bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of the TNFa2 microsatellite allele with the presence of colorectal cancer
Authors: Gallagher G. , Lindemann M. , Oh H.‐H. , Ferencik S. , Walz M. K. , Schmitz A. , Richards S. , Eskdale J. , Field M. , Grosse‐Wilde H.
Published: December 2008

Independently, our two groups collaborating in this report performed association studies to consider the influence of the TNF region within the human MHC on the presence of colorectal cancer. In the Glasgow Study, 84 colorectal cancer patients were compared with 91 controls at the TNFa microsatellite locus. Analysis of individual alleles by Fisher's exact test revealed a significant overrepresentation of the TNFa2 allele in the colorectal cancer group, after correcting for multiple comparisons. In the Essen Study, 47 colorectal cancer patients were compared with 117 matched controls, and the hypothesis of TNFa2 overrepresentation in colorectal cancer was confirmed. These data provide evidence for the involvement of the TNF locus in the pathogenetic etiology of colorectal cancer.
Identification by polymerase chain reaction and hybridization with sequence‐specific oligoprobes and complete exon‐2 cDNA sequencing of a new DRB1 (DRB1*1131) allele
Authors: Balas A. , Santos S. , García‐Sánchez F. , García‐Escribano C. , Merino J. L. , Vicario J. L.
Published: December 2008

HLA‐DRB1 is the most polymorphic gene described so far, and their encoded molecules disclose a major role in allogeneic responses. We describe in this report a new DRB1 allele in a Spanish Caucasian bone marrow donor, initially defined by PCR‐SSO as a DRB1*11‐like allele. Complete exon 2 cDNA‐sequencing reveled that this allele was identical to DRB1*1119 except for a single substitution at position 178, which generates an amino acid change (Tyr‐His) at position 60. This residue is shared by several DRB1*14 subtypes and DRB1*0808. The new allele was officially named DRB1*1131.
Idiopathic and secondary membranous nephropathy and polymorphism at TAP1 and HLA‐DMA loci
Authors: Chevrier D. , Giral M. , Perrichot R. , Latinne D. , Coville P. , Muller J. Y. , Soulillou J. P. , Bignon J. D.
Published: December 2008

In a previous study on the effects of TAP1 and TAP2 gene polymorphism in kidney allograft recipients, we found no association between graft outcome and recipient/donor TAP1 and TAP2 allele polymorphism or compatibility, but we observed a surprising increased frequency of the TAP1*0201 allele among kidney recipients. This increase was restricted to patients with glomerulopathy. We now report on a larger cohort of 178 patients with membranous nephropathy who were typed for their HLA‐DPB1, ‐DRB1, ‐DMA, ‐DMB, LMP2, LMP, TAP1 and TAP2 genes compared with 100 random ethnically matched and healthy unrelated individuals used as controls. The results show a significant increased frequency of two markers in membranous nephropathy patients as compared with controls: firstly the previously recognized increase in HLA‐DR3 (59% vs 18%: Pc < 1 × 10‐9, RR=6.6), secondly a new association with two TAP1 amino acid variants displaying respectively a valine in amino acid position 333 (TAP1‐Val‐333) and consequently a glycine in position 637 (TAP1Gly‐637) due to its strong linkage disequilibrium with Val‐333. No linkage disequilibrium was found between TAP1‐Val‐333 and HLA‐DR3. Moreover, we also noticed a decrease of the DMA*0102 phenotype in membranous nephropathy patients. The other HLA‐DPB, ‐DMB, LMP2, LMP7 and TAP2 phenotype frequencies were roughly similar between patients and controls. These results show that the TAP1‐Val‐333 like HLA‐DR3 phenotype is positively associated with membranous nephropathy and that these two risk factors are not cumulative in membranous nephropathy pathophysiology.
High‐resolution HLA‐DRB typing using denaturing gradient gel electrophoresis and direct sequencing
Authors: Knapp L. A. , Lehmann E. , Hennes L. , Eberle M. E. , Watkins D. I.
Published: December 2008

High‐resolution HLA‐DRB typing is required for bone marrow transplantation between unrelated donors and recipients and also for identification of novel HLA‐DRB alleles. Here we describe a method for the unambiguous identification of HLA‐DRB alleles using the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The highly variable second exon of all HLA‐DRB1, ‐DRB3, ‐DRB4, ‐DRB5, ‐DRB6 and ‐DRB7 alleles was amplified using a single pair of generic DRB‐specific primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and the sequences of these alleles were determined using fluorescent‐based sequencing and allele‐assignment software. The validity of this typing procedure was confirmed by identification of HLA‐DRB alleles for 17 individuals previously characterized by PCR‐SSP and/or cloning and sequencing techniques. We identified 34 different HLA‐DRB alleles in these 17 unrelated individuals. Importantly, our analysis revealed HLA‐DRB1 alleles which had not been identified using the PCR‐SSP typing technique. Additionally, alleles from the HLA‐DRB3, ‐DRB4 and ‐DRB5 loci were identified. Whereas traditional HLA‐DRB typing methods provide limited information or require the use of multiple oligonucleotide primers or probes, our technique provides a reliable, specific and relatively rapid way of identifying all HLA‐DRB alleles for high‐resolution tissue typing.
Dissimilar evolution of B‐locus versus A‐locus and class II loci of the HLA region in South American Indian tribes
Authors: Fernández‐Vin̈a M. A. , Lázaro A. M. , Marcos C. Y. , Nulf C. , Raimondi E. , Haas E. J. , Stastny P.
Published: December 2008

Native American populations have a limited HLA polymorphism compared with other ethnic groups. In spite of this, many novel HLA‐B locus alleles, not observed in other populations, have been identified in South American tribes, and rapid evolution of this locus has been suggested. We have studied unrelated subjects of the Toba (TOB n=116), Wichi (WIC n= 46) and Pilaga (PIL n=14) tribes from northeastern Argentina to investigate the extent of the HLA polymorphism and obtain clues of selective forces that may have acted in these populations. In these tribes the number of HLA alleles is small at all loci except HLA‐B, which presents 22 alleles. Seven novel alleles were characterized including 5 of HLA‐B (B*35092, B*3518, B*3519, B*4009, B*4803) 1 at HLA‐A (A*0219) and 1 at DRB1 (DRB1*0417). All these variants may have arisen by gene conversion events. Some of the novel variants represent the most frequent alleles of these populations (B*4803 in TOB and PIL; B*3519 in WIC) or are the most frequent subtypes in their lineages. HLA‐A, B, DRB1.DQA1 and DQB1, but not DPB1, display relatively similar gene frequencies. This results in high heterozygosity in all the tribes for all the loci studied except HLA‐DPB1. The larger polymorphism and the generation and maintenance of novel alleles at the HLA‐B locus suggests a more specialized response of this locus to evolutionary forces. These effects may be related to the nature of the polymorphism, to the number of founder alleles and to the functional characteristics of the individual alleles.
High‐resolution HLA typing for the DRB3/4/5 genes by sequence‐based typing
Authors: Voorter C. E. M. , de Bruyn‐Geraets D. , van den Berg‐Loonen E. M.
Published: December 2008

The high degree of polymorphism of the HLA genes at the nucleotide sequence level has proven sequence‐based typing a major typing strategy. For DRB1 the allelic variability is predominantly present in the second exon and by DNA sequencing of exon 2 all hitherto known DRB1 alleles can be detected. For the associated genes DRB3, DRB4 and DRB5 the situation is slightly different. Allelic differences are not limited to exon 2 and the sequence of exon 3 and sometimes exon 4 is needed for complete subtyping. Oligonucleotides to amplify the exons needed for subtyping of DRB3, DRB4 and DRB5 were designed. Gene‐specific products were generated to make simultaneous detection of alleles in heterozygous combinations possible. In this way 238 individuals were fully typed for their DRB3, 4 and 5 subtypes. Additional samples were typed for only one of the genes. All samples had been previously typed by PCR‐SSP. Concordant typing results were obtained for all individuals tested. The DRB3 alleles typed for included *0101, *0201, *0202 and *0301, for DRB4 they were *01011, *0102 and *0103 and for DRB5 *0101, *0102, *0103, *0105, *0201, *0202 and *0203. All alleles were easily detected by the protocol described except for DRB5*0201. Sequencing of exon 3 and 4 of the DRB5*0201 allele showed this allele to be a sequencing error and the sequences obtained were identical to the exon 2, 3 and 4 sequences of DRB5*0202. Two new alleles were identified in the samples studied, DRB4*0105 and DRB3*0207. Sequence based typing has been recognized as a valuable tool for HLA typing of DRB1, DQB1 and DPB1 since several years. It is shown to be a superior typing method as well in the detection of the different DRB3, 4 and 5 subtypes.
A three‐step allele‐level DRB1‐DRB3‐DRB4‐DRB5 genotyping assay using polymerase chain reaction with immobilized sequence‐specific oligoprobes
Authors: Buyse I. M. , Couture C. , Sargent M. D. , Hashemi‐Tavoularls S.
Published: December 2008

A three‐step reverse hybridization assay for allele level‐resolution DRB1‐DRB3‐DRB4‐DRB5 genotyping is described. Samples are initially amplified using a generic primer pair for all DRB1‐DRB3‐DRB4‐DRB5 alleles and PCR products are hybridized to generic typing membranes. An intermediate‐resolution level genotyping is obtained at this point. Depending on the phenotype, samples are then subjected to a DR1, DR2, DR4, DR52A, DRB3 and/or DRB5 type‐specific amplification and hybridization. A third step, involving sequence‐specific PCR followed by type‐specific hybridization, is only performed to solve certain DR4 and DR52A heterozygous combinations. The assay allows 100% allele level‐resolution DRB genotyping. Hybridization membranes contain panels of SSO probes that were optimized to all react specifically under identical stringency conditions. A computer program was written to assist in analysis of the hybridization patterns. The assay was thoroughly evaluated and has been used to type over 10,000 donors from the Canadian Unrelated Bone Marrow Donor Registry (UBMDR) at allele level‐resolution. This method proved to be flexible, easy to update for newly described alleles, easy to perform, fast, and safe. It is also reliable and specific, as 9 novel DRB alleles so far have been detected as aberrant hybridization patterns.
Tumor necrosis factor locus polymorphisms in rheumatoid arthritis
Authors: Field M. , Gallagher G. , Eskdale J. , McGarry F. , Richards S. D. , Munro R. , Oh H.‐H. , Campbell C.
Published: December 2008

We examined six polymorphic elements in the tumor necrosis factor (TNF) locus and determined their allelic distribution in 98 Caucasian rheumatoid arthritis patients in comparison with 91 ethnically‐matched controls. Polymorphic elements at four biallelic sites were distributed similarly between patients and controls, irrespective of the presence or absence of DR4. Differences were observed between the two groups at the TNFa and TNFe loci, but these were consistent with extended MHC haplotypes known to be present in rheumatoid arthritis patients. Therefore, this study suggests that there is little, if any, independent contribution of the TNF locus to the genetic background for rheumatoid arthritis susceptibility.
An intronic mutation responsible for a low level of expression of an HLA‐A*24 allele
Authors: Laforet M. , Froelich N. , Parissladis A. , Bausinger H. , Pfeiffer B. , Tongio M.‐M.
Published: December 2008

HLA class I typing performed in parallel by molecular biology and serology has revealed cases where an HLA class I allele was identified but the corresponding antigen on the cell surface was not detected. In the present report, we describe three members of a family in whom an HLA‐A24 allele identified at the molecular level was typed as A “blank” by lymphocytotoxicity. This serologically blank antigen was nevertheless faintly detectable by isoelectric focusing (IEF) and FACS analyses. Sequencing of the HLA‐A*24 allele from the promoter region to the eighth exonic region revealed a point mutation in the acceptor site of the second intron as compared to the normal HLA‐A*24 allele. This mutation could lead to incorrect processing of mRNA through a cryptic acceptor site located at the beginning of the third exon and hence to alternative splicing with a frame shift introducing an early stop codon into the fourth exon.
Accurate typing of HLA‐A antigens and analysis of serological deficiencies
Authors: Yu N. , Ohashi M. , Alosco S. , Granja L. C. , Salazar M. , Hegland J. , Yunis E.
Published: December 2008

We are reporting the results of HLA‐A typing by PCR‐SSOP complemented by PCR‐SSP of samples obtained from the National Marrow Donor Program (NMDP). These samples were a representative group from 2486 tested in duplicate by serology. A total of 390 samples gave HLA‐A discrepant results. Comparing the molecular typing results of 238 samples (samples with available DNA) with the serological typing results, 54 homozygotes and 184 heterozygotes produced a total of 422 assignments by molecular methods. We found assignment discrepancies in 147/422 (35%) in laboratory 1 and 144/422 (34%) in laboratory 2 (a combined group of 4 NMDP laboratories; laboratory 1 is not included). The serological discrepancies found were of 3 categories: a) false negatives, b) incomplete typing (discrepancies due to the level of resolution within a cross‐reactive or CREG group) and c) false positives. Major problems were identified using serology for typing HLA‐A antigens: a) inability to identify all WHO‐recognized specificities, more frequently in non‐Caucasians or in HLA‐A specificities known to be found more frequently in non‐Caucasians for laboratory 1 and incorrect assignments of A19 specificities in laboratory 2, b) incorrect assignments in cells with poor viability and c) false‐positive assignments in homozygotes. We propose a possible strategy to type HLA‐A specificities with two steps: a) a minimum of serology for typing specificities for common CREG groups: A1, A2, A3, A11, A9, A10, A28, A19. However, a given laboratory can determine the level of serological assignments needed as a first step. And b) molecular methods to identify splits: A23, A24, A29, A30, A31, A32, A33, A34, A36, A66, A74 and A80. The technique described is useful for large‐scale bone marrow donor typings for cells with poor viability, and for resolving ambiguous results including false‐positive assignments of homozygous cells.
Immunologic phenotype of cultured endothelial cells: quantitative analysis of cell surface molecules
Authors: Mutin M. , Dignat‐George F. , Sampol J.
Published: December 2008

Endothelial cells express a wide spectrum of surface molecules involved in multiple vascular functions. We quantitatively determined an extensive immunologic phenotype of endothelial cells through a large panel of antibodies directed against i) well‐known endothelial molecules CD31, CD34, CD49b, e, f, CD51, CD54, CD55, CD62E and P, CD105, CD106, HLA class I and HLA class II; ii) molecules defined by monoclonal antibodies newly clustered during the 6th workshop of Human Leukocyte Differentiation Antigen (HLDA) CD109, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146 and CD147; iii) molecules defined by unclustered monoclonal antibodies. The expression of these molecules was quantified on human umbilical vein endothelial cells (HUVEC) cultured in resting conditions and after stimulation with IL‐ip (10 U/ml), TNF‐a (10 ng/ml) and phorbol myristate acetate (60 ng/ml). Some molecules were constitutively expressed, and others were negative, which served to determine the basal phenotype. After cell stimulation, the molecules showed weak or strong expression modulation, leading to the definition of an activated phenotype. Changes in the kinetics and the amplitude of expression served to characterize poorly defined molecules and may be useful to determine their physiologic role. Also, we compared the phenotypes of endothelial cell lines EA.hy 926 and ECV 304 to that of HUVEC to assess their reliability as an endothelial cell model. Each cell line displayed a specific repertoire of molecules expressed at different levels, which could have significant im‐I plications for cell line behavior as endothelial cells.
Molecular genetic studies on DNA polymorphism of the HLA class II genes associated with human longevity
Authors: Akisaka M. , Suzuki M. , Inoko H.
Published: December 2008

Okinawan Japanese are well known for their longevity; the population rate of centenarians in Okinawa is about 3.8 times higher than that of the whole Japan, where the average life expectancies both among men and among women are the highest in the world. In this study, we analyzed HLA class II alleles of Okinawan centenarians by the polymerase chain reaction‐restriction fragment‐length polymorphism (PCR‐RFLP) method for the purpose of clarifying the presence of primary genetic factors in the major histocompatibility complex (MHC) region associated with human longevity. DRB1*1401, DQB1*0503, DQA1*0101=0104 and DQA1*05 were significantly increased in the centenarians. The significant increase of HLA‐DQB 1*0503 and/or DQA 1*0101=0104 in the centenarians can be explained by a linkage disequilibrium with DRB 1*1401, or vice versa. Further, the tendency was observed toward increase with respect to DRB 1*0101 and DRB1*1201. These data suggest that several alleles of the HLA‐DRB1 and/or HLA‐DQ genes are involved in human longevity.
The HLA A1‐B8 haplotype extends 6 Mb beyond HLA‐A: associations between HLA‐A, B, F and 15 microsatellite markers
Authors: Worwood M. , Chowdhury R. Raha , Robson K. J. H. , Pointon J. , Shearman J. D. , Darke C.
Published: December 2008

Allele frequencies for HLA‐A, B and F and 15 microsatellite markers located from 100 kb telomeric to HLA‐A to 6 Mb telomeric have been determined in a group of 60 blood donors. Linkage disequilibrium analysis revealed significant haplotype associations even after correction for the number of comparisons made. The HLA‐A1, B8 haplotype extends as far as D6S276 (6.0 Mb telomeric to HLA‐A). It is important to realize that this common haplotype extends beyond the HLA region, especially when evaluating haplotype associations with particular disorders.
HLA‐C heavy chains free of ß2‐microglobulin: distribution in normal tissues and neoplastic lesions of non‐lymphoid origin and interferon‐γ responsiveness
Authors: Giacomini P. , Beretta A. , Nicotra M.R. , Ciccarelll G. , Martayan A. , Cerbonl C. , Lopalco L. , Bini D. , Delfino L. , Ferrara G.B. , Siccardi A.G. , Natall P.G.
Published: December 2008

Lacking monospecific antibodies to HLA‐C, the expression and synthesis of these molecules have been difficult to evaluate. Using biochemical and flow cytometry approaches, the present report demonstrates that the reactivity of the murine monoclonal antibody L31 is restricted to naturally occurring HLA‐C (HLA‐Cwl through‐Cw8), HLA‐B8 and HLA‐B51 heavy chains not associated with ß2‐microglobin (ß2m). This is due to two properties of HLA‐C heavy chains: (a) they share the L31 epitope which distinguishes them from all the HLA‐A and most HLA‐B molecules; (b) they accumulate intracellularly, in a ß2m‐free form, in much greater amounts than most L31‐reacting HLA‐B heavy chains. On the basis of this restricted reactivity, a representative panel of normal and neoplastic human tissues and cells derived from HLA‐B8‐B51‐individuals was selected and employed to assess the tissue distribution, surface expression and IFN‐γ responsiveness of ß2m‐free HLA‐C heavy chains. At variance from antibody W6/32 to ß2m‐associated heavy chains, L31 stains normal and neoplastic tissues with a ground‐glass pattern and weakly binds to the surface of viable cells, even after treatment with interferon γ (IFN‐γ). Thus, ß2m‐free HLA‐C heavy chains are, for the most part, located intracellularly. In spite of their distinct cellular localization, L31‐and W6/32‐reacting molecules have an overlapping tissue distribution, undergo concordant changes upon transformation and are upregulated in their synthesis by IFN‐γ to a similar extent. These observations demonstrate a coordinate regulation of HLA‐C with HLA‐A and‐B molecules. In addition, they indicate that the assembly of HLA‐C is impaired in most body districts and IFN‐γ is unable to completely reverse this impairment. The present results are consistent with a low surface expression of HLA‐C and with a privileged role of these molecules in signaling class I loss to cytotoxic effectors in pathological conditions.
Screening of the TAP1 gene by denaturing gradient gel electrophoresis in insulin‐dependent diabetes mellitus: detection and comparison of new polymorphisms between patients and controls
Authors: Yan G. , Shi L. , Fu Y. , Wang X. , Schoenfeld D. , Ma L. , Penfornis A. , Gebel H. , Faustman D.L.
Published: December 2008

New protective or disease‐associated polymorphisms in the TAP1 gene were sought in insulin‐dependent diabetes mellitus (IDDM) patients with the use of denaturing gradient gel electrophoresis (DGGE) screening of genomic DNA. The TAP1 gene is located in the human leukocyte antigen (HLA) class II region of the genome and encodes components of a peptide transporter essential for antigen presentation by HLA class I molecules. Fragments of TAP1 corresponding to the 5′ promoter, each of the 11 exons (with portions of adjacent intronic regions) and the 3′ flanking region were amplified by the polymerase chain reaction and then subjected to DGGE. DNA fragments of TAP1 yielded DGGE bands with patterns whose frequencies differed between IDDM patients and controls. Specific DGGE band patterns with fragments corresponding to the promoter, exons or introns 3, 6, 7, 8, 9 or 10 of TAP1 were detected exclusively in either patients or controls. Sequencing of TAP1 fragments encompassing exon 7 gave rise to a DGGE band pattern exclusively observed in an IDDM patient and sequencing revealed a previously unidentified polymorphisms at codon 518 (GTCATC, ValIle). Another unique polymorphism uncovered by DGGE revealed by sequencing a polymorphism in intron 2 in a diabetic patient. The genotypes of additional HLA class II matched patients and controls were determined with regard to five exonic and one intronic TAP1 polymorphism. A 10 base pair intronic insertion in intron 9 was exclusively identified in controls and missing from patients (P = 0.017). Further large population‐based studies may reveal whether these newly identified at risk or protective TAP1 variants confer markers of statistical risk in diverse population groups.
Triplet repeat polymorphism within the NOTCH4 gene located near the junction of the HLA class II and class III regions in narcolepsy
Authors: Ando A. , Shigenari A. , Naruse T. K. , Sugaya K. , JujI T. , Honda Y. , Ikemura T. , Inoko H.
Published: December 2008

A polymorphic (CTG)n, microsatellite repeat was found in the signal peptide domain of the NOTCH4 gene located near the junction of the class II and class III regions of the human major histocompatibility complex. This gene belongs to a multigenc family of NOTCH originally identified as a differential factor of neuronal cells. To ascertain whether the NOTCH4 gene is involved in the development of neurogenic disease, narcolepsy, which is known to be tightly associated with HLA‐DR15, this microsatellite polymorphism of the (CTG)n repeat was analyzed in Japanese patients with narcolepsy. One allele, 9 repetitions of CTG (Leu) was significantly increased in the patient group. However, the significant increase of this allele in the patient group could be explained by a strong linkage disequilibrium with the HLA class II alleles, DRB1*1501, DQA1*0102 and DQB1*0602, which were more strongly associated with the disease. These results suggest that the (CTG)n repeat polymorphism in NOTCH4 does not primarily determine the susceptibility to narcolepsy.
Involvement of CD44 exon v10 in B‐cell activation
Authors: Rösel M. , Föger N. , Zöller M.
Published: December 2008

Abstract: The family of CD44 glycoproteins has been suggested to be involved in lymphocyte homing, maturation and activation. Using in vitro blocking studies with a monoclonal antibody, we here addressed the question of functional activity of CD44 variant exon v10 (CD44v10) in B‐cell activation. We became interested in this question by the observation that CD44v10 was transiently expressed on activated T cells, B cells and mono‐cytes as well as on a subpopulation of bone marrow cells. A potential ligand, as revealed by staining with a CD44v10 receptor globulin, was only detected on monocytes. Anti‐CD44v10 had no major impact on T‐cell activation and no influence on primed B cells, but interfered with the mounting of a primary B‐cell response to T‐independent and T‐dependent antigens. Addition of anri‐CD44v10 at different stages during the activation process revealed that CD44v10 was not engaged in B‐cell‐T‐cell interactions. The antibody exerted some effect on monocyte activation as defined by a slight decrease in IL‐1 production, but most efficiently inhibited antigen‐specific as well as mitogen‐induced B‐cell activation when present during the co‐culture of virgin B cells with monocytes. These findings, together with the observation that a CD44v10 ligand was only detected on monocytes but not on lymphocytes, point towards a requirement for CD44v10 in a B‐cell‐monocyte interaction. Furthermore, since activation of B cells by engagement both of the B‐cell receptor and of mitogen receptors was inhibited by anti‐CD44v10, the data suggest that a costimulatory function of CD44v10 proceeds independent of the B‐cell receptor.
The PCR‐SSP Manager computer program: a tool for maintaining sequence alignments and automatically updating the specificities of PCR‐SSP primers and primer mixes
Authors: Bunce M. , Barnardo M.C.N.M. , Welsh K.I.
Published: December 2008

Abstract: An emerging problem of molecular typing methods such as PCR amplification using sequence‐specific primers (PCR‐SSP) is that they frequently require updating as new alleles are constantly being described which potentially affect the specificity of every PCR‐SSP reaction. PCR‐SSP uses pairs of primers to detect cis‐linked polymorphisms and thus each new allele described must be compared to each individual primer pair. Furthermore, sequence homology between the various loci for class I and class Il means that, for example, new HLA‐A sequences have to be compared with HLA‐B and HLA‐C primer mixes to rule out cross‐locus amplification. We have developed a computer program known as SSP Manager which is capable of aligning HLA class I and class TI sequences obtained from Internet‐accessible databases such as GenBank. The program then updates all individual primer specificities held in its database before updating the specificities of all primer mixes. Sets of primer mixes can then be combined from the primer mix directory to create PCR‐SSP typing trays which are subsequently analysed by the program. A report is generated which stipulates whether all known sequences are amplified and the reason for apparent failure to test for individual alleles, e.g. a lack of relevant sequence information. SSP Manager has the flexibility to cope with unusual sequences (deletions and insertions), primers with internal mismatches and primers with a deliberate mismatch. The program also has many tools for developing new primer mixes, such as the facility to search for novel reactions using Boolean operators. The organisation and operational use of the SSP Manager program is described and its uses are illustrated with an updated allele list for our previously described Phototyping PCR‐SSP class I and class TI typing set. The SSP Manager is available on request from the authors.
HLA‐B*27 typing by group‐specific hybridization in microtiter plates
Authors: Wagner T. , Oberkanins C. , Weinmayr B. , Helmberg W. , Kury F. , Lanzer G.
Published: December 2008

Abstract: We have developed and evaluated a test for HLA‐B*27 based on PCR and DNA hybridization in microtiter plates. A region within exon 2 of the HLA‐B gene is amplified and labeled by PCR and the amplification product is hybridized to a group‐specific HLA‐B*27 and a generic control oligonucleotide probe in two separate cavities of the plate. Bound sequences are detected using an ELISA‐like protocol. The assay has been evaluated on 254 DNA samples routinely received for B27 testing in parallel with sero‐logical and SSP‐PCR typing. Results were concordant in typing 102 HLA‐B27‐positive and 152 HLA‐B27‐negative individuals except for two samples containing HLA‐B*73, which stained B27 positive in the microwell test. The new procedure is rapid and simple to perform, and the microwell format is particularly suitable for automation.
New TAP2 polymorphisms in Africans
Authors: Tang J. , Allen S. , Karita E. , Musonda R. , Kaslow R.A.
Published: December 2008

Abstract: Polymorphisms in genes encoding transporters associated with antigen processing (TAP) have been associated with heterogeneity of disease progression in HTV‐l‐infected homosexual men. In our recent AIDS‐related studies of cohorts from Rwanda and Zambia, four new polymorphic sites in the TAP2 coding region were detected by single‐strand conformation polymorphism (SSCP) and confirmed by bi‐directional nucleotide sequencing and restriction enzyme digestion. The first, a substitution of Thr (GCC) for Ala (ACC) at codon position 374 in exon 5, was found in about 13% of Rwandans and Zambians (n=213). The remaining 3 new polymorphisms were seen in the 7th exon with changes of 458Thr‐ACG to ACA, 466Gly‐GGG to GGA, and 467Val‐GTT to Ile‐ATT, respectively. These 3 variants occurred exclusively on the same chromosome and appeared to have arisen together from the 374Thr‐bearing allele. Analyses of the relationship between the 374Thr‐457Ile segment and the nearby markers in DQB1 and DRB1 suggested the existence of a unique extended haplotype related to these newly identified variants.
Mapping of the monoclonal antibody W6/32: sensitivity to the amino terminus of β2‐microglobulin
Authors: Shields M.J. , Ribaudo R.K.
Published: December 2008

Abstract: The monoclonal antibody W6/32 is one of the most commonly used pan‐HLA‐ABC antibodies in studying human MHC I structure and function. We have discovered that the reactivity of W6/32 is absolutely sensitive to the amino terminus of human β2‐microglobulin (hβ2m). Bac‐terially expressed recombinant forms of hβ2m that have been extensively used in structural and biochemical studies of MHC I molecules often have an additional methionine at their amino terminus. Cell surface MHC I molecules reconstituted with allele‐specific peptides and recombinant hβ2m are reactive with various HLA‐specific mAbs, but not W6/32. In contrast, cell surface HLA molecules reconstituted with peptide and native hβ2m, which has no amino terminal methionine, are recognized by W6/32 as well as other HLA‐specific mAbs. Thus, the specificity of W6/32 includes the amino terminus of hβ2m.
HLA‐DRB1, ‐DQB1 and ‐DPB1 polymorphism in the Slovak population
Authors: Čechová E. , Fazekašová H. , Ferenčik S. , Shawkatová I. , Buc M.
Published: December 2008

Abstract: HLA‐DRB1, ‐DQB1 and ‐DPB1 allele frequencies were investigated in a sample of the Slovak population by PCR‐SSP and PCR‐RFLP methods. The most frequent DRB1 alleles were DRBl*1101–5 (0.2038), DRBl*0701–2 (0.1423), and DRBl*1501–2 (0.1231). The most rare alleles found were DRBl*0901 (0.0038), and DRB1*1201 (0.015). The most common DQB1 alleles were DQBl*0301 (0.2448), DQB1*0201 (0.2098), and DQB1*0501 (0.1119), respectively. The alleles with the least occurrence rate were DQBl*0601 (0.0035) and DQB1*0401 (0.007). The most common DPB1 alleles found were DPBl*0401 (0.4329), DPBl*0402 (0.2089), and DPB1*0201 (0.1438), respectively. The least frequent alleles were DPBl*0601, *1101, and *1501 (0.0034). Allele frequencies found in our study were compared to those in Czech, Austrian, and German populations. No statistically significant differences were observed.
Identification of new DRB1*07 (DRBl*0703), DRB1*08 (DRB1*0817) and two DRB3* (DRB3*0302 and DRB3* 01014) alleles
Authors: Hashemi‐Tavoularis S. , Ouellet S. , Buyse I.M.
Published: December 2008

Abstract: We report here the identification of four novel DRB alleles using a reverse hybridization (CANTYPE) assay. Molecular cloning and sequencing confirmed the initial unusual hybridization patterns. All four new alleles were detected during routine HLA typing for the Canadian Unrelated Bone Marrow Donor Registry. DRBl*0703 is identical to DRB1*0701 except for a single nudeotide substitution (AGA→AGT), changing codon 29 from Arg to Ser, a so far undetected DRB polymorphism. DRB1*0817 differs from DRBl*0801 by a single nucleotide substitution (TAC→TTC), changing codon 47 from Tyr to Phe. This polymorphism has not, until now, been identified in DRB1*08 alleles. Compared with DRB3*0301, DRB3*0302 contains a single nucleotide substitution (TAC→CAC) at codon 30, changing the encoded Tyr to His. This polymorphism is typical for DRB3*02 alleles. DRB3*01014 is identical to DRB3*0101 except for a single silent nucleotide substitution (GGG→GGA) at codon 84. This polymorphism has previously only been described for the DRB1*15012 allele. DRB1*0817, DRB3*0302 and DRB3*01014 may have arisen from gene conversion, but DRB1*0703 most likely was generated by a point mutation event. The DRB3*0302 allele was detected in two unrelated subjects, while the other three have each only been detected once.
DQCAR microsatellite polymorphism and susceptibility to rheumatoid arthritis
Authors: Singal D.P. , Li J. , Ye M.
Published: December 2008

Abstract: Rheumatoid arthritis (RA) is a chronic inflammatory joint disease associated with HLA‐DR genes that share a five amino acid sequence motif, QKRAA or QRRAA, from position 70 to 74 in the third hypervariable region of the DRβ1 molecule. Since the associations between DRB1 genes and susceptibility to RA are incomplete, in this study we examined the CA repeat polymorphic marker DQCAR, located between DQA1 and DQB1 genes, alleles in 98 adult patients with seropositive RA and 100 normal healthy controls. The prevalence of the DQCAR 117 allele was significantly higher in RA patients as compared to normal controls. On the other hand, the frequency of DQCAR 99 was lower in patients than in normal subjects. Analysis of the data suggested that DRB1 genes sharing the QKRAA/ QRRAA epitope have the primary association with disease susceptibility and DQCAR alleles do not provide an additional risk for the development of RA.
Sequencing‐based typing reveals six novel MHC class I chain‐related gene B (MICB) alleles
Authors: Visser C.J.T. , Tilanus M.G.J. , Schaeffer V. , Tatari Z. , Tamouza R. , Janin A. , Charron D.
Published: December 2008

Abstract: The MHC class I chain‐related gene A (MICA) is highly polymorphic. In this paper we demonstrate polymorphism in the other expressing member of the MIC family of genes: MICB. Using a sequencing‐based typing approach on cDNA, analysis of exons 2 through 6 revealed eight polymorphic sites resulting in six unique MICB sequences. Although MICB has high nucleotide homology with MICA, its polymorphism is restricted and at different sites compared to those in MICA.
Identification of novel DRBI*13 (DRB1*1333), DRB1*04(DRB1*0426) and DRB5* (DRB5*0109) alleles
Authors: Buyse I.M. , Ouellet S. , Hashemi‐Tavoularis S.
Published: December 2008

Abstract: Three novel HLA class H alleles (DRB1*1333, DRB1*O426, DRB5*0109) are described here. The 3 novel alleles were initially detected as previously unidentified SSO hybridization patterns using the CANTYPE reverse hybridization assay. Sequences were determined by cloning/sequencing. DRB1*1333 is identical to DRB1*1303 except for a single nucleotide substitution (ACC→AAC), changing codon 77 from Thr to Asn. This polymorphism is typical for DRB1*03 alleles. DRB1*0426 is identical to DRB1*0401 except for a single nucleotide substitution (GCC→ACC) at codon 58, changing the encoded Ala to Thr. DRB5*0109 is identical to DRB5*0101, except for a single nucleotide substitution (GAC→AAC), changing codon 70 from Asp to Asn. Both latter polymorphisms were so far undetected in DRB alleles. DRB1*1333 could have arisen from a gene conversion event, but DRB1*0426 and DRB5*0109 most likely were generated by point mutation events. For all 3 alleles, the sequence was confirmed by the original hybridization pattern (DRB1*1333) or by hybridization to a newly designed probe (DRB1*0426 and DRB5*0109). Ethnic backgrounds were Lebanese for DRB1*1333 and Caucasian for DRB1*0426 and DRB5*0109.
CD36 is rapidly and transiently upregulated on phytohemagglutinin (PHA)‐stimulated peripheral blood lymphocytes. Analysis by a new monoclonal antibody (UN7)
Authors: Tassone P. , Tuccillo F. , Bonelli P. , Turco M.C. , Cecco L. , Cerra M. , Bond H.M. , Barbieri V. , Venuta S.
Published: December 2008

Abstract: The monoclonal antibody (mAb) UN7, clustered as an anti‐CD36 mAb, has been used to test the cell surface expression of CD36 on peripheral blood lymphocytes (PBL) following mitogenic stimulation. CD36, scarcely expressed on resting cell membranes, was rapidly upregulated on PBL after phytohemagglutinin (PHA) stimulation. The antigen was detected on the cell surface after 15 min of stimulation, increased rapidly by 60 min and peaked between 3 and 12 h, declining thereafter. The inhibition of protein synthesis by cycloheximide did not modify the PHA‐induced expression of CD36. Neither the anti‐CD3 0KT3 mAb nor the anti‐CD2 BIL 2.29 and 9.1 mAbs induced any significant upregulation of the molecule. The addition of anti‐CD28 15E8 mAb or IL‐2 or IFN‐γ to PHA or anti‐CD3 or anti‐CD2 mAbs did not influence the pattern of CD36 expression. The phorbol‐2‐myr‐istate‐13‐acetate (PMA), alone or in combination with ionomycin, was unable to activate the expression of CD36, while it inhibited the PHA‐induced upregulation. The PHA‐induced upregulation of CD36 was partially inhibited by the addition of LY294002 or wortmannin, while not affected by that of calphostin C. Thus, CD36 was found to be early and transiently upregulated by PHA stimulation on PBL. The rapid modulation of the molecule was not related to new protein synthesis, but was probably due to the insertion into the plasma membrane of a presynthetized protein pool.
Microsatellite single nucleotide polymorphisms in the HLA‐DQ region
Authors: Lin L. , Jin L. , Lin X. , Voros A. , Underhill P. , Mignot E.
Published: December 2008

Abstract: Sequencing studies were performed in three previously described microsatellite and minisatellite markers located within the HLA‐DQ region, DQCAR, DQCARII and G51152. Multiple nucleotide substitutions that did not change size polymorphisms were observed in all three markers. In all loci, the number of core repeats did not correlate with neighboring DQ allele sequence motifs while single nucleotide changes within or flanking the microsatellite sequence did. This result indicates higher mutation rates for microsatellite expansions/contractions than for nucleotide substitutions in these loci. Further analysis indicated an almost complete phylogenetic correspondence between DQCAR single nucleotide polymorphisms (SNPs) and DQB1 sequences on one side (1.0–1.5 kb apart) and a complete relationship between DQCARII and DQA1 sequences on the other (4.5 kb apart). In contrast, G51152 sequences did not correspond perfectly with DQB1 allelic sequences, thus suggesting the existence of several ancestral crossovers between this marker and DQB1 (20–25 kb). Sequencing microsatellites might be useful in disease mapping studies by increasing marker informativeness and by helping in the interpretation of association study results. It is also proposed that SNPs within the flanking region of CA repeats could be used to develop biallelic markers from already available mapped microsatellite markers.
HLA‐DPp residue 69 plays a crucial role in allorecognition
Authors: Diaz G. , Catálfamo M. , Coiras M.T. , Álvarez A.M. , Jaraquemada D. , Nombela C. , Sánchez‐Pérez M. , Arroyo J.
Published: December 2008

Abstract: To investigate the contribution to allorecognition of the individual polymorphic positions Glu 69 and Val 36 from the DPB1*02012 allele, DPB1*02012 cDNA was subjected to site‐directed mutagenesis and alleles expressing Lys at 69 and Ala at 36 were generated. The Iymphoblastoid cell line (LCL) 45.EM1, a previously generated mutant B‐LCL which expresses normal levels of DPA mRNA but is not able to transcribe DPB, was transfect‐ed with wild‐type or mutant DPBl*02012 cDNAs. The ability of two HLA‐DPw2 alloreactive CD4+ cytotoxic T‐lymphocyte (CTL) clones to lyse the panel of DPBl*02012 wild‐type and site‐directed mutant B‐cell lines was tested. Both CTL clones (8.3 and 8.9) lysed the B‐LCL 45.1, which is haploid for HLA and expresses wild‐type DPBl*02012, and transfectants expressing Ala at 36 instead of Val, indicating that this polymorphic residue is not critical for T‐cell recognition. However, the change of Glu to Lys at 69 prevented recognition by clones 8.3 and 8.9. These data demonstrate that the residue at peptide‐binding position 69 is crucial for T‐cell receptor recognition and suggest the requirement for a negatively charged residue at this position for allostimulation of these T‐cell clones. The side chain of DP(3–69 is predicted to point into the peptide‐binding groove, and the existence of positive (Lys) or negative (Glu) residues probably leads to substantial differences in the allo‐ or auto‐DP‐bound peptides or to differences in the conformation of the peptide‐MHC complex, which would therefore be responsible for specific DPw2 allorecognition. The binding of a panel of monomorphic and polymorphic anti‐HLA‐DP monoclonal antibodies (mAbs) to these transfectants was also tested by flow cytometry. The changes at Glu 69 and Val 36 did not affect recognition by any of the monomorphic antibodies tested. However, the binding pattern of some of the polymorphic mAbs was clearly modified. Therefore, even though it is not crucial for T‐cell allorecognition, polymorphic residue 36 must be involved in epitopes recognized by some polymorphic anti‐DP antibodies, while residue 69 of the DPB molecule is crucial both for T‐cell allorecognition and recognition by some mAbs.
High resolution HLA class I typing by reference strand mediated conformation analysis (RSCA)
Authors: Argüello J.R. , Little A.‐M. , Bohan E. , Goldman J.M. , Marsh S.G.E. , Madrigal J.A.
Published: December 2008

Abstract: We describe the application of RSCA for the high resolution typing of alleles encoded within the highly polymorphic HLA class I system. RSCA differs from other sequence based typing methodologies in that the HLA type is assigned on the basis of differences in DNA conformation between different HLA alleles. HLA class I locus specific PCR was performed on the sample to be HLA typed and the PCR product mixed with a fluorescent labelled locus specific reference strand. DNA molecules were allowed to reanneal and the duplexes formed were separated by non‐denaturing PAGE. Only duplexes which possess the fluorescent labelled reference strand were detected using an automated DNA sequencer. The mobilities of duplexes formed for 131 HLA class I alleles have been calculated and HLA type assigned. The use of a laser scanning system for accurate detection of migrating DNA, in combination with specific software for data analysis and correction of gel variablility, make RSCA sufficiently robust to detect alleles differing by as little as one nucleotide, resulting in a simple and automated technique for high resolution HLA typing.
A novel PCR‐based methodology to determine TAP allele frequencies in population studies
Authors: Perry C.L. , Mijovic C.H. , Cockram C.S. , Yeung V.T.F. , Jenkins D. , Barnett A.H.
Published: December 2008

Abstract: The TAP genes have been extensively studied as candidate susceptibility genes for autoimmune and infectious diseases. TAP1 and TAP2 have two and three polymorphic sites respectively which are used for the allele assignment following WHO nomenclature. The usual techniques employed to determine the TAP alleles cannot unequivocally ascertain the alleles present in subjects which are heterozygous at more than one polymorphic position within the genes. This results in an inability to determine absolute TAP allele frequencies in population studies. The aim of this study was to devise a PCR‐based method to unambiguously assign TAP alleles to all subjects. The novel method was tested in Oriental Type I diabetic and control subjects. The technique is a valuable tool for allele assignment in heterozygous subjects solely using PCR.
On the nature of “HLA‐B41+B42” alloantibodies. Specific reagents for HLA‐Cw*17?
Authors: Herrero M.J. , Bunce M. , van Dam M. , de Pablo R. , Vilches C.
Published: December 2008

Abstract:An almost complete and bidirectional association exists between HLA‐Cw*17 and the HLA‐B antigens B41 and B42. Serological and molecular analysis of an individual in which HLA‐B*4101 was identified in the absence of Cw*17 provides experimental evidence to prove a previously proposed hypothesis predicting that alloantisera classified as “B41+B42” are instead specific reagents for HLA‐Cw*17.
HLA‐C high resolution typing: analysis of exons 2 and 3 by sequence based typing and detection of polymorphisms in exons 1–5 by sequence specific primers
Authors: Delfino L. , Morabito A. , Longo A. , Ferrara G.B.
Published: December 2008

Abstract: HLA‐C high resolution sequence based typing developed in this study involves a unique DNA amplification encompassing exon 1 to intron 3 and four fluorescent sequencing reactions covering exon 2 and 3. Both dye primer and dye terminator sequencing techniques were performed and results compared. This approach allowed the identification of all of the 50 HLA‐C allelic variants so far described, except for two allele pairs that are distinguished by non‐coding nucleotide changes (Cw*12021 = 12022, Cw*15051 = 15052) and three allele pairs (Cw*0701=0706, Cw*1701 = 1702 and Cw*1801 = 1802) that share the same nucleotide sequence in exon 2 and 3. For complete subtyping of these allelic variants, an amplification based on sequence specific primers (PCR‐SSP) was used. No ambiguous heterozygous combinations of alleles were detected in our panel so far. HLA‐C typing data obtained by this method were compared with data from serological and low resolution PCR‐SSP typing, which had been performed previously on the samples sequenced.
A novel DRB3 allele (DRB3*0208), a new allelic variant of DRB1*1502 (DRB1*15023) and two new DQB1 (DQBl*03012 and DQB1*0614) alleJes
Authors: Hashemi‐Tavoularis S. , Ouellet S. , Sengar D.P.S. , Buyse I.M.
Published: December 2008

Abstract: Four novel HLA Class II alleles were identified using CANTYPE reverse hybridization assay. The initial unusual SSO hybridization patterns were confirmed by cloning and sequencing analysis. DRB3*0208 allele is identical to DRB3*0202 except for three nucleotide substitutions (GAT→ AGC) changing codon 57 from Asp to Ser. This polymorphism has so far been undetected in DRB3 alleles. DRB1*15023 differs from DRB1*15021 by a single silent nucleotide substitution (AAC→AAT, both encoding for Asn) at codon 33. This polymorphism has not, until now, been identified in DRB alleles. Compared with DQB1*03011, the novel DQB1*03012 contains a single silent nucleotide substitution (GCA→GCG, both encoding for Ala) at codon 38. Finally, DQB1*0614 allele is identical to DQB1*0603 except for a single nucleotide substitution (TAC→ TTC), changing codon 9 from Tyr to Phe. Polymorphisms observed here in the DQB1*03012 and DQB1*0614 alleles are present in several of the known DQB1 alleles. DRB3*0208, DQB1*03012 and DQB1*0614 may have arisen from gene conversion, but the DRB1*15023 most likely was generated by a point mutation event. DQB1*0614 was detected in three related subjects, while each of the other three new alleles has only been detected once.