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C polymorphism in the promoter region of the beta T-cell receptor gene
Molecular and Cellular Probes (2000) 14, 195–197 doi: 10.1006/mcpr.2000.0301, available online at http://www.idealibrary.com on
Polymorphism Report
A common T/C polymorphism in the promoter region of the beta T-cell receptor gene P. Tripputi,∗ D. Graziani, R. M. Alfano, B. Cassani, G. Coggi Department of Pathology, University of Milan, Ospedale S. Paolo via A. di Rudinı` 8, 20142 Milano, Italy (Received 8 February 2000, Accepted 17 April 2000) Polymorphisms in the T-cell receptor genes can provide important information for the study of the immune response system, particularly for autoimmune diseases. This report characterizes a common T to C polymorphism in the promoter of the beta 2 constant chain of the T-cell receptor, which abolishes a recognition site for BglII restriction endonuclease. 2000 Academic Press KEYWORDS: T-cell receptor, TCR beta, TCR polmorphism, BglII polymorphism.
INTRODUCTION T-cell receptors (TCRs) are heterodimers of either alpha and beta chains or gamma and delta chains. Unlike antibodies that recognize antigens as such, TCRs recognize antigens as a complex of a short peptide bound to a major histocompatibility complex (MHC) molecule on the surface of antigen presenting cells. Regardless of the composition of their ligandbinding variable components, TCRs initiate an extensive array of temporally ordered biological responses involving all T-cell(s) of a given functional phenotype.1,2,3 Each TCR chain has a variable region, which is involved in antigen recognition, and a constant region, which interacts with the CD3 complex on the T-cell surface.4 Antigenic specificity of T-cell is due to the diversity that originates in the TCR chains by random juxtaposition of variable, diversity, and joining genes. Several studies, using Southern blotting, have shown an association between the presence of a
BglII polymorphism of TCR beta chain with insulindependent diabetes,5,6,7 autoimmune hepatitis,8 IgA nephropathy9 and membranous nephropathy.10 However, the position of the nucleotide change has never been characterized. Because this change occurs within the promoter region of the TCR beta gene (300 bp before the beta 2 chain transcription site) it could influence gene expression and play a functional role in these diseases. Direct nucleotide sequence analysis of the TCR polymorphism was performed following PCR amplification with primers flanking the BglII site in the promoter region of the TCR beta 2 constant chain.
MATERIALS AND METHODS DNA from 117 unrelated healthy Italian adults (60 males and 57 females) was extracted from peripheral blood by proteinase K digestion, phenol extraction and ethanol precipitation as described earlier.11
∗ Author to whom all correspondence should be addressed at: Department of Pathology, University of Milan, Ospedale S. Paolo via A. di Rudinı` 8, 20142 Milano, Italy. Tel: +39 02 8135366; Fax: +39 02 89122032; E-mail: [email protected]
0890–8508/00/030195+03 $35.00/0
2000 Academic Press
196
P. Tripputi et al.
bp 603 – bp 400 –
bp 203 – 1
2
3
4
5
6
7
8
9
10
Fig. 1. Bgl II restriction digestion of TCR beta promoter. Lanes 1–6 are heterozygous samples. Lanes 7, 9, 10 are samples homozygous for the C allele. Lane 8 is a sample homozygous for the T allele.
DNA sequence for the TCR beta 2 constant region was download from the web site NCBI (GenBank accession number U66061; TCR B map position 7q35; OMIM number 186930) and appropriate primers were designed flanking the polymorphic BglII site:
Table 1. Allele frequencies calculated according to Hardy–Weinberg equilibrium from genotype data of our population
Forward primer: 5′ TAA TTT TGA AAT AAG GGA AGA TGA C 3′ Reverse primer: 5′ TTT TGT ATC CAC CCT ATG GGT TGG C 3′
Genotype frequencies A1 A2 (T C) 0·5214 A1 A1 (T T) 0·2820 A2 A2 (C C) 0·1966
The resulting product of 603 bp was amplified in a 50 l volume using 10 pmol of each primer, 1·5 mM MgCl2, 200 M dNTPs, and 1·25 U Taq polymerase. PCR conditions were as follows: 94°C for 1 min, 55°C for 1 min, 72°C for 1 min for 40 cycles. Restriction digestion was done as described earlier12 with BglII endonuclease in 20 l volume using 10 l of PCR product (about 2 g) and 5U enzyme at 37°C overnight. Digested samples were run on an ethidium bromide stained 2% (u/v) agarose gel. Fragments obtained after restriction were recognized as two bands of 203 and 400 bp. In order to characterize the polymorphism, fluorescent sequencing of homozygous alleles was done using ABI BigDyeTM chemistry and an ABI prism 310 sequencer.
Allele frequencies: A1 (T) 0·5427 A2 (C) 0·4573
(19·7%) were homozygous for the 603-bp allele (Fig. 1). Sequencing of the PCR product from homozygous samples revealed the transition from AGATCT to AGATCC that abolishes the BglII site. As shown in Table 1, allele frequencies estimated according to Hardy–Weinberg equilibrium are 0·5427 for the T allele and 0·4573 for the C allele. Mendelian inheritance was observed in three families.
ACKNOWLEDGEMENTS This work was supported by CNR-ACRO and University of Milan.
REFERENCES RESULTS Restriction fragment analysis for TCR beta BglII polymorphism was performed on 117 non-related Caucasians from Italy. In the presence of the recognition site the 603-bp PCR product was digested into two bands of 203 and 400 bp. The following results were obtained: 61/117 showed three bands, being heterozygous for the polymorphism (52·1%), 33/117 (28·2%) were homozygous for the two digestion fragments and 23/117
1. Ashwell, J. D. & Klausner D. R. (1990). Genetic and mutational analysis of the T-cell antigen receptor. Annual Review of Immunology 8, 139–67. 2. Lanzavecchia, A. (1993). Identifying strategies for immune intervention. Science 260, 937–44. 3. Marrack, P. & Kappler, J. (1987). The T cell receptor. Science 238, 1073–979. 4. Schwartz, R. H. (1985). T-lymphocyte recognition of antigen in association with gene products of the major histocompatibility complex. Annual Review of Immunology 3, 237–61. 5. Hibberd, M. L., Millward, B. A., Wong, F. S. et al.
BglII polymorphism in TCR (1992). T-cell receptor constant beta chain polymorphisms and susceptibility to type 1 diabetes. Diabetic Medicine 9, 929–33. 6. McMillan, S. A., Graham, C. A., Hart, P. J. et al. (1990). A T cell receptor beta chain polymorphism is associated with patients developing insulin-dependent diabetes after the age of 20 years. Clinical Experimental Immunology 82, 538–41. 7. Millward, B. A., Welsh, K. I., Leslie, R. D. G. et al. (1987). T cell receptor beta chain polymorphisms are associated with insulin-dependent diabetes. Clinical Experimental Immunology 70, 152–7. 8. Manabe, K., Hibberd, M. L., Donaldson, P. T. et al. (1994). T cell receptor constant beta germline gene polymorphisms and susceptibility to autoimmune hepatitis. Gastroenterology 106, 1321–5.
197
9. Tatsuma, N., Tsugu, H., Murakami, M. (1997). HLA-DQ region and TCR gene polymorphism associated with primary IgA nephropathy in Japanese children. Japanese Journal of Nephrology 39, 734–9. 10. Demaine, A. G., Vaughan, R. W., Taube, D. H. et al. (1988). Association of membranous nephropathy with T-cell receptor constant beta chain and immunoglobulin heavy chain switch region polymorphisms. Immunogenetics 27, 19–23. 11. Tripputi, P., Blasi, F., Verde, P. et al. (1985). The human urokinase gene is located on the long arm of chromosome 10. Proceedings of the National Academy of Sciences USA 82, 4448–52. 12. Graziani, D., Romagnoli, S., Cassani, B. et al. (1999). An AvaI polymorphism in the TP53 gene. Molecular and Cellular Probes 13, 393–5.
Polymorphism Report
A common T/C polymorphism in the promoter region of the beta T-cell receptor gene P. Tripputi,∗ D. Graziani, R. M. Alfano, B. Cassani, G. Coggi Department of Pathology, University of Milan, Ospedale S. Paolo via A. di Rudinı` 8, 20142 Milano, Italy (Received 8 February 2000, Accepted 17 April 2000) Polymorphisms in the T-cell receptor genes can provide important information for the study of the immune response system, particularly for autoimmune diseases. This report characterizes a common T to C polymorphism in the promoter of the beta 2 constant chain of the T-cell receptor, which abolishes a recognition site for BglII restriction endonuclease. 2000 Academic Press KEYWORDS: T-cell receptor, TCR beta, TCR polmorphism, BglII polymorphism.
INTRODUCTION T-cell receptors (TCRs) are heterodimers of either alpha and beta chains or gamma and delta chains. Unlike antibodies that recognize antigens as such, TCRs recognize antigens as a complex of a short peptide bound to a major histocompatibility complex (MHC) molecule on the surface of antigen presenting cells. Regardless of the composition of their ligandbinding variable components, TCRs initiate an extensive array of temporally ordered biological responses involving all T-cell(s) of a given functional phenotype.1,2,3 Each TCR chain has a variable region, which is involved in antigen recognition, and a constant region, which interacts with the CD3 complex on the T-cell surface.4 Antigenic specificity of T-cell is due to the diversity that originates in the TCR chains by random juxtaposition of variable, diversity, and joining genes. Several studies, using Southern blotting, have shown an association between the presence of a
BglII polymorphism of TCR beta chain with insulindependent diabetes,5,6,7 autoimmune hepatitis,8 IgA nephropathy9 and membranous nephropathy.10 However, the position of the nucleotide change has never been characterized. Because this change occurs within the promoter region of the TCR beta gene (300 bp before the beta 2 chain transcription site) it could influence gene expression and play a functional role in these diseases. Direct nucleotide sequence analysis of the TCR polymorphism was performed following PCR amplification with primers flanking the BglII site in the promoter region of the TCR beta 2 constant chain.
MATERIALS AND METHODS DNA from 117 unrelated healthy Italian adults (60 males and 57 females) was extracted from peripheral blood by proteinase K digestion, phenol extraction and ethanol precipitation as described earlier.11
∗ Author to whom all correspondence should be addressed at: Department of Pathology, University of Milan, Ospedale S. Paolo via A. di Rudinı` 8, 20142 Milano, Italy. Tel: +39 02 8135366; Fax: +39 02 89122032; E-mail: [email protected]
0890–8508/00/030195+03 $35.00/0
2000 Academic Press
196
P. Tripputi et al.
bp 603 – bp 400 –
bp 203 – 1
2
3
4
5
6
7
8
9
10
Fig. 1. Bgl II restriction digestion of TCR beta promoter. Lanes 1–6 are heterozygous samples. Lanes 7, 9, 10 are samples homozygous for the C allele. Lane 8 is a sample homozygous for the T allele.
DNA sequence for the TCR beta 2 constant region was download from the web site NCBI (GenBank accession number U66061; TCR B map position 7q35; OMIM number 186930) and appropriate primers were designed flanking the polymorphic BglII site:
Table 1. Allele frequencies calculated according to Hardy–Weinberg equilibrium from genotype data of our population
Forward primer: 5′ TAA TTT TGA AAT AAG GGA AGA TGA C 3′ Reverse primer: 5′ TTT TGT ATC CAC CCT ATG GGT TGG C 3′
Genotype frequencies A1 A2 (T C) 0·5214 A1 A1 (T T) 0·2820 A2 A2 (C C) 0·1966
The resulting product of 603 bp was amplified in a 50 l volume using 10 pmol of each primer, 1·5 mM MgCl2, 200 M dNTPs, and 1·25 U Taq polymerase. PCR conditions were as follows: 94°C for 1 min, 55°C for 1 min, 72°C for 1 min for 40 cycles. Restriction digestion was done as described earlier12 with BglII endonuclease in 20 l volume using 10 l of PCR product (about 2 g) and 5U enzyme at 37°C overnight. Digested samples were run on an ethidium bromide stained 2% (u/v) agarose gel. Fragments obtained after restriction were recognized as two bands of 203 and 400 bp. In order to characterize the polymorphism, fluorescent sequencing of homozygous alleles was done using ABI BigDyeTM chemistry and an ABI prism 310 sequencer.
Allele frequencies: A1 (T) 0·5427 A2 (C) 0·4573
(19·7%) were homozygous for the 603-bp allele (Fig. 1). Sequencing of the PCR product from homozygous samples revealed the transition from AGATCT to AGATCC that abolishes the BglII site. As shown in Table 1, allele frequencies estimated according to Hardy–Weinberg equilibrium are 0·5427 for the T allele and 0·4573 for the C allele. Mendelian inheritance was observed in three families.
ACKNOWLEDGEMENTS This work was supported by CNR-ACRO and University of Milan.
REFERENCES RESULTS Restriction fragment analysis for TCR beta BglII polymorphism was performed on 117 non-related Caucasians from Italy. In the presence of the recognition site the 603-bp PCR product was digested into two bands of 203 and 400 bp. The following results were obtained: 61/117 showed three bands, being heterozygous for the polymorphism (52·1%), 33/117 (28·2%) were homozygous for the two digestion fragments and 23/117
1. Ashwell, J. D. & Klausner D. R. (1990). Genetic and mutational analysis of the T-cell antigen receptor. Annual Review of Immunology 8, 139–67. 2. Lanzavecchia, A. (1993). Identifying strategies for immune intervention. Science 260, 937–44. 3. Marrack, P. & Kappler, J. (1987). The T cell receptor. Science 238, 1073–979. 4. Schwartz, R. H. (1985). T-lymphocyte recognition of antigen in association with gene products of the major histocompatibility complex. Annual Review of Immunology 3, 237–61. 5. Hibberd, M. L., Millward, B. A., Wong, F. S. et al.
BglII polymorphism in TCR (1992). T-cell receptor constant beta chain polymorphisms and susceptibility to type 1 diabetes. Diabetic Medicine 9, 929–33. 6. McMillan, S. A., Graham, C. A., Hart, P. J. et al. (1990). A T cell receptor beta chain polymorphism is associated with patients developing insulin-dependent diabetes after the age of 20 years. Clinical Experimental Immunology 82, 538–41. 7. Millward, B. A., Welsh, K. I., Leslie, R. D. G. et al. (1987). T cell receptor beta chain polymorphisms are associated with insulin-dependent diabetes. Clinical Experimental Immunology 70, 152–7. 8. Manabe, K., Hibberd, M. L., Donaldson, P. T. et al. (1994). T cell receptor constant beta germline gene polymorphisms and susceptibility to autoimmune hepatitis. Gastroenterology 106, 1321–5.
197
9. Tatsuma, N., Tsugu, H., Murakami, M. (1997). HLA-DQ region and TCR gene polymorphism associated with primary IgA nephropathy in Japanese children. Japanese Journal of Nephrology 39, 734–9. 10. Demaine, A. G., Vaughan, R. W., Taube, D. H. et al. (1988). Association of membranous nephropathy with T-cell receptor constant beta chain and immunoglobulin heavy chain switch region polymorphisms. Immunogenetics 27, 19–23. 11. Tripputi, P., Blasi, F., Verde, P. et al. (1985). The human urokinase gene is located on the long arm of chromosome 10. Proceedings of the National Academy of Sciences USA 82, 4448–52. 12. Graziani, D., Romagnoli, S., Cassani, B. et al. (1999). An AvaI polymorphism in the TP53 gene. Molecular and Cellular Probes 13, 393–5.