of allelic polymorphism at the major histocompatibility complex loci has been demonstrated in a number of species. Estimating the substitution rates and the age of trans-specifically evolving alleles requires detailed information about the alleles in related species. We provide such information for the chimpanzee DRB genes. DNA fragments encompassing exon 2 were amplified in vitro from genomic DNA of ten chimpanzees. The nucleotide sequences were determined and their relationship to the human DRB alleles was evaluated. The alleles were classified according to their positioni in dendrograms and the presence of lineage-specific motifs. Twenty alleles were found at the expressed loci Patr-DRB1,-DRB3, -DRB4, -DRB5, and at the pseudogenes Patr-DRB6, -DRB7; of these, 13 are new alleles. Two other chimpanzee sequences were classified as members of a new lineage tentatively designated DRBX. Chimpanzee counterparts of HLA-DRB1 ^* 01 and ^* 04 were not detected. The number of alleles found at individual loci indicates asymmetrical distribution of polymorphism between humans and chimpanzees. Estimations of intra-lineage divergence times suggest that the lineages are more than 30 million year old. Predictions of major chimpanzee DRB haplotypes are made.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M94937-M94954.     

MHCDB: database of the human MHC (release 2)

 The second release of the human major histocompatibility complex (MHC) database is now publicly available. It contains an updated physical map and considerably more genomic sequence. cDNA sequences of all current alleles are accessible as individual sequence entries. The variability of different genes is displayed graphically as static and dynamic images accessible from the database. Known disease-serotype associations have also been incorporated, together with data from the MHCPEP database of eluted peptides. Received: 13 February 1996 / Revised: 12 August 1996     

Allelic variants of the human MHC class I chain-related B gene (MICB)

 The human major histocompatibility complex (MHC) is located within a 4 megabase segment on chromosome 6p21.3. Recently, a highly divergent MHC class I chain-related gene family, MIC was identified within the class I region. The MICA and MICB genes in this family have unique patterns of tissue expression. The MICA gene is highly polymorphic, with more than 20 alleles identified to date. To elucidate the extent of MICB allelic variations, we sequenced exons 2 (α1), 3 (α2), 4 (α3), and 5 (transmembrane) as well as introns 2 and 4 of this gene in 46 HLA homozygous B-cell lines. We report the identification of eleven alleles based on seven non-synonymous, two synonymous, and four intronic nucleotide variations. Interestingly, one allele has a nonsense mutation resulting in a premature termination codon in the α2 domain. Thus, MICB appears to have fewer alleles than MICA, not unlike the allelic ratio between the HLA-C and -B loci. A preliminary linkage analysis of the MICB alleles with those of the closely located MICA and HLA-B genes revealed no conspicuous linkage disequilibrium between them, implying the presence of a potential recombination hotspot between the MICB and MICA genes. Received: 16 April 1997 / Revised: 19 May 1997     

Identification of new Mamu-DRB alleles using DGGE and direct sequencing

 Rhesus macaques represent important animal models for biomedical research. The ability to identify macaque major histocompatibility complex (Mhc) alleles is crucial for fully understanding these models of autoimmune and infectious disease. Here we describe a rapid and unambiguous way to distinguish DRB alleles in the rhesus macaque using the polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE), and direct sequencing. The highly variable second exon of Mamu-DRB alleles was amplified using generic DRB primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and alleles were determined using fluorescent-based sequencing. Validity of this typing procedure was confirmed by identification of all DRB alleles for three macaques previously characterized by cloning and sequencing techniques. Importantly, our analysis revealed DRB alleles not previously identified in the three reference animals. Using this technique, we identified 40 alleles in fifteen unrelated macaques. On the basis of phylogenetic tree analyses, 14 new DRB alleles were assigned to 10 different Mhc-DRB lineages. Interestingly, two of the new DRB6 lineages had previously been identified in prosimians and pigtailed macaques. Whereas traditional DRB typing methods provide limited information, our new technique provides a simple and relatively rapid way of identifying DRB alleles for tissue typing, determining individual identification and studies of disease association and susceptibility. This new technique should also contribute to ongoing studies of Mhc function and evolution in many different species of nonhuman primates. Received: 29 May 1996 / Revised: 8 August 1996