Epidemiology/Health Services/Psychosocial Research ORIGINAL ARTICLE Concordance for Type 1 Diabetes in Identical Twins Is Affected by Insulin Genotype 1 2 KARL A. METCALFE, MRCP XIAOJIAN HUANG, PHD cated in disease risk, only a few with 1 2 GRAHAM A. HITMAN, MD TIMOTHY STEWART, PHD known function have been identified, in- 1 1 RACHEL E. ROWE, MD R. DAVID G. LESLIE, MD 1 cluding the insulin gene hypervariable re- MOHAMMED HAWA, BSC gion (2–5), the interleukin gene cluster (6,7), CTLA4 (8–10), and the vitamin D receptor (11,12). However, the only con- sistent association between type 1 diabe- tes and a non-MHC region is with the OBJECTIVE — Monozygotic twins are usually discordant (only one twin affected) for type 1 insulin gene. diabetes. Discordance for disease between such twins implies a role for nongenetically deter- The study of monozygotic twins con- mined factors but could also be influenced by a decreased load of diabetes susceptibility genes. The aim of this study was to determine whether two susceptibility genes were less prevalent in cordant or discordant for type 1 diabetes discordant twins compared with concordant twins. is a potentially powerful method to deter- mine whether disease penetrance is a ran- RESEARCH DESIGN AND METHODS — We studied 77 monozygotic twin pairs dom effect or influenced by genetic (INS), 40 concordant and 37 discordant, for type 1 diabetes at polymorphism of the insulin gene factors. Monozygotic twins tend to de- region on chromosome 11p and HLA-DQBI. velop type 1 diabetes within 6 years of each other (13), so that twin pairs discor- RESULTS — The disease-associated INS genotype (Hph I) was identified in 87.5% of the ϭ dant for diabetes are likely to remain dis- concordant twins but only in 59.5% (P 0.005) of the discordant twins. Neither DQB1*0201 cordant. We identified such discordant nor DQB1*0302 was seen in 2 of 40 (5%) concordant twins compared with 8 of 37 (22%) twin pairs in a previous HLA phenotype discordant twins (P ϭ 0.04). No statistical differences were seen between concordant and discordant twins at individual alleles of DQB1. Combining insulin and DQ data, 5% of concor- study and showed that they were less dant twins compared with 32.4% of discordant twins had neither DQB1*0201/DQB1*0302 nor likely than concordant pairs to have both the high-risk Hph I INS “ϩϩ” genotype (P ϭ 0.002). HLA-DR3 and HLA-DR4 antigens (14). Subsequent studies indicated that pairs CONCLUSIONS — We conclude that the possession of the high-risk Hph I insulin genotype remaining discordant are more accurately increases the likelihood of identical twins being concordant for type 1 diabetes and that the defined as nondiabetic twins more than “load” of both major histocompatibility complex (MHC) and non-MHC susceptibility genes has 11 years from the diagnosis of the index an impact on the disease penetrance of type 1 diabetes. twin, with normal glucose tolerance and without antibody markers (15–17). Be- Diabetes Care 24:838–842, 2001 cause there are no previous genotype studies of HLA-DQB1 or non–MHC- encoded susceptibility genes to type 1 di- oncordance rates for type 1 diabetes factors also influence susceptibility to dis- abetes in identical twins, we have now are higher in monozygotic than ease. tested discordant as well as concordant C dizygotic twins, which is consistent At least 30% of the genetic suscepti- pairs using this definition of discordance. with a role for genetic factors determining bility to type 1 diabetes can be explained Our hypothesis was that discordance, as the disease (1). Nevertheless, ϳ50% of by an association with the major histo- compared with concordance, for type 1 monozygotic twins are discordant for compatibility complex (MHC) (2). Al- diabetes in monozygotic twins is caused type 1 diabetes, providing strong evi- though a large number of non-MHC by a decreased load of both MHC and dence that nongenetically determined chromosomal regions have been impli- non-MHC susceptibility genes. ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● RESEARCH DESIGN AND From the 1Department of Diabetes and Metabolic Medicine, St. Bartholomew’s and the Royal London School METHODS — We have studied and of Medicine and Dentistry, Queen Mary and Westfield College, University of London, London, U.K.; and 2 compared 40 concordant and 37 discor- Genentech, South San Francisco, California. dant monozygotic twins for type 1 diabe- Address correspondence and reprint requests to Graham A. Hitman, MD, Department of Diabetes and Metabolic Medicine, Medical Unit, The Royal London Hospital, Whitechapel, London E1 1BB, U.K. E-mail: tes at both HLA-DQBI and the insulin [email protected]. gene (INS) loci. Twins studied were se- Received for publication 8 May 2000 and accepted in revised form 21 December 2000. lected blind to those included in our pre- X.H. and T.S. are employed by and hold stock in Genentech. Genentech is engaged in the development vious study 15 years ago of HLA-DR of pharmaceuticals for the treatment of diabetes and its complications. Abbreviations: ICA, islet cell antibody; MHC, major histocompatibility complex. phenotype (14). A total of 54% of twin A table elsewhere in this issue shows conventional and Syste`me International (SI) units and conversion pairs were common to both studies. factors for many substances. Twins were ascertained from 1967 to 838 DIABETES CARE, VOLUME 24, NUMBER 5, MAY 2001 Metcalfe and Associates 1997 by referral to the British Diabetic Table 1—HLA-DQB1 allele positivity in monozygotic twins with type 1 diabetes Twin Study; monozygosity was estab- lished in each twin pair as described pre- % Concordant % Discordant viously (18,19). DNA was not available Allele (n ϭ 40) (n ϭ 37) P value from all twins. All twins were invited to attend the Diabetes Department for blood 0201 82.5 (n ϭ 33) 65 (n ϭ 24) 0.08 sampling, but in the study period, only a 0301 2.5 (n ϭ 1) 5.4 (n ϭ 2) NS fraction were able to do so. We therefore 0302 60 (n ϭ 24) 40.5 (n ϭ 15) 0.09 selected twin pairs who had attended the 0402 — 5.4 (n ϭ 2) NS Diabetes Department consecutively from 0501 — 2.7 (n ϭ 1) NS 1995 to 1997, who were either concor- 0502 13 (n ϭ 5) 5.4 (n ϭ 2) NS dant or discordant for type 1 diabetes. 0601 — 2.7 (n ϭ 1) NS The diabetic index twin from both con- 0602/3 2.5 (n ϭ 1) 5.4 (n ϭ 2) NS cordant and discordant pairs had a com- 0604 — 5.4 (n ϭ 2) NS parable age at diagnosis, and nondiabetic 0201/0302 47.5 (n ϭ 19) 27 (n ϭ 10) 0.06 twins from discordant pairs had a low risk Negative for DQB1*201 and/or *0302 5 (n ϭ 2) 22 (n ϭ 8) 0.04 of developing diabetes. Discordant pairs NS, not significant. likely to remain discordant were identi- fied by being Ͼ11 years from the diagno- sis of the index twin and, at 11 years by oped type 1 diabetes but was not part of Nomura et al. (22). DNA was extracted having normal glucose tolerance and the this study) to further evaluate the cutoff from blood (23), and a 241-bp region absence of islet cell antibody (ICA), GAD level for positivity for GAD65 and IA-2ic. from the polymorphic second exon of the Ͼ antibody, or IA-2ic antibody. Levels 3 SD higher than that of a control DQB1 gene was amplified in two separate Of 247 concordant twin pairs, we se- population were considered positive, as de- PCRs, specific for DQwI and DQw2,3,4 lected a consecutive series of 40 pairs at- scribed previously (17). In the latest IA- alleles. Seven DQwI and six DQw2,3,4 tending the Diabetes Center (mean age of 2ic (unpublished) and GAD65 antibody alleles can be identified after restriction diagnosis of index twin 16 Ϯ 9 years, proficiency workshops, our assays had a against panels of seven and five restriction range 1–39; 16 male pairs; median time to sensitivity, specificity, validity, and con- enzymes, respectively (22,24). The di- concordance 3.95 years, range 0.4–25). sistency of 100% in each (21). gested and undigested products were vi- Of 109 discordant twin pairs, we identi- Undiluted sera were screened for sualized by ethidium bromide staining fied a consecutive series of 37 pairs (mean ICA. The presence of ICA was established after electrophoresis in a 4.2% agarose age of diagnosis of index twin 19 Ϯ 11 by testing serum with indirect immuno- gel. years, range 2–59; 20 male pairs) in fluorescence on a fresh group O cryofixed which the index twin had a comparable human pancreas (16). Positive tests were INS Ն age at diagnosis to the index twin of the defined as 4 Juvenile Diabetes Founda- INS was studied at the 1,127 Pst I poly- Ј concordant pairs. Nondiabetic twins un- tion units. The ICA assay assessed in the morphic restriction site in the 3 untrans- derwent oral glucose tolerance testing ENDIT workshop (unpublished) had a lated region of the insulin gene and at the Ϫ (glucose was given as 75 g or 1.75 g/kg, sensitivity and specificity of 100%. 23 Hph I polymorphic restriction site whichever was less) to confirm that they (3). The digested and undigested prod- had neither diabetes nor impaired glucose HLA-DQBI typing ucts were visualized by ethidium bromide tolerance both initially and at intervals HLA-DQBI was studied by a polymerase staining after electrophoresis in a 4.2% thereafter; diagnosis of type 1 diabetes chain reaction restriction fragment– agarose gel.