bluuebunny/crossref_metadata_2025 huggingface dataset:
HLA‐DRB1 genotyping by modified PCR‐RFLP method combined with group‐specific primers
Authors: Ota Masao. , Seki Takeshi , Fukushima Hirofumi , Tsuji Kimiyosi , Inoko Hidetoshi
Published: December 2008

Abstract: We previously introduced HLA‐DQA1,‐DPB1 and DQB1 genotyping with the modified PCR‐RFLP method using some informative restriction enzymes which have either a single cleavage site or alternatively no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. In this study, 43 HLA‐DRB1 alleles, excluding DRB1*1103 and *1104 for which no restriction enzymes are available to distinguish each from the other, could be defined by this modified PCR‐RFLP method combined with 7 pairs of group‐specific primers. It is impossible to distinguish DRB 1*0701 and DRB1*0702 as they are identical for the second exon of DRB1. For DRI‐DRB1, DR2‐DRB1, DR4‐DRB1, DR7‐DR1, DR9‐DRB1, DRw10‐DRB1 or DRw52 associated antigens (DR3, w11, w12, w13, w14, and DRw8)‐DRB1 gene amplification, the second exon of the DRB1 gene was selectively amplified using each group‐specific primer from genomic DNAs of 70 HLA‐homozygous B‐cell lines and healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA, although some alleles can be distinguished only after examination of RFLP band patterns generated and in some cases using double digestion technique with two restriction enzymes. This modified PCR‐RFLP method can be successfully applied to all possible DRB1 heterozygotes, despite the fact that 15 pairs of heterozygotes among them cannot be distinguished theoretically by the PCR‐SSO method, because the PCR‐RFLP method can tell whether two polymorphic sites are linked to each other (cis position) or located on a different chromosome (trans position) by checking the length of RFLP bands generated with double digestion. Thus, the PCR‐RFLP method is technically simple, practical and inexpensive for determination of the HLA‐DRB1 alleles for routine HLA typing work.
HLA‐DR typing by PCR amplification with sequence‐specific primers (PCR‐SSP) in 2 hours: An alternative to serological DR typing in clinical practice including donor‐recipient matching in cadaveric transplantation
Authors: Olerup Ole , Zetterquist Henrik
Published: December 2008

Abstract: In most PCR‐based tissue typing techniques the PCR amplification is followed by a post‐amplification specificity step. In typing by PCR amplification with sequence‐specific primers (PCR‐SSP), typing specificity is part of the amplification step, which makes the technique almost as fast as serological tissue typing. In the present study primers were designed for DR “low‐resolution” typing by PCR‐SSP, i.e. identifying polymorphism corresponding to the serologically defined series DR1‐DRw18. This resolution was achieved by performing 19 PCR reactions per individual, 17 for assigning DR1‐DRw18 and 2 for the DRw52 and DRw53 superspecificities. Thirty cell lines and 121 individuals were typed by the DR “low‐resolution” PCR‐SSP technique, TagI DRB‐DQA‐DQB RFLP analysis and serology. The concordance between PCR‐SSP typing and RFLP analysis was 100% The reproducibility was 100% in 40 samples typed on two separate occasions. No false‐positive or false‐negative typing results were obtained. All homozygous and heterozygous combinations of DR1‐DRw18 could be distinguished. Amplification patterns segregated according to dominant Mendelian inheritance. DNA preparation, PCR amplification and post‐amplification processing, including gel detection, documentation and interpretation, were performed in 2 hours. In conclusion, PCR‐SSP is an accurate typing technique with high sensitivity, specificity and reproducibility. The method is rapid and inexpensive. DR “low‐resolution” typing by the PCR‐SSP technique is ideally suited for analyzing small numbers of samples simultaneously and is an alternative to serological DR typing in routine clinical practice including donor‐recipient matching in cadaveric transplantations.
The molecular basis for reactivity of anti‐Cw1 and anti‐Cw3 alloantisera with HLA‐B46 haplotypes
Authors: Zemmour Jacqueline , Gumpert Jenny E. , Hlldebrand William H. , Ward Frances E. , Marsh Steven G. E. , Williams Robert C. , Parham Peter
Published: December 2008

Abstract: HLA haplotypes containing the HLA‐B46 allele react with both anti‐Cw1 and anti‐Cw3 alloantisera, a pattern of reactivity defined as the Cwl1 antigen and postulated to involve either a distinctive Cw11 allele or a duplicated HLA‐C locus. From serological characterization of CIR cells transfected with B46 cDNA we now demonstrate that the anti‐Cw3 reactivity with these haplotypes is solely due to the B46 molecule and not to an HLA‐C molecule. Furthermore, isolation and characterization of HLA‐C mRNA from cells expressing B46 strongly suggest that anti‐Cw1 reactions are directed against the product of a conventional Cw1 allele. The antigenic cross‐reactivities of B46 with B62 and Cw3 correlate with its chimaeric primary structure, which is identical to that of B62, except in the a, helix where it is identical to both Cw3 and Cw1. The structure, distribution and genetic linkage of B46 indicate it is of recent, Asian origin and is the result of a gene conversion, involving Cw1 as the donor gene and B62 as the recipient. These results demonstrate that the Cw11 antigen neither dorresponds to a novel HLA‐C allele nor a duplicated HLA‐C locus, but to a combination of epitopes contributed by linked Cw1 and B46 alleles. The nucleotide sequence we previously and erroneously attributed to a distinct Cwl1 allele is now demonstrated to encode Cw8. Isolation of the cDNA clone with this sequence from a library made from a cell homozygous for the B46 haplotype was probably an artefact of contamination.
A cytotoxic monoclonal human hybridoma antibody (TrJ3) against HLA‐B44(12) and ‐B45(12)
Authors: Ge Jun , Bratlie Anne , Hannestad Kristian
Published: December 2008

Abstract: We have generated a human monoclonal cytotoxic IgMlD antibody (TrJ3) that reacted specifically with all lymphoblastoid B‐cell lines expressing HLA‐B44(12) and B45(12). TrJ3 hybridoma supernatant was suitable for HLA‐B IZ typing of freshly isolated blood mononuclear cells. Analysis of available amino acid sequences of HLA‐B molecules indicated that the al domain does not contain the TrJ3 serological epitope. Since HLA‐B44 is associated with a unique serine residue at position 167 that points towards the peptide binding groove, we propose that S167 of the α2 domain helix is a critical part of the TrJ3 epitope.
DNA typing of HLA Class II genes in B‐lymphoblastoid cell lines homozygous for HLA
Authors: Kimura Akinori , Dong Rui‐Ping , Harada Haruhito , Sasazuki Takehiko
Published: December 2008

Abstract: The HLA class II genotypes were determined in the B‐lymphoblastoid cell lines selected for the Tenth International Histocompatibility Workshop. The HLA class II genes were determined by the PCR‐SSOP method using the reagents provided by the Eleventh Histocompatibility Workshop. Additional studies have been performed for further characterization of HLA class II polymorphism on these cell lines. It is observed that several cell lines have HLA class II haplotypes with the same DRB1, DQA1 and DQB1 alleles on both haplotypes but different alleles at the other class II loci, confirming that these cell lines are not truly HLA class II‐homozygous. Other cell lines carried HLA class II haplotypes which were only different at the DRB1 gene. These results suggest double recombination events or gene conversion‐like events in generation of HLA DR, DQ haplotypes. These cell lines provide an important tool as references for HLA DNA typing.
HLA‐DP polymorphism in Sudanese controls and patients with insulin‐dependent diabetes mellitus
Authors: Magzoub M. M. A. , Stephens H. A. F. , Sachs J. A. , Biro P. A. , Cutbush S. , Wu Z. , Bottazzo G. F.
Published: December 2008

Abstract: Human leukocyte antigen (HLA) genes are candidates for susceptibility to insulin‐dependent diabetes mellitus (IDDM). The association of IDDM with particular DR and DQ alleles has been reported in all populations studied, but its association with HLA‐DP alleles has been controversial. To address this question we analyzed 19 DPB1 and 2 DPA1 alleles and their associations in well‐characterized Sudanese (an admixture of Arab and Black) IDDM patients (n = 71) and ethnically matched controls (n = 86) using polymerase chain reaction‐sequence specific oligonucleotide (PCR‐SSO) typing. There were no significant differences between the patient and control groups in the DPB1 frequencies. DPB1*0201, *0401 and DPA1*01 were the most frequent alleles in both IDDM patients and control subjects. Significant positive and negative associations between DPB1 and DPA1 alleles were detected in both groups. A novel DPB1 allele included in DPB1*1701 was identified.
Monoclonal antibodies specific for canine CD4 and CD8 define functional T‐lymphocyte subsets and high‐density expression of CD4 by canine neutrophils
Authors: Moore P. F. , Rossitto P. V. , Danilenko D. M. , Wielenga J. J. , Raff R. F. , Severns E.
Published: December 2008

Abstract: The characteristics of canine homologues of CD4 and CD8, defined by murine monoclonal antibodies CA13.1E4 (IgG1) and CA9.JD3 (IgG2a) respectively, are described. These antibodies identify mutually exclusive subpopulations of non‐B lymphocytes in peripheral lymphoid organs and blood. However, in thymus the antibodies defined populations of double‐positive, double‐negative and single‐positive cells that showed a progressive maturation consistent with that described for CD4 and CD8 in other mammalian species. Furthermore, functional studies clearly associated cytotoxic effector cell function with the subpopulation reactive with CA9.JD3 (CD8). In contrast, proliferation stimulated by allogeneic cells and mitogens was more pronounced in the subpopulation reactive with CA13.1E4 (CD4). Cell and tissue distribution studies revealed that CA13.1E4 stained neutrophils with equivalent intensity to the staining of peripheral T cells. CA13.1E4 precipitated a 60 kD protein from the surface of T cells and highly purified neutrophils under both reducing and non‐reducing conditions. CA9.JD3 precipitated a heterodimer of 32 kd and 36 kD under reducing conditions, and a 70 kD protein under non‐reducing conditions. The expression of CD4 by canine neutrophils is without precedent in other mammalian species; the functional significance of neutrophil CD4 expression is puzzling in light of the current understanding of the functions of CD4 which include it's role as a receptor for non‐polymorphic regions of MHC class II molecules.
Improvement in HLA‐DQB typing by PCR‐RFLP: Introduction of a constant restriction site in one of the primers for digestion control
Authors: Mercier Bernard , Ferec Claude , Dufosse Françoise , Huart Jean‐Jacques
Published: December 2008

Abstract: The PCR‐RFLP method previously reported by Inoko is a powerful technique for HLA class II typing. The reliability of RFLP interpretation depends on complete digestion by restriction endonucleases using a modified primer with restriction sites as an internal digestion control. The use of restriction enzymes which recognize specific HLA DQB allelic variations makes HLA DQB genotyping possible.
HLA Class II nucleotide sequences, 1992
Authors: Marsh Steven G. E. , Bodmer Julia G.
Published: December 2008

The HLA Class II sequences included in this compilation are taken from publications listed in the papers: Nomenclature for factors of the HLA system, 1991 (1), Nomenclature for factors of the HLA system, 1990 (2), and Nomenclature for factors of the HLA system, 1989 (3). Where discrepancies have arisen between reported sequences, the original authors have been contacted, where possible, and necessary amendments to published sequences have been incorporated into this alignment. Future sequencing may identify errors in this list and we would welcome any evidence that helps to maintain the accuracy of this compilation. In the sequence alignments, identity between residues is indicated by a hyphen (‐). An unavailable sequence is indicated by an asterisk (*), gaps in the sequence are inserted to maintain the alignment between different alleles showing variation in amino acid number.
HLA‐DR1 and rheumatoid arthntis in Israeli Jews: Sequencing reveals that DRB1*0102 is the predominant HLA‐DR1 subtype
Authors: de Vries Niek , Renningen Kjersti S. , Tilanus Marcel G. J. , Bouwens‐Rambouts Anne , Segal Raphael , Egeland Torstein , Thorsby Erik , Van De Putte Lea B. A. , Brauthar Chaim
Published: December 2008

Rheumatoid arthritis (RA) is associated with the HLA‐DR4 cellular subtypes Dw4 and Dw14 in Caucasians, with Dw15 in Japanese, and possibly with HLA‐DR1 in Israeli Jews. Sequencing studies in Caucasians have shown that these molecules share a common amino acid sequence in the third hypervariable region of the DR molecule (AA 67‐74: LLEQRRAA or LLEQKRAA), suggesting that this sequence is primarily associated with RA. An important argument in favor of this shared‐epitope hypothesis has been the reported association between DR1 and RA in Israeli Jews. However, a later report did not confirm this association, and cellular typing showed that Israeli DR1 consists of three or more subtypes, suggesting that new subtypes might be present. Since no sequencing data on Israeli HLA‐DR1 genes have been reported, we sequenced the first domain (AA 10–91) of the DRB1 gene in 12 DR1‐positive Israeli RA patients, 5 healthy controls and a homozygous typing cell (HTC), defining the major Jewish cellular HLA‐DR1 subtype. DRB1*0102 (DR1 Dw20) was found in 8 RA patients, 3 controls and the HTC “LVA”. DRB1*0101 (DR1 Dw1) was found in 4 RA patients and 2 controls. No other DR1 subtypes were encountered. In all 20 DR1 haplotypes, the DRB1*0101 or 0102 allele was associated with DQA1*0101 and DQB1*0501, being identical to the Caucasian DR1 haplotypes. Thus, at the sequence level, we found no basis for the reported extensive cellular heterogeneity of DR1 in the Israeli population. Both alleles encountered, DRB1*0102 or DRB1*0101, encode a third hypervariable region sequence (AA 67–74) shared with DRB1*0404, 0405, 0408 and 0410.
Combinatorial diversity in DR2 haplotypes
Authors: Wade Judith A. , Hurley Carolyn Katovich , Hastings Alice , Ehrenberg Philip , Johnson Armead H. , Martell Robert W. , du Toit Ernette D.
Published: December 2008

Sequence analysis has identified multiple alleles at two loci that encode for the DR2 specificity. The loci, DRB1 and DRB5, are in linkage disequilibrium which can extend to alleles of the DQ loci. Serologic, cellular, and sequence‐specific oligonucleotide probe (SSOP) typing techniques have been used to identify the DR2 haplotypes. In this report, we have characterized by SSOP typing and cDNA/DNA sequence analyses the combinatorial diversity of DR2 haplotypes. Cells were selected on the basis of unique serologic reactivity, unique associations of alleles of DR and DQ loci, and/or presence in populations which have not been extensively characterized for HLA diversity. An asymmetric polymerase chain reaction (PCR) amplification was applied to rapidly screen unique cells and to characterize DNA sequence in conjunction with more conventional cDNA sequence analysis. The sequence data confirm the lack of a DRB5 locus in the DR2“LUM” specificity, the unexpected association of DRB1*1602 and DRB5*010 alleles in a nonCaucasoid population, and the association of the allele DRB1*1503 with DRB5*0101 in black African, African American and native American individuals. The DRB1*1503 and DRB5*0101 alleles were identified in an unusual haplotype, DR2,DQ2. The combinatorial diversity of the DR2 haplotypes is extended by these studies in nonCaucasoid populations.
Isolation and characterization of two new functional subtypes of HLA‐B35
Authors: Theiler Graciela , Pando Marcelo , Delfino José M. , Takiguchi Masafumi , Satz M. Leonardo
Published: December 2008

Human minor histocompatibility antigen‐specific, HLA‐B*3501‐restricted cytotoxic T‐cell clones were assayed on a panel of 25 different target cells previously typed by serology as HLA‐B35. Cells from 6 donors were not killed and cells from 2 of these were further studied by molecular cloning to characterize their HLA class I alleles. Two new HLA‐B35 subtypes were identified. The sequence of one of them differs from B*3501 by one nucleotide change at codon 156, replacing a Leu for an Arg. The sequence of the other new subtype also shows a single nucleotide change compared to B*3501, with Gly Val substitution at residue 16. With these new variants, the allelic complexity of HLA‐B35 extends now to eight subtypes.
Analysis of HLA‐DQA1 and ‐DQB1 genes in Mexican Americans with insulin‐dependent diabetes mellitus
Authors: Sanjeevi C. B. , Zeidler A. , Shaw S. , Rotter J. , Nepom G. T. , Costin G. , Raffel L. , Eastman S. , Kockum I. , Wassmuth R. , Lernmark Å.
Published: December 2008

Abstract: Mexican American patients (n = 35) with insulin‐dependent diabetes mellitus (IDDM) and control subjects (n = 39) were HLA‐DQA and DQB typed by the polymerase chain reaction technique combined with allele‐specific oligonucleotide probes. Either DQBl*0302 or DQB1*0201 was present among 91% (32/35) of the patients compared to 67% (26/39) of controls. Either DQA1*0501 or DQA1*0301 was present in all patients (100% or 35/35) compared to 29/39 (74%) (OR 12.06 Pc<0.05) of controls. All four of these genes, in cis or trans, were present in 15/35 (43%) of the patients compared to 3/39 (8%) of controls (OR 9.0; Pc<0.01). The presence of one or more non‐susceptibility alleles showed a dose‐related decrease in relative risk. Presence of aspartic acid (Asp) at position 57 of the DQ β chain did not confer protection and non‐Asp homozygozity did not confer susceptibility to IDDM in this ethnic group. In conclusion, susceptibility to IDDM in Mexican Americans is associated with particular DQA and DQB combinations, illustrates dose‐dependent parameters and contradicts the critical residue hypothesis.
DNA typing of HLA‐B gene in Takayasu's Arteritis
Authors: Yoshida Masayuki , Kimura Akineri , Katsuragi Kiyanari , Numano Fujia , Sasazuki Takohlko
Published: December 2008

Abstract: Sixty‐four patients with Takayasu's arteritis and 156 healthy individuals in the Japanese population were examined for HLA‐B specificity at the DNA level by DNA typing using polymerase chain reaction (PCR)/sequence‐specific oligonucleotide probe (SSOP) analysis and by subsequent sequencing analysis. The frequency of epitope combination group‐B52 (EC‐B52) corresponding precisely to HLA‐B52 specificity and that of EC‐B39.2 which is a newly‐identified subtype of HLA‐B39 specificity were increased in the patient group. These two disease‐associated HLA‐B alleles share an epitope composed of 63Glu and 67Ser. Because two HLA‐B alleles, HLA‐B51 and B39.1, which are similar but different at the epitope from HLA‐B52 and B39.2, respectively, are not associated with Takayasu arteritis, 63Glu and 67Ser are supposed to be involved in the pathogenesis.
Study of HLA class II alleles by PCR oligotyping in leprosy patients from North India
Authors: Rani Rajni , Fernandez‐Vina M. A. , Zaheer S. A. , Beena K. R. , Stastny Peter
Published: December 2008

Abstract: Host factors seem to play an important role in determining the immune response and the differential manifestations of lepromatous (LL) and tuberculoid (TT) leprosy. In order to investigate the role of immunogenetic factors in determining the form of leprosy, the HLA class II alleles of DRB1, DRB3, DRB5, DQA1, DQB1 and DPB1 were studied by a PCR oligotyping technique in 93 patients and 47 healthy controls. DRB1*1501 and DRB1*1502 (two of five tested subsets of the serologically defined DR2) accounted for 81.5% of the multibacillary patients (relative risk 16.3) and 60.7% of the TT patients (relative risk 5.7) compared to 21.3% in normal, ethnically‐ and geographically‐matched controls. The much stronger association of DRB1*1501 with the multibacillary form than with the TT type of leprosy suggests a possible role in the differential immune response to M. leprae antigens. DQB1*0601 was found significantly more often than in controls throughout the leprosy spectrum, while DQA1*0103 was most frequent in the LL group and DQA1*0102 was selectively increased in the borderline lepromatous (BL) patients. On the other hand, DRB1*0701, DQB1*0201 and DQA1*0201 were decreased in the multibacillary leprosy patients (MLP) compared to TT patients and controls, and DQB1*0503 was selectively decreased in TT patients, suggesting that these HLA alleles might play a role in modulating the immune response that determines the form of leprosy that develops in each patient.
Direct detection of PCR products for HLA class II typing
Authors: Chia David , Terasaki Paul , Chan Henry , Tonai Richard , Siauw Pee‐An
Published: December 2008

Abstract: Direct detection of the PCR, or DD‐PCR is proposed as an efficient method for performing PCR assays. Following the PCR reaction, ethidium homodimer dye is added to the reaction mixture and read by fluorescence. The dye step circumvents the necessity of running reactions on agarose gel electrophoresis, which is the current standard. This simple modification should find wide application for assays utilizing the PCR reaction. Here we show the ready detection of HLA class II polymorphism.
HLA‐B locus DNA typing: Detection of B*7801 and seven additional alleles by BW6‐specific exon 2 amplification
Authors: Andrien M. , Tiercy J‐M. , Defleur V. , Bouillenne C. , Toungouz M. , Jeannet M. , Dupont E.
Published: December 2008

Abstract: A molecular approach to type a new HLA‐B5 antigen, HLA‐BSNA, characterized by its unusual association with the public determinant BW6, referred to as B*7801, has been designed. Antigens disclosing serological identity with SNA, BXI and Te76 were also investigated. Based upon HLA‐B exon 2 group‐specific PCR, the following procedure was established: 5′ and 3′ primers were designed by targetting the codons 11‐12 (AM) and 81‐83 (LRG), respectively, in exon 2 (α1 domain). The 5′ primer discriminates with HLA‐A, ‐C genes and pseudogenes, while the 3′ primer detects the sequence encoding the BW6 epitope (NLRG) and discriminates it from the BW4 epitope. The combination of this pair of primers specifically amplifies 26 HLA‐B alleles. Oligotyping for B*7801 was performed using a combination of two non‐radioactively labeled SSO probes identifying positions T45 and D74 in exon 2. To resolve ambiguous hybridization patterns, an additional set of probes was used. The specificity of this BW6‐group‐specific amplification procedure was investigated on 150 genomic DNA samples. Among them, we obtained 94 amplified DNA products which were tested with eight SSOs. Beside B*7801, the following B alleles could be defined: B*0801, B*35, B*5401, B*5501‐2, B*5601‐2. B*1501‐7 (including 862. B75 and B72) and B*4601. SNA, BXI and Te76 DNA samples gave similar hybridization pattern providing a clue to the identity of these antigens. This “one PCR and two probes” procedure represents a simple oligotyping strategy which can also be applied to type many other HLA‐B specificities.
Structural heterogeneity in HLA‐B70, a high‐frequency antigen of black populations *
Authors: Demsna Jobi D. , Uttld Ann‐Mawant , Maddgal Ahjaadm J. , Hildabrand Ynlllam H. , Jshasten‐Dow Leslie , du Teit Ernotta , Bias Wilma B. , Parham Patar
Published: December 2008

Abstract: Although the B70 antigen exhibits allele frequencies of 8‐23% in African and American black populations, it remains poorly defined. Cloning and sequencing of cDNA encoding B70 antigens from six cell lines has identified a group of three closely related alleles: B*1503, B*1509 and B*1510, that form a subgroup of the B15 family. The sequences of these alleles and, in particular, B*1503, are close to that of the HLA‐B consensus consistent with the difficulty in their serological definition. The products of the three alleles correspond to three electrophoretically detected variants of the B70 antigen and some correlation with the B71 and B72 subspecificities of the B70 antigen can be made. A fourth allele, B*7901. previously described by Choo et al. (J. Immunol. 147: 174‐180, 1991) that was not serologically typed as B70, differs by a single nucleotide substitution from B* 1510. The sensitivity of alloantibodies to single differences in peptide binding residues suggest a role for bound peptides in the HLA‐970 alloantigenic specificities. The heavy chains encoded by the four alleles differ at four peptide binding residues of the antigen recognition site, the evolutionary modification of which can be explained in terms of interallelic recombination events.
HLA‐DRB1, DQA1 and DQB1 DNA polymorphisms in healthy Algerian and genetic relationships with other populations
Authors: Djoulah S. , Sanchez‐Mazas A. , Khalil I. , Benhamamouch S. , Degos L. , Deschamps I. , Hors J.
Published: December 2008

Abstract: This study presents the results of HLA‐DRB1, ‐DQA1, and ‐DQB1 sequence‐specific oligonucleotide probe (SSOP) typings for a population sample of 47 individuals originating from Western Algeria. Allele and haplotype frequencies, as well as linkage disequilibria are computed by the standard methods used for the XIth International Histocompatibility Workshop data. A total of 24 alleles are detected at the DRB1 locus, where a very high heterozygosity level (0.914) is found. The highest DRB1 frequencies are 0.160, DRB1*1101, and 0.138, for DRB1*0301 and DRB1*0701. The DQA1 and DQB1 loci are less polymorphic. Among the 8 DQA1 alleles detected, DQA1*0501 is highly predominant with a frequency of 0.383. Thirteen DQB1 alleles are observed among which DQB1*0301 and DQB1*0201 are the most frequent (0.351 and 0.245, respectively). Three haplotypes predominate clearly: DRB1*1101‐DQA1*0501‐DQB1*0301 (0.138), DRB1*0701‐DQA1*0201‐DQB1*0201 (0.128) and DRB1*0301‐DQA1*0501‐DQB1*0201 (0.117). The two latter are among the most frequent haplotypes found in European and North American Caucasoid populations, but the DQA1*0501‐DQB1*0201 association is not significant in Algerians. The genetic distances computed for each locus among a set of populations from different continents are significantly correlated to geography. They indicate that the Algerians are very close to South European populations, particularly to Sardinians, Italians, Romanians and French, with some intermediate characteristics between Europeans and sub‐Saharan Africans. These results may serve as reference for future studies of HLA and disease in the Algerian population.
Molecular genetic studies of HLA class II alleles in sarcoidosis
Authors: Ishihara Mami , Ohno Shigeaki , Ishida Takako , Ando Hitoshi , Naruse Taeko , Nose Yoshisuke , Inoko Hidetoshi
Published: December 2008

Abstract: Previous HLA serological studies showed positive associations of the DR52 antigen, the DR52‐associated antigens (DR3, DR5 and DR6) and the DR8 antigen with sarcoidosis. To investigate the HLA alleles that may contribute to the genetic susceptibility to sarcoidosis at the DNA level, HLA‐DRB1, ‐DRB3, ‐DQA1 and DQB1 genotyping using the polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method was performed in 63 Japanese patients with sarcoidosis. The frequencies of the DR52‐associated DRB1 alleles (DRB1*11, DRB1*12 and DRB1*14 except DRB1*1302), DRB1*08, DRB3*0101, DQA1*0501 and DQB1*0301 were significantly increased in patients compared with healthy controls. The significant increase of DRB3*0101, DQA1*0501 and DQB1*0301 could be explained by linkage disequilibrium with the DR52‐associated DRB1 alleles. It must be noted that the DR8 haplotype, which does not possess the DRB3 gene, also showed a significant increase in sarcoidosis. These results suggest that the HLA‐alleles responsible for the susceptibility to sarcoidosis are located at the HLA‐DRB1 locus rather than the HLA‐DRB3, ‐DQA1 and ‐DQB1 loci. In contrast, DRB1*1302 may confer resistance to the disease.
A simple and economical DRB1 typing procedure combining group‐specific amplification, DNA heteroduplex and enzyme restriction analysis
Authors: D'Amato Mauro , Sorrentino Rosa
Published: December 2008

Abstract: A new procedure for HLA‐DRB1 typing is proposed. The method combines six group‐specific amplifications with heteroduplex analysis and, in some cases, enzyme restriction analysis. This technique, which is as discriminative as oligotyping, is simple, economical and does not require probes. These characteristics make this approach a valid alternative to other HLA genomic typing procedures, especially in those cases such as donor‐recipient pairs matching where a small number of samples has to be managed at once.
Molecular diversity of the HLA‐C locus in unrelated marrow transplantation
Authors: Petersdorf Effie W. , Stanley Jon F. , Martin Paul J. , Hansen John A.
Published: December 2008

Abstract: The diversity of HLA‐C exon‐2 alleles in 56 HLA‐A, B, DRB and DQB1‐matched patient‐unrelated marrow donor pairs was examined by non‐cloning polymerase chain reaction‐based sequencing of genomic DNA. This method allows simultaneous analysis of both alleles in heterozygous samples. All Cw5‐positive individuals encoded a sequence which differed from the published Cw*0501 sequence at position 61. Among 82 samples assigned a single antigen by serologic testing, 64 (78%) were heterozygous for two distinct alleles when tested by sequencing. Cw*1202, 1601 and 15 were identified in samples for which no phenotype could be assigned (C “blank”). Finally, 7 of the 56 HLA‐A, B, DRB, DQB1‐matched pairs (12.5%) were mismatched for one or both HLA‐C alleles. We conclude that sequence‐based methods constitute the optimal strategies for typing HLA‐C alleles in the unrelated marrow transplant population.
Biochemical characterization of bovine MHC DQ allelic variants by one‐dimensional isoelectric focusing
Authors: Bissumbhar B. , Nilsson Ph. R. , Hensen E. J. , Davis W. C. , Joosten I.
Published: December 2008

Abstract: Previous studies on expressed bovine MHC class II polymorphism using one‐dimensional isoelectric focusing (1D‐IEF) allowed the identification of at least 12 allelic variants of the DRB3 gene. So far, only limited data have been available on the expression of other class II genes. The present study involved biochemical analysis of bovine MHC class II molecules using a set of monoclonal antibodies presupposed to be bovine DR and DQ reactive. After essential modification of the standard electrophoresis conditions used for 1D‐IEF typing of bovine DR products, biochemical polymorphism was observed for non‐DR molecules, revealing polymorphic sets of basic and acidic focusing bands. Because of the extensive DNA polymorphism described for bovine DQA and DQB genes, and the apparent similarity with the focusing pattern of human DQ products, these molecules were considered to be the bovine DQ homologues. The definition of the DQ‐associated banding patterns was made possible by using two half‐sib sire families. Four different DQA‐like patterns and nine DQB‐like patterns were detected. Segregation of the DQ types was supported by serological class I and class II typing. These results show that it is now possible to discriminate between expressed bovine DR and DQ polymorphism.
Gene frequencies and haplotypic associations within the HLA region in 916 unrelated Japanese individuals
Authors: Hashimoto Mitsuo , Kinoshita Tomoko , Yamasaki Miho , Tanaka Hidenori , Imanishi Tadashi , Ihara Hideari , Ichikawa Yasuji , Fukunishi Takanobu
Published: December 2008

Abstract:The gene frequencies and haplotypic associations within the HLA region have been investigated in 916 unrelated Japanese individuals. HLA class I and class II antigens were studied by conventional serology, and class II alleles, DRB1, DRB3, DQA1, DQB1 and DPB1 were typed by using polymerase‐chain reaction amplification and sequence‐specific oligonucleotide probe (PCR‐SSOP) method. Thirty DRB1, 3 DRB3, 8 DQA1, 15 DQB1 and 13 DPB1 alleles were found in our population. DR‐NJ25, a characteristic antigen in the native American and Asian populations, was observed at 3.0%. This antigen was observed mainly with the DRB1*1403 and 1406 alleles. Twenty‐seven out of 30 DRB1 alleles found in this study had a high positive linkage disequilibrium with DQB1 alleles and 20 of them had an exclusive association with one specific DQA1‐DQB1 combination. The strong association between DRB1 alleles and HLA‐B antigens was the most striking finding in this study. Twenty‐eight out of 30 DRB1 alleles had a positive linkage disequilibrium with 24 HLA‐B antigens (p<0.01). The other two alleles, DRB1*0404 and 1402, were very rare, and their frequencies were 0.2% and 0.1%, respectively. The data presented in this population study should be useful for the studies on anthropology, organ transplantation and disease susceptibility.
HLA class II alleles confer susceptibility to recurrent fetal losses in Danish women
Authors: Christiansen O. B. , Rasmussen K. L. , Jersild C. , Grunnet N.
Published: December 2008

Abstract: HLA‐DR and ‐DQ typings were performed by a combination of RFLP and PCR‐SSP techniques in 234 Danish women with at least three consecutive unexplained fetal losses (recurrent fetal losses) and 360 controls and the DRB1, DQA1 and DQB1 alleles were deduced. In the total group of patients, the frequency of no DRB1‐DQA1‐DQB1 haplo‐type was significantly increased compared with controls. In the subgroup of 97 women with four or more fetal losses (multiple fetal loss group), the frequency of women carrying the DRB1*0101, DQA1*0101, DQB1* 0501; DRB1*0102, DQA1*0101, DQB1*0501 and DRB1*0103, DQA1*0101, DQB1*0501 haplotypes or the DRB1*0301, DQA1*0501, DQB1*0201 haplotype were significantly increased compared with controls (RR=2.1; pc<0.05 with regard to former three haplotypes combined and RR=2.2; pc<0.05 for the latter). The frequency of women with at least one of the four haplotypes was significantly (p<0.002) increased with the number of previous fetal losses in the women's history. Analysis of the DQA1 and DQB1 phenotypes in women with at least four fetal losses showed that DQA1*0501 and DQB1*0501 were increased compared with controls (RR=1.9; pc<0.05 and RR = 2.2; pc<0.025, respectively). Analysis of DRB1‐DQA1‐DQB1/DRB1‐DQA1‐DQB1 genotypes suggested that genotypes comprising both DQA1*0501 and DQB1*0501 alleles (in trans) exhibited a higher RR for experiencing at least four fetal losses (RR=3.4, p=0.002) than each of the alleles did alone. Genotypes comprising a DQB1 allele encoding a β chain negative for aspartate in position 57 were associated with an increased RR (2.3; p<0.01) for multiple fetal losses and the etiologic fraction was high (0.49). The results suggest that genes or gene‐sequences, linked to the DRB1*0101, DQA1*0101, DQB1*0501; DRB1*0102, DQA1*0101, DQB1*0501; DRB1*0103, DQA1*0101, DQB1*0501 and DRB1*0301, DQA1*0501, DQB1*0201 haplotypes, confer susceptibility to multiple fetal losses. These alleles/gene‐sequences are presumably located in the DQA1 and DQB1 loci and may elicit their effect by mediating an autoimmune reaction against one or more trophoblast antigens.
Striking conservation of three extended HLA‐DR13 haplotypes in the Japanese population
Authors: Kinoshita Tomoko , Hashimoto Mitsuo , Yamasaki Miho , Ihara Hideari , Ichikawa Yasuji , Fukunishi Takanobu
Published: December 2008

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.
HLA class II gene polymorphism in Tunisians
Authors: Hmida S. , Gauthier A. , Dridi A. , Quillivic F. , Genetet B. , Boukef K. , Semana G.
Published: December 2008

Abstract: The polymorphism of HLA clas II genes (HLA‐DRB, DQB, DPB) was investigated in 101 Tunisians using polymerase chain reaction (PCR) amplification and reverse dot blot (RDB) hybridization. Allele and haplotype frequencies, as well as DRB1‐DQB1 linkage disequilibria, were calculated. A total of 26 DRB1 alleles were detected and the most prevalent variant was DRB1*0301 with an allelic frequency at 21.87%. In the DR1 group, DRB1*0102 was most frequent than DRB1*0101. In the DR4 group, DRB1*0403 was the most common allele and was associated with DQB1*0402. Interestingly this DRB 1‐DQB1 association has not been observed in other populations. With regard to the DR8 group, DRB1*0804 was the unique variant detected, whereas with the DR13 specificity, the most common variant was DRB1*1303 in Algerians also. Although the DQB1 polymorphism analysis showed an allelic distribution very close to that observed in caucasoids, many DRB1‐DQB1 associations which have not been reported in studies of other populations, were described. Finally at the DPB1 locus DPB1*1701 and *1301 allele frequencies distinguish clearly this Tunisian sample from a French caucasoïd panel of 83 subjects. In conclusion, a specific distribution of HLA components in terms of gene and haplotype frequencies characterizises this Tunisian population. This specific pattern may reflect the great ethnic diversity of this community. All these informations may be helpful in the future for HLA and disease association studies.
A description of a new DR allele, DRB1*1113
Authors: Rosenberg S. M. , Wollenzien T. F. , Johnson M. M. , Eberly L. , Hurley C. K. , Reinsmoen N. L. , Steiner N. , Goeken N. E.
Published: December 2008

Abstract: We have discovered a previously unpublished HLA‐DRB1 allele, observed in a patient (SB), his mother, and one sibling. The undefined allele gave sporadic positive reactions with sera in the DR52‐associated group. SSOPH analysis utilizing both generic and group specific primers and probes also gave ambiguous results. SB typed clearly as a DRB 1*0301 (paternal allele) but the DNA from SB also bound probes specific for DRB 1*14 and DRB1*11. Sequencing revealed that the undefined allele was similar to a DRB 1*14 allele with a segment of sequence found in DRB1*11 alleles. The patient was MLC reactive with donors who express DRB1*0301, *1401 and *0301, *11 and was nonreactive solely to DRB 1*0301 (Dw3) homozygous typing cells.
Differential antigen presentation of hepatitis B surface antigen on cell membranes of responder and nonresponder mice
Authors: Rajadhyaksha M. M. , Thanavala Y. M.
Published: December 2008

Abstract: In several systems it has been shown, that non‐responsiveness to an antigen in mice of a particular haplotype is due to the lack of binding of an immunogenic peptide to class II molecules. Such studies have been done using detergent solubilized, affinity purified class II molecules. It has been reported, that the presence of certain phospholipids around class II molecules dramatically alters the extent of peptide binding to these molecules. It thus appears that the milieu in which the class II molecules are inserted influences to a considerable extent the level of peptide binding. Hence it is likely that the kinetics of binding of immunogenic peptides to class II on the cell surface, may be different from that of molecules inserted in detergent micelles. We therefore decided to test this by studying the binding of radiolabeled peptides to class II molecules on cell membranes. We report here a rapid and sensitive assay for peptide binding to murine class II molecules on cell membranes. Further, we have used this assay to study the nature of the interaction of immunogenic peptides and class II molecules on cell membranes of mice who are responders and non‐responders to Hepatitis B surface Antigen (HBsAg). Interestingly, we find that immunogenic peptides bind in good correlation with their MHC restriction. We also observe that the HBsAg derived peptide which is unable to elicit a T cell response in the non‐responder B10.M strain actually binds to the class II of the non‐responder but fails to make a stable class II‐peptide complex. We thus conclude that defective antigen presentation may be one of the possible explanations, underlying the mechanism of non‐responsiveness.
RG1, a new murine monoclonal antibody recognizing a “supertypic” determinant on HLA‐A molecules
Authors: Pettersen R. D. , Hestdal K. , Lee N. , Geraghty D. E. , Valø E. T. , Lie S. O. , Gaudernack G.
Published: December 2008

Abstract: Monomorphic and polymorphic anti‐HLA monoclonal antibodies (mAb) are valuable reagents for assessment of the structural and functional importance of different class I determinants. We have generated a new mAb, RG1, reacting with an epitope variably expressed on normal and leukemic hematopoietic cells of different lineages. Immunoprecipitation of the RG1 antigen disclosed a bimolecular complex characteristic of class I proteins. The RG1 epitope was expressed on an HLA‐A2 transfected cell line but not on cells transfected with HLA‐E, ‐F or ‐G molecules. MAb reactivity with reference B‐lymphoblastoid cell lines and HLA typing of RG1 reactive and unreactive cells demonstrated that the epitope was expressed in conjunction with defined HLA‐A molecules. Cells expressing HLA‐A2, ‐A24(9) and ‐A68(28) proteins were brightly stained with RG1 whereas mAb binding to HLA‐A1, ‐A11 and a split of A3 molecules was significantly lower. In contrast, the RG1 epitope was apparently not expressed on HLA‐A23(9), ‐A25(10), ‐A26(10), ‐A29(19), ‐A30(19), ‐A31(19), ‐A32(19), ‐A33(19) and some HLA‐A3 molecules. Based on class I α sequence data, these results suggest that the RG1 epitope is localized to a region of the α2 helix accessible to the T cell receptor for antigen on cytotoxic T lymphocytes. Lys in position 144 and His in position 151 are apparently critical for RG1 binding.
HLA—DPB1 alleles in a population from North India and description of a new variant (DPBl*5601)
Authors: Rani R. , Fernandez‐Viñna M. A. , Zhang S. , Stastny P.
Published: December 2008

Abstract: HLA‐DPB1 alleles were studied in 51 normal individuals and 93 leprosy patients from North India using a PCR‐oligotyping technique. Hybridization patterns could identify 47 alleles of which 20 were found in the population studied. DPB1*0401 was found to be the most frequent allele with a frequency of 66.7% followed by DPB1*0402 (21.6%), DPB1*0201 (21.6%), DPB1*1301 (15.7%) and DPB1*0301 (13.7%). Besides the common alleles, DPB1*0101, *1701, *2601, *1001, *1601, *0901, *2901, *1501, *0501, *1401, and *3301 were observed at low frequencies. DPB1*2101, DPB1*2801, DPBP3201 and DPB1*3501 were not found in the normal individuals studied but were observed in the group of leprosy patients. DPB1*0202, *0601, *0801 and *1101 were not found in this population. Two alleles with apparent new hybridization patterns were isolated and sequenced. The nucleotide sequences obtained have confirmed the hybridization patterns. One of them (DPB1 * 4601) confirms a sequence recently reported. The other has been given the official designation of DPB1*5601.
Identification of woodchuck class I MHC antigens using monoclonal antibodies
Authors: Michalak T. I. , Churchill N. D. , Codner D. , Drover S. , Marshall W. H.
Published: December 2008

Abstract: Two class I major histocompatibility complex (MHC) proteins with molecular masses of 43‐ and 39‐kDa were identified in the cell surface membranes of normal woodchucks using a newly developed anti‐woodchuck class I monoclonal antibody (mAb) B1b.B9 and immuno‐blotting. B1b.B9 was generated by immunizing mice with viable wood‐chuck peripheral blood mononuclear cells and was selected for anti‐class I MHC reactivity using a cellular enzyme–linked immunoassay, indirect immunofluorescence on tissue sections and flow cytofluorimetry. The distribution pattern of class I MHC antigen on woodchuck lymphoid cells was found to be similar to that reported in other species. Also, the antigen expression on normal woodchuck hepatocytes was comparable to that observed on normal human liver parenchymal cells; thus, the antigen was not detected on hepatocytes by staining of liver tissue sections, but was found by indirect immunofluorescence staining of isolated liver cells. Western blot analysis of the plasma membranes from normal woodchuck hepatocytes revealed the presence of a single species of class I MHC heavy chain protein with a molecular mass of 43‐kDa, whereas splenocyte plasma membranes showed intense expression of a 43‐kDa species, as well as the presence of a 39‐kDa protein. The 39‐ and 43‐kDa proteins were extracted with Triton X‐114 to the hydrophobic protein phase, suggesting that they both contain a hydrophobic transmembrane domain. The data obtained indicate that the B1b.B9 identifies a nonpolymorphic epitope of woodchuck class I MHC heavy chains, providing an important reagent for the study of the pathogenesis of hepatitis B virus infection in a woodchuck model.
CD50 (intercellular adhesion molecule‐3) is expressed at higher levels on memory than on naive human T cells but induces a similar calcium mobilization on both subsets
Authors: Pino‐Otín M. R. , Juan M. , de la Fuente M. A. , Viñas O. , Martínez‐Cáceres E. , Fernández M. D. , Miralles A. , Vilella R. , Yagüe J. , Vives J. , Gayà A.
Published: December 2008

Abstract: CD50, the intercellular adhesion molecule‐3 (ICAM‐3), is expressed almost exclusively on hematopoietic cells. T lymphocytes display a bimodal distribution on CD50 expression levels. It was observed that CD45RO+ cells expressed higher levels of CD50 than CD45RA+ T lymphocytes. A similar situation was observed when CD4 and CD8 sub‐populations were analyzed, with CD8+ cells expressing higher levels of CD50 than CD4+ cells. When adult T lymphocytes were analyzed by three‐color flow cytometry in CD8+CD45RA+ cells both CD50low and CD50high expressing cells were detected, in accordance with several memory markers on T lymphocytes, whereas only cells with a low level of CD50 were observed in CD4+CD45RA+. The different level of CD50 expression was confirmed by analyzing purified CD45RA+ and CD45RO+ T cells. Moreover, after the comparison of CD50 expression level in thymocytes, cord blood and adult T lymphocytes, a progressive increase was observed. When T cells were sorted by their intensity of CD50 expression, only CD50high cells proliferated in response to tetanus toxoid. Therefore, the phenotypic and functional analysis of adult and cord blood T lymphocytes as well as thymocytes indicates that CD50 expression increases during the maturation process of T lymphocytes: from the lowest CD50 levels present on CD1+ thymocytes, to the highest levels of CD50 on human memory T cells. In addition, we have observed that after CD50 cross‐linking on human T lymphocytes, a transient increase in intracellular calcium concentration ([Ca2+]i) is produced. When CD45RA+ and CD45RO+ T cells were analyzed, in spite of the level of CD50 expression, the stimulation through CD50 induced a similar level of Ca2+ mobilization in both subpopulations, contrasting with the higher rise in [Ca2+]i induced by CD3 stimulation on CD45RA+ versus CD45RO+. These data suggest that the signal transduction pathways activated after CD50 cross‐linking are, at least partially, independent of those involved after CD3 stimulation.