MICB Alleles Research Articles

Characterization of the major histocompatibility complex class I chain-related gene B (MICB) polymorphism in a northern Chinese Han population: The identification of a new MICB allele, MICB*023

Major histocompatibility complex class I chain-related gene B (MICB) has only been characterized for allelic variation in very few human populations. The MICB polymorphism remains largely unknown in Chinese populations. In this study, 104 healthy unrelated Han subjects recruited from central Inner Mongolia Autonomous Region, northern China, were investigated by sequence-based typing for MICB allelic variation, the association of MICB alleles with AluyMICB insertion/deletion dimorphism located in MICB intron 1, linkage disequilibrium of MICB with human leukocyte antigen (HLA)-B and MICA, and HLA-A-C-B-MICA-MICB haplotypic diversity. Ten kinds of MICB alleles were observed, among which MICB*005:02/010, MICB*002:01, and MICB*004:01 were the most frequent alleles with frequencies of 51.44, 16.35, and 11.54%, respectively. Significant linkage disequilibrium (LD) was observed for 9 of the 21 HLA-B-MICB haplotypes and 6 of the 17 MICA-MICB haplotypes with a frequency >1.5%. In particular, HLA-B*13:01 and HLA-B*13:02, both of which were frequently represented in this population, exhibited a distinct LD pattern with the MICB allele. A new MICB allele, MICB*023, was identified, which differed from MICB*005:02/010 by a single mutation of G to A at position 86 in exon 2, resulting in an amino acid change from arginine to histidine at codon 6. HLA-A*30-C*06-B*13:02-MICA*008:01-MICB*005:02/010 was the most common haplotype, with a frequency of 8.64% in this population. HLA-A*02-C*08-B*48-MICA*Del-MICB*009N demonstrated a frequency of 2.4% in this population. Our results provide for the first time data regarding the MICB genetic polymorphism in northern Chinese Han populations and will form the basis for future studies of the potential role of MICB in allogeneic organ transplantation and disease association in related ethnic groups.

Association study of MICA and MICB in Alzheimer’s disease

Following the replication of the association of the human leucocyte antigen (HLA) allele, HLA-B*07, with Alzheimer's disease (AD) in the cohort of the Oxford Project to Investigate Memory and Ageing (OPTIMA) in a previous study, we examined whether that association could be due to linkage disequilibrium with MICA or MICB alleles. We found a possible association of MICA*00801 heterozygotes with AD in subjects positive for the epsilon 4 allele of apolipoprotein E. This finding was supported by Hardy-Weinberg analysis, by stratified association analysis and by interaction analysis, but did not survive correction for multiple testing. In any case, these results do not explain our previously reported association of HLA-B*07 with AD.

Allelic Variation within the NKG2DL COMPLEX Influences Event Free Survival Following Stem Cell Transplantation.

NKG2D, an important activating receptor on NK cells, is capable of transmitting costimulatory signals by certain subsets of T cells. Viruses and tumours have evolved strategies to evade the NKG2D-mediated immune recognition thus illustrating the importance of NKG2D in mediating anti-tumour and anti-viral responses. Although NKG2D is a highly conserved molecule, it interacts with several structurally diverse ligands that are upregulated by cellular stress. The currently identified Ligands (NKG2DL) are the MHC class I-chain related proteins A and B (MICA and -B) and the ULBP/RAET1 families. MICA and MICB are structurally similar to MHC class I proteins, but share only 27% identity and do not associate with β2-microglobulin or bind peptides. MICA and MICB are very polymorphic with more than 50 MICA and 13 MICB alleles so far reported. Some of their alleles have been shown to be associated with auto-immune diseases such as Behcet's disease and psoriasis vulgaris. Furthermore, there are currently six identified ULBPs and RAET genes, which are related to MHC class I in their α1 and α 2 domains, but lack the α3 domain and are located in a linked cluster on the long arm of chromosome 6. These proteins are closely related and polymorphic; however, the functional significance of this polymorphism remains unclear. We developed a novel polymerase chain reaction sequence specific primer (PCR-SSP) method for typing NKG2DL using genomic DNA and newly designed primers. First, we established the population genetics of 29 SNPs of NKG2DL, both in the promotor and coding regions in a control population of 223 Healthy controls and confirmed the presence of 24 of these SNPs. For our clinical studies we have limited the analysis to 10 NKG2DL SNPs with allele frequencies of at least 10%, which were used in subsequent analysis of association with clinical outcome of allogeneic stem cell transplantation. The allele and haplotype frequencies were determined in 211 patient and donor pairs who had undergone sibling stem cell transplantation (SSCT) for a range of diseases. Interestingly, a strong association between event free survival (EFS) and a specific SNP within this group of ligands was observed, which resulted in greater than a 25% difference in Event Free Survival at 6 years between its two allelic forms, (p=0.001). Initial analysis of the effect of matching of the SNPs between donor and recipient does not indicate any clear association with SSCT outcome. This is the first study of genetic variation within NKG2D ligands and effect on stem cell transplantation outcome and the findings of the significant associations of their SNPs with EFS indicates a critical role for the NKG2D-ligand interactions with transplant outcome.

Human microRNAs regulate stress-induced immune responses mediated by the receptor NKG2D

MICA and MICB are stress-induced ligands recognized by the activating receptor NKG2D. A microRNA encoded by human cytomegalovirus downregulates MICB expression by targeting a specific site in the MICB 3' untranslated region. As this site is conserved among different MICB alleles and a similar site exists in the MICA 3' untranslated region, we speculated that these sites are targeted by cellular microRNAs. Here we identified microRNAs that bound to these MICA and MICB 3' untranslated region sequences and obtained data suggesting that these microRNAs maintain expression of MICA and MICB protein under a certain threshold and facilitate acute upregulation of MICA and MICB during cellular stress. These microRNAs were overexpressed in various tumors and we demonstrate here that they aided tumor avoidance of immune recognition.

Associations of MICB with cervical cancer in north-eastern Thais: identification of major histocompatibility complex class I chain-related gene B motifs influencing natural killer cell activation

The expression of MICB, a member of the major histocompatibility complex class I chain-related gene B family, is induced in response to cellular stress. It is one of the ligands to the NKG2D receptor. MICB is polymorphic, but the distribution of MICB polymorphism in north-eastern Thais and their potential associations with cancer have not yet been elucidated. In this study, polymerase chain reaction-sequence-specific primers were developed to identify 15 MICB alleles and one group of alleles. We performed MICB typing in 100 healthy north-eastern Thai females (NETF) and 99 cervical cancer patients to evaluate the association of MICB polymorphisms and the risk of developing cervical cancer. Eight and nine alleles were detected in the NETF and cervical cancer respectively. MICB*00502 was associated negatively with a corrected P-value of 0.0009, suggesting the existence of a protective allele in cervical cancer. Amino acid substitutions carried by this allele were investigated for their potential involvement in natural killer (NK) cell activation. Although lysine at amino acid position 80 (Lys80) and aspartic acid at position 136 (Asp136) were associated negatively with cervical cancer, only MICB carrying Asp136 could induce NK cell killing more efficiently than MICB-Lys80 when the NK cells were blocked by anti-NKG2D. This result suggested that aspartic acid at position 136 may affect NKG2D binding, leading to different degrees of immune cell activation.

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

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. 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. 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 = 0.08, MICA-STR P = 0.76, MICB P = 0.03, after conditioning on HLA-DRB1 and HLA-DQB1). Common MICA and MICB genetic variations including the MICA-STR are not associated, in a primary way, with susceptibility to type 1 diabetes.

MHC Class I Chain-Related Gene B Promoter Polymorphisms and Celiac Disease

The possibility that susceptibility to celiac disease (CD) might be influenced by the MHC class I chain-related gene family, MICA and MICB, has been previously reported. In this study, we analyzed the MICB promoter and examined the association of the polymorphisms found within such in a group of CD patients. To study the MICB promoter we sequenced the 5′ flanking region of MICB gene in DNA from homozygous B-lymphoblastoid cell lines corresponding to the most frequent MICB alleles found in our population (MICB*00502, MICB*002, MICB*004, and MICB*008). DNA from a MICB*003 homozygous individual was also analyzed. Sequence analysis revealed six single nucleotide polymorphisms located at positions 45860 C/A, 45862 G/C, 45877 C/G, 46113 A/C, 46219 G/C, and 46286 G/C and an insertion of 2 bp --/AG at position 45944 according to the published genomic sequence. Those polymorphisms were found to be associated in four different haplotypes corresponding to different MICB alleles. Subsequently, 126 CD subjects and 117 healthy controls were typed by polymerase chain reaction using sequence-specific primers for these polymorphisms. MICB promoter polymorphism haplotypes were also found in our population and showed strong linkage disequilibrium with MICB alleles. MICB promoter polymorphism Haplotype 3, included in MICB*002 and MICB*008 alleles, was found to be overrepresented in CD patients (79.4% CD patients vs 45.3% healthy controls; p c < 0.0001; OR = 4.64; CI 95% = 2.64–8.16). Both MICB*008 and MICB*002 alleles were found as part of the CD susceptibility extended haplotypes B8/DR3/DQ2, B18/DR3/DQ2, and DR4/DQ8.

Associations of major histocompatibility complex class I chain‐related molecule polymorphisms with Behcet's disease in Caucasian patients

HLA-B*51 is known to be associated with Behcet's disease (BD) in many ethnic groups. The pathogenic gene, however, may lie close to the HLA-B locus and therefore be in linkage disequilibrium with HLA-B*51. On the basis of the proximity of MIC genes to HLA-B, their expression pattern and their affinity for the activating NKG2D receptor on natural killer (NK) cells and gammadelta T cells, these molecules have been postulated as susceptibility factors in BD. DNA from 56 western European Caucasians with BD and 90 Caucasian controls were analysed by polymerase chain reaction using allele-specific primers for MICA and MICB alleles. An increased allele frequency of MICA*009 was found in the BD patient group (25.0%) when compared with the controls (7.2%). This was associated with a corresponding decrease in MICA*008 in the BD patients (36.6%) compared with the controls (46.7%), which was not significant. MICA*009 was strongly associated with the presence of HLA-B*51 in patients and controls. No significant difference in frequency of MICB alleles was found between patients and controls. Both HLA-B*51 and MICA*009 are strongly associated with BD in a pure Caucasian BD patient group, and the two alleles are in linkage disequilibrium. No MICB allele was found to associate significantly with the disease, an unexpected finding considering the close proximity of the MICA and MICB loci. Our results suggest that while MICB does not influence the development of BD, polymorphisms in MICA may be pathogenic, perhaps through the interaction with NK and gammadelta T cells.

A Typing System for the Major Histocompatibility Complex Class I Chain Related Genes A and B Using Polymerase Chain Reaction with Sequence-Specific Primers

The Major Histocompatibility Complex (MHC) class I chain related (MIC) A and B genes are important additional loci within the MHC. We have developed a MICA and MICB typing system using the polymerase chain reaction with sequence-specific primers (PCR-SSP), which operates under the same conditions as our routine HLA-A, -B, and -C typing method. We designed 95 primers in 84 SSP mixtures for MICA and 39 primers in 29 mixtures for MICB. This detected and differentiated all 55 MICA and 19 MICB alleles (except MICA*00701 from MICA*026, MICA*00201 from MICA*020, and three MICB alleles, which are intronic variations). A computer program confirmed the MICA amplification reactivity of each SSP mixture and evaluated the typing set for MICA allele combination ambiguities. Seventy-six "reference" DNA samples were used for validation: 50 from International Histocompatibility Workshop B lymphoblastoid cell lines (IHW BCLs) and 26 MICA-typed samples from two laboratories. The reference material identified 28 out of the 55 MICA alleles and 13 of the 19 MICB alleles, and directly validated 62 of the 84 MICA and 20 of the 29 MICB SSP mixtures. Our genotyping agreed with 283 out of the 286 (98.95%) MICA and MICB reference laboratories' allele assignments or the consensus assignments. Two of the discrepancies remain unresolved, whereas one was probably due to a reference laboratory's failure to differentiate alleles differing in exon 5 of the MICA gene. A comparison of the MICA and MICB allele assignments between laboratories identified a "disagreement rate" of 19.4% for MICA alleles and 13.1% for MICB alleles. Accordingly, we have compiled "consensus" MICA and MICB genotypes for the 50 IHW BCLs tested, which have been confirmed by our typing. We also typed 166 random blood donors. Their MICA and MICB carriage and allele frequencies and HLA-B, MICA, MICB linkage disequilibrium parameters and haplotype frequencies largely concurred with other published data on United Kingdom subjects, further supporting the validity of our typing system. This PCR-SSP system is a simple, reliable and rapid technique for typing MICA and MICB alleles. It is easily updated as new alleles are identified but clearly requires a continuing validation review until all known MICA and MICB alleles have been identified.

Nomenclature for factors of the HLA system, 2004

Nomenclature for factors of the HLA system, 2004

Eight novel MICB alleles, including a null allele, identified in gastric MALT lymphoma patients

Eight novel MICB alleles, including a null allele, identified in gastric MALT lymphoma patients

Eight novel MICB alleles, including a null allele, identified in gastric MALT lymphoma patients.

MICA and MICB, as members of the major histocompatibility complex (MHC) class I-chain-related genes (MIC), encode stress-inducible glycoproteins that act as activating ligands for NKG2D and gammadelta T-cell receptor-bearing cells. We here describe the identification of eight novel MICB variants, including a null allele, which were identified in peripheral blood leukocytes of gastric MALT lymphoma patients. Only two of the novel alleles are characterized by point mutations, whereas the other variants display a recombination of known exonic MICB sequences that may be best explained by intragenic conversions. The novel MICB null allele is characterized by a Cytosin (C) deletion in a stretch of four Cs beginning from nucleotide 135 of exon 2 that leads to a premature stop codon (TGA) at codon 66.

Association of MHC dimorphic Alu insertions with HLA class I and MIC genes in Japanese HLA-B48 haplotypes.

A large proportion of Japanese with the HLA-B48 allele have a MICA gene deletion associated with a MICB null allele within the class I region of the Major Histocompatibility Complex (MHC). Here, we report for the first time a novel positive association between the presence of a polymorphic Alu insertion, AluyMICB, within the first intron of the MICB gene and the MICAdel/MICBnull/HLA-B48 haplotype for five of six well-characterized Japanese cell-lines. The AluyMICB insertion was found to be present at a frequency of 0.242 in 86 Japanese tissue donors and in four of the five individuals with the HLA-B48 allele. The AluyMICB insertion was also associated with at least three different MICB alleles, *0102, *0107N and *0105, and three different HLA-B alleles, B13, B48 and B57, respectively, in the seven Workshop cell-lines (the 4th Asia-Oceania Histocompatibility Workshop, and the 10th International Histocompatibility Workshop) and the six Japanese cell-lines that were selected for this study. Based on the analysis of associations between different polymorphic markers within the beta block, the MICB*0102 allele was inferred to be the ancestral form of the MICB*0105 and MICB*0107N alleles. The AluyMICB polymorphism can now be used to further investigate its relationship with other MICB alleles and consequently their origins. In addition, we have examined the absence and presence of three other polymorphic Alu markers distributed within the alpha block of the class I region of the HLA-B48/AluyMICB haplotype. We conclude that the extended HLA-B haplotypes are best defined by considering multiple genomic sites including the four polymorphic Alu insertions described in this study.

Possible polyphyletic origin of major histocompatibility complex class I chain-related gene A (MICA) alleles.

Phylogenetic relationships among 23 nonhuman primate (NHP) major histocompatibility complex class I chain-related gene (MIC) sequences, 54 confirmed human MICA alleles, and 16 human MICE alleles were constructed with methods of sequence analysis. Topology of the phylogenetic tree showed separation between NHP MICs and human MICs. For human MICs, the topology indicated monophyly for the MICB alleles, while MICA alleles were separated into two lineages, LI and LII. Of these, LI MICA alleles shared a common ancestry with gorilla (Ggo) MIC. One conservative amino acid difference and two nonconservative amino acid differences in the alpha3 domain were found between the MICA lineages. The nonconservative amino acid differences might imply structural and functional differences. Transmembrane (TM) trinucleotide-repeat variants were found to be specific to the MICA lineages such as A4, A9, and A10 to LI and A5 to LII. Variants such as A5.1 and A6 were commonly found in both MICA lineages. Based on these analyses, we postulate a polyphyletic origin for MICA alleles and their division into two lineages, LI and LII. As such, there would be 30 alleles in LI and 24 alleles in LII, thereby reducing the current level of polymorphism that exists, based on a presumed monophyletic origin. The lower degree of polymorphism in MICA would then be in line with the rest of the human major histocompatibility complex nonclassical class I genes.

MICB typing by PCR amplification with sequence specific primers.

MICB is a member of the MIC (MHC class I chain-related gene) family. Sixteen MICB alleles have been described; however, the functional relevance and population distribution of MICB alleles or their potential association to disease has not yet been evaluated. In this study, we have developed a PCR system using sequence-specific primers (PCR-SSP) that allows unambiguous amplification of all MICB alleles. This approach has been applied to type 100 healthy unrelated individuals from the Spanish population. The extent of polymorphism in this population is lower than that initially expected, and only nine alleles were detected. The alleles MICB01021 (46%), MICB0103101 (13.5%), MICB0104 (13.5%) and MICB0106 (12.5%) were found to be the most frequent alleles. HLA-B and MICA transmembrane polymorphism typing were also performed in this population. Our data showed that MICB is in linkage disequilibrium with MICA and even with HLA-B. Thus, the linkage disequilibrium with MICA and HLA-B suggests that MICB is a potential candidate for those diseases classically associated with HLA class I alleles.

Alternatively spliced forms of MICA and MICB lacking exon 3 in a human cell line and evidence of presence of similar RNA in human peripheral blood mononuclear cells.

MICA and MICB genes encode MHC class I chain-related proteins, which are polymorphic, do not appear to present peptides or associate with beta(2)-microglobulin, and are expressed predominantly in epithelial cells, endothelial cells, fibroblasts and several cultured cell lines. Alternatively spliced isoforms are known to exist for HLA-A and B, as well as HLA-G and the MHC class I-related gene, MR1. In the course of cloning MICA and MICB cDNA from the colon carcinoma cell line HCT 116, it was observed that two kinds of cDNAs were obtained: a 1161-bp cDNA, representing full-length MICA or MICB, and a shorter variant of 873 bp. The sequences of these short cDNAs were those of the correct MICA or MICB alleles but lacking exon 3. They were found in 7 of 72 clones examined or about 10% and were called MICA2 and MICB2. MICA1 and MICA2 were transfected into Chinese hamster ovary cells and found to be expressed both in the cells and on their surface. PCR with a primer based on a sequence formed by the joining of exons 2 and 4 allowed detection of the isoform RNA in different cells including freshly prepared normal PBMC.

High resolution molecular phototyping of MICA and MICB alleles using sequence specific primers

Major histocompatibility complex (MHC) class I chain-related genes, MICA and MICB, are located centromeric to human leukocyte antigen B ( HLA-B) on chromosome 6. In response to stress stimuli, MIC is expressed on epithelial, endothelial and fibroblast cells, but not lymphocytes and has been demonstrated to ligate the natural killer (NK) cell receptor, NKG2D. Nucleotide sequences of MICA and MICB are highly polymorphic and several methods have been established to identify these polymorphisms, including sequence-based typing and sequence-specific oligonucleotide probing. In this study we have developed a high-resolution polymerase chain reaction–sequence-specific primer (PCR-SSP) phototyping scheme that detects all WHO-recognized MICA alleles and all 12 MICB alleles. Our method will also recognize a MICA deletion haplotype and distinguish between MICA alleles with different binding affinities for NKG2D, encoded by a non-synonymous nucleotide substitution in codon 129. Furthermore, our scheme targets almost 90% of the dimorphic codon positions in exons 2, 3, and 4, which result in non-synonymous amino acid changes. This method can be used to determine MIC allele frequencies within different populations, as well as investigate MIC associations in cohorts of patients with autoimmune and infectious diseases and explore the impact of MIC on the survival of solid organ and stem cell transplants.

Linkage disequilibria between HLA-B, C1_4_1, MICA and MICB.

The polymorphisms of MICA exon 5 (5 alleles), MICB intron 1 (13 alleles), C1_4_1 (6 alleles), HLA-B (29 alleles) and HLA-A (15 alleles) were investigated in a healthy German population. Sequencing was performed for the MICB alleles CA14, CA15, CA17, CA23 and CA26 isolated from different cell lines. Variation to the published sequence was observed for CA14, CA15 and for CA17. At the C1_4_1 locus a new allele (CAAA)9 was identified and confirmed by sequencing. Linkage disequilibria were investigated for two-point- and three-point-haplotypes. Although the average relative delta value correlates loosely with the physical distance from HLA-B to MICB: HLA-B-C1_4_1>HLA-B-MICA>HLA-B-MICB, there are several exceptions to this rule. Analyzing three-point-haplotypes for the segment MICB to HLA-A a wide variation of linkage disequilibria for some of the classical HLA-A, B haplotypes has been observed. While the HLA-A1, B8 haplotype displays strong relative delta values over the entire distance from HLA-A to MICB, other haplotypes have linkage disequilibria only in a limited region.

Primary sclerosing cholangitis is associated to an extended B8-DR3 haplotype including particular MICA and MICB alleles

Primary sclerosing cholangitis is associated to an extended B8-DR3 haplotype including particular MICA and MICB alleles

Primary sclerosing cholangitis is associated to an extended B8-DR3 haplotype including particular MICA and MICB alleles.

Susceptibility to primary sclerosing cholangitis (PSC) is associated with HLA-B8, -DR3, -DR2, and -DR6. It is not established whether these HLA genes or closely linked genes confer the primary disease susceptibility. MICA and MICB genes are found in the class I region between HLA-B and DRB. MICA is expressed in gastrointestinal epithelium and activates gammadelta T cells in the gut. Because PSC is strongly associated with inflammatory bowel disease, we investigated whether MICA and MICB contribute to the HLA-associated genetic susceptibility to develop PSC. The study included 130 PSC patients and 306 healthy controls, previously typed for HLA class I and II genes, typed for 5 MICA and 15 MICB microsatellite alleles. The phenotype frequencies of MICA5.1 and MICB24 were significantly increased among PSC patients compared with controls (90% vs. 74%; odds ratio [OR] = 3.2; P(c) = 3 x 10(-3) and 58% vs. 29%; OR = 3.3; P(c) < 1 x 10(-7), respectively). When stratified for B8- or DR3-positive and -negative individuals, the association of these markers to PSC was no longer evident. However, we observed that B8 and DR3 were associated to PSC only in the presence of both MICA5.1 and MICB24 markers. The frequency of individuals carrying all 4 alleles was significantly increased among the PSC patients compared with controls (49% vs. 18%; OR = 4.5; P(c) < 1 x 10(-7)). Our data indicate that PSC is associated to the extended B8-MICA5.1-MICB24-DR3 haplotype.