Am. J. Hum. Genet. 47:45-52, 1990
T-Cell Receptor Genes and Insulin-dependent Diabetes Mellitus (IDDM): No Evidence for Linkage from Affected Sib Pairs Patrick Concannon, * Jocyndra A. Wright, * Lawrence G. WrightT Daniel R. Sylvestert and Richard S. Spielmant *Virginia
Mason
Research Center, Seattle; and fUniversity of Pennsylvania School of Medicine, Philadelphia
Summary Several investigators have reported an association between insulin-dependent diabetes mellitus (IDDM) and an RFLP detected with a probe for the constant region of the fi chain (CO) of the human T-cell receptor (TCR). A likely hypothesis is that the closely linked TCR variable (VD) region genes contribute to IDDM susceptibility and that the association with the TCR Co3 locus reflects this contribution, via linkage disequilibrium between VD3 and CoI. The products of the E-chain genes might be expected to be involved in the etiology of IDDM because of the autoimmune aspects of IDDM, the known involvement of HLA, and the necessity for TCR and HLA molecules to interact in an immune response. In order to investigate the hypothesis, we tested for linkage between IDDM and V genes encoded at either the TCRPi locus on chromosome 7 or the TCRa locus on chromosome 14, using 36 families with multiple affected sibs. No excess sharing of haplotypes defined by Va or VPi gene RFLPs was observed in affected sib pairs from IDDM families. We also studied unrelated IDDM patients (N = 73) and controls (N = 45) with the COi RFLP but were unable to confirm the reported association even when the sample was stratified by HLA-DR type. Our results are incompatible with close linkage, in the majority of families, between either the TCRa or TCRI3 locus and a gene making a major contribution to susceptibility to IDDM.
Introduction Family and population studies have greatly increased our understanding of the genetic components of susceptibility to insulin-dependent diabetes mellitus (IDDM). Associations between various HLA markers and IDDM were first observed in population studies, which revealed different distributions of HLA haplotypes among unrelated IDDM patients and normal controls. These initial observations were later confirmed and extended through family studies in multiplex families, both by studies of affected sib pairs and by conventional linkage analysis. As a result of these studies it is now well established that the HLA class II region contains at least one gene locus that contributes strongly to genetic susReceived January 5, 1990; revision received March 14, 1990. Address for correspondence and reprints: Dr. Patrick Concannon, Division of Immunology, Virginia Mason Research Center, 1000 Seneca Street, Seattle, WA 98101. i 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4701-0007$02.00
ceptibility to IDDM (for review, see Svejgaard et al. 1983; Svejgaard and Ryder 1989). Despite the strength of the HLA class II locus association with IDDM, family studies of identical twin pairs and pairs of HLA identical siblings who are discordant for IDDM indicate that other factors, environmental and/or genetic, may play a role in the etiology of IDDM. In the latter category, a number of genes not linked to the HLA complex have been suggested as candidate loci whose products might play a role in the pathogenesis of diabetes. Such candidate loci include the insulin gene (Owerbach and Nerup 1982; Bell et al. 1984; Thomson et al. 1989), the immunoglobulin heavy- and lightchain genes (Rich et al. 1986; Field 1989), and, recently, the genes that encode the T-cell receptor (TCR) for antigen (Hoover et al. 1986; Millward et al. 1987; Ito et al. 1988). The most abundant form of the TCR for antigen is a heterodimer of one a and one 0i chain expressed on the surface of a mature T cell in conjunction with a series of proteins that make up the CD3 complex. The 45
Concannon et al.
46
genes that encode the a and 0i chains are located on human chromosomes 14 and 7, respectively, and are composed of variable (V), diversity (D), joining (J), and constant (C) gene segments. During T-cell development, a process of chromosomal rearrangement juxtaposes one VN3, one Do3, and one Joi gene segment to create a mature TCRO1 chain gene. The mature TCRa gene is formed by rearrangement of one Va and one Ja. The rearranged V, D, and J gene segments form the variable domains of the TCR molecules, responsible for antigen recognition, while the C gene segments, which are separated from the J gene segments by an intron in germline DNA, presumably play a role in signal transduction. It has been estimated that there are more than 60 human VDB genes extending over at least 1,000 kb of DNA on the long arm of chromosome 7 and that there are a larger number of Va genes on the long arm of chromosome 14 (for review, see Wilson et al. 1988). There is ample evidence suggesting a role for T cells in the development of IDDM. T cells are found infiltrating the pancreas undergoing diabetic islet-cell destruction (Bottazzo et al. 1985). Evidence from animal models of IDDM indicates that the disorder is mediated by T cells and that, if recognition of presented antigen is blocked by monoclonal antibodies directed against major-histocompatability-complex molecules (Boitard et al. 1985) or the T-cell surface-marker CD4 (Shizuru et al. 1988), the disease process is abrogated. Further, the strong HLA association with IDDM, the organ-specific nature of the autoimmune response in IDDM, and the general requirement for physical interaction between an HLA molecule and the TCR of a specific T cell in the process of antigen presentation all suggest a role for specific TCR molecules in the disease. There are several mechanisms by which TCR V genes and their products might play a role in IDDM. One simple model is that germ-line polymorphism of TCR V genes might lead to the existence of allelic V gene products in the population, products which, when expressed in the context of predisposing HLA alleles and an unidentified environmental stimulus, would break tolerance and react with a specific pancreatic 13-cell antigen. Alternatively, V genes capable of recognizing this 13-cell antigen could be present in all individuals in the population, but, in most cases, their P1-cell reactivity might be masked by either cross-tolerance due to clonal elimination of T cells bearing these potentially autoreactive receptors during thymic education or the presence of specific suppressor cells. The first of these models explicitly proposes that the TCR loci are candidates for the location of non-HLA IDDM sus-
ceptibility genes. Such considerations have led a number of investigators to test for a population-level association between IDDM and RFLPs of the TCR13 locus. Several of these studies (Hoover et al. 1986; Millward et al. 1987; Ito et al. 1988) have revealed associations between IDDM and alleles of an RFLP detected with a Co3-gene probe in random unrelated individuals. The aim of the present study was to test the hypothesis that germ-line variation at or near loci encoding either of the two chains of the TCR influences susceptibility to IDDM. The hypothesis requires linkage between IDDM susceptibility and the TCRa and TCR13 locus. A formal demonstration that linkage is present requires family studies. We have carried out an analysis of TCR a and 13 haplotype sharing in affected sib pairs (ASPs) from multiplex IDDM families, using a panel of RFLPs detected with probes for various V and C gene segments of these loci. Despite the plausibility of the hypothesis and the promising published data on associations at the population level, our studies provide no evidence for linkage, at the germ-line level, between IDDM and the TCR a or 13 locus. Furthermore, in our own sample of unrelated insulin-dependent diabetics, we find no evidence for a population association with the TCR Co3 marker. Material and Methods Patients and Controls
The patients studied belong to two groups. All diabetic patients were classical juvenile-onset, insulindependent diabetics with onset before age 22 years. In the 36 "multiplex" families (i.e., those with two or more affected sibs), we obtained specimens from both parents and from all available affected and unaffected sibs. In one family with two affected sibs and in another with three affected sibs, the father was also affected with IDDM. These were the only parents with IDDM. In addition, we studied 37 diabetics who were the only affected member of their sibship ("simplex" families). Thus, for the population analysis, DNA was available from a total of 73 unrelated diabetics, who included an "index" case from each multiplex family. The 43 controls were healthy individuals from the donor panel of the Tissue-Typing Laboratory of the hospital of the University of Pennsylvania. Although our diabetic and control samples included a number of blacks and Asians, we report data only from Caucasians, as they constituted by far the largest component. Among the 36 families, 29 had exactly two affected
T-Cell Receptor Genes and IDDM
47
sibs, six had three affected sibs, and one had four affected sibs. (Many of the families have been described, with HLA genotype data, by Spielman et al. [1980].) All genotype assignments for the members of these families were made without knowledge of their disease status or their family relationships. All 36 families were studied for TCR1i sharing. Twenty-five of the families were studied for TCRa sharing. Probes
The following probe and enzyme combinations were used to detect RFLPs in the genomic DNA derived from IDDM families in the present study. For the TCRI3 locus, a C13 probe (provided by T. Mak) was used to detect two previously described polymorphisms, one with Bg1II and one with KpnI. Polymorphisms were also detected in BamHI-digested DNA by a VfP8 probe, a V111 probe, and a V137.2 probe and in TaqI-digested DNA by a Vo1 probe. For the TCRa locus, we used TaqI RFLPs detected by a Va17 probe, a ValS probe, a Val.2 probe, and a Ca probe. A complete list of probes, allele sizes detected, and references is given in table 1. Since recombination is rare (
T-Cell Receptor Genes and Insulin-dependent Diabetes Mellitus (IDDM): No Evidence for Linkage from Affected Sib Pairs Patrick Concannon, * Jocyndra A. Wright, * Lawrence G. WrightT Daniel R. Sylvestert and Richard S. Spielmant *Virginia
Mason
Research Center, Seattle; and fUniversity of Pennsylvania School of Medicine, Philadelphia
Summary Several investigators have reported an association between insulin-dependent diabetes mellitus (IDDM) and an RFLP detected with a probe for the constant region of the fi chain (CO) of the human T-cell receptor (TCR). A likely hypothesis is that the closely linked TCR variable (VD) region genes contribute to IDDM susceptibility and that the association with the TCR Co3 locus reflects this contribution, via linkage disequilibrium between VD3 and CoI. The products of the E-chain genes might be expected to be involved in the etiology of IDDM because of the autoimmune aspects of IDDM, the known involvement of HLA, and the necessity for TCR and HLA molecules to interact in an immune response. In order to investigate the hypothesis, we tested for linkage between IDDM and V genes encoded at either the TCRPi locus on chromosome 7 or the TCRa locus on chromosome 14, using 36 families with multiple affected sibs. No excess sharing of haplotypes defined by Va or VPi gene RFLPs was observed in affected sib pairs from IDDM families. We also studied unrelated IDDM patients (N = 73) and controls (N = 45) with the COi RFLP but were unable to confirm the reported association even when the sample was stratified by HLA-DR type. Our results are incompatible with close linkage, in the majority of families, between either the TCRa or TCRI3 locus and a gene making a major contribution to susceptibility to IDDM.
Introduction Family and population studies have greatly increased our understanding of the genetic components of susceptibility to insulin-dependent diabetes mellitus (IDDM). Associations between various HLA markers and IDDM were first observed in population studies, which revealed different distributions of HLA haplotypes among unrelated IDDM patients and normal controls. These initial observations were later confirmed and extended through family studies in multiplex families, both by studies of affected sib pairs and by conventional linkage analysis. As a result of these studies it is now well established that the HLA class II region contains at least one gene locus that contributes strongly to genetic susReceived January 5, 1990; revision received March 14, 1990. Address for correspondence and reprints: Dr. Patrick Concannon, Division of Immunology, Virginia Mason Research Center, 1000 Seneca Street, Seattle, WA 98101. i 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4701-0007$02.00
ceptibility to IDDM (for review, see Svejgaard et al. 1983; Svejgaard and Ryder 1989). Despite the strength of the HLA class II locus association with IDDM, family studies of identical twin pairs and pairs of HLA identical siblings who are discordant for IDDM indicate that other factors, environmental and/or genetic, may play a role in the etiology of IDDM. In the latter category, a number of genes not linked to the HLA complex have been suggested as candidate loci whose products might play a role in the pathogenesis of diabetes. Such candidate loci include the insulin gene (Owerbach and Nerup 1982; Bell et al. 1984; Thomson et al. 1989), the immunoglobulin heavy- and lightchain genes (Rich et al. 1986; Field 1989), and, recently, the genes that encode the T-cell receptor (TCR) for antigen (Hoover et al. 1986; Millward et al. 1987; Ito et al. 1988). The most abundant form of the TCR for antigen is a heterodimer of one a and one 0i chain expressed on the surface of a mature T cell in conjunction with a series of proteins that make up the CD3 complex. The 45
Concannon et al.
46
genes that encode the a and 0i chains are located on human chromosomes 14 and 7, respectively, and are composed of variable (V), diversity (D), joining (J), and constant (C) gene segments. During T-cell development, a process of chromosomal rearrangement juxtaposes one VN3, one Do3, and one Joi gene segment to create a mature TCRO1 chain gene. The mature TCRa gene is formed by rearrangement of one Va and one Ja. The rearranged V, D, and J gene segments form the variable domains of the TCR molecules, responsible for antigen recognition, while the C gene segments, which are separated from the J gene segments by an intron in germline DNA, presumably play a role in signal transduction. It has been estimated that there are more than 60 human VDB genes extending over at least 1,000 kb of DNA on the long arm of chromosome 7 and that there are a larger number of Va genes on the long arm of chromosome 14 (for review, see Wilson et al. 1988). There is ample evidence suggesting a role for T cells in the development of IDDM. T cells are found infiltrating the pancreas undergoing diabetic islet-cell destruction (Bottazzo et al. 1985). Evidence from animal models of IDDM indicates that the disorder is mediated by T cells and that, if recognition of presented antigen is blocked by monoclonal antibodies directed against major-histocompatability-complex molecules (Boitard et al. 1985) or the T-cell surface-marker CD4 (Shizuru et al. 1988), the disease process is abrogated. Further, the strong HLA association with IDDM, the organ-specific nature of the autoimmune response in IDDM, and the general requirement for physical interaction between an HLA molecule and the TCR of a specific T cell in the process of antigen presentation all suggest a role for specific TCR molecules in the disease. There are several mechanisms by which TCR V genes and their products might play a role in IDDM. One simple model is that germ-line polymorphism of TCR V genes might lead to the existence of allelic V gene products in the population, products which, when expressed in the context of predisposing HLA alleles and an unidentified environmental stimulus, would break tolerance and react with a specific pancreatic 13-cell antigen. Alternatively, V genes capable of recognizing this 13-cell antigen could be present in all individuals in the population, but, in most cases, their P1-cell reactivity might be masked by either cross-tolerance due to clonal elimination of T cells bearing these potentially autoreactive receptors during thymic education or the presence of specific suppressor cells. The first of these models explicitly proposes that the TCR loci are candidates for the location of non-HLA IDDM sus-
ceptibility genes. Such considerations have led a number of investigators to test for a population-level association between IDDM and RFLPs of the TCR13 locus. Several of these studies (Hoover et al. 1986; Millward et al. 1987; Ito et al. 1988) have revealed associations between IDDM and alleles of an RFLP detected with a Co3-gene probe in random unrelated individuals. The aim of the present study was to test the hypothesis that germ-line variation at or near loci encoding either of the two chains of the TCR influences susceptibility to IDDM. The hypothesis requires linkage between IDDM susceptibility and the TCRa and TCR13 locus. A formal demonstration that linkage is present requires family studies. We have carried out an analysis of TCR a and 13 haplotype sharing in affected sib pairs (ASPs) from multiplex IDDM families, using a panel of RFLPs detected with probes for various V and C gene segments of these loci. Despite the plausibility of the hypothesis and the promising published data on associations at the population level, our studies provide no evidence for linkage, at the germ-line level, between IDDM and the TCR a or 13 locus. Furthermore, in our own sample of unrelated insulin-dependent diabetics, we find no evidence for a population association with the TCR Co3 marker. Material and Methods Patients and Controls
The patients studied belong to two groups. All diabetic patients were classical juvenile-onset, insulindependent diabetics with onset before age 22 years. In the 36 "multiplex" families (i.e., those with two or more affected sibs), we obtained specimens from both parents and from all available affected and unaffected sibs. In one family with two affected sibs and in another with three affected sibs, the father was also affected with IDDM. These were the only parents with IDDM. In addition, we studied 37 diabetics who were the only affected member of their sibship ("simplex" families). Thus, for the population analysis, DNA was available from a total of 73 unrelated diabetics, who included an "index" case from each multiplex family. The 43 controls were healthy individuals from the donor panel of the Tissue-Typing Laboratory of the hospital of the University of Pennsylvania. Although our diabetic and control samples included a number of blacks and Asians, we report data only from Caucasians, as they constituted by far the largest component. Among the 36 families, 29 had exactly two affected
T-Cell Receptor Genes and IDDM
47
sibs, six had three affected sibs, and one had four affected sibs. (Many of the families have been described, with HLA genotype data, by Spielman et al. [1980].) All genotype assignments for the members of these families were made without knowledge of their disease status or their family relationships. All 36 families were studied for TCR1i sharing. Twenty-five of the families were studied for TCRa sharing. Probes
The following probe and enzyme combinations were used to detect RFLPs in the genomic DNA derived from IDDM families in the present study. For the TCRI3 locus, a C13 probe (provided by T. Mak) was used to detect two previously described polymorphisms, one with Bg1II and one with KpnI. Polymorphisms were also detected in BamHI-digested DNA by a VfP8 probe, a V111 probe, and a V137.2 probe and in TaqI-digested DNA by a Vo1 probe. For the TCRa locus, we used TaqI RFLPs detected by a Va17 probe, a ValS probe, a Val.2 probe, and a Ca probe. A complete list of probes, allele sizes detected, and references is given in table 1. Since recombination is rare (
