Identification and physical mapping of a polymorphic human T cell receptor V beta gene with a frequent null allele.

Germline variation in genes that encode the human T cell receptors (TCRs) may have an important influence in shaping the immune T cell repertoire. In this report we describe a frequent null allele of the human V beta 18 gene, resulting from a nucleotide substitution that creates a stop codon (CGA<-->TGA). Approximately 11% of the population tested was homozygous for this null allele, indicating that this is a frequent "hole in the repertoire." We confirmed that there is a greatly reduced (undetectable) level of V beta 18 mRNA in peripheral blood lymphocytes from an individual homozygous for this null allele. In addition, all heterozygous individuals expressed detectable levels of only the functional V beta 18 allele in their peripheral blood lymphocytes. Two other DNA polymorphisms were identified in V beta 18, one of which would result in an amino acid substitution in an expressed V beta 18 gene. Genotypes for all three of these V beta 18 DNA polymorphisms were determined in a group of unrelated individuals. Statistical analyses of the associations between alleles of the V beta 18 polymorphisms and those of other DNA polymorphisms in the TCR beta locus suggested a close physical proximity between the V beta 18 gene and the 3' end of the C beta 2 region. This localization of human V beta 18 had been previously predicted by the sequence homology between human V beta 18 and mouse V beta 14, a V gene segment previously mapped to 3' of the mouse C beta genes. We confirmed this localization of the human V beta 18 gene by isolating a cosmid clone that contains both the V beta 18 and C beta 2 segments. Mapping by restriction enzyme digestion and by the polymerase chain reaction indicated that the V beta 18 gene segment is approximately 9 kb 3' of the C beta 2 gene, making this the only known human V beta gene 3' of the C beta region.

The repertoire of T cell antigen receptor beta-chain variable regions associated with psoriasis vulgaris.

We investigated whether the pattern of T-cell receptors expressed by T cells in inflamed psoriatic skin differed substantially from the pattern seen in T cells from the peripheral blood. A bias or restriction in the repertoire of T-cell receptors found in the lesional skin of different patients might imply that specific subsets of T cells were causally associated with initiating or maintaining the lesions. By using a polymerase chain reaction-based assay of T-cell receptor beta-chain variable region mRNA, we found that the patterns of beta-chain mRNAs displayed in 14 samples of lesional skin or six samples of noninvolved skin were not significantly less diverse than the patterns found in matched peripheral blood samples. There was no evidence that the active lesions of multiple patients showed overexpression of T cells expressing one or a few T-cell receptor forms. The pattern of T-cell receptors displayed in clinically normal skin from normal control individuals showed about the same diversity as normal blood. While these results may not exclude either classical antigen or superantigen-based T-cell activation mechanisms in active plaques, the absence of a simple pattern of Vbeta usage in different patients suggests than other aspects of T-cell biology including trafficking, proliferation, co-stimulation, or responses to cytokines must also be considered.

Further localization of a multiple sclerosis susceptibility gene on chromosome 7q using a new T cell receptor beta-chain DNA polymorphism.

Multiple sclerosis (MS) has been associated with particular HLA haplotypes and has recently been reported to also be associated with the T cell receptor (TCR) beta-chain complex. We have tried to determine the source of the TCR-beta/MS association by exploiting the pattern of linkage disequilibrium within the TCR-beta complex. We describe a new DNA polymorphism with the TCR variable region gene segment V beta 15 which appears to localize between the constant region and V beta 11. When the distribution of V beta 11-V beta 15 haplotypes in MS patients was compared to healthy controls, the strength of the V beta 11-V beta 15 MS association (p = 0.107) was much less than the MS association with the adjacent V beta 8-V beta 11 haplotype (p = 0.0010). On the basis we exclude an MS susceptibility gene telomeric to V beta 11. The reported MS association with the TCR-beta gene complex therefore does not appear to be due to genes within the diversity, joining, or constant region but more likely involves a specific gene(s) within the variable region.

The germline repertoire of T cell receptor beta-chain genes in patients with chronic progressive multiple sclerosis.

The T cell receptor (TcR) beta-chain germline gene repertoire of multiple sclerosis (MS) patients was compared to that of 100 normal individuals. No differences in the number of gene segments defined by probes representing 14 different human V beta subfamilies and the constant region genes were found. The distribution of haplotypes defined by restriction fragment length polymorphism (RFLP) alleles detected with V beta 8, V beta 11, and C beta probes in the MS patients was significantly different from that found in normal individuals. Because 84% of the MS patients were DR2+, the findings in these patients were compared to a second group of 43 normals who were DR2+. The distribution of TcR haplotypes in MS patients was also significantly different from that in the DR2+ normals. The data suggest that an MS susceptibility gene(s) may be located in the region of the TcR beta-chain gene complex.

T-cell receptor polymorphisms in Tlingit Indians with rheumatoid arthritis.

Rheumatoid arthritis (RA) develops as a result of the interaction of both genetic and environmental factors. Among the genes in humans that have been suggested as candidate susceptibility genes in RA are those encoding the T cell receptor for antigen (TCR). A high prevalence and early age of onset of RA has previously been reported in Alaskan Tlingit Indians. In this study, the frequency of seven different restriction fragment length polymorphisms (RFLPs) in the TCR alpha and beta gene complexes were measured in a population of Alaskan Tlingit Indians. No statistically significant differences were noted when the frequencies of these RFLPs were compared between Tlingits with RA and healthy controls (p > 0.05). These results do not support the hypothesis of an RA-susceptibility allele in the vicinity of these TCR alpha or beta genes. Since TCR RFLPs have not been extensively studied in native American populations, TCR polymorphism frequencies in the Tlingits were also compared to the frequencies observed in a second control group of healthy Caucasians. Statistically significant differences were observed in these comparisons implying a different distribution of individuals in these populations with different TCR repertoires.

Search for the psoriasis susceptibility gene: the Newfoundland Study.

The authors review early pioneering research on the genetics of psoriasis and recently published independent and collaborative investigations searching for the psoriasis susceptibility genes. We describe the research design and current plans for a joint pursuit between the Psoriasis Research Institute, the Memorial University of Newfoundland, and Chiroscience R&D, Inc., for susceptibility genes. A unique study sample from Newfoundland, drawn from affected and unaffected members of multiplex families and relatives, provides a nearly homogeneous isolated population. Families reflect English, Scottish, and Irish ancestry, and have been in residence in Newfoundland for over 300 years. The prevalence of psoriasis is estimated to be 2 to 3%. Familial psoriasis occurs in over 85% of families, with at least one affected member. The experimental strategy using linkage analysis and linkage disequilibrium analysis of the collected tissue bank are discussed, emphasizing prospects for the future outcome of the research findings.

Polymorphism and phylogeny of dinucleotide repeats in human T-cell receptor Vb6 genes.

The Vb6 subfamily is the largest reported subfamily of human T-cell receptor (Tcr) genes, with as many as 14 possible members based on variation in reported DNA sequences. A study of the genomic organization of four distinct Vb6 genes indicated that they contained within their introns the uninterrupted dinucleotide repeat (GT)n, with n > 8. DNA amplification primers and conditions were determined which amplified the intron of these four different Vb6 gene segments. All four Vb6 genes tested showed length polymorphism when examined in a group of unrelated individuals. Careful sizing and DNA sequencing showed that the alleles of each gene differed in size by multiples of two base pairs (bp), due to different repeat numbers of the dinucleotide (GT)n. These four microsatellite polymorphisms had from three to ten alleles, and individual heterozygosities of 26% to 83%. The large number of alleles and the high heterozygosity make these polymerase chain reaction (PCR)-based polymorphisms very attractive genetic markers for segregation studies which postulate the presence of autoimmune susceptibility genes within the Tcrb region. Vb6 hybridization to genomic DNA confirmed the relatively large size of the Vb6 subfamily in several hominoid species. Nucleotide sequencing of an intron of the Vb6 genes from other primates revealed the presence of dinucleotide repeats similar to those found in human Vb6 genes. Thus, the (GT)n microsatellite was not only present in the Vb6 intron before Vb6 gene duplication, but was present before speciation of the hominoids.

PCR-based genotyping and haplotype analysis of human TCRBV gene segment polymorphisms.

There are at least 63 tandemly arranged human T-cell receptor (Tcr) beta-chain variable region (BV) gene segments, which have presumably arisen by repeated gene duplication events. The 5'-most half of the TCRBV gene loci is particularly complex in organization due to the presence of multiple interspersed members of the largest BV subfamilies, BV5, BV6, and BV13. Polymorphism and linkage relationships among these genes has been poorly characterized in part due to the high similarity of these duplicands. Germline DNA polymorphisms were specifically examined in the exons and introns of these and other BV gene segments distributed across 240 kilobases (kb) in this 5'-most region. Polymerase chain reaction restriction enzyme-based assays were used to genotype ten point mutations in seven of the BV gene segments. Eight of these polymorphisms altered an amino acid of the BV gene segment. In addition, length polymorphisms due to simple sequence repeats were noted in the introns of six BV6 subfamily members. Approximately 250 unrelated haplotypes were constructed by segregation analyses of fifteen of these TCRBV polymorphisms. Linkage disequilibrium analyses indicated that haplotypic relationships are not detectable over a distance of more than 55 kb in this genomic region. These TCRBV polymorphisms, and the haplotypic analysis, provide important resources and guidance for future attempts to associate Tcr germline DNA differences in the human population with immune response differences, such as might occur in some autoimmune diseases.

Polymorphism detection and sequence analysis of human T-cell receptor V alpha-chain-encoding gene segments.

The T-cell receptor (Tcr) provides specificity for antigen recognition by its variable domain, primarily consisting of two germline encoded variable (V) region gene segments. Thus it has been suggested that inherited polymorphisms in the TCRV gene segments could contribute to differential immune responsiveness (e.g., autoimmunity) in human populations. In the present study, we have sought potentially functional polymorphisms in the germline TCRAV gene segments. Using denaturing gradient gel electrophoresis on polymerase chain reaction (PCR)-amplified products from the pooled DNA of many individuals, we identified polymorphisms in the TCRAV2S1, AV4S1, AV7S1, and AV8S1 gene segments. A complete DNA sequence analysis of these PCR products identified polymorphisms that affected amino acids in the predicted antigen-binding regions of the Tcr alpha chain, as well as polymorphisms in the introns. Genotype analysis of all nine DNA point mutations showed a 5%-50% range (averaging 35%) of minor allele frequencies, often resulting in individuals homozygous for the alternate allele forms. All possible haplotype combinations of the amino acid-affecting polymorphisms were found, indicating that in human populations there are a large number of different germline haplotypes encoding V gene segment alleles. These TCRAV coding region polymorphisms provide the rationale for, and allow the direct testing of, hypotheses concerning inherited polymorphisms within the T-cell receptor genes that may contribute to autoimmune susceptibility.

Organization, polymorphism, and expression of the human T-cell receptor AV1 subfamily.

At least 32 mostly single-member subfamilies of T-cell receptor alpha variable (TCRAV) genes have been described in humans. The AV1 subfamily is the largest, estimated by hybridization to contain as many as five members. However, a search of nucleotide sequence databases reveals a much greater number of unique sequences corresponding to this subfamily. In order to resolve this discrepancy between hybridization and nucleotide sequencing data, and to better understand the nature of variability among variable genes within a large subfamily, a genomic characterization of the AV1 subfamily in humans was carried out. Total genomic DNA, as well as isolated genomic clones spanning the TCRA region were screened for members of the AV1 subfamily by polymerase chain reaction (PCR) and nucleotide sequencing as well as by hybridization. A total of eight AV1 genes were identified and their nucleotide sequences were determined. Three of the sequences represent new genes. Based on structural features and the results of PCR screening of cDNA, none of these new genes appear to be functional. Several additional previously reported AV1 sequences were determined to represent alleles of AV1 genes, and simple PCR restriction digest assays were established for their detection. Use of each of the identified AV1 genes as hybridization probes failed to reveal any additional hybridizing bands. Thus the AV1genes represent the largest TCRAV subfamily with a maximum of eight members, several of which have common allelic forms.

Genetic linkage analysis and homology relationships of genes located on human chromosome 11q.

We have used DNA polymorphisms detected by probes for 11q to order 16 genes and to determine the genetic distances between them. Our map includes the genes for CD20, tyrosinase, progesterone receptor, stromelysin, collagenase, N-CAM, dopamine-D2 receptor, apolipoproteins AI-CIII-AIV, CD3-epsilon, -delta, and -gamma, porphobilinogen deaminase, thy-1, and ets-1. These genes have previously been sequenced as well as placed on the 11q cytogenetic map, which now makes them anchor points between the cytogenetic, genetic, and physical maps of this region. The ordering and distances between these genes are of immediate use in testing hypotheses of candidate genes for human genetic diseases associated with chromosome 11q. A comparison between our genetic map and similar maps from other species defines regions of homologous synteny that may be useful in mapping human genetic disease genes localized to the 11q region. Analysis of such homology provides additional bases for speculation of the evolutionary histories of gene families in this region.

Human T-cell receptor V beta gene polymorphism and multiple sclerosis.

Population-based genetic associations have been reported between RFLPs detected with probes corresponding to the genes encoding the beta chain of the T-cell receptor for antigen (TCRB) and a variety of autoimmune disorders. In the case of multiple sclerosis (MS), these studies have localized a putative disease-associated gene to a region of approximately 110 kb in length, located within the TCRB locus. In the current study, all 14 known TCRBV (variable region) genes within the region of localization were mapped and identified. The nucleotide sequences of these genes were determined in a panel of six MS patients and six healthy controls, who were human-leukocyte antigen and TCRB-RFLP haplotype matched. Nine of the 14 TCRBV genes studied showed evidence of polymorphism. PCR-based assays for each of these polymorphic genes were developed, and allele and genotype frequencies were determined in a panel of DNA samples from 48 MS patients and 60 control individuals. No significant differences in allele, genotype, or phenotype frequencies were observed between the MS patients and controls for any of the 14 TCRBV-gene polymorphisms studied. In light of the extensive linkage disequilibrium across the region studied, the saturating numbers of polymorphisms examined, and the direct sequence analysis of all BV genes in the region, these results suggest that it is unlikely that germ-line polymorphism in the TCRBV locus makes a major contribution to MS susceptibility.(ABSTRACT TRUNCATED AT 250 WORDS)

T-cell receptor beta-chain DNA polymorphism frequencies in healthy HLA-DR homozygotes.

In view of numerous recent reports of T-cell receptor (TCR) beta-chain/disease associations with HLA-associated diseases, we tested the possibilities that associations might exist directly between these two gene complexes at the level of the germline DNA. We determined frequencies of five TCR-beta DNA polymorphisms in 33 HLA-DR2/2 homozygotes, 29 HLA-DR3/3 homozygotes and 42 HLA-DR4/4 homozygotes. The control population (n = 74) was chosen without "bias toward" their HLA-DR genes. We selected DR2, DR3 and DR4 homozygotes because they have been the most frequently involved in HLA-DR associated diseases. Our results indicate that the recent reports in the literature of TCR-beta/disease associations can not be explained by a significantly different distribution of TCR-beta genes in HLA-DR2+, -DR3+, or -DR4+ subpopulations. Our results also suggest that if co-evolution between TCR-beta and MHC haplotypes does exist, the selective pressures in recent generations have not been strong enough to significantly alter the germline TCR-beta gene frequencies in HLA-DR2+, -DR3+, or -DR4+ subpopulations.

Frequency and polymorphism of simple sequence repeats in a contiguous 685-kb DNA sequence containing the human T-cell receptor beta-chain gene complex.

The human T-cell receptor beta-chain (TCRB) gene complex spans 575 kb in chromosome region 7q35 and has been the subject of a large-scale DNA sequencing effort. A contiguous 685-kb DNA sequence from this region was searched by computer analysis for the occurrence of simple sequence repeats (microsatellites) with core sequence lengths of 2-5 nucleotides. Twenty-nine such microsatellites of repeat number n > or = 9 were found, with the majority being dinucleotide repeats. By PCR analysis, 19 were found to be polymorphic in repeat number, thus averaging one per 36 kb. These polymorphic di-, tri-, and tetranucleotide repeats had between 3 and 15 differently sized alleles each. The potential usefulness of these TCRB microsatellites for detecting disease susceptibility alleles was examined by measuring the linkage disequilibrium between these markers and flanking biallelic mutations. All but 4 microsatellites (79%) demonstrated significant linkage disequilibrium (P < 0.0001). This present study highlights the utility and potential outcomes of large-scale DNA sequencing for the identification of polymorphic simple sequence repeats.

HLA and T cell receptor beta-chain DNA polymorphisms identify a distinct subset of patients with pauciarticular-onset juvenile rheumatoid arthritis.

OBJECTIVE: To evaluate and extend upon a reported association of a T cell receptor (TCR) V beta coding region polymorphism with pauciarticular-onset juvenile rheumatoid arthritis (JRA). METHODS: TCR V beta 6.1 genotypes and haplotypes in JRA and control groups were determined by DNA amplification. RESULTS: Haplotypes of the V beta 6.1 gene which encode a nonfunctional form of V beta 6.1 were significantly associated with pauciarticular JRA in patients possessing the HLA-DQA1*0101 allele (P = 0.0073). CONCLUSION: A TCR V beta gene segment in the vicinity of V beta 6.1, possibly V beta 6.1, is apparently involved in the pathogenesis of pauciarticular-onset JRA in DQA1*0101-positive individuals.

Influence of coding region polymorphism on the peripheral expression of a human TCR V beta gene.

A number of human TCR V beta gene segments are reported to be polymorphic, with alleles differing by one or a small number of amino acid substitutions. In the absence of detailed structural information regarding the interaction of specific positions in the TCR with Ag or MHC, the significance of such variation is difficult to assess. In this report the relative use of the two common alleles of the human V beta 6.7 gene, 6.7a and 6.7b, which differ by two non-conservative amino acid substitutions, and the use of two common alleles of the V beta 12.2 gene, which differ by only silent substitutions, were measured in PBL derived from individuals heterozygous for these alleles. Equal use of V beta 12.2 alleles was observed, consistent with the inability of selection mechanisms to discriminate between the products of these alleles that are indistinguishable at the amino acid level. However, statistically significant skewing in the use of V beta 6.7 alleles was observed in 15 of 16 individuals studied. Expression levels for each allele ranged from 16 to 84% of the total V beta 6.7 signal in heterozygous individuals, with either the 6.7a or the 6.7b allele predominant in different individuals. Based on segregation studies in families, it seems unlikely that other unidentified polymorphism in the TCR beta locus, such as in the V beta 6.7 promoter, was responsible for the differential allele expression. Family studies provided no evidence for an association between specific HLA haplotypes and V beta 6.7 allele use. These results indicate that even modest allelic variation in human TCR V beta coding regions can have a significant impact on the expression of human V beta genes in the peripheral repertoire.

The extent of the human germline T-cell receptor V beta gene segment repertoire.

An assessment of the size of the human TCRBV gene segment repertoire based on the identification of TCRBV gene segments in genomic DNA was undertaken. PCR amplification from cloned and uncloned genomic DNA sources, nucleotide sequencing, Southern blot hybridization, and cosmid cloning were used to identify TCRBV gene segments in multiple unrelated individuals. The key advantages to this approach were: 1) TCRBV gene segments which are expressed only at very low levels in cDNA libraries were still detectable, and 2) it was possible to discriminate between alleles at the same locus vs products of different loci. A total of 63 unique TCRBV gene segments were identified and sequenced. Six of these TCRBV gene segments had not been previously described. Thirty-four cosmid clones containing 51 of the 63 identified TCRBV gene segments were isolated and screened for the presence of additional novel TCRBV subfamily members. These results, obtained by a variety of complementary approaches, indicate that the human TCRBV germline repertoire encodes at least 63 TCRBV gene segments of which 52 are functional. The availability of the majority of these TCRBV gene segments on cosmid clones should facilitate further investigation of germline TCRBV gene segment polymorphism and putative disease associations.

Differences in T cell receptor restriction fragment length polymorphisms in patients with rheumatoid arthritis.

OBJECTIVE: The purpose of this study was to determine whether a T cell receptor (TCR) polymorphism, either by itself or in combination with particular HLA polymorphism, leads to susceptibility to rheumatoid arthritis (RA). METHODS: Eight restriction fragment length polymorphisms (RFLPs) detected with TCR gene segments were investigated in 46 individuals with RA and were compared with data from normal control subjects. RESULTS: A statistically significant difference in the genotype frequencies of a Taq I RFLP detected with the TCR alpha constant region (C alpha) gene was noted. In addition, when the DR4+ subpopulations were examined, the allelic frequency of a 2-kb Bam HI fragment detected with a V beta 8 gene was increased in the samples from RA patients (P less than 0.0086). CONCLUSION: The results of this study suggest that germline differences in the TCR repertoire may be associated with RA, and that there is a contributory effect of DR4+ haplotypes with certain TCR haplotypes in susceptibility to RA.