Estimation of Diabetes Risk in Brazilian Population by Typing for Polymorphisms in HLA-DR-DQ, INS and CTLA-4 Genes

139 Disease Markers 21 (2005) 139–145 IOS Press Estimation of diabetes risk in Brazilian population by typing for polymorphisms in HLA-DR-DQ, INS and CTLA-4 genes Omar M. Hauachea,b, André F. Reisa,b , Carolina S.V. Oliveiraa , José Gilberto H. Vieiraa,b , Minna Sjöroosc and Jorma Ilonend,∗ a Escola Paulista de Medicina/UNIFESP, Sao Paulo, SP, Brazil Fleury Institute, Sao Paulo, SP, Brazil c PerkinElmer Life and Analytical Sciences, Turku, Finland d JDRF Centre for Prevention of Type 1 Diabetes in Finland and Department of Virology, University of Turku, Turku, Finland b Abstract. The study aimed to further characterise HLA encoded risk factors of type 1 diabetes (T1D) in Brazilian population and test the capability of a low resolution full-house DR-DQ typing method to find subjects at diabetes risk. Insulin and CTLA-4 gene polymorphisms were also analysed. The method is based on an initial DQB1 typing supplemented by DQA1 and DR4 subtyping when informative. Increased frequencies of both (DR3)-DQA1*05-DQB1*02 and DRB1*04-DQA1*03-DQB1*0302 haplotypes were detected among patients. DRB1*0401, *0402, *0404 and *0405 alleles were all common in DQB1*0302 haplotypes and associated with T1D. (DRB1*11/12/1303)-DQA1*05-DQB1*0301, (DRB1*01/10)-DQB1*0501, (DRB1*15)-DQB1*0602 and (DRB1*1301)-*0603 haplotypes were significantly decreased among patients. Genotypes with two risk haplotypes or a combination of a susceptibility associated and a neutral haplotype were found in 78 of 126 (61.9%) T1D patients compared to 8 of 75 (10.7%) control subjects (P < 0.0001). Insulin gene −2221 C/T polymorphism was also associated with diabetes risk: CC genotype was found among 83.1% of patients compared to 69.3% of healthy controls (P = 0.0369, OR 1.98) but CTLA-4 gene +49 A/G polymorphism did not significantly differ between patients and controls. Despite the diversity of the Brazilian population the screening sensitivity and specificity of the used method for T1D risk was similar to that obtained in Europe. 1. Introduction Type 1 diabetes is a result of immune mediated destruction of insulin producing beta-cells in pancreatic islets. The central role of HLA region or human MHC associated genetic susceptibility has been known for thirty years and polymorphisms of class II region encoded molecules, especially DQ molecules, seem to be the single most important genetic disease risk determinant. In addition several loci in other chromosomes are contributing to the genetic risk, but only the ef∗ Corresponding author: Jorma Ilonen, University of Turku/ Medicity, Tykistökatu 6A, Turku, Finland. Tel.: +358 2 3337028; Fax: +358 2 3337000; E-mail: . fects of insulin and perhaps CTLA-4 gene region polymorphisms can be considered as firmly established [1]. DQ molecules associated with different strength with both disease risk and protection against it can be discerned, and interaction of the molecules encoded by the genotype is important as demonstrated by a clear dominant effect of strong protective molecules. In addition to HLA-DQ molecules there are other loci within HLA region affecting disease susceptibility. The role of DR molecules on DQ mediated susceptibility has been clearly demonstrated in DQA1*03-DQB1*0302 positive DR4 haplotypes where some particular DR4 subtype encoding alleles are associated with disease risk and others are abolishing the DQA1*03-DQB1*0302 associated susceptibility. There is also evidence for the ISSN 0278-0240/05/$17.00 © 2005 – IOS Press and the authors. All rights reserved 140 O.M. Hauache et al. / Estimation of diabetes risk in Brazilian population by typing for polymorphisms role of class I alleles or loci in close vicinity in certain haplotypes as well as multiple other loci like DP, TNF region and a locus telomeric to HLA close to D6S2223 microsatellite marker [2–6]. MHC is the most polymorphic mammalian gene region and gene density there is extremely high. Complete high-resolution typing for HLA region is practically impossible, although much effort is made in tissue transplantation for exact matching of the donor recipient pairs for all molecules important in possible induction of immunological rejection. Estimation of HLA related genetic risk for type 1 diabetes is at the moment used for selection of subjects for clinical prevention studies and studies aiming to identify environmental factors contributing to the betacell damaging autoimmune process [7,8], but in future the identification of individuals at risk might be part of clinical routine if preventive measures will be developed. Study subjects in these projects have been either selected among first degree relatives or among general population. In the latter case the need for a simple and inexpensive screening protocol is accentuated as great number of samples must be processed. In addition to the huge polymorphism of HLA gene region it is also characterised by strong linkage disequilibrium, which makes testing for disease risk associated determinants more simple. Recombinations between DRB1, DQA1 and DQB1 genes are extremely rare and alleles in these loci are found in fixed combinations. Variability in major allele combinations still exists between various populations due to some ancient recombinatory events. Definition of either DRB1 or DQB1 alleles is in several haplotypes thus enough for low resolution typing, whereas in other cases more diverse combinations exist. For purposes of diabetes screening an emphasis can be put on polymorphisms known to be relevant for disease susceptibility. We have developed a semiautomated sequence specific oligonucleotide hybridisation method for diabetes screening based on microtitre plate format and timeresolved fluorometry reading of hybridisation reactions of lanthanide chelate labelled oligonucleotides. PCR amplification of gene regions of interest directly from punched pieces of dried blood spots makes DNA extraction unnecessary and use of various lanthanide labels allows simultaneous measurement of three different signals in each microtitre plate well. The method was originally developed for testing the presence of T1D associated DQB1*02 and DQB1*0302 as well as protective DQB1*0301 and DQB1*0602/3 alleles [9] and was used in the Finnish Diabetes Prediction and Prevention (DIPP) study [7]. It has later on been applied also for typing DQA1 alleles informative for diabetes risk when used in combination with DQB1 data and also for relevant DR4 subtypes [10]. These additional steps have been shown to increase the sensitivity and specificity of risk estimation considerably especially in more diverse Greek and Hungarian populations, whereas the effect in the less diverse Finnish populations was only modest [11]. Addition of a series of new DQB1 probes made possible a low-resolution full-house genotyping of all common Caucasian haplotypes [12,13]. An assay based on similar technology has also been developed for typing of insulin gene and CTLA-4 polymorphisms [1 (...truncated)