PCR SSCP REVEALS HAPLOTYPE RELATED POLYMORPHISM OF PERB 1: A NEW MARKER FOR MHC β BLOCK TYPING

Many new Major Histocompatibility Complex (MHC) genes have been discovered in the last 5 years. Defining the polymorphism of these new genes may elucidate their function and their relevance to diseases with MHC associations. Polymerase chain reaction and single stranded conformation polymorphism (PCR SSCP) analyses were used to detect sequence polymorphisms of PERB1 demonstrated by comparing the available genomic sequence of four haplotypes. This study showed that PCR SSCP of PERB 1 is reproducible. In addition, PERB1 alleles segregate within families together with MHC haplotypes. Typing results from the Forth Asia and Oceania Histocompatibility Workshop (4AOHW) cell panel indicate that the identified polymorphisms of PERB 1 are ‘haplotypic’, i.e., unrelated individuals carrying the same MHC ancestral haplotypes carry the same PERB1 SSCP pattern. Interestingly, PERB1 SSCP patterns allow the distinction of ancestral haplotypes which share HLA-B serological specificities, such as HLA-B44 and therefore this analysis can be used to further define MHC haplotypes and thus to improve our understanding of the evolution of this complex.     

Sequence analysis of the MHC class I region reveals the basis of the genomic matching technique

The genomic matching technique (GMT) improves survival following bone marrow transplantation (BMT) between unrelated donor and recipient pairs correlating with a decrease in incidence and severity of graft-versus-host disease (GvHD). The principles of this technique are based on the duplication and polymorphic characteristics of the major histocompatibility complex (MHC). Specifically, the beta block GMT matches for a 300 kb region that contains the human leukocyte antigen (HLA-B and -C) genes as well as other non-HLA genes such as the natural killer cell receptor ligand PERB11 (MIC). The block contains two large segmental duplications. One results in two PERB11 genes (11.1 and 11.2), the other in two class I genes (HLA-B and -C). With the complete sequencing of the class I region of the MHC in different haplotypes, we can now show that the beta block GMT profiles reflect amplification of the duplicated PERB11 segments and not the duplicated segments containing HLA-B and -C, and yet provide a signature that characterizes the entire block rather than individual loci.     

PERB11 (MIC): a polymorphic MHC gene is expressed in skin and single nucleotide polymorphisms are associated with psoriasis

The susceptibility genes for psoriasis remain to be identified. At least one of these must be in the major histocompatibility complex (MHC) to explain associations with alleles at human leucocyte antigen (HLA)-A, -B, -C, -DR, -DQ and C4. In fact, most of these alleles are components of just two ancestral haplotypes (AHs) designated 13.1 and 57.1. Although relevant MHC gene(s) could be within a region of at least 4 Mb, most studies have favoured the area near HLA-B and -C. This region contains a large number of non-HLA genes, many of which are duplicated and polymorphic. Members of one such gene family, PERB11.1 and PERB11.2, are expressed in the skin and are encoded in the region between tumour necrosis factor and HLA-B. To investigate the relationship of PERB11.1 alleles to psoriasis, sequence based typing was performed on 97 patients classified according to age of onset and family history. The frequency of the PERB11.1*06 allele is 44% in type I psoriasis but only 7% in controls (Pc = 0.003 by Fisher's exact test, two-tailed). The major determinant of this association is a single nucleotide polymorphism (SNP) within intron 4. In normal and affected skin, expression of PERB11 is mainly in the basal layer of the epidermis including ducts and follicles. PERB11 is also present in the upper keratin layers but there is relative deficiency in the intermediate layers. These findings suggest a possible role for PERB11 and other MHC genes in the pathogenesis of psoriasis.     

Phylogenetic analysis of primate MIC (PERB11) sequences suggests that the representation of the gene family differs in different primates: comparison of MIC (PERB11) and C4.

Duplication of segments within the MHC has led to numerous multicopy families such as class I, class II, C4 and MIC (PERB11). Different copy numbers between haplotypes and species may be explained by the extent of duplication and subsequent deletion. There are at least five copies of MIC (PERB11) in humans, but MICA (PERB11.1) appears to have been deleted from the chimpanzee. By comparing the sequences of primates (chimpanzee, gorilla, gibbon, orang-utan, pygmy chimpanzee, Patas monkey, Aethiops and baboon) we conclude that the gorilla has a copy of PERB11.1, whereas the baboon and Patas possess MICD (PERB11.4) and/or MICE (PERB11.5) rather than MICA (PERB11.1). These findings indicate that the primate MHC is more plastic than has been appreciated.     

Phylogenetic analysis of primate MIC (PERB11) sequences suggests that the representation of the gene family differs in different primates: comparison of MIC (PERB11) and C4

Duplication of segments within the MHC has led to numerous multicopy families such as class I, class II, C4 and MIC (PERB11). Different copy numbers between haplotypes and species may be explained by the extent of duplication and subsequent deletion. There are at least five copies of MIC (PERB11) in humans, but MICA (PERB11.1) appears to have been deleted from the chimpanzee. By comparing the sequences of primates (chimpanzee, gorilla, gibbon, orang-utan, pygmy chimpanzee, Patas monkey, Aethiops and baboon) we conclude that the gorilla has a copy of PERB11.1, whereas the baboon and Patas possess MICD (PERB11.4) and/or MICE (PERB11.5) rather than MICA (PERB11.1). These findings indicate that the primate MHC is more plastic than has been appreciated.     

MICA polymorphisms and haplotypes with HLA-B and HLA-DRB1 in Koreans

Abstract
Major histocompatibility complex (MHC) class I chain-related gene A ( MICA ) is located within the human MHC, centromeric to HLA-B and telomeric to HLA-DRB1 . The location of MICA in the MHC indicates the presence of linkage disequilibrium with human leukocyte antigen (HLA). Like HLA, MICA is highly polymorphic; however, the information available for MICA polymorphisms is not as comprehensive as that for HLA polymorphisms. We estimated the allelic frequencies of MICA and haplotypes with HLA-B and HLA-DRB1 at high-resolution in a population of 139 unrelated Korean individuals by applying the newly developed method of sequence-based typing (SBT). A total of 17 MICA alleles were identified. The most frequent allele was MICA*010 (19.4%), followed by alleles *00201 (17.6%), *00801 (14.7%), *01201 (9.4%), *004 (8.3%) and *049 (7.9%). The most common two- and three-locus haplotypes were HLA-B*1501-MICA*010 (10.4%), MICA*010-HLA-DRB1*0406 (5.8%) and HLA-B*1501-MICA*010-HLA-DRB1*0406 (5.8%). This is the first study to provide such high-resolution information on the distribution of haplotypes comprising MICA , HLA-B and HLA-DRB1 in Korean individuals, a level of resolution made possible by use of the SBT method. The results of this study should help determine the mechanisms underlying diseases associated with MICA polymorphisms in Korean individuals.     

Genomics of the major histocompatibility complex: haplotypes, duplication, retroviruses and disease

Summary: The genomic region encompassing the Major Histocompatibility Complex (MHC) contains polymorphic frozen blocks which have developed by local imperfect sequential duplication associated with insertion and deletion (indels), In the alpha block surrounding HLA-A, there are ten duplication units or beads on the 62,1 ancestral haplotype. Each bead contains or contained sequences representing Class 1, PERB11 (MHC Class I chain related (MIC)) and human endogenous retrovirus (HERV) 16, Here we consider explanations for co-occurrence of genomic polymorphism, duplication and HERVs and we ask how these features encode susceptibility to numerous and very diverse diseases. Ancestral haplotypes differ in their copy number and indels in addition to their coding regions. Disease susceptibility could be a function of all of these differences. We propose a model of the evolution of the human MHC. Population-specific integration of retroviral sequences could explain rapid diversification through duplication and differential disease susceptibility. If HERV sequences can be protective, there are exciting prospects for manipulation. In the mean-while, it will be necessary to understand the function of MHC genes such as PEKB11 (MIC) and many others discovered by genomic sequencing.     

SNP profile within the human major histocompatibility complex reveals an extreme and interrupted level of nucleotide diversity

The human major histocompatibility complex (MHC) is characterized by polymorphic multicopy gene families, such as HLA and MIC (PERB11); duplications; insertions and deletions (indels); and uneven rates of recombination. Polymorphisms at the antigen recognition sites of the HLA class I and II genes and at associated neutral sites have been attributed to balancing selection and a hitchhiking effect, respectively. We, and others, have previously shown that nucleotide diversity between MHC haplotypes at non-HLA sites is unusually high (>10%) and up to several times greater than elsewhere in the genome (0.08%-0.2%). We report here the most extensive analysis of nucleotide diversity within a continuous sequence in the genome. We constructed a single nucleotide polymorphism (SNP) profile that reveals a pattern of extreme but interrupted levels of nucleotide diversity by comparing a continuous sequence within haplotypes in three genomic subregions of the MHC. A comparison of several haplotypes within one of the genomic subregions containing the HLA-B and -C loci suggests that positive selection is operating over the whole subgenomic region, including HLA and non-HLA genes. [The sequence data for the multiple haplotype comparisons within the class I region have been submitted to DDBJ/EMBL/GenBank under accession nos. AF029061, AF029062, and AB031005-AB031010. Additional sequence data have been submitted to the DDBJ data library under accession nos. AB031005-AB03101 and AF029061-AF029062.]     

The MHC type 1 diabetes susceptibility gene is centromeric to HLA-DQB1

HLA-DQB1 is widely considered to be the major histocompatibility complex (MHC) susceptibility gene for type 1 diabetes (T1D). However, since inheritance of the gene in T1D is recessive, the presence of the protective HLA-DQB1 0602 allele with normal nucleotide sequence in some patients raises the question of whether HLA-DQB1 is not the susceptibility locus itself but merely a good marker. HLA-DQB1 0602 is part of a conserved extended haplotype (CEH) [HLA-B7, SC31, DR2] (B7, DR2) with fixed DNA over more than 1Mb of genomic DNA that normally carries a protective allele at the true susceptibility locus. We postulated that, in patients with HLA-DQB1 0602, the protective allele at the susceptibility locus has been replaced by a susceptibility allele through an ancient crossover at meiosis centromeric to HLA-DQB1. We analyzed single nucleotide polymorphisms (SNPs) distinguishing the HLA-DQA2 (the first expressed gene centromeric to HLA-DQB1) allele on the normal HLA-B7, DR2 CEH from those on susceptibility CEHs in T1D patients and controls with HLA-DQB1 0602. All but 1 of 20 healthy control HLA-DQB1 0602 haplotypes had identical (consensus) first intron HLA-DQA2 5-SNP haplotypes. Fifteen of 19 patients with HLA-DQB1 0602 were homozygous for 1 or more HLA-DQA2 SNPs differing from consensus HLA-DQA2 SNPs, providing evidence of crossover involving the HLA-DQA2 locus. The remaining 4 patients were heterozygous at all positions and therefore uninformative. The loss of dominant protection usually associated with HLA-DQB1 0602 haplotypes is consistent with a locus centromeric to HLA-DQB1 being a major determinant of MHC-associated susceptibility, and perhaps the true T1D susceptibility locus.     

Ancestral haplotypes: conserved population MHC haplotypes

We describe here a number of Caucasoid MHC haplotypes that extend from HLA-B to DR and that have been conserved en bloc. These haplotypes and recombinants between any two of them account for 73% of unselected haplotypes in our Caucasoid population. The existence of ancestral haplotypes implies conservation of large chromosomal segments. Irrespective of the mechanisms involved in preservation of ancestral haplotypes, it is clear that these haplotypes carry several MHC genes, other than HLA, which may be relevant to antigen presentation, autoimmune responses, and transplantation rejection. In light of the existence of ancestral haplotypes, it is critical to evaluate MHC associations with disease and transplantation outcome in terms of associations with ancestral haplotypes rather than individual alleles.     

Diversity of MICA (PERB11.1) and HLA haplotypes in Northeastern Thais

MICA or PERB11.1 is a polymorphic major histocompatibility complex (MHC) class I-related gene located 46 kb centromeric of the HLA-B gene in the HLA class I region. It is expressed mainly in gut epithelial cells, keratinocytes, endothelial cells, fibroblasts and monocytes, and is upregulated by heat stress. MICA has been found to interact with gamma delta T cells, alpha beta CD8(+) and natural killer (NK) cells bearing the NKG2D/DAP10 receptor. The MICA gene displays a high degree of polymorphism with at least 54 alleles. In the present study, polymorphic exons 2, 3 and 4 of the MICA gene were analyzed using sequencing based typing (SBT) in 255 unrelated healthy northeastern Thais. Thirteen previously reported MICA alleles were detected. MICA*008, *010, *002 and *019 were highly predominant with the allele frequencies of 21.4%, 18.2%, 17.6% and 15.3%, respectively. Five of these 13 MICA alleles show significantly different frequencies from those of the Japanese and Caucasian populations. Interestingly, MICA052, which is a very rare allele in other populations, was prevalent with the allele frequency of 8.2%, mainly on the HLA haplotype carrying HLA-B*13 in this population. Strong linkage disequilibria were observed between MICA and HLA-B, as similarly observed in other populations, namely MICA*010-B*4601, MICA052-B*13, MICA*002-B*5801, and MICA*019-B*15 (1502, 1508, 1511, 1515, 1528, 1530). A large variety of three-locus (MICA - HLA-B - HLA-Cw) and six-locus (HLA-DQB1 - HLA-DRB1 - MICA - HLA-B - HLA-Cw - HLA-A) haplotypes were recognized in the northeastern Thai population. This is the first report on MICA allelic distribution in Southeast Asian populations. These data will provide the important basis for future analyses on the potential role of the MICA gene in disease susceptibility and transplantation matching in Southeast Asian populations.     

Further characterization of MHC haplotypes demonstrates conservation telomeric of HLA-A: update of the 4AOH and 10IHW cell panels.

Cell panels have been used extensively in studies of polymorphism and disease associations within the major histocompatibility complex (MHC), but the results from these panels require continuous updates with the increasing availability of novel data. We present here an updated table of the typings of the 10IHW and 4AOH panels. Local data included are HFE, HERV-K(C4) and six microsatellites telomeric of HLA-A. Typings for class I, MICA (PERB11.1), MICB (PERB11.2), XA, XB, LMP2 and 10 microsatellites reported by others have also been consolidated in this table. The tabulation shows that the length of conservation in the human MHC is even more extensive than previously thought. Human MHC ancestral haplotypes are inherited as a conserved region of genomic sequence spanning some 6-8 megabases from the HLA class II region and beyond the HLA class I region up to and including the HFE gene. Numerous examples of historical recombinations were also observed.     

C4 polymorphism and major histocompatibility complex haplotypes in IgA deficiency: association with C4A null haplotypes.

IgA-deficient individuals (n = 110) and six families comprising 9 cases of IgA deficiency were typed for HLA-A, -B, -DR, C4 and factor B. Phenotype frequencies were increased for HLA-B8 (p = 0.004), HLA-DR3 (p = 0.001) and homozygous C4AQ0 (p = 0.01) and decreased for HLA-B7 (p = 0.004), HLA-DR2 (p = 0.0001) and C4A3 (p = 0.00007) compared to controls. Homozygous C4A deficiency was found in 20% of IgA-deficient persons. As clearly suggested by investigation of families, the findings could be attributed to high prevalence of the extended major histocompatibility complex (MHC) haplotype [HLA-A1, B8, C4AQ0, C4B1, BfS, DR3] in IgA deficiency. All but 1 of the 9 IgA-deficient persons included in the family study carried this haplotype and 4 of them were homozygous. In the families, 3 persons with normal serum IgA concentrations had the same MHC haplotypes as their IgA-deficient relatives. The findings were also consistent with possible overrepresentation of other MHC haplotypes with aberrant C4 gene organization in IgA deficiency. As previously suggested, the presence of two MHC haplotypes associated with IgA deficiency appears to be a necessary but not sufficient requirement for manifestation of the condition. The putative existence of a recessive gene in the MHC with regulatory function with regard to IgA gene expression is consistent with the findings.     

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.     

ANALYSIS OF THE MAJOR HISTOCOMPATIBILITY COMPLEX MICROSATELLITE CL1 IN DIFFERENT HUMAN HAPLOTYPES

Characterization of the region between HLA-B and the TNF loci in the human MHC revealed the presence of duplicated loci, named CL1 and CL2, that included repeat sequences. Development and use of a PCR typing methodology that amplified both CL microsatellites simultaneously indicated that PCR product patterns analysed on native agarose gels were allelic (Abraham et al., 1992). The purpose of the current study was to determine the molecular explanation for the unique patterns achieved. Sequence analysis of the CL1 locus from 32 chromosomes representing 10 ancestral haplotypes indicated that six alleles were present. The CL microsatellites also provided an opportunity to study the evolutionary relationships between MHC haplotypes from different racial groups. Sequence comparison of closely related ancestral haplotypes from different racial groups suggested that the CL1 microsatellite has not changed in the period since divergence.     

Common HLA-B8-DR3 haplotype in Northern India is different from that found in Europe

The aim of this study was to determine whether a common diabetic haplotype, including human leukocyte antigen (HLA)-B8 and HLA-DR3, in Northern India is the same haplotype as the European HLA-B8-DR3 haplotype. DNA samples from Northern Indian subjects selected on the basis of HLA-B8 and HLA-DR3 were tested for microsatellite and single nucleotide polymorphism alleles throughout the major histocompatibility complex (MHC). It was found that the Indian samples represent a conserved haplotype in which all alleles were shared by Indian subjects with HLA-B8 and HLA-DR3, but were different to those that are characteristic of the European 8.1 ancestral haplotype. The Indian and European haplotypes share HLA-B*0801, HLA-DRB1*0301 and HLA-DQB1*02 but differ for subtypes of HLA-Cw*07 and HLA-DRB3 and all central MHC alleles tested. In contrast, Indian subjects selected on the basis of HLA-B58 ( 1-17) and HLA-DR3 shared the same alleles at other MHC loci as have been described in the common Chinese haplotype with HLA-B58/17 and HLA-DR3. A third haplotype, HLA-B50/21 and HLA-DR3, was also found to be highly conserved but shares little in common with the other two HLA-DR3-containing Indian haplotypes.     

Orientation of major histocompatibility (MHC) genes relative to the centromere of human chromosome 6

Linkage analysis of the data obtained from a three-generation Dutch family segregating for genetic variants of centromeric heterochromatic region in the band pi 1 on chromosome 6 (6ph), major histocompatibility (MHC) genes HLA-A, HLA-B and HLA-C, glyoxalase I (GLO) and phosphoglucomutase-3 (PGMg) showed that the genetic distance between the HLA gene cluster and 6ph is about 6 cM (at an estimated peak lod score of 3.466), that GLO is closer than HLA to the centromere and that PGM₃ is probably not situated on the same chromosomal arm as HLA.     

成纤维细胞生长因子受体1配体精细结合位点的确定

目的 确定人成纤细胞生长因子受体1（FGFR1）的配体精细结合位点。方法 合成肽文库筛选，定向点突变,源核细胞重组蛋白质表达及受体－配体结合实验。结果 采用^125I标记的酸性成纤维细胞生生长因子（aFGF)，自合成肽文库中筛选到一个五肽序列（WGPGM），其序列及立体结构和FGFR1细胞外段的一个基序（WISPEKM）相似。为了证明FGFR1的WTSPEKM基序是结合FGF的重要结合，采用定向突变技术将WITSPEKM基序中的P（CCA）突变成A（GCA），并在原核细胞中表达了野生型和突变型FGFR1细胞外段重组蛋白质，受体－配体结合实验显示突变的GFFR1细胞外段结合aFGF的能力明显降低。结论 FGFR1的WISPEKM基序是配体结合的一个重要部位。     

MHC microsatellite diversity and linkage disequilibrium among common HLA-A, HLA-B, DRB1 haplotypes: implications for unrelated donor hematopoietic transplantation and disease association studies

Twenty-two human major histocompatibility complex (MHC) region microsatellite (Msat) markers were studied for diversity and linkage disequilibrium (LD) with HLA loci in hematopoietic cell transplant recipients and their HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 allele-matched unrelated donors. These Msats showed highly significant LD over much of the MHC region. The Msat diversity of five common Caucasian haplotypes (HLA-A1-B8-DR3, A3-B7-DR15, A2-B44-DR4, A29-B44-DR7, and A2-B7-DR15) was examined using a new measure called 'haplotype specific heterozygosity' (HSH). Each of the five haplotypes had at least one Msat marker with an HSH value of zero indicating that only one Msat allele was observed for the particular HLA haplotype. In addition, the ability of Msats to predict HLA-A-B-DRB1 haplotypes was studied. Over 90% prediction probability of two common haplotypes (HLA-A1-B8-DR3 and HLA-A3-B7-DR15) was achieved with information from three Msats (D6S265/D6S2787/D6S2894 and D6S510/D6S2810/D6S2876, respectively). We demonstrate how the HSH index can be used in the selection of informative Msats for transplantation and disease association studies. Markers with low HSH values can be used to predict specific HLA haplotypes or multilocus genotypes to supplement the screening of HLA-matched donors for transplantation. Markers with high HSH values will be most informative in studies investigating MHC region disease-susceptibility genes where HLA haplotypic effects are known to exist.