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.

Evolution and selection of primate T cell antigen receptor BV8 gene subfamily.

The set of potential T cell receptor specificities is highly diverse. The relative contributions of T cell receptor (TCR) V beta gene segment polymorphisms, duplications, deletions, and gene conversions to this final T cell receptor protein diversity are unknown. To study these mechanisms, we sequenced and compared closely related primate TCR gene segments from BV8S1, S2, and S5. Interspecies comparisons show that these gene segments have sustained multiple duplication, gene conversion, and deletion events during the last 35 million years of anthropoid primate evolution. BV8 coding sequences are generally conserved with respect to their flanking noncoding sequences, but we find no evidence for positive or negative selection in sequences coding for the first two putative complementarity-determining (ligand-binding) regions. Sequences of TCRBV8 gene segments from unrelated humans demonstrate no nonsynonymous substitutions in nonleader regions of either the BV8S1 or S2 gene segments. We conclude that gene duplication, deletion, and conversion mechanism contribute in a substantial way to the overall diversity of the TCRBV8 gene segment repertoire in primate evolution and that germline substitutions and consequent polymorphisms in CDRs 1 and 2 of these gene segments probably do not play an active role in generating TCR beta chain protein variation.

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.

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.

Synergy between T cell receptor beta gene polymorphism and HLA-DR4 in susceptibility to rheumatoid arthritis.

OBJECTIVE: To investigate the etiologic significance of germline polymorphisms in the T cell receptor beta variable region 6S7 (TCRBV6S7) gene segment and adjacent loci in susceptibility to rheumatoid arthritis (RA). METHODS: Ten TCRB allelic polymorphisms were analyzed from 3 groups of white women: 112 with RA, 72 with systemic lupus erythematosus, and 70 healthy controls. All participants were also HLA typed. RESULTS: HLA-DR4+ RA patients showed significantly increased frequencies of TCRBV6S7*1, 13S5P*1 (an allelic variant of BV13S5 promoter), and 12S4*2, compared with healthy controls. The combination of DR4 with either BV6S7*1, 13S5P*1, or 12S4*2 conferred greater risk for RA than HLA-DR4 alone. Pairwise analyses showed a high degree of linkage disequilibrium (P = 10(-5)-10(-8)) between these 3 TCRBV loci that span 47 kilobases (kb). CONCLUSION: Our data suggest that a TCR gene segment in or linked to this 47-kb region may be involved in genetic susceptibility to RA through an interaction with HLA-DR4.

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.

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)

Relative size and evolution of the germline repertoire of T-cell receptor beta-chain gene segments in nonhuman primates.

The mammalian T-cell receptor (TCR) gene complexes exist as multiple tandemly arrayed gene segments that have apparently arisen by gene duplication mechanisms. A study of the number of TCR germline gene segments in several primate species might provide insight into the relative rate and patterns of gene duplication and deletion within these gene complexes. DNA probes from the TCR beta-chain variable (TCRBV) region gene segment subfamilies 1 through 25 and the constant region gene segment were sequentially hybridized under low stringency to Southern blots containing genomic DNA of human, gorilla, orangutan, and pig-tailed macaque. The number of gene members in each subfamily was estimated from the number of hybridizing DNA fragments. The results show apparent examples of both TCRB V gene duplication and deletion since speciation of the Hominoids from Cercopithecoid (Old World) primates. For one putative duplication/deletion event involving six TCRBV gene segments, derivation and comparison of germline DNA sequence from macaque and human as well as Southern blot analysis of additional primates demonstrated that this event was a duplication that occurred after the divergence of the family Pongidae (Greater Apes) from Hylobatidae (Lesser Apes). Southern blot analysis of multiple pig-tailed macaques and their offspring suggests a degree of DNA sequence variability in these gene segments similar to that observed in humans. An appreciation of the size and variability of each TCRBV subfamily will be useful when considering the DNA primers and probes necessary to measure the relative usage of these TCRBV genes as part of the immune response in these nonhuman primates.

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.

T-cell receptor repertoire in tumor-infiltrating lymphocytes. Analysis of melanoma-specific long-term lines.

Cytotoxic T-lymphocytes (CTLs) can be isolated from human melanoma biopsies that specifically lyse autologous melanoma in vitro and can be effective therapeutic agents for patients with advanced disease. Recent evidence indicates that HLA-A2-restricted, melanoma-specific tumor-infiltrating lymphocytes (TILs) recognize melanomas obtained from different HLA-A2+ patients, suggesting the presence of one or more common melanoma antigens. Furthermore, T-cell receptor (TCR) repertoire analysis by other groups of TILs from fresh melanoma biopsies suggests that there is limited TCR V gene usage in TILs. One serious limitation in analyzing the TCR repertoire in fresh tumors has been the inability to correlate TCR usage with immune function. Therefore, the TCR repertoire was determined in long-term TIL cultures that specifically lysed autologous melanoma in vitro and in many cases mediated in vivo regression of metastatic cancer in patients with advanced disease. The TCR repertoire in cultured melanoma-specific TILs was diverse, with each TIL containing an average of 9.5 +/- 5.7 of the 23 V alpha and 11.2 +/- 5.9 of the 23 V beta subfamilies. Despite the large diversity observed, several V alpha and V beta genes (V alpha 1, V alpha 2, V alpha 22, V beta 13, V beta 14, and V beta 18) are very commonly found in melanoma-specific TILs. No statistically significant associations were observed between the presence of a TCR V gene subfamily in TILs and clinical response, HLA haplotype, or age of the culture. Even though the results in this study suggest that certain TCR V gene segments may be involved in immune responses to human melanoma, we were unable to demonstrate functionally that a particular T-cell clonotype recognizes melanoma tumor-associated antigens. Only the analysis of melanoma-specific CTL clones can determine which clonotypes are important in lysis of human melanoma.

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.

T-cell receptor usage by melanoma-specific clonal and highly oligoclonal tumor-infiltrating lymphocyte lines.

Tumor-infiltrating lymphocytes (TIL) obtained from human melanomas can specifically lyse autologous tumor in vitro and mediate tumor regression in vivo. To develop more effective therapeutic reagents and to further understand the T-cell response to tumors, the diversity of T-cell receptors (TCRs) involved in melanoma antigen recognition has been studied. The TCR variable (V) genes, joining (J) segments, and N diversity regions used by five clonal lines and one highly oligoclonal, melanoma-specific, CD8+ TIL line were examined utilizing PCR amplification with V gene subfamily-specific primers and anchor PCR. The TIL lysed multiple allogeneic melanomas expressing matched surface major histocompatibility complex class I molecules. TCR analysis confirmed the clonal nature of the TIL lines; however, the TCR repertoire was diverse. Even among the three HLA-A2 restricted TIL (TIL 1200, TIL F2-2, and TIL-5), no common V gene usage was found. Comparison of the third complementarity-determining regions of the TCRs from the HLA-A2 restricted TIL revealed no homology. Results presented here identify T-cell clonotypes that recognize epitopes on highly prevalent, shared melanoma tumor-associated antigens presented in the context of HLA-B55, HLA-A1, and HLA-A2. These T cells and the antigens they recognize represent important components for the design of new immunotherapies for patients with advanced melanoma.

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.

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.

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.

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.

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.