bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparative study of HLA‐DRB1 typing by standard serology and hybridization of non‐radioactive sequence‐specific oligonucleotide probes to PCR‐amplified DNA
Authors: Mickelson Eric , Smith Anajana , McKinney Susan , Anderson Ganet , Hansen John A.
Published: December 2008

A double‐blind study was carried out to evaluate the relative performance and reliability of the PCR/SSOP assay compared to conventional serological typing in identifying HLA‐DR alleles. A total of 268 consecutive samples were entered into the study. In 14 (5.2%) of the cases, HLA‐DR serology could not be performed due to poor cell viability, while in seven (2.6%) of the cases, PCR/SSOP typing could not be performed due to poor amplification or to contamination with exogenous DNA. Among samples that were successfully typed by both methods, serologic typing correctly identified 455/465 (97.9%) DR antigens, while PCR/SSOP correctly identified 464/465 (99.8%) DR alleles (p = 0.0117, McNemar's test). The majority of discrepancies in serologic typing resulted from a lack of discriminative alloantisera to identify DR6 or DR103. For the overall sample set (N = 268), serology provided accurate results in 244 (91.0%) cases, while PCR/SSOP provided accurate results in 260 (97.0%) cases (p = 0.0037). The results of this study demonstrate that PCR/SSOP typing for HLA‐DRB1 alleles provides results that are equal to or surpass serological typing for HLA‐DR antigens. In addition, the PCR/SSOP approach offers the advantages of better reagent availability, lower cost, more rapid turn‐around time, and greater accuracy, all of which would warrant its use as an HLA typing method of choice.
Sequence‐specific oligonucleotide typing in Shona patients with rheumatoid arthritis and healthy controls from Zimbabwe
Authors: Cutbush S. , Chikanza I. C. , Biro P. A. , Bekker C. , Stein M. , Lutalo S. , Garcia‐Pacheco J. M. , McCloskey D. S. , Lanchbury J. S. , Sachs J. A.
Published: December 2008

Seventy‐two patients with rheumatoid arthritis (RA) and 82 controls have been typed with the XI Histocompatibility Workshop DRB1 and DQB1 sequence‐specific oligonucleotide probes. The increase of DRB1*04 corresponds to an increase of the serologically defined DR4, previously found in a small group of Zimbabwean RA patients and we now show that this increase is due to the subtype DRB1*0405 in association with DQB1*0302. In addition there is a clearcut increase of DRB1*1001 equivalent to the serologically defined DR10. There was no increase amongst RA patients of DRB1*0102 which was the predominant DR1 sub‐type amongst controls. In the course of our investigation, we observed a DRB1*04 variant which corresponds to DRB1*0412, newly defined in the XIth Histocompatibility Workshop.
On the HLA‐DQ(α1*0501, β1*0201)‐associated susceptibility in celiac disease: A possible gene dosage effect of DQB1*0201
Authors: Ploski Rafal , Ek Johan , Thorsby Erik , Sollid Ludvig M.
Published: December 2008

The HLA‐associated susceptibility to develop celiac disease (CD) seems mainly to be conferred by a particular HLA‐DQ heterodimer encoded by the DQA1*0501 and DQB1*0201 genes either in cis or in trans position. To study the possible influence of DRB1 or other DQA1 and DQB1 alleles on the CD susceptibility conferred by these DQ genes, we performed genomic HLA typing of 94 CD patients, selected those who carried at least one copy of the DRB1*0301‐DQA1*0501‐DQB1*0201 haplotype (N = 89) and compared them to 47 random, healthy Norwegians matched with the patients to carry at least one copy of the above haplotype. We found an excess of DQB1*0201 homozygosity in the patients. This was due to an increased frequency of the DRB1*0301‐DQA1*0501‐DQB1*0201 and DRB1*0701‐DQA1*0201‐DQB1*0201 haplotypes present on the other chromosome. We propose that, in individuals carrying the DQA1*0501 and DQB1*0201 alleles, the presence of a second copy of the DQB1*0201 allele increases susceptibility to CD.
The HLA polymorphsm of two distinctive South‐American Indian tribes: The Kaingang and the Guarani
Authors: Luiza Maria , Erler Petzl , Luz Roberto , Sotomaior Vanessa Santos
Published: December 2008

The HLA‐A, B, C, DR and DQ antigens of 240 Kaingang and 98 Guarani individuals have been characterized. The most frequent antigens found among the Kaingang are A31, 2, 24; B35, 51, 39, 48; Cw4, 7, 3, 1; DR8, 4, 2; DQ blank, 3. In the Guarani, they are A2, 28, 31; B40, 62, “53G”; Cw3, 4; DR2, 4, 8, 6; DQ3, blank. B “53G” is an unusual antigen of the B5 cross‐reactive group. DQ blank possibly corresponds to DQ4, not tested in this study. The reaction patterns of B35, B40 and DR4 indicate intra‐tribal (of B35 and B40), and inter‐tribal (DR4, B40 and B35) heterogeneity of these antigens. 408 Kaingang and 141 Guarani haplotypes were defined by segregation analysis. Of the commonest 10 Guarani and 9 Kaingang haplotypes, only one is shared by both tribes. Significant, positive linkage disequilibrium values for HLA‐A,B; HLA‐A,C; HLA‐B,DR and most HLA‐B,C antigen pairs were also different for the two populations. Genetic distance estimates between these two and another seven South‐American Indian populations, and relative to the major human races (negroids, caucasoids, and mongoloids) reveal a comparatively high degree of divergence between the Kaingang and the Guarani, which is uncommon for Amerindian populations living close one to another.
Summary report from the first international workshop on soluble HLA antigens. Paris, August 1992
Authors: Pouletty Philippe , Ferrone Soldano , Amesland Française , Cohen Nadine , Westhoff Ulriko , Charron Dominique , Shimizu Robert M. , Grosse‐Wilde Hans
Published: December 2008

Abstract: The First International Workshop on Soluble HLA antigens focused on the comparison of immunoassay procedures for quantitation of soluble HLA (sHLA) class I antigens and the selection of a sHLA class I antigen international standard. Several sets of serum, plasma, and cell culture supernatant specimens were assayed blindly for levels of sHLA class I antigens by 15 participating laboratories using different immunoassay formats. The sandwich ELISA using (i) for antigen capture: an anti‐HLA class I heavy chain monoclonal antibody (mAb) specific for a monomorphic epitope, and (ii) for antigen detection: an anti‐β2 micro‐globulin antibody‐enzyme conjugate, was the assay format of choice. There was a high inter‐laboratory correlation among the majority of laboratories. All serum and plasma specimens from normal donors, and from a single transplant patient, had detectable levels of sHLA class I antigens. Paired serum and plasma specimens had similar levels of sHLA class I antigens, although plasma sHLA antigens seemed more stable than serum sHLA antigens. sHLA‐A2 and sHLA‐B7 antigens were detected in all specimens from HLA‐A2 and HLA‐B7 donors, respectively, using allele‐specific ELISAs. No difference in reactivity was observed for quantitation of native sHLA class I antigens whether the capture mAb was TP25.99 (alpha 3 domain‐specific) or W6/32 (alpha 2+alpha 3‐specific). However, a human‐mouse chimeric sHLA class I antigen reacted weakly in assays which used TP25.99 mAb. The wide variation among laboratories in their reporting of μg/ml units pointed to the need for an inter‐laboratory standardization based on a calibrated sHLA antigen preparation. T.sB7, an sHLA‐B7 antigen derived from a cell line transfected within human β2 microglobulin and HLA‐B7 genes, was accepted as the First sHLA class I Antigen International Standard at the workshop meeting.
A significant percentage of normal T lymphocytes express HLA‐DP in the peripheral blood
Authors: Baudeau C. , Falkenrodt A. , Parissiadis A. , Tongio M. M.
Published: December 2008

Abstract: HLA‐DP expression has been widely investigated on T lymphocytes activated under different conditions. In the present study, a double staining procedure was used in flow cytometric experiments to define DP expression on normal peripheral blood T lymphocytes. In about two‐thirds of the case analyzed, DP was expressed on a higher percentage of normal peripheral T lymphocytes than DR was. This was particularly true for 1 of the 16 cases investigated in which the percentage of T lymphocytes expressing DP was 46%) and in which DP expression was mainly the prerogative of CD8+ and CD56+ lymphocytes.
Unique associations between HLA‐B and HLA=DRB1 *04 gene variants in Japanese
Authors: Nithimeto T. , Matsrki K. , Islam S. M. M. , Hirata A. , Maeda H.
Published: December 2008

Abstract: HLA‐DRB1 *04 gene variants were determined in apparently healthy Japanese with PCR‐SSCP and PCR‐RFLP, and HLA‐B‐DRB1 *04 haplotype frequencies were estimated statistically. DRB l *0405, which consisted of 60% of the Japanese DRB1 *04 gene pool, was associated strongly with HLA‐B54. This association was observed only for DRB1 *0405, and not for any other DR4 variants. DRB1 *0403 and DRB1 *0406, which share the same peptide sequence except for amino acid 37. were both associated with B35 and B62. DRB1 *0407 was associated exclusively with B35. DRB1 *0410 was associated with B60 and B61. 860 and B61, on the other hand, were associated only with DRB1 *0410 and DRB1 *0405. B35 was associated with all DR4 microvariants. With this possible exception, our calculation suggests that unique B‐DR associations were present even for DR4 microvariants. The knowledge of HLA‐B and DRB1 *04 associations would be useful in clinical settings.
Restriction fragment length polymorphism of HLA‐DQ and ‐DR allogenotypes in normal Southern Chinese
Authors: Taa Li Phillp Kam , Poon Angela S. Y. , Hawkins Brian R , Yaung Vincent T. F. , Bo Yu Hong , Hi Kar Neng
Published: December 2008

Abstract: We have examined the Taq I restriction fragment length polymorphism (RFLP) of the MHC class II regions of 96 normal Southern Chinese with respect to the HLA‐DRB. ‐DQA, and ‐DQB genes. The data were compared with the DR and DQ serotyping. RFLP correlates well with the serological typing of the DR types of Chinese. Variants are seen in DR2 and DR12. The variant in DR12 with a 10 kb at the upper band (DR12b) is not found in Caucasoids and is more frequent in Chinese (19.4% of the alleles). With the help of DQA and DQB RFLP the assignment of genotyping of DRB types is facilitated. Some of the linkage disequilibrium patterns among DRB, DQA and DQB are different from Caucasoids. This is particularly obvious in the DR2 and DR5 haplotypes. In the 96 Southern Chinese, the 3 commonest haplotype frequencies are: DR12b. DQα1b, DQβ3b (18.3%); DR9, DQα3. DQβ3α (15.7%); DR4. DQα3. DQβ3a (14.2%). The various DRβ. DQα. DQβ genes can be accurately defined in Chinese by RFLP. Polymerase chain reactions using sequence‐specific primers were performed to confirm the various HLA‐DRB and ‐DQB alleles. The use of RFLP is important in the study of HLA matching and HLA‐disease association in Southern Chinese.
HLA and disease associations: Detecting the strongest association
Authors: Svejgaard Arne , Ryder Lars P.
Published: December 2008

Abstract: A major aim of HLA and disease association studies is to identify the causative HLA factor truly responsible for the association. This is usually difficult due to the pronounced linkage disequilibrium between most HLA determinants. The causative factor must show the strongest association compared to all other factors. Here we describe a simple analysis which can be used to identify which of two factors, say A and B, shows the strongest association. The basic data for the analysis are the entries of the two‐by‐four table giving the four phenotypic combinations of A and B in patients and controls, respectively. These data are analyzed in various two‐by‐two tables involving stratification of each of the two factors against the other. A stronger increase of factor A is established if A is significantly associated with the condition both in B‐positives and in B‐negatives, when this is not true for B in A‐positives and A‐negatives. Using simulation with control data, it is demonstrated how linkage disequilibrium may influence secondary associations. The analysis may also be used to investigate interaction between HLA factors, but linkage disequilibrium complicates the interpretation in such cases. The method is exemplified using various published data. Finally, some statistical recommendations are given. Thus, we advise that phenotype (marker) frequencies are generally used instead of gene (i.e. allele, or haplotype) frequencies. The importance of correcting p‐values, the levels of significance, and the power of Fisher's exact test are discussed.
HLA‐B67: A member of the HLA‐B16 family that expresses the ME1 epitope
Authors: Little Ann‐Margaret , Domena John D. , Hildebrand William H. , Shen Susan Y. , Barber Linda D. , Marsh Steven G. E. , Bias Wilma B. , Parham Peter
Published: December 2008

Abstract: HLA‐B67 is an uncommon antigen that has been defined by serological crossreactivity with the HLA‐B7 and HLA‐B16 (B38 and B39) antigens. It is found at highest frequency in certain Oriental populations and has been best defined in the Japanese. Nucleotide sequencing of cDNA encoding B67 reveals the B*6701 allele to be a subtype of B39 which differs from B*39011 by substitution at residues 67–71 of the α1 helix. In the region of difference B*6701 is identical in sequence to B7, B22, B27 and related molecules that express the epitope recognized by the ME1 monoclonal antibody. That the HLA‐B67 molecule binds strongly to the ME1 antibody was demonstrated by immunoprecipitation and cell surface binding assays. Identical B*6701 nucleotide sequences were obtained for the B67 alleles isolated from 2 unrelated Japanese and 1 North American caucasoid.
Identification of two different HLA B49 DR4 extended haplotypes in the Sardinian population
Authors: Vacca A. , Carcassi C. , La Nasa G. , Mulargia M. , Pizzati A. , Ledda A. , Floris L. , Baldini G. , Porcella R. , Contu L.
Published: December 2008

Abstract: The HLA‐B49 DR4 haplotype, which is rare in Caucasoid populations, has a frequency in Sardinia of approximately 6% and a very strong linkage disequilibrium (LD). To better understand its genetic structure, the Bf, C4A and C4B Class III alleles were studied in 56 healthy unrelated Sardinian subjects of B49 DR4 phenotype and in 24 of their families. Moreover, 14 sick subjects with the same phenotype were examined together with five of their families. A group of 285 haplotypes belonging to randomly selected individuals was used as a control population sampling. The distribution of the Bf, C4A and C4B alleles among the healthy probands revealed two main groups of association: one major group of 36 subjects (64.3%) with the BfF, C4A3 and C4B4 alleles and one minor group of 14 subjects (25%) with BfS, C4AQ0 and C4B1. A similar subdivision was also observed in the small group of patients. The family analysis confirmed these results and showed two different B49 DR4 extended haplotypes: one with the F34 complotype in 79.1% of the probands (Δ × 1000 = 49.0) and the other with the SO1 complotype in 20.8% of the probands (Δ × 1000= 17.3). The first one, in LD with the A1 and Cw7 alleles, has not yet been reported in other populations. The second one seems to be identical to a haplotype already reported in the Spanish population. The molecular analysis of the DRB1 locus carried out in 20 of the 70 probands demonstrated that, in both haplotypes, DR4 was represented by the DRB1*0405 allele. On the other hand, the study of the DQA1 and DQB1 loci in 11 probands revealed differences between the two haplotypes.
An estimate on the frequency of duplicated haplotypes and silent alleles of human C4 protein polymorphism. II. Investigations in healthy Negro families
Authors: Steuer Miriam Katharina , Oudshoorn Machteld , Brenden Martina , Fimmers R. , Neugebauer M. , du Toit Ernette , Baur M. P. , Mauff G.
Published: December 2008

Abstract: The first investigation of complete MHC marker data in South African Negroes by segregation analysis in 11 families with up to three generations is presented, including quantitative evaluation of C4 allotype patterns and C4β chain determinations according to Steuer et al. (1). The frequency of homo‐ and heteroduplicated, hybrid, and non‐expressed C4 alleles was determined from C4 protein phenotyping, including C4 alpha and beta chains, quantitative estimates of the relative electrophoretic C4 banding patterns by scanning densitometry, and from the other classical MHC markers by submitting all results to the family analysis program (FAP). From unrelated non‐diseased individuals (n = 105) in these families with 62 haplotypes, the following frequencies were observed for non‐expressed alleles: C4A*Q0 0.1189, C4B*Q0 0.2552, and for the total of heteroduplicated alleles: C4A 0.0645, C4B 0.0608. Applying additionally quantitative determinations of C4 banding patterns, homoduplications such as C4A*3 A*3, C4B*1 B*1, C4B*3 B*3, and the heteroduplication C4A*3 A*2 were assumed. In the investigated individuals the heteroduplications of C4A*12 and C4A*3 with the A*91 allele and of C4B*2 with C4B*92 were observed. It was concluded that not only allele frequencies but also the frequency of heteroduplications seems to be of specific ethnic character. Furthermore, the prior hypothesis that deletion or non‐expression at one C4 locus is accompanied by duplication at the other was only confirmed for non‐expressed B‐alleles with C4A*3 A*91 or C4A*12 A*91. For the correlation of C4 genes with other class III markers no linkage disequilibria with p‐values less than 0.001 as in Caucasoid populations were seen. The presented data may form a basis for further investigations in African Negroes on characteristic MHC haplotypes in defined diseases.
Recognition of HLA‐A1 by murine monoclonal antibodies
Authors: Carrel Stefan , Salvi Suzanne , Hartmann Françoise , Rimoldi Donata
Published: December 2008

Abstract: Balb/c mice were immunized with cells from the mouse mastocytoma line P815 transfected with an HLA‐A1 gene. The splenocytes of the immunized mice were fused with cells from the murine myeloma NS‐1. In an initial screening, supernatants of growing cultures were tested for their binding capacity to the immunizing P815/A1+ cells as well as to P815/A2+ cells. Three out of 756 hybrids produced antibodies which bound to P815/A1+ cells only. They were cloned and further analyzed for their binding reactivity to reference B‐lymphoblastoid cells from the Tenth International Histocompatibility Workshop. One monoclonal antibody, designated 6B11, reacted only with HLA‐A1+ cells, while the two other antibodies, 3G3 and 7F10, appeared to detect antigenic determinants shared by HLA‐A1, A3, A11, A26, and A30 (3G3) and by HLA‐A1, A3, A11, A26, A28 and A30 (7F10). Flow cytometric studies on B‐lymphoblastoid cell lines as well as on a series of tumor cell lines, including melanoma and colon carcinoma lines, confirmed the specificity of these antibodies. Monoclonal antibodies 7F10 and 6B11 were found to be of the IgM class and 3G3 of the IgG1 class. By complement‐dependent 51Cr release experiments it was further shown that the two IgM antibodies 7F10 and 6B11 were able to lyse all cell lines of the HLA‐A1 haplotype tested.
Serological definition of bovine MHC class II polymorphism in Holstein‐Friesians
Authors: Nilsson Ph. R. , Schreuder G. M. Th. , D'Amaro J. , Joosten I. , Buis R. C. , Davies C. J. , van der Poel J. J.
Published: December 2008

Abstract: Analysis of the reaction patterns of 57 putative class II MHC alloantisera revealed 18 B‐cell serum clusters, named Ds01–Ds18. These clusters were grouped together in such a manner that 17 provisional serological class II haplotypes were defined. Segregation in half‐sib families of these class II haplotypes with serologically defined class I and ID‐IEF defined DRB3 types indicated that the 18 clusters identify class II polymorphisms in cattle and can be considered as local specificities.
HLA class II associations with idiopathic nephrotic syndrome in children
Authors: Konrad Martin , Mytilineos Joannis , Bouissou Francois , Scherer Sabine , Gulli Marie‐Pierre , Meissner Ingeborg , Cambon‐Thomsen Anne , Opelz Gerhard , Schärer Karl
Published: December 2008

Abstract: The occasional familial occurrence of idiopathic nephrotic syndrome (NS) points to a genetic predisposition. Reports on associations with certain HLA class II antigens support this hypothesis. In order to define the immunogenetic background of NS more precisely, HLA class II allele frequencies in 161 children with NS were studied by restriction fragment length polymorphism (RFLP) typing. The patient cohorts consisted of 87 children from Southwest‐France and 74 from Southwest‐Germany. The control group consisted of 118 French and 101 German unrelated individuals from the same geographical areas. HLA alleles were defined in patients with steroid‐sensitive (SS) and steroid‐resistant (SR) NS and in controls. RFLP typing revealed that the previously reported association between SSNS and HLA‐DR7 is confined to the RFLP split 7.1 (DRB1*07) with a combined relative risk (RRcomb) of 6.2. HLA‐DQB typing showed an increased frequency of the allele DQB2b (DQB1*0201) (RRcomb = 7.8). HLA‐DQA typing showed an association of SSNS with DQA3 (DQA1*0201,0301,0302) (RRcomb = 4.1). The highest RR (16.5) for SSNS was found in German patients who carried the two DRB1 specificities 17.1 (DRB1*0301) and 7.1 (DRB1*07). All associations were stronger in SS patients with frequent relapses or steroid dependency than in non‐ or infrequent relapsers. SR patients exhibited no significant associations with HLA class II alleles.
Comprehensive typing of DR52 (DRB3)‐associated DRB1 and DRB3 alleles by PCR‐RFLP
Authors: Sengar Dharmendra P. S. , Goldstein Rose , Toye Baldwin , Hampton Nancy
Published: December 2008

Abstract: The DR52‐associated DRB1 and DRB3 alleles were resolved by PCR‐RFLP. Second exon was amplified using four primer pairs (groups 1–4) for DRB1 and a pair for DRB3 alleles. Except for three endonucleases, all others had either none or only one site for a specific amplified product. Group 1 primers amplify 10 DRB1 alleles (DRB1*0302, 1101, 1302, 1303, 1305, 1307, 1402, 1403, 1407 and 1409). All but one pair, DRB1*1402 from 1409, could be resolved using seven endonucleases (ApaI, SacII, FokI, AvaII, BsaAI, BsrBI and SfaNI). Group 2 consisted of four alleles (DRB1*1201, 1202, 1404 and 1411) that can be resolved along with co‐amplified DRB1*0804 and 0806 using five endonucleases (AvaII, SacII, FokI, HaeII and RsaI). Group 3 primers amplify 15 DRB1 alleles (DRB1*0301, 0303, 1102, 1103, 1104, 1107, 1301, 1304, 1306, 1308, 1401, 1405, 1406, 1408 and 14‐New), which can be resolved using nine enzymes (KpnI, AvaII, FokI, SacII, HaeII, BsrBI, SfaNI, DdeI and RsaI). BsrBI, a new endonuclease, can resolve DRB1*1301 from 1306 and the previously unresolved allele DRB1*1103 from 1104. DRB1*1410, co‐amplified with DR4 group‐specific primers, is resolved with PstI which cleaves all DR4 alleles but not DRB1*1410. All four DRB3 alleles (DRB3*0101, 0201, 0202 and 0301) and their heterozygotes are resolved using two endonucleases, RsaI and HphI. Thirty‐four DR52‐associated alleles and their heterozygotes can be unambiguously resolved, except for DRB1*1402 from 1409. Thus, PCR‐RFLP remains an effective method for high‐resolution HLA‐DR typing. Furthermore, PCR‐RFLP can complement the evolving PCR‐SSP method for allele‐specific typing using a minimal number of restriction endonucleases.
A one‐step monoclonal antibody typing procedure that simplifies HLA class I and class II typing
Authors: Lee Jar‐How , Lias Mamie , Deng Chun‐Tsan , Etessami Soudabeh , Loon Jimmy , Chen Mike , Banh Leninh , Conger Nathan , Connors Dan , Manzo Alex , Ayoub George , Terasaki Paul I.
Published: December 2008

Abstract: We have developed monoclonal antibodies to most HLA specificities, making it possible for us to devise a simple, rapid, one‐step micro‐cytotoxicity test. The test is performed by adding 1 μl of cells to 1 μl of antibody‐complement mixture predotted on the microtest tray. The reactions are read following a 1‐hour incubation period (30 minutes in some instances). The analysis of reactions seen on testing 105 class I antibodies and 50 class II antibodies is shown. A comparison of typing by the standard NIH method and the new one‐step procedure showed a > 96% concordance in the 500 T cells and 200 B cells we examined. Class I and class II typing could be performed using B cells, thus obviating the need to isolate both T and B cells for HLA typing.
PCR‐RFLP‐based DQA1 typing of rhesus monkeys: sequence analysis of a new allele
Authors: Christ Ricarda , Hunsmann Gerhard , Sauermann Ulrike
Published: December 2008

Abstract: In spite of the widespread use of rhesus monkeys (Macaca mulatta) in biomedical research, MHC typing of this species is not yet routine. Since suitable antibodies are lacking, serological typing of Mamu‐DQA1 is not feasible. We developed a typing protocol for MhcMamu‐DQA1 from published sequences of the second exon of Mamu‐DQA1. This protocol is based on the amplification of the second exon of Mamu‐DQA1 with one specific primer pair followed by a “diagnostic” digestion of the PCR products with, at most, 5 different restriction endonucleases. This modified PCR‐RFLP permits the rapid identification of 11 out of 13 Mamu‐DQA1 alleles in homozygous and heterozygous combinations. The protocol was validated by cloning and sequencing the PCR‐products of several animals of different geographic origin. In addition, an as yet unknown allele was detected by PCR‐RFLP and was subsequently cloned and its nucleotide sequence determined. All examined sequences except the new allele were identical to those previously published. Therefore, we assume that many of the Mamu‐DQA1 alleles of rhesus monkeys have been identified molecularly and that the typing technique presented here can reliably identify Mamu‐DQA1 alleles.
Genotyping of the granulocyte‐specific NA antigens from small quantities of blood or serum
Authors: Stein E.‐L. , Santoso S. , Behrens G. , Mueller‐Eckhardt C. , Bux J.
Published: December 2008

Abstract: To avoid the well‐known shortcomings of phenotyping granulocytes for the NA antigens using NA‐specific human sera, a DNA‐based method to determine the NA genotype was developed. Genomic DNA was isolated from blood cells or serum, amplified by polymerase chain reaction (PCR), immobilized on nylon membrane and genotyped using digoxigenin‐labeled, sequence‐specific oligonucleotides (SSO). The genotyping results of whole blood samples from 54 and of serum from 20 individuals correlated perfectly with our phenotyping using the antigen capture assay MAIGA. In three cases with the phenotype “NA‐null” no hybridization of the NA‐specific oligonucleotides occurred. These data show that SSO is a reliable method for NA genotyping especially if only small volumes of blood or even only serum probes are available.
Distribution of HLA antigens and haplotypes in the Buryat population of Siberia
Authors: Tokunaga K. , Sideltseva E. W. , Tanaka H. , Uchikawa C. , Nieda M. , Sideltsev V. V. , Zhuravleva E. , Imanishi T. , Itoh K. , Akaza T. , Takahashi K. , Khalturin V. , Alexeev L. P. , Juji T.
Published: December 2008

Abstract: Polymorphisms of HLA‐A, B, C, DR, and DQ antigens were investigated in a Mongoloid population named Buryat living in Siberia. HLA gene and haplotype frequencies were calculated from the population data obtained from 141 unrelated healthy Buryat adults. Gene frequencies of class I antigens A2, A24, A1, B61, Cw10, and Cw6 were estimated to be more than 10%. For class II, DR4, DR7, DR13, DQ7, and DQ1 antigens were predominant. A phylogenetic tree was constructed based on HLA gene frequencies, and the Buryat population was clustered with the Mongoloid groups in Northeast Asia. In the analysis of HLA‐A, C, B, DR, and DQ five‐locus haplotype frequencies, seven kinds of haplotypes were calculated to occur at frequencies of more than 2%. Five of the seven common haplotypes have also been described in the other human populations thus far. Some of the haplotypes have been described in European populations, while the others were shared with Northeast Asian Mongoloids as well as Amerindians. Similar situation was also found in the analysis of class I (HLA‐A, C, B) three locus haplotypes. These observations suggest the unique genetic background of this Buryat population.
Comparison of HLA‐DRB1 typing by DNA‐RFLP, PCR‐SSO and PCR‐SSP methods and their application in providing matched unrelated donors for bone marrow transplantation
Authors: Jordan F. , McWhinnie A. J. , Turner S. , Gavira N. , Calvert A. A. , Cleaver S. A. , Holman R. H. , Goldman J. M. , Madrigal J. A.
Published: December 2008

Abstract: The aim of the study was to devise a strategy for large batch analysis to determine HLA Class II alleles exhibited by candidate bone marrow transplant donors and prospective recipients using previously published DNA‐based typing techniques. Special attention was directed towards the technical aspects of procedures, the level of typing resolution and the speed of data analysis. 200 blood samples from volunteer bone marrow transplant donors typed serologically for HLA‐DR and DQ were further investigated using three DNA‐based typing methods: (i) restriction fragment length polymorphism (RFLP) analysis, (ii) polymerase chain reaction (PCR) amplification and subsequent hybridisation with sequence specific oligonucleotide probes (PCR‐SSO), and (iii) PCR amplification with sequence specific primers (PCR‐SSP) to resolve the DRB1* specificity of each individual. In general, the HLA‐DR results obtained using PCR‐SSO and PCR‐SSP correlated well with each other. However, discordant results were obtained between PCR and RFLP based typing in 21 cases, especially in relation to DRB3* alleles associated with the DRB1 gene. These differences were due to three problems pertaining to RFLP analysis: i) alleles with identical DRB, DQA and DQB fragment sizes, ii) reliance on DQA and DQB results to assign the DRB genotype, and iii) a “new polymorphism” of DR7, in a DR7 homozygous, exhibiting a fragment similar in size to DR8. Our findings suggested a strategy requiring PCR‐SSO analysis for initial low resolution class II typing involving large numbers of samples, while the use of PCR‐SSP is reserved for small numbers of samples, for urgent samples or for situations where higher resolution is required. As the PCR‐based methods are relatively quick, precise, simple and inexpensive, they have replaced RFLP analysis and become the routine approach for DR‘ typing in our laboratory.
HLA‐DRB1*01 subtyping by heteroduplex analysis
Authors: Savage D. A. , Wood N. A. P. , Bidwell J. L. , Hui K. M.
Published: December 2008

Abstract: In this report we describe an alternative method for the identification of the four known HLA‐DRB1*01 alleles, which is based on the generation of unique heteroduplex patterns between the different DRB1*01 alleles and a synthetic DNA heteroduplex generator (DHG) molecule. The method is technically simple, rapid and cost‐effective, as it essentially involves only a single polymerase chain reaction (PCR) followed by polyacrylamide gel electrophoresis. This technique allows the rapid discrimination of the various known HLA‐DRB1*01 subtypes, both in homozygous and heterozygous situations. We propose that this technology can potentially be applied to most HLA class I and class II subtyping.
Population diversity of B‐locus alleles observed by high‐resolution DNA typing
Authors: Fernandez‐Viña M. , Lazaro A. M. , Sun Y. , Miller S. , Forero L. , Stastny P.
Published: December 2008

Abstract: HLA B‐locus typing by group‐specific PCR and hybridization with SSOP was performed in 81 10th IHWS B cell lines and 334 selected subjects of our local panel, from four ethnic groups. Most of the B‐locus serological specificities were well defined. However, some antigens like B41, B58, B56, the splits of B14, and some subtypes of B5, were not accurately assigned by serology. In the panel studied, we found 17 hybridization patterns that corresponded to probable new alleles. New patterns occurred in the four ethnic groups examined. Multiple subtypes of B35, B5, B15, B41, B44, B57, B58, B70, B14, B40, B22 were found in subjects of the same ethnic group. In view of the poor serological definition of some alleles, and the occurrence of multiple subtypes in the same ethnic population, it appears that high resolution B‐locus typing may be an important addition for detection of potentially relevant HLA incompatibilities in transplantation. It should also be valuable for population studies and for the investigation of HLA associations with diseases.
Genetic polymorphism within HLA‐A*02: significant allelic variation revealed in different populations
Authors: Krausa P. , III M. Brywka , Savage D. , Hui K. M. , Bunce M. , Ngai J. L. F. , Teo D. L. T. , Ong Y. W. , Barouch D. , Allsop C. E. M. , Hill A. V. S. , McMichael A. J. , Bodmer J. G. , Browning M. J.
Published: December 2008

Abstract: HLA‐A2 is present at high frequency in most populations, as identified by serological and biochemical means. The value of these methods is limited by their failure to discriminate between the products of the 14 known allelic HLA‐A*02 variants. The great majority of genetic polymorphism which defines the allelic variants is found in exons 2 and 3 of the A*02 genes. These exons encode the α‐1 and α‐2 domains of the HLA Class I molecules, and variation within the genes may influence the peptide binding specificity of the gene products of each allele. Failure to accurately assign the allelic types has implications in transplantation, in interpretation of cellular assays and in the understanding of HLA disease associations. We have developed a method for determining the 14 Known alleles of HLA‐A*02 by use of ARMS–PCR to determine the degree of variation of HLA‐A*02 alleles in 3 different population groups. Considerable variation was found in the relative frequencies of particular A*02 alleles between Caucasian, oriental and black individuals. Our results indicate the importance of ethnic origin in terms of the expected HLA‐A*02 allelic profile, and emphasize the functional significance of allele supecific subtyping of HLA‐A*02.
Polymorphism in the upstream regulatory region of DQA1 gene in the Italian population
Authors: Petronzelli F. , Kimura A. , Ferrante P. , Mazzilli M. C.
Published: December 2008

Abstract: Polymorphism in the 5′‐upstream regulatory region of the DQA1 gene has been recently described. Using PCR‐SSO method and SSCP analysis we have investigated this polymorphism in a group of 111 Italian blood donors which had been oligotyped for DRB1, DQA1 and DQB1 genes. Eight allelic variants were detected. Looking at the relationships among QAP sequences and DQA1 and DRB1 genes, three alternative situations were found: 1. a one‐to‐one relation between QAP and DQA1 alleles, independently of the other class II genes; 2. the same QAP allele in association with different DQA1‐DRB1 haplotypes; 3. the same DQA1 allele with different QAP sequences according to the DRB1 specificity. No unexpected associations with DQB1 gene were found. These results must be interpreted considering that DQA1 and DRB1 genes are transcribed in opposite directions so that the promoter region of DQA1 gene lies between DQA1 and DRB1, close to the former but several hundreds kb away from the latter.
Early‐onset pauciarticular juvenile chronic arthritis is associated with a mutation in the Y‐box of the HLA‐DQA1 promoter
Authors: Haas J. P. , Kimura A. , Truckenbrodt H. , Suschke J. , Sasazuki T. , Volgger A. , Albert E. D.
Published: December 2008

Abstract: Early‐onset pauciarticular juvenile chronic arthritis (EOPA‐JCA) has associations with different alleles of the MHC region (HLA‐A2, DR5, 6, 8, DQA 1*0401, *0501, *0601 and DPB1*0201). All susceptible DQA1 alleles carry an exclusive sequence motif. MHC‐class II gene expression is controlled by 5′ flanking upstream regulatory regions (URR). A hypervariable region in the promoter region of the HLA‐DQA1 gene (‐240 and‐200 base pairs upstream) defines ten different QAP (DQA1‐Promoter) alleles, which are associated with certain DQA1‐alleles. The Y‐Box in the DQA1 promoter (YC‐Box‐125 to‐115 upstream from the ATG) differs from the consensus sequence (‐123 A for G) of all other MHC class II Y‐Boxes, resulting in a lower affinity to the NF‐Y transcription factor and in a reduced expression of DQA1. A second substitution in the Y‐Box of QAP 4.1 and 4.2 (‐119 A for G) is found in the promoter alleles of the DQA 1‐alleles (DQA 1*0401, *0501, *0601) and is strongly associated with susceptibility to EOPA‐JCA.
Anchored PCR cloning of the novel HLA‐Cw*0704 allele detected by PCR‐SSP
Authors: Vilches C. , Bunce M. , de Pablo R. , Herrero M. J. , Kreisler M.
Published: December 2008

Abstract: The novel HLA‐Cw*0704 allele, previously detected as the PCR‐SSP variant Cw7/8v, has been cloned and sequenced from the homozygous typing cell KR03/4 after amplification by anchored PCR. The nucleotide sequence of Cw*0704 is closely related to those of other Cw*07 alleles, but carries specific changes in exon 3 consistent with its serological behavior ‐ a short Cw7 cross‐reactive with antibodies directed against HLA‐Cw8. Some of the substitutions of Cw*0704 have not been previously described for HLA‐C but are found in HLA‐B alleles and in published C sequences of non‐human primates. The new allele carries a novel polymorphism in its 5′ untranslated region (5′ut) that could be shared by all Cw*07 alleles. By PCR‐SSP, Cw*0704 is a relatively common allele in English Caucasoids at a frequency of 4.6%. It is most often observed on HLA‐B44 haplotypes previously described as HLA‐C “blank”, although linkage disequilibria with other HLA‐B specificities have been found.
An evaluation of a multicenter study on HLA‐DPB1 typing using solid‐phase Taq‐cycle sequencing chemistry
Authors: Rozemuller E. H. , Eliaou J. F. , Baxter‐Lowe L. A. , Charron D. , Kronick M. , Tilanus M. G. J.
Published: December 2008

Abstract: In HLA Class II genes, polymorphism is mainly located in the second exon. Most DNA based typing methods are confined to the identification of specific sequence motifs in the second exon. In contrast, Sequencing Based Typing (SBT) elucidates the entire exon 2 sequence for typing. Comparison of the obtained exon 2 sequence with an allele sequence library results in allele assignment. We tested the applicability of SBT using a protocol for amplification followed by solid phase Taq‐cycle sequencing for HLA‐DPB1 typing. A panel of 32 samples were typed by SBT at five test sites which are participating in the Sequencing Based Typing component of the 12th International Histocompatibility Workshop. The panel represents the existing polymorphism at all known polymorphic positions of exon 2, both in homozygous and heterozygous combinations. In this multicenter study we focused on the reliability of analyzing heterozygous sequences for HLA typing. A multi‐sequence analysis approach, Polall, was developed to evaluate sequences obtained. The assignment of homozygosity and heterozygosity was validated by cluster analysis of chromatographic data of all sequences. Sequence characteristics were examined and considered for appropriate assignment. Differences in sequence characteristics that occurred between the test sites are considered in detail. The evaluation of data of 5 test sites reveals that Taq‐cycle sequencing can reliably be performed for HLA‐DPB1 SBT.
ZAP‐70 and p72syk are signaling response elements through MHC class II molecules
Authors: Kanner S. B. , Grosmaire L. S. , Blake J. , Schieven G. L. , Masewicz S. , ødum N. , Ledbetter J. A.
Published: December 2008

Abstract: Ligation of major histocompatibility complex (MHC) class II antigens expressed on antigen‐activated human CD4+ T‐lymphocytes induces early signal transduction events including the activation of tyrosine kinases, the tyrosine phosphorylation of phospholipase‐Cγl and the mobilization of intracellular calcium. Similar responses have been observed in B‐cells following stimulation of MHC class II molecules, including the increased production of intracellular cAMP. In this report, we demonstrate that the ZAP‐70 tyrosine kinase is a responsive signaling element following cross‐linking of HLA‐DR in class II+ T‐cells, and that the homologous tyrosine kinase p72syk is stimulated in B‐cells following ligation of class II antigens. Antibody mediated co‐ligation of the T‐cell antigen receptor (TCR/CD3) with class II molecules resulted in augmented tyrosine phosphorylation of ZAP‐70. Comparable to antibody induced receptor ligation, bacterial superantigen (SEA and SEB) treatment of HLA‐DR+ T‐cells stimulated ZAP‐70 tyrosine phosphorylation, consistent with class II transmembrane signaling by ligation of HLA‐DR and Vβ in cis. Modulation of the TCR/CD3 led to abrogation of class II induced ZAP‐70 tyrosine phosphorylation, but did not result in sequestering of ZAP‐70 from the cellular cytoplasm. Hyperphosphorylated ZAP‐70 was associated with TCR/CD3 ζ‐chain following cross‐linking of HLA‐DR, suggesting a mechanism for the TCR/CD3‐dependence of class II induced signals in alloantigen‐activated human T‐cells. In both tonsillar B‐lympho‐cytes and B‐cell leukemia lines, p72syk was rapidly phosphorylated on tyrosine residues following HLA‐DR cross‐linking. Tyrosine phosphorylation of p72syk induced through ligation of either the B‐cell antigen receptor or class II molecules was potently inhibited by herbimycin A. MHC class II ligation on B‐lymphocytes resulted in cell death, which was both qualitatively distinct from Fas‐induced apoptosis and partially protected by herbimycin A pretreatment. Thus, ligation of MHC class II molecules expressed on human lymphocytes stimulates the ZAP‐70/p72syk family of tyrosine kinases, leading functionally to a tyrosine kinase‐dependent pathway of receptor‐induced cell death.
The HLA‐A3, Cw6, B47, DR7 extended haplotypes in salt losing 21‐hydroxylase deficiency and in the Old Order Amish: identical class I antigens and class II alleles with at least two crossover sites in the class III region
Authors: Donohoue P. A. , Guethlein L. , Collins M. M. , Van Dop C. , Migeon C. J. , Bias W. B. , Schmeckpeper B. J.
Published: December 2008

Abstract: The HLA‐B47, DR7 haplotype in congenital adrenal hyperplasia (CAH) due to 21–hydroxylase deficiency contains a deletion of most of the active CYP21 gene and the entire adjacent C4B gene. The C4A gene produces a protein which is electrophoretically C4A but anti‐genically C4B. In the Old Order Amish, the HLA‐B47. DR7 haplotype contains no deletion, but is immunologically identical to the CAH haplotype in both areas flanking the crossover region. We compared some of the genes in the MHC Class II and Class III regions in the Amish and CAH‐linked haplotypes to define further the relationships between the two. The complement factor B (Bf) proteins differed, but no Bf RFLPs were identified. The complement factor 2 genes exhibited different BamHI RFLPs. Analyses of the tumor necrosis factor‐α genes revealed the same Ncol restriction patterns. The RD genes contained microsatellites of the same size. Portions of the MHC Class II DR and DQ, and Class III CYP21 and C4 alleles were sequenced. The exon 2 sequences of DQ2 and DR7 were identical in the two haplotypes. In the Amish haplotype, both CYP21 and C4 gene pairs were present and functionally normal. The CAH haplotype had two sequence crossovers: from CYP21P to CYP21 in the 7th intron, and from C4A to C4B between codons 1106 (exon 26) and 1157 (exon 28). A model is proposed which accounts for the CAH‐linked mutant haplotype arising from a nonmutant homologue via three crossings‐over.
Sequenase sequence profiles used for HLA‐DPB1 sequencing‐based typing
Authors: Rozemuller E. H. , Chadwick B. , Charron D. , Baxter‐Lowe L. A. , Eliaou J. F. , Johnston‐Dow L. , Tilanus M. G. J.
Published: December 2008

Abstract: Sequencing‐based HLA typing (SBT) is a PCR based high resolution HLA typing method in which polymorphic regions of the gene are sequenced and directly used for typing. Currently, for class II SBT, alleles are identified by comparison of the exon 2 sequence with their corresponding allele sequence library. Routine SBT requires reliable identification of heterozygosity, and automated assignment of the alleles. In sequencing strategies different enzymes can be used for primer extension. The most characteristic difference between sequences obtained by two protocols using Sequenase®, or Taq‐cycle sequencing, respectively, is a difference in incorporation of nucleotides in the primer extension leading to different sequence profiles. In Taq‐cycling sequencing variable nucleotide incorporation results in irregular, but reproducible peak patterns, whereas Sequenase® incorporates nucleotides in nearly equal amounts, resulting in more even peak patterns. In a previously published multi‐center study we evaluated HLA‐DPB1 SBT using Taq‐cycle sequencing, and showed that typing can reliably be performed, considering the specific sequence profiles. In this study the applicability of Sequenase® for HLA‐DPB1 SBT was tested. A panel of samples were typed by SBT at five test sites which participate in the Sequencing Based Typing component of the 12th International Histocompatibility Workshop. The panel represents the existing polymorphism at all known polymorphic positions of exon 2, both in homozygous and heterozygous combinations. The assignment of homozygosity and heterozygosity was validated by Multi‐Sequence Analysis, performing cluster analysis of chromatographic data of all sequences at each position. Sequence characteristics were examined and considered for appropriate assignment. Data reveals that Sequenase® sequencing can also reliably be used for HLA‐DPB1 typing.
Further molecular diversity in the HLA‐B15 group
Authors: Lin L. , Tokunaga K. , Tanaka H. , Nakajima F. , Imanishi T. , Kashiwase K. , Bannai M. , Mizuno S. , Akaza T. , Tadokoro K. , Shibata Y. , Juji T.
Published: December 2008

In order to further clarify the diversity of the HLA‐B15 antigens and the correspondence of serological types with alleles in Asians, we screened various B15 serological splits by means of a polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) method. Subsequently, the genes encoding various B15 variants were sequenced. Two novel alleles, B*1528 and B*1529, were identified: the nucleotide sequence of the former contained a single‐base substitution at position 263 in exon 2 as compared to that of the B*1501 allele, which results in an amino acid change at position 64 in the α1 domain, and the nucleotide sequence of the latter differs from that of B*1518 by a single‐base substitution at position 272 of exon 2 which results in an amino acid change at position 67 of the α1 domain. One new allele, B*1521, described recently in Australian Aborigines was also identified in Asians in the present study. Moreover, the results of sequencing demonstrated that Asian HLA‐B62, B70, and B77 antigens are encoded by B*1501, B*1518, and B*1513, respectively. Two splits of B75 antigens, B75V (TS‐1) and B15N, which have been proposed to exist in the Japanese population were encoded by B*1511 and B*1502, respectively. Most of the B15 alleles detected in the present study showed positive associations with other locus antigens. Especially, B*1502 was strongly associated with Cw8, while B*1521 was strongly associated with A34 and Cw6.