Major histocompatibility complex haplotypes in Spanish immunoglobulin A deficiency patients: a comparative fine mapping microsatellite study

The most consistent finding in Immunoglobulin A deficiency (IgAD) genetics is the presence of susceptibility factors located in the major histocompatibility complex (MHC). We have described the existence of at least two distinct susceptibility genes in the MHC present in different haplotypes. The aim of the present study was to locate with precision the susceptibility genes present in DR1- and DR7-positive haplotypes, taking advantage of their structural diversity, as opposed to the conserved nature of the DR3-extended susceptibility haplotype (DR3/B8), that hampers a more exhaustive scrutiny. A detailed analysis with 20 markers along the MHC in the 400 haplotypes present in 100 IgAD families, with special density at Class II locations, was performed to define the minimal shared susceptibility region present in all haplotypes carrying DR1 and, on the other hand, in all DR7-positive haplotypes. A comparison of the fine microsatellite allele structure of DR-extended haplotypes in the Spanish population with those described for Swedish and British families revealed no difference in DRB1*0101 and DRB1*0102 haplotypes between both populations. Our data suggest that the etiologic mutation present in DRB1*0101 and DRB1*0102 in North Europe (Sweden and UK) is missing in the Spanish DRB1*0101 haplotypes but is present in the DQB1/DRB1 region in DRB1*0102 haplotypes. The results obtained also indicated that the most likely susceptibility gene in the DR7 haplotypes is either DQA1 or DRB1.     

High resolution HLA-DRB1 SSP typing for cadaveric donor transplantation

Abstract: An HLA-DRB1 typing procedure by means of sequence-specific primer (SSP) amplification was developed for 65 different DRB1 subtypes. Subtyping is achieved by the performance of two subsequent PCR assays (PCR-1 assay and PCR-2 assay) using a limited number of reactions. The PCR-1 assay determined low-resolution HLA-DRB1 typing, i.e. the serologically defined specificities DR1, 2, 3, 4, 11, 12, 6, 7, 8, 9 and 10. The second exon of the DRB1 gene is amplified also in this PCR-1 assay. High-resolution subtyping for positively identified alleles was performed in the PCR-2 assay with the exon-2 product from PCR-1 assay as DNA template. PCR reactions were carried out using unpurified primers in reaction volumes of 20 μ1 and 100 ng of chromosomal DNA. After 3 hours, the results of the PCR-1 assay were analyzed and subsequently subtyping results in the PCR-2 assay were obtained in another 1.5 hours. A total of 249 DNA samples was typed by this method. No false positive nor false negative results were obtained in DRB1 typing of 32 homozygous cell lines, 56 serologically well-defined panel cells and 125 unrelated individuals. Segregation of the amplification patterns was investigated in 36 members of 7 two-generation families. DRB1 subtyping revealed codominant Mendelian segregation for all subtypes investigated. In conclusion, LR-HR-PCR-SSP typing is a fast and reliable typing technique for routine DNA typing purposes which gives complete DRB1 subtyping within 4.5 h. Besides low-resolution DRB typing, also high-resolution DRB subtyping for prospective HLA-DR matching in cadaveric renal transplantation is possible by this method.     

Polymorphic MHC ancestral haplotypes affect the activity of tumour necrosis factor-alpha.

It remains unclear which MHC loci are involved in susceptibility to autoimmune diseases and immune deficiencies. We have chosen to evaluate whether different alleles of tumour necrosis factor-alpha (TNF-alpha) are important, as TNF has been implicated in the etiology of many immunological disorders. We have shown previously that a restriction fragment length polymorphism in the TNF region correlates with MHC ancestral haplotypes. We therefore examined the effect of ancestral haplotype on the activity of TNF-alpha in culture supernatants of lymphoblastoid cell lines. The results demonstrate that TNF-alpha activity in supernatants of 8.1 (A1, B8, DR3) cell lines was higher than that present in the supernatants from cells homozygous for eight different MHC ancestral haplotypes, and indicate that polymorphisms in TNF-alpha, or in other MHC genes that regulate TNF, may be responsible for the 8.1 phenotype.     

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.     

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.     

Additional factor in some HLA DR3/DQ2 haplotypes confers a fourfold increased genetic risk of celiac disease

Although HLA-DQ genes are the major celiac disease (CD) susceptibility genes, results from Finnish families suggest that not all DQ2-encoding haplotypes confer equal susceptibility to CD, implying the effect of other gene(s) in the HLA region. The aim of the present work was to extend and confirm the aforementioned results in a southern European population ( Italian) and to better localize the additional risk factor/s. The association of nine loci spanning the HLA region from DR to HFE, 4.5-Mb telomeric of HLA-A, was tested. The analysis was performed by comparing marker frequencies in DR3-DQ2 haplotypes transmitted and non-transmitted to the affected offspring in 156 Italian CD families selected for having at least one DR3-positive parent. The same analysis was performed independently in 101 Finnish CD families selected with the same criteria. Three alleles, MICA-A5.1, MICB-CA24 and MIB-350, all characteristic of the B8-DR3 extended haplotype, showed a significantly increased frequency in DR3 transmitted haplotypes in the Italian families. DR3 haplotypes carrying the combination of these alleles conferred an approximate fourfold increased CD risk. B8-DR3 transmitted haplotypes were significantly more conserved telomerically down to the MIC-Class I region. Similar results were seen in the Finnish families. The major conclusion that holds true in both populations is that, while DQ2 is an absolute requirement for the development of CD, the presence of an additional genetic factor within the MIC-Class I region confers an approximate 4-fold increased risk of the disease.     

Extensive interbreed,but minimal intrabreed,variation of DLA class II alleles and haplotypes in dogs

The DLA class II genes in the dog major histocompatibility complex are highly polymorphic. To date, 52 DLA-DRB1, 16 DLA-DQA1 and 41 DLA-DQB1 allelic sequences have been assigned. The aim of this study was to examine the intrabreed and interbreed variation of DLA allele and haplotype frequencies in dogs, and to ascertain whether conserved DLA class II haplotypes occur within and between different breeds. One thousand and 25 DNA samples from over 80 different breeds were DLA class II genotyped, the number of dogs per breed ranging from 1 to 61. DNA sequence based typing and sequence specific oligonucleotide probing were used to characterize dogs for their DLA-DRB1, DQA1 and DQB1 alleles. The high frequency of DLA class II homozygous animals (35%), allowed the assignment of many haplotypes despite the absence of family data. Four new DLA alleles were identified during the course of this study. Analysis of the data revealed considerable interbreed variation, not only in allele frequency, but also in the numbers of alleles found per breed. There was also considerable variation in the number of breeds in which particular alleles were found. These interbreed variations were found in all three DLA class II loci tested, and also applied to the three-locus haplotypes identified. Within this data set, 58 different DLA-DRB1/DQA1/DQB1 three-locus haplotypes were identified, which were all found in at least two different animals. Some of the haplotypes appeared to be characteristic of certain breeds. The high interbreed, and relatively low intrabreed, variation of MHC alleles and haplotypes found in this study could provide an explanation for reports of interbreed variation of immune responses to vaccines, viruses and other infections.     

Polymorphism and distribution of HLA-DR2 alleles and haplotypes in a Greek population

Abstract: HLA-DR2 serological subtyping has indicated that the DR16 serotype appears at a higher frequency relative to the DR15 serotype in the Greek population, differing from the distribution observed in most other Caucasian groups. In this study, we have analyzed by the PCR-SSP technique a DR2-positive group of unrelated Greek individuals selected from our normal control panel for the different DRB1, DRB5, DQB1 and DQA1 DR2-associated alleles present. Six of the 50 individuals analyzed were homo-zygous for DR2, contributing a total of 56 haplotypes for DR2. The observed frequencies of the DR2-related DRB1 alleles were as follows: 58.9% for the DRB1*1601, 7.1% for the DRB1*1602, 25.0% for the DRBl*1501 and 7.1% for the DRB1*1502 allele. The rare allele DRB1*1605 was detected in one heterozygous sample and its presence was definitively established by DNA sequencing. The alleles *1503, *1504, *1505, *1603 and *1604 were not detected. Three DRB5 alleles were identified: DRB5*0202 (67.8%), DRB5*0101 (25.0%) and DRB5*0102 (7.1%). Ten different DRB1/DQB1/ DQA1 DR2-associated haplotypes were denned. The most frequently observed haplotype was DRBl*1601-DQBl*0502-DQAl*0102 (relative frequency =57%) followed by DRB1*1501-DQB1*0602-DQA1*0102 (relative fre-quency=14.3%). In conclusion, the refined analysis of the DR2-associated DRB1 alleles in the Greek population revealed the prevalence of the DRB1*1601 allele. The rare allele DRB1*1605 was demonstrated once. A considerable variety of different DR2-related DR/DQ haplotypes was detected and the overall haplotypic frequencies in the Greek population are distributed differently compared to those reported for most other Caucasian populations.     

High resolution HLA-DRB1 identification of a Caucasian population

Polymerase chain reaction-sequence-specific oligonucleotide probes typing methods have been applied to 1000 individuals from the Northern Ireland population to give human leukocyte antigen DRB1 (HLA-DRB1) allele assignment. HLA-DRB1 allele frequencies and four-locus haplotypes (A/B/C/DR) for this Caucasian population, based on HLA class I and class II allele assignment, are now presented. No significant deviations from Hardy-Weinberg proportions were observed. The HLA-C locus exhibited marginal evidence of selection (p<0.03, uncorrected one-sided test) in the direction of balancing selection; the HLA-A, -B, and -DRB1 allele frequency distributions were compatible with expectations under a neutral model (which does not mean that selection is not operating). Evidence for selection was seen on haplotypes HLA-A*010101-B*0801-DRB1*030101 and HLA-A*290201-B*440301-DRB1*070101 based on their patterns of linkage disequilibrium.     

High-Density SNP genotyping defines 17 distinct haplotypes of the TNF block in the Caucasian population: implications for haplotype tagging

The region spanning the tumor necrosis factor (TNF) cluster in the human major histocompatibility complex (MHC) has been implicated in susceptibility to numerous immunopathological diseases, including type 1 diabetes mellitus and rheumatoid arthritis. However, strong linkage disequilibrium across the MHC has hampered the identification of the precise genes involved. In addition, the observation of "blocks" of DNA in the MHC within which recombination is very rare, limits the resolution that may be obtained by genotyping individual SNPs. Hence a greater understanding of the haplotypes of the block spanning the TNF cluster is necessary. To this end, we genotyped 32 human leukocyte antigen (HLA)-homozygous workshop cell lines and 300 healthy control samples for 19 coding and promoter region SNPs spanning 45 kb in the central MHC near the TNF genes. The workshop cell lines defined 11 SNP haplotypes that account for approximately 80% of the haplotypes observed in the 300 control individuals. Using the control individuals, we defined a further six haplotypes that account for an additional 10% of donors. We show that the 17 haplotypes of the "TNF block" can be identified using 15 SNPs.     

Striking conservation of three extended HLA-DR13 haplotypes in the Japanese population

Abstract: HLA class II DNA typing was conducted for 1335 unrelated Japanese individuals. The study on the linkage disequilibrium revealed a striking conservation of HLA DR13 haplotypes. Among these Japanese, 155 were typed for HLA-DR13 serologically, and they were correspondent to three DRB1 alleles, DRB1*1301, 1302 and 1307 defined by using the polymerase-chain reaction and sequence-specific oligonucleotide probe (PCR-SSOP) method. The two alleles, DRB1*1301 and 1307 were exclusively associated with each specific DRB3-DQA1-DQB1 combination which was DRB1*1301-DRB3*0101-DQA1*0103-DQB1*0603, and DRB1*1307-DRB3*0202-DQA1*0501-DQB1*0301, respectively. DRB1* 1302, the most common DR13 allele in Japanese, had two significant associations with DRB3*0301-DQA1*0102-DQB1*0604 (DRB1*1302A) and with DRB3*0301-DQA1*0102-DQB1*0605 (DRB1*1302B). In this study, no other DR13 class II combinations were found. Ony the DRB1*1302A halotype was associated with the DPB1*0401 allele while the DRB1*1302B haplotype was not. The complete conservation of these DR13 class II haplotypes was found to extend toward the HLA class I region. They were HLA A3-B44-DRB1*1301, A33-B44-DRB1*1302A and A33-B17-DRB1*1302B. Japanese could be characterized with these three extended haplotypes which were remakrably different from those in Caucasian, Black and Asian other than Korean populations.     

The study of the extended haplotypes of rare HLA-B*2730 allele using microsatellite loci

The aim of the present study was to compare haplotypes of the most frequent B*27 alleles among Croatians (B*2702 and *2705) and the rare B*2730 allele. For this purpose, 37 families with members carrying human leukocyte antigen (HLA)-B27 were selected. All individuals were analysed for eight microsatellites (Msats): D6S2927, short tandem repeat - MHC class I-related gene (STR_MICA), D6S2793, D6S2811, tumor necrosis factor a (TNFa), tumor necrosis factor d (TNFd), D6S273 and D6S1014, while individuals carrying the HLA-B27 specificity were subtyped. Of 39 analysed haplotypes, 20 individuals had B*2702, 15 subjects were positive for the B*2705 allele, the B*2730 allele was found in three haplotypes from different families, while one individual carried the B*2703 allele. HLA-A3 and -DRB1*16 were shared by all three B*2730 haplotypes. The DRB1*16 allele was also observed in the majority of B*2702 haplotypes (76.5%), while HLA-A3 was, after HLA-A2, the second most frequent HLA-A specificity in B*2702 haplotypes. No such correlation was found for the B*2705 haplotypes. Msat analysis showed that B*2730 haplotypes also share the same allele at all tested Msats. The D6S2927, D6S2793, MICA and TNFd Msats were not useful in distinguishing B*2702 and B*2705 alleles because D6S2927-213bp, STR_MICA-179bp, D6S2793-206bp, D6S2811-83bp and TNFd-130bp were detected in almost all cases. Conversely, for the TNFa, D6S273 and D6S1014 loci, haplotypes carrying B*2702 and B*2730 shared a single Msat allele in the majority of cases (TNFa-113bp, D6S1014-134bp and D6S273-134bp), which was not observed for B*2705 haplotypes. In conclusion, the similarity between B*2702 and B*2730 DNA sequences as well as their sharing of the same haplotypic combinations corroborates the proposed mechanism of B*2730 evolution from B*2702 by interallelic recombination.     

MICA exon 5 microsatellite typing by DNA heteroduplex analysis: a new polymorphism in the transmembrane region

Abstract: MICA (MHC class I chain-related gene A) is localized 47 kb upstream from HLA-B on the short arm of chromosome 6. It has been postulated that MICA protein folds similarly to the class I chain and may have the capacity to bind short ligands. Short tandem repeats (STR) within the transmembrane (TM) region of this gene have been described and five alleles consisting of 4 to 9 GCT codons, each encoding an alanine residue have been defined. We have applied DNA heteroduplex analysis to type MICA trinucleotide repeats in order to develop a simple and reliable method for their identification. This approach allowed the characterization of all MICA alleles. Moreover, a new polymorphism within the TM region was identified.     

Application of microsatellite typing for the investigation of a cluster of cases of Candida albicans candidaemia

A cluster of cases of Candida albicans candidaemia in a surgical intensive care unit was investigated. The probability of such a cluster during a single month was highly significant compared with the frequency of candidaemia in the previous year. A molecular typing method, based on length analysis of three (EF3, CDC3, HIS3) microsatellite-containing regions, was used to investigate isolates from patients in and outside the ward. This demonstrated the involvement of different strains, indicating the absence of cross-transmission among patients. Results of microsatellite typing can be obtained almost in real-time, which is particularly useful in an outbreak context.     

Population and family studies of HLA-DR4 by use of oligonucleotide typing

Using PCR and DR4 group-specific primers and SSO's we have examined DRB1*04 nucleotide polymorphisms in a population of 123 DR4-positive individuals (86 NAC, 27 Hispanics and 10 African Americans) from New York carrying a total of 134 DR4 haplotypes. We found that the distribution of DRB1*04 alleles on DR4 haplotypes differs in these three ethnic groups. In this relatively small population, certain alleles such as DRB1*0406 and 0411 were encountered only in Hispanics, while others such as DRB1*0403, 0408 and 0409 were found only in NAC (North American Caucasians). Such differences may be important in studies of HLA-DR4 and disease associations. Evidence from MLC and PLT studies of an HLA-B/DR crossover family, which was informative for the segregation of HLA-DRB1*0406 and DRB1*0407, supports the concept that subtypes of HLA-DR4 and/or associated HLA-DP alleles elicit T-cell alloreactivity, and may thus play a role in transplantation.     

Two major histocompatibility complex haplotypes influence susceptibility to sporadic inclusion body myositis: critical evaluation of an association with HLA-DR3

Previous studies of sporadic inclusion body myositis (sIBM) have shown a strong association with HLA-DR3 and other components of the 8.1 ancestral haplotype (AH) (HLA-A1, B8, DR3), where the susceptibility locus has been mapped to the central major histocompatibility complex (MHC) region between HLA-DR and C4. Here, the association with HLA-DR3 and other genes in the central MHC and class II region was further investigated in a group of 42 sIBM patients and in an ethnically similar control group (n = 214), using single-nucleotide polymorphisms and microsatellite screening. HLA-DR3 (marking DRB1*0301 in Caucasians) was associated with sIBM (Fisher's test). However, among HLA-DR3-positive patients and controls, carriage of HLA-DR3 without microsatellite and single-nucleotide polymorphism alleles of the 8.1AH (HLA-A1, B8, DRB3*0101, DRB1*0301, DQB1*0201) was marginally less common in patients. Patients showed no increase in carriage of the 18.2AH (HLA-A30, B18, DRB3*0202, DRB1*0301, DQB1*0201) or HLA-DR3 without the central MHC of the 8.1AH, further arguing against HLA-DRB1 as the direct cause of susceptibility. Genes between HLA-DRB1 and HOX12 require further investigation. BTL-II lies in this region and is expressed in muscle. Carriage of allele 2 (exon 6) was more common in patients. BTL-II(E6)*2 is characteristic of the 35.2AH (HLA-A3, B35, DRB1*01) in Caucasians and HLA-DR1, BTL-II(E6)*2, HOX12*2, RAGE*2 was carried by several patients. The 8.1AH and 35.2AH may confer susceptibility to sIBM independently or share a critical allele.