European Journal of Clinical Investigation (2004) 34, 543–548 © 2004 Blackwell Publishing Ltd Blackwell Publishing, Ltd. Type 1 diabetes is insulin -2221 MspI and CTLA-4 +49 A / G polymorphism dependent K. Haller * , K. Kisand * , V. Nemvalts † , A.-P. Laine ‡ , J. Ilonen ‡ and R. Uibo * * University of Tartu, Tartu, Estonia, † Kuressaare Hospital, Kuressaare, Estonia, ‡ University of Turku, Turku, Finland Abstract Background Several studies have demonstrated an association of type 1 diabetes with specific alleles of HLA class II molecules, as with polymorphisms of insulin gene region. The aim of our study was to evaluate the interaction of insulin -2221 MspI polymorphism to type 1 diabetes susceptibility in connection with autoimmunity associated gene – CTLA- 4 polymorphism. Materials and methods Insulin - 2221 MspI C / T and CTLA-4 +49 A / G polymorphisms were detected by restriction fragment-length polymorphism analysis or oligonucleotide hybridization in type 1 (n = 69), type 2 diabetes (n = 301) patients and 158 healthy controls. Regression model adjusted for age, gender and gene polymorphisms was studied. Results C-allele of insulin -2221 MspI and G-allele of +49 CTLA-4 were significant risk factors for type 1 diabetes (crude OR 3·53 and 1·59, respectively) and this impact increased in the homozygous form of both alleles. The regression model supported the idea of insulin CC and CTLA-4 GG genotypes for an independent and clearly significant risk for developing type 1 diabetes. We could not detect any significant correlation between investigated poly- morphisms and type 2 diabetes. Conclusions There exists a significant association between the C-allele of - 2221 MspI in the insulin gene and type 1 diabetes. The CTLA-4 G-allele is also positively correlated with type 1 diabetes. According to the regression model the investigated gene polymorphisms are independent risk factors for development of type 1 diabetes in the Estonian population. We propose that - 2221 MspI is a good marker for evaluation of risk of insulin gene haplotype in type 1 diabetes patients. Keywords Case – control study, CTLA-4 gene, Estonian population, insulin gene, type 1 diabetes. Eur J Clin Invest 2004; 34 (8): 543–548 Introduction Diabetes mellitus represents a heterogeneous group of dis- orders with a multifactorial origin involving the interaction of environmental exposures with the genetic make-up of the individual. It has been estimated that approximately 60% of the genetic susceptibility to type 1 diabetes (T1D) is conferred by HLA class II molecules [1]. However, there are also other genes that may have an effect on the pathogenesis and development of diabetes. Polygenic susceptibility to diabetes includes two important gene regions: insulin (11p15) and the cytotoxic T-lymphocyte antigen-4 (CTLA-4) (2q33). Ten different polymorphisms have been described in the insulin gene [2–4]. These polymorphisms are all in strong linkage disequilibrium [3] spanning a restricted number of insulin haplotypes [2]. The - 2221 MspI at the promoter region has been shown to be in strong linkage with three main haplotypes of insulin associated with T1D susceptibility in Caucasian populations [2]. CTLA-4 gene also contains several polymorphisms [5,6], among which the +49 A / G is the only SNP resulting from substitution of an amino acid in the peptide, thereby influ- encing the structure of the CTLA-4 molecule [7]. This polymorphism has been demonstrated to associate with development of T1D [3,8–19] and other autoimmune disorders [7,20–22]. It has been speculated that polymorphisms of the insulin gene influence insulin expression in the pancreas and thymus Department of Immunology, Institute of General and Molecular Pathology, Centre of Molecular and Clinical Medicine, University of Tartu, Tartu, Estonia (K. Haller, K. Kisand, R. Uibo); Kuressaare Hospital, Kuressaare, Estonia (V. Nemvalts); Department of Virology, University of Turku, Turku, Finland (A.-P. Laine, J. Ilonen). Correspondence to: Raivo Uibo, Ravila str.19, Biomedicum, Tartu 50411, Estonia. Tel.: +372 7 374231; fax: +372 7 374232; e-mail: raivo.uibo@ut.ee Received 21 May 2004; accepted 28 June 2004