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A Novel Polymorphism in the Pseudogene TCRBV5S5 Combines with TCRBV6S1 to Define Three Haplotypes

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A Novel Polymorphism in the Pseudogene TCRBV5S5 Combines with TCRBV6S1 to Define Three Haplotypes Genes and Immunity (2001) 2, 290–291 2001 Nature Publishing Group All rights reserved 1466-4879/01 $15.00 www.nature.com/gene ALLELE REPORT A novel polymorphism in the pseudogene TCRBV5S5 combines with TCRBV6S1 to define three haplotypes JL Brzezinski1, DN Glass2 and E Choi1 1Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA; 2Department of Pediatrics, William S. Rowe Division of Rheumatology, Children’s Hospital Medical Center, Cincinnati, OH, USA In the current study, we report a G to A single nucleotide polymorphism at base pair 396 of the TCRBV5S5P gene. This polymorphism has a frequency of 0.20 in a cohort of Caucasian controls. In addition, we provide evidence for linkage disequilibrium between TCRBV5S5P and the TCRBV6S1 gene. Genes and Immunity (2001) 2, 290–291. Keywords: TCR; polymorphism; haplotype The TCRBV genes encode the variable region of the ␤ A1/A2 heterozygotes 0.24 chain of the T cell receptor (TCR). There are 46 known A2 homozygotes 0.08 functional TCRBV genes, a number of which have been identified as having coding region polymorphisms.1 The It has been previously reported that TCRBV5S5 is a current study reports the presence of a single nucleotide pseudogene based on a mutation of the highly conserved polymorphism in a TCRBV pseudogene, TCRBV5S5P, GT dinucleotide to GA in the 5Ј donor splice site in the and the identification of strong linkage disequilibrium to intron following exon one, corresponding to base pair the closely linked gene, TCRBV6S1. The G to A transition 203960 of GenBank accession number U66059.2 Sequence in the second exon of the TCRBV5S5P gene corresponds analysis of TCRBV5S5A2 from two homozygotes confirm to base pair 204306 of GenBank accession number that this mutation is present in the novel TCRBV5S5P U66059. This polymorphism can be detected by the poly- allele as well. The inability to PCR amplify TCRBV5S5 merase chain reaction amplification and single-stranded transcripts from peripheral blood lymphocyte cDNA conformational polymorphism (PCR-SSCP). PCR ampli- from four heterozygous individuals supports the fication (35 cycles, 94° 1 min, 58° 2 min, 72° 30 s) was car- assertion that TCRBV5S5P is a pseudogene (data not ried out using forward and reverse primers 5Ј shown). CCCCAGTTTATCTTTGAATATGC and 5Ј CACAGAG The TCRBV6S1 gene is 4.8 kb upstream of the TTCCAGTGACT. The 190 base pair products labeled TCRBV5S5P gene. The TCRBV6S1 gene has three com- with 35S ␣dATP were separated on a 0.5× MDE gel in mon alleles, two of which are null alleles due to an alter- 0.8× TBE according to the manufacturer’s protocols (FMC ation of a conserved cysteine residue involved in a disul- Bioproducts, Rockland, ME, USA). The following allele fide loop that is essential in the formation of and genotype frequencies were determined in a popu- immunoglobulin domains.3 The TCRBV6S1 null alleles lation of 63 unrelated Caucasians from the Cincinnati, are not expressed in the peripheral T cell repertoire. The OH area. Genotype frequencies to not deviate signifi- cohort of 63 Caucasians were typed for the various cantly from Hardy–Weinberg equilibrium (␹2 = 2.78, TCRBV6S1 alleles to determine the relationship between P = 0.25, 2df). the polymorphisms in both TCRBV genes. The observed allele frequencies of TCRBV6S1 are in agreement with Observed allele frequency: previously published data.4 TCRBV5S5A1 (G) 0.80 TCRBV5S5A2 (A) 0.20 Observed allele frequency: TCRBV6S1A1 functional 0.71 Observed genotype frequency: TCRBV6S1A2 null 0.09 A1 homozyotes 0.68 TCRBV6S1A3 null 0.20 The distribution of TCRBV6S1 and TCRBV5S5P geno- Correspondence: Dr Edmund Choi, Department of Molecular Genetics, types in this cohort are nonrandom and provide compel- Biochemistry and Microbiology, University of Cincinnati College of Medi- Ȱ ling evidence for strong linkage disequilibrium (LD) cine, Cincinnati, OH 45267-0524, USA. E-mail: Edmund.Choi uc.edu between the two genes (Table 1a). All carriers of This work was supported, in part, by National Institutes of Health grants AR44059 and AR44566, the Schmidlapp Foundation, and the TCRBV6S1A3 are also carriers of TCRBV5S5A2. This Children’s Hospital Research Foundation of Cincinnati relationship is even more striking in that all five of the Received 22 December 2000; revised and accepted 13 February 2001 TCRBV6S1A3 homozygotes are TCRBV5S5A2 homozy- TCRBV5S5P Polymorphisms JL Brzezinski et al 291 Table 1 TCRBV6S1 and TCRBV5S5 genotypes demonstrates that linkage disequilibrium is present between these loci. The DЈ statistic, a standardized meas- (a) Healthy controls (b) JRA patients ure of linkage disequilibrium,6 was used to quantify the LD between TCRBV6S1 and TCRBV5S5 alleles based on BV6S1 BV5S5 n BV6S1 BV5S5 n the expected haplotype frequencies computed by the EH Genotype Genotype Genotype Genotype program. TCRBV6S1A1 and A2 demonstrate maximum LD with TCRBV5S5A1 (DЈ = 1). Maximum LD was also 1/1 1/1 32 1/1 1/1 92 observed between TCRBV6S1A3 and TCRBV5S5A2 1/2 1/1 10 1/2 1/1 34 (DЈ = 1). 2/2 1/1 1 2/2 1/1 2 Previous studies have reported the association between 1/3 1/2 15 1/3 1/2 65 TCRBV6S1 null alleles and clinical subsets of juvenile 3/3 2/2 5 3/3 2/2 4 rheumatoid arthritis subsets.7,8 The data presented here 2/3 1/2 12 suggest that the TCRBV6S1 alleles are in strong linkage disequilibrium with TCRBV5S5P and are associated with distinct TCR haplotypes. The relationship to other TCR polymorphisms in these haplotypes could be important Table 2 Expected haplotypes frequencies as derived by the EM a for interpreting associations between these genes and dis- algorithm ease. TCRBV6S1 Allele TCRBV5S5 Allele Frequency Acknowledgements 1 1 0.68 The authors wish to thank Mehdi Keddache from the 1 2 0.00 2 1 0.12 Department of Pediatrics, Division of Human Genetics, 2 2 0.00 Children’s Hospital Research Foundation of Cincinnati 3 1 0.00 for assistance with the EH software analysis. 3 2 0.20 References aDeviation of expected haplotype frequencies from random associ- ation (␹2 = 420.21, P = 1.3 × 10−88, 5df). 1 Arden B, Clark SP, Kabelitz D, Mak TW. Human T-cell receptor variable gene segment families. Immunogenetics 1995; 42: 455– 500. gotes. The other null allele, TCRBV6S1A2, appears to be 2 Wei S, Charmley P, Robinson MA, Concannon P. The extent of concordant with TCRBV5S5A1, as is the functional the human germline T-cell receptor V beta gene segment reper- TCRBV6S1A1 allele. The genotypes obtained from a toire. Immunogenetics 1994; 40:27–36. cohort of 209 JRA patients are summarized in Table 1b, 3 Luyrink L, Gabriel CA, Thompson SD et al. Reduced expression of a human V␤6.1 T-cell receptor allele. Proc Natl Acad Sci USA which are included to show a greater representation of 1993; 90: 4369–4373. all possible TCRBV6S1 genotypes. Overall, there are no 4 Barron KS, Robinson MA. The human T-cell receptor variable significant differences between the allele and genotype gene segment TCRBV6S1 has two null alleles. Hum Immunol frequencies in the control and JRA cohorts. These data 1994; 40:17–19. are consistent with the existence of three haplotypes: 5 Terwilliger J, Ott J. Handbook of Human Genetic Linkage. Johns TCRBV5S5A1 and TCRBV6S1A1, TCRBV5S5A2 and Hopkins University Press: Baltimore, 1994. TCRBV6S1A3, and TCRBV5S5A1 and TCRBV6S1A2. This 6 Lewontin RC. The interaction of selection and linkage I. General strengthens the notion that the two TCRBV6S1 null considerations, heterotic models. Genetics 1964; 49:49–67. alleles are found on distinct TCRBV haplotypes. Haplo- 7 Grom AA, von Knorre C, Murray KJ, Donnelly PA, Glass DN, type frequency estimates from the combined data set Choi E. T-cell receptor BV6S1 null alleles and HLA-DR1 haplo- types in polyarticular outcome juvenile rheumatoid arthritis. were determined using the estimation of haplotype (EH) 5 Hum Immunol 1996; 45: 152–156. software package, which employs the expectation-maxi- 8 Charmley P, Nepom BS, Concannon P. HLA and T-cell receptor mization (EM) algorithm (Table 2). A highly significant ␤-chain DNA polymorphisms identify a distinct subset of deviation from random association (␹2 = 420.21 patients with pauciarticular-onset juvenile rheumatoid arthritis. − P = 1.3×10 88, 5df) was observed in our samples, which Arthritis Rheum 1994; 37: 695–701. Genes and Immunity.
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