Sequencing-Based Genotyping and Association Analysis of the MICA and MICB Genes in Type 1 Diabetes

Abstract

OBJECTIVE-The nonclassical major histocompatibility complex (MHC) class I chain-related molecules (MICs), encoded within the MHC, function in immunity. The transmembrane polymorphism in MICA (MICA-STR) has been reported to be associated with type 1 diabetes. In this study, we directly sequenced both of the highly polymorphic MIC genes (MICA and MICB) in order to establish whether they are associated with type 1 diabetes independently of the known type 1 diabetes MHC class II genes HLA-DRB1 and HLA-DQB1. RESEARCH DESIGN AND METHODS-We developed a sequencing-based typing method and genotyped MICA and MICB in 818 families (2,944 individuals) with type 1 diabetes from the U.K. and U.S. (constructing the genotype from single nucleotide polymorphisms in exons 2-4 of MICA and 2-5 of MICB) and additionally genotyped the MICA-STR in 2,023 type 1 diabetic case subjects and 1,748 control subjects from the U.K. We analyzed the association of the MICA and MICB alleles and genotypes with type 1 diabetes using regression methods.RESULTS-We identified known MICA and MICB alleles and discovered four new MICB alleles. Based on this large-scale and detailed genotype data, we found no evidence for association of MICA and MICB with type 1 diabetes independently of the MHC class II genes (MICA P ϭ 0.08, MICA-STR P ϭ 0.76, MICB P ϭ 0.03, after conditioning on HLA-DRB1 and HLA-DQB1). A pproximately 50% of the familial clustering of type 1 diabetes is attributable to the major histocompatibility complex (MHC) region on human chromosome 6p21, and the MHC class II genes HLA-DRB1 and HLA-DQB1 account for a large proportion of this clustering (1). However, association studies in the MHC region indicate that other genes have effects in type 1 diabetes independently of the class II genes (2-4). Identification of these genes is complicated because genes in the MHC have multiple alleles and exhibit extensive linkage disequilibrium (LD), and the class II genes HLA-DQB1 and HLA-DRB1 show complicated dominance and epistatic effects. Importantly, any putative new effect must be distinguished from the effect of these class II genes (2,3). CONCLUSIONS-CommonThe nonclassical MHC class I chain-related molecule (MIC) genes MICA and MICB are located 46.4 and 141.2 kb, respectively, centromeric of HLA-B and ϳ2 Mb telomeric of HLA-DRB1 (5) and have been associated both with immunity and with autoimmune diseases (5). In common with most MHC HLA genes, alleles of MICA and MICB are named based on haplotypes of nonsynonymous single nucleotide polymorphisms (nsSNPs). However, the most studied polymorphism in either of these genes is a short tandem repeat (STR) in the transmembrane domain of MICA (MICA-STR). Whether these genes are associated with type 1 diabetes is uncertain, with multiple conflicting reports in the literature (3,6 -14). Most of these studies failed to demonstrate convincingly that the association is independent of the MHC class II genes, and all were restricted to analyses of the MICA-STR. The MICA-STR is a GCT repeat microsatellite in MICA ex...

Association of MICA and MICB alleles with symptomatic dengue infection

“…The same alleles were reported with similar frequencies in other populations with different genetic backgrounds [14,18,19].…”

“…To determine the polymorphism of MICA and MICB, polymerase chain reaction (PCR) amplification of the MICA and MICB genes was carried out in a total volume of 30 L containing 1ϫ buffer, 0.3 and 0.8 mM MgCl 2 , respectively, 0.2 mM dNTPs, 0.5 mM each of forward and reverse primers [14], 0.75 U of Taq polymerase (Takara Bio, Inc., Shiga, Japan), and 150 ng of sample DNA. Cycling was performed under the following conditions: denaturing at 95ЊC for 95 minutes, followed by 6 cycles of denaturing at 95ЊC for 45 seconds, annealing at 65ЊC for 45 seconds, and extension at 72ЊC for 2 minutes; 6 cycles of denaturing 95ЊC for 45 seconds, annealing at 62ЊC for 45 seconds, and extension at 72ЊC for 2 minutes; 6 cycles of denaturing at 95ЊC for 45 seconds and annealing at 58ЊC for 2 minutes; 18 cycles of denaturing at 95ЊC for 45 seconds, annealing at 55ЊC for 45 seconds, and extension at 72ЊC for 2 minutes; and a final extension at 72ЊC for 10 minutes.…”

“…Briefly, sequencing reactions were carried out in a total of 10-L reaction containing 3 L PCR product, 2 L 5ϫ buffer (BigDye Terminator AB, Austin, TX), 2 L of 0.8 pmol primer, 0.25 L of Big Dye V1.1, and 3 L of distillated water. Primers for MICA and MICB have been published previously [14]. Sequencing PCR was performed under the following conditions: 25 cycles of denaturing at 96ЊC for 10 seconds, annealing at 50ЊC for 5 seconds, and extension at 60ЊC for 4 minutes.…”

Correlation of major histocompatibility complex class I related A (MICA) polymorphism with the risk of developing breast cancer

“…Structurally, allele 5.1 contains five triple repeat plus one additional insertion as GGCT/ AGCC and plays an important role in immature production (truncated form) of MICA intracellular protein [18]. The truncated protein lacks transmembrane domain and therefore could be released to cytoplasm which lead to the solubility of MICA protein [11,19]. The soluble MICA instead has been shown unable to flag the malignant cells for natural killer cells and CD8 T cells through NKG2D immune receptors [20].…”

“…Elevated release of MICA protein has been shown to be significantly correlated with malignancies [14]. In relation to the release of soluble MICA protein, two mechanisms have been explained: (1) the involvement of allele 5.1 [11,19] and (2) the activity of metalloproteinase on intracellular MICA protein, by proteolytic removal external domain [21,22]. Recently, Salih and coworkers showed that human tumor cells could spontaneously release a soluble form of MICA, which lacks transmembrane domain of mature MICA protein [13].…”