ASSESSMENT OF THE RISK FOR TYPE 1 DIABETES MELLITUS CONFERRED BY HLA CLASS II GENES Summary Irina Durbală CELL AND MOLECULAR BIOLOGY DEPARTMENT FACULTY OF MEDICINE, OVIDIUS UNIVERSITY CONSTANŢA Class II HLA genes, particularly HLA-DRB1 and DQB1 genes, are well established risk genes for type 1 diabetes mellitus and other autoimmune diseases. In type 1 diabetes mellitus, however, both susceptible and protective alleles have been described, influencing the development of autoimmunity and progression to overt diabetes. This study determines the class II genes in a group of children with type 1 diabetes mellitus registered at the Pediatric Clinic of the Emergency Clinical Hospital Constanţa and their healthy first degree relatives. The method used for the assignment of alleles at HLA-DRB1 and DQB1 loci was molecular genotyping, primarily by the sequence specific oligonucleotide hybridization method, and when required, by the sequence specific primers method. The presence of different alleles in patients and control groups has been analyzed for statistical significance and predisposing and protective alleles have been defined. Keywords: class II HLA genes, type 1 diabetes, predisposing and protective alleles. Introduction Insulin-dependent diabetes mellitus (IDDM), or type 1 diabetes, is a chronic disease characterized by the autoimmune destruction of pancreatic ß-cells and severe insulin deficiency (Gavin et al., 1992). Type 1 diabetes frequently develops in children, adolescents and young adults. Inherited genetic factors influence both susceptibility to and resistance to the disease. Association studies and linkage analysis have been used to identify IDDM susceptibility loci. These are conventionally noted using the abbreviation IDDM and a number, e.g. IDDM1, IDDM2, etc., with the number usually reflecting the order in which such loci were reported. Using the candidate gene approach, association studies provided evidence for the first two susceptibility loci, the HLA region (IDDM1) and the insulin gene (INS) locus (IDDM2). These two loci contribute the great majority of familial clustering; MHC alone is estimated to contribute 41% of the familial clustering of type 1 diabetes of the 48% estimated to be accounted for with all known susceptibility genes (Cox et al., 2001; Steck et al., 2007). 123 The major locus for type 1 diabetes susceptibility is located within the HLA (Human Leukocyte Antigen) region on the short arm of chromosome 6. In this region, HLA class II genes (especially DQ and DR) were shown to have the strongest association with the disease, with both susceptible and protective alleles at the DRB1 and DQB1 loci. Both loci are highly polymorphic, but only few of the alleles are demonstrated to be associated with type 1 diabetes. There were found susceptible alleles encoding for the antigens DR3, DR4, DQ2 and DQ8, and protective alleles encoding for DR2 (DR15), DR6 (DR11), DQ6 and DQ7 (Kelly et al., 2003). Our country has the lowest incidence of type 1 diabetes in Europe (4 ), almost 10 times lower than Finland which has the highest incidence (Karvonen et al., 2000). Since the environmental factors are not so different between various regions in Europe, it appears that this difference in incidence is due to difference in genetic background in European populations. Previous studies reported lower frequencies of diabetogenic alleles in Romanian populations, providing an
explanation for the lower incidence of type 1diabetes in Romania (Guja et al., 2004). This work aimed to analyze the distribution of HLA-DRB1 and DQB1 alleles in a group of children with type 1 diabetes from Constanţa County. Materials and methods The subjects included in this study comprise 62 patients with type 1 diabetes registered at the Pediatric Clinic of the Emergency Clinical Hospital Constanţa and 59 healthy subjects, first degree relatives of the patients parents and siblings. All the patients were diagnosed with type 1 diabetes based on the onset with ketoacidosis and/or required insulin treatment from the first month since diagnosis. Genotyping methods. Total genomic DNA was extracted from EDTAanticoagulated venous blood with the QIAmp DNA Blood Mini Kit. HLA genotyping was performed with PCR-based sequence-specific oligonucleotide probe assays, the RELI SSO HLA typing systems from Invitrogen, and the ambiguities were solved with two sequence-specific primers systems, Domino HLA SSP System from Protrans and AllSet Gold SSP System from Invitrogen. The sequence-specific oligonucleotide probe assay was based on oligonucleotide probes corresponding to known polymorphic sequence motifs in second exon of HLA-DRB1 and HLA- DQB1genes immobilized onto a nylon strip. The relevant regions of the HLA genes were amplified with byotinilated primers, denatured and hybridized to the immobilized probe array. a) b) Figure 1. a) Image of a batch of strips after the completion of the RELI SSO HLA-DRB1 assay. 17 amplifications followed by colorimetric detection were run simultaneously. b) Interpretation of the result the strip from the image shows the following specificities: 3, 4, 20, 22, 25, 27, 36, 37, 41, 45, 48, 59, 60 and the genotype assigned by the Pattern Matching Program were DRB1*0401 & 1101. 124
After hybridization and wash, strips were incubated with streptavidinhorseradish peroxidase, followed by a chromogenic substrate (figure 1a). The results of the colorimetric reactions were analyzed with the Pattern Matching Program v5.42 from Invitrogen and the HLA-DRB1 and HLA-DQB1alleles were assigned (figure 1b). The sequence-specific primers assays were based on the amplification of the target regions with 20 to 96 primer pairs complementary to known polymorphisms in the target genes. If one. polymorphism is present in the sequence of the gene, the amplicon is synthesized in the corresponding reaction tube. All the 20 to 96 reactions were then analyzed on an agarose gel electrophoresis system, and the presence or absence of the amplicons was demonstrated by ethidium bromide staining on a UV transillumination system (figure 2). The images were digitally captured and analyzed with the UniMatch software for Invitrogen and SCORE software for the Protrans systems, respectively. Figure 2. Image of four amplification reactions migrated simultaneously on a 96-well agarose gel. The targeted gene is HLA-DRB1, and the system used is Protrans HLA-DRB1*03 high resolution. The test employs 19 different pair of primers and a negative control reaction. The first reaction was used to solve the following ambiguous DRB1 result from the SSO assay: DRB1*0301 & 0402 or DRB1*0422 & 1327. The positive lanes are 1, 3, 4, 5, 7 and 16 and the *0301 allele was assigned based on the amplification pattern. This solved the ambiguity, and the genotype assigned was DRB1*0301 & 0402. 125
Statistical analysis. The study was designed as an association study with affected family-based controls. This control population provide an unbiased estimate of the overall population HLA alleles, under the assumption of zero recombination between the marker an the disease loci. The statistical significance of differences in allele/genotype frequencies between type 1 diabetes cases and controls was assessed using a χ 2 test. Bonferoni correction for multiple comparisons has been applied to p values 0.05 and corrected p values (p c ) 0.05 have been considered statistically significant. Results and discussion The frequencies of the main genotypes, and HLA-DRB1 and DQB1 alleles are presented in tables I, II and III, respectively. As expected, frequency of the high-risk genotype DR3/DR4 was significantly higher in patients than in controls (p c <10 3 ), while the genotypes non-harboring high-risk genes were better represented in controls (p c <0.01). There was no difference between patients and controls where the moderate risk genotype DR4/Y and the mild risk genotype DR3/X were concerned. The frequency of the highrisk genotype in diabetics (30.64%) was close to that found in other studies in Caucasian populations (Erlich et al., 2008). Table I. Frequencies of high-risk, moderate-risk and low-risk genotypes in diabetic patients and controls. Genotype Pacients Controls χ 2 p value p c no. freq. no. freq. DR3/DR4 * 19 30.64 2 3.39 15.66 7.59 10 5 3.04 10 4 DR3/X 17 27.42 17 28.81 0.029 0.86 DR4/Y 23 37.1 24 40.68 0.16 0.69 nondr3/nondr4 3 4.84 16 27.12 11.34 7.59 10 4 3.04 10 3 * Association with the disease (either positive or negative) is shown in bold characters. The analysis of the DRB1 alleles frequencies showed DRB1*0301 as a predisposing allele (p c <0.025), but failed to demonstrate DR4 alleles as a predisposing alleles due to the overrepresentation of the DR4 non-transmitted alleles in our control group. DR11 and DRB1*1104 were demonstrated to be protective, being significantly better represented in controls than in patients (p c <0.01). Other known DRB1 protective alleles, such as *1501 and *1401, although having higher frequency in diabetics than in controls, failed to reach statistical significance because of the low number of cases. DQ2 group showed a significantly higher frequency in patients than in controls (p c <0.013) due to a higher representation of DQB1 alleles *0201 and *0202. However, these alleles failed to reach the statistical significance independently, which is a surprising finding since DQB1*0201 in known to be in linkage disequilibrium with DR3 alleles and we expected their representation to parallel that of DR3. The analysis of the haplotypes in controls showed the presence of DQB1*0201 on three untransmitted DRB1*0701/DQB1*0201haplotypes from the control group, and this accounted for a 126
slightly higher frequency than expected of DQB1*0201 in controls, which explained the why DQB1*0201 failed to be assigned as a predisposing. DQB1*0302, known also as DQ8, is in linkage disequilibrium with DR4 alleles and, similarly, failed to be assigned as a predisposing allele. We demonstrated DQ7 group and DQB1*0301 (an allele of this group) as being protective for the disease, having significantly higher frequencies in control than in the patients group (p c <10 3 in both cases). Table II. Frequencies of DRB1 alleles and groups of alleles in diabetic patients and controls. /locus Pacients Controls χ 2 p value p c no. freq. no. freq. DRB1 01 6 4.84 8 6.78 0.42 0.51 0101 * 4 3.23 7 5.93 1.02 0.31 0102 2 1.61 1 0.85 0.29 0.59 0301 41 33.06 19 16.10 9.33 0.0023 0.025 04 49 39.51 30 25.42 5.46 0.019 0.214 0401 21 16.94 14 11.86 1.26 0.26 0402 10 8.06 6 5.08 0.87 0.35 0403 2 1.61 1 0.85 0.29 0.59 0404 6 4.84 2 1.69 1.87 0.17 0405 7 5.74 2 1.69 2.63 0.10 0407 2 1.61 3 2.54 0.26 0.61 0408 1 0.81 2 1.69 0.39 0.53 0701 1 0.81 3 2.54 1.12 0.29 0801 1 0.81 1 0.85 0.001 0.97 0901 2 1.61 1 0.85 0.29 0.59 11 2 1.61 19 16.10 16.02 6.28 10 5 6.91 10 4 1101 1 0.81 5 4.24 2.94 0.086 1104 1 0.81 14 11.86 12.72 3.62 10 4 4.35 10 3 13 6 4.84 12 10.17 3.95 0.047 0.517 1301 4 3.23 9 7.63 3.86 0.049 0.588 1302 2 1.61 3 2.54 0.26 0.61 1401 2 1.61 6 5.08 2.28 0.13 1501 1 0.81 4 3.39 1.99 0.16 1601 13 10.48 15 12.71 0.29 0.59 * Individual alleles are shown in italic characters Association with the disease (either positive or negative) is shown in bold characters. This paralleled the presence of the DR11 alleles which are in linkage disequilibrium with DQB1*0301, although the later were also found on DR4 haplotypes. DQB1*0602 known to confer dominant protection for type 1 diabetes 127 (Ettinger and Kwok, 2008), failed to be demonstrated as protective in this dataset because of the low number of cases, similar with DRB1*1501 which has been found on the same haplotypes.
Table III. Frequencies of DQB1 alleles and groups of alleles in diabetic patients and controls. /locus Pacients Controls χ 2 p value p c no. freq. no. freq. DQB1 DQ2 * 45 36.29 23 19.49 9.40 0.0022 0.013 0201 40 32.26 22 18.64 5.88 0.015 0.105 0202 5 4.03 1 0.85 2.54 0.11 DQ7 6 4.84 25 21.19 14.47 1.43 10 4 8.56 10 4 0301 4 3.23 22 18.64 14.99 1.08 10 4 7.56 10 4 0304 2 1.61 3 2.54 0.26 0.61 DQ8 0302 43 34.68 24 20.34 6.21 0.013 0.076 DQ9 0303 2 1.61 1 0.85 0.29 0.59 DQ5 21 16.94 29 24.58 2.15 0.14 0501 6 4.84 8 6.78 0.42 0.41 0502 13 10.48 15 12.71 0.29 0.59 0503 2 1.61 6 5.08 2.28 0.13 DQ6 7 5.65 16 13.56 4.40 0.036 0.216 0602 1 0.81 4 3.39 1.99 0.16 0603 4 3.23 9 7.63 2.30 0.13 0604 2 1.61 3 2.54 0.26 0.61 * Association with the disease (either positive or negative) is shown in bold characters. Individual alleles are shown in italic characters. Overall, the frequencies of the alleles and genotypes showed concordance with the most recent studies that analyzed HLA-DRB1 and DQB1 alleles in type 1 diabetes. One previous Romanian study demonstrated a lower representation than expected of high-risk DR3/DR4 genotypes (21% compared with over 30% cited by other authors) (Guja et al., 2004; Erlich et al., 2008), but that finding was not reproduced here. We found an overrepresentation of the DR4 alleles not only in patients, but also in controls (39.51% and 25.42% respectively) which represents a deviation from the standard frequency of 16.90% for Caucasian populations (Klitz et al., 2003); this fact explains why the positive association of DR4 alleles with type 1 diabetes could not be demonstrated on this dataset. 128 Conclusion This study demonstrated the positive association of DRB1*0301 allele as well as negative association of DRB1*1104/DQB1*0301 haplotypes with type 1 diabetes. We failed to demonstrate association with the disease for other known susceptible and protective alleles, fact that is probably due, when such polymorphic loci were involved, to the relatively low number of cases analyzed. References Cox, N.J., Wapelhorst, B., Morrison, V.A., Johnson, L., Pinchuk, L., Spielman, R.S., et al.: Seven regions of the genome show evidence of linkage to type 1 diabetes in a consensus analysis of 767 multiplex families. Am. J. Hum. Genet., 69, 820-830, 2001 Erlich, H., Valdes, A.M., Noble, J., Carlson, J.A., Varney, M., Concannon, P., et al.: HLA DR-DQ
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