bluuebunny/crossref_metadata_2025 huggingface dataset:
The HLA‐A,B,C genotype of the class I negative cell line Daudi reveals novel HLA‐A and ‐B alleles
Authors: Browning M. J. , Madrigal J. A. , Krausa P. , Kowalski H. , Allsopp C. E. M. , Little A‐M. , Turner S. , Adams E. J. , Arnett K. L. , Bodmer W. F. , Bodmer J. G. , Parham P.
Published: December 2008

Abstract:Daudi, a lymphoblastoid B cell line derived from an African Burkitt lymphoma does not express HLA‐A,B,C antigens at the cell surface. Although HLA‐A,B,C heavy chains are made normally they do not assemble into functional molecules because β2‐microglobulin is absent. Previous serological analysis of somatic cell hybrids indicated that the HLA haplotypes of Daudi encoded HLA‐A1, A10(A26), B17, and B16(38) antigens. Here we describe the application of molecular methods: ARMS‐PCR, cDNA cloning and sequencing, immunoprecipitation and gel electrophoresis, to define the class I genotype of the Daudi cell line which is HLA‐A*0102, A*6601, B*5801, B*5802, Cw*0302 and Cw*0602. With the exception of the B38 antigen, which is not a product of the alleles defined, the genotype is consistent with the serological description. Two previously undiscovered alleles emerged from this analysis: A*0102 and B*5802. The A*0102 allele differs from A*0101 by 5 nucleotide substitutions within exon 2 where it has a motif shared with A*30 alleles; the B*5802 allele differs from B*5801 by 3 substitutions in exon 3 where it has a motif shared with B*14 alleles. Subtyping HLA‐A 1 alleles showed A*0102 was well represented amongst individuals typed serologically as Al in an African population but was absent from caucasoids. B*5802 has been found in a second individual. Thus the novel A and B alleles are not specific to the Daudi tumor. Overall, this analysis of a single East African cell illustrates the power of molecular methods to define new class I HLA alleles in non‐caucasoid populations.
An investigation of HLA‐encoded genetic susceptibility to multiple sclerosis in subjects of Asian Indian and Afro‐Caribbean ethnic origin
Authors: Kelly M. A. , Jacobs K. H. , Penny M. A. , Mijovic C. H. , Nightingale S. , Barnett A. H. , Francis D. A.
Published: December 2008

Abstract: The association of multiple sclerosis (MS) with the HLA class II loci DR and DQ was investigated in populations of Asian Indian and Afro‐Caribbean ethnic origin, resident in the United Kingdom. The putative haplotype, DRB1*150l.DQA1*0102.DQB 1*0602, was weakly positively associated with MS in both races. The overall contribution to disease susceptibility of this marker was small. Over 80% of the MS patients in both racial groups did not possess this haplotype. The data suggest that other genetic and/or environmental factors may be more important in predisposing to MS in these two races. Our study also raises the possibility that genetically distinct forms of the disease may be expressed in white Caucasian and non‐Caucasian populations.
HLA class II typing and idiopathic IgA nephropathy (IgAN): DQB1*0301, a possible marker of unfavorable outcome
Authors: Raguénès O. , Mercier B. , Clèdes J. , Whebe B. , Férec C.
Published: December 2008

Abstract: Previous studies have reported associations between HLA antigens and Idiopathic IgA Nephropathy (IgAN). Nevertheless most of the studies were performed by serology. Thus we decided to perform the HLA class II typing of 58 patients by molecular biology techniques. We report a small increase of DRB1*04. But the main result of our study is the identification of a strong association between HLA DQB1*0301 and IgAN patients with an unfavorable outcome.
DR4Dw4/DR53 molecules contain a peptide from the autoantigen calreticulin
Authors: Verreck F. A. W. , Elferink D. , Vermeulen C. J. , Amons R. , Breedveld F. , de Vries R. R. P. , Koning F.
Published: December 2008

Abstract: Rheumatoid arthritis (RA) occurs more frequently in HLA‐DR4+ individuals than in those who do not express this MHC class II molecule. Although the role of this genetic factor in the immunopathology of this autoimmune disease is unclear, the association of RA with HLA‐DR4 may indicate that DR4 molecules present autoantigen(s) to T cells. Here we report the analysis of naturally processed peptides, eluted from a mixture of HLA‐DR4Dw4 (DRB1*0401) and DR53 (DRB4* 0101) molecules isolated from an RA patient‐derived EBV‐transformed B cell line. Several (size variants of) self‐peptides originating from the autologous molecules HLA‐A2, HLA‐Cw9, HLA‐B62, HLA‐DR4Dw4 and HLA‐DR53, were identified. We also found a sequence that has no homology to any protein in the SwissProt protein sequence databank, and a peptide identical to an internal fragment of the autoantigen calreticulin. The association of the identified peptides with cells expressing HLA‐DR4Dw4/DR53 was confirmed by peptide binding analysis. In agreement with previously described peptide binding motifs for DR4Dw4, most peptides contained an aromatic residue (Phe, Tyr, Trp) at relative position i and a small hydroxyl‐containing residue (Ser, Thr) at i+5. Our findings indicate that in RA patient‐derived EBV‐transformed B cells DR4Dw4/ DR53 molecules present a peptide from the autoantigen calreticulin. Interestingly, autoantibodies against calreticulin have been found in various rheumatic diseases, including rheumatoid arthritis. Thus, the analysis of HLA class II‐bound peptides can lead to the identification of putative T helper epitopes, which might be involved in the immunopathology of autoimmune diseases.
The relative distribution of B35 alleles and their IEF isotypes in a HLA‐B35‐positive population
Authors: Ragupathi G. , Cereb N. , Yang S. Y.
Published: December 2008

Abstract: The HLA‐B35 serotype represents a group of antigens detectable by IEF, cytotoxic T cells, and by sequencing analysis. Four isotypes and eight alleles have been thus far reported. We have determined the relative frequencies of these B35 subtypes in a group of 203 unrelated people. Dot blot hybridization of PCR amplified products was performed using 23 sequence‐specific oligo probes designed based on the EMBL HLA class I sequence database. The amplification was achieved by a pair of group‐specific primers, producing ˜600 bp fragments. By hybridization pattern analysis, we found that four alleles represent over 95% of the B35+ population, with relative frequency of 48.2% for B*3501, 23.7% for B*3502, 15.2% for B*3503, and 8.0% for B*3508. We also identified 3 individuals with B*3504 and one with B*3505, and seven samples with new patterns. B*3501 and B*3503 exactly correlated with the most common isotype B35.3, B*3502 and B*3504 with B35.2, B*3508 may be the B35.1 IEF isotype. The B*3505 was identified from an individual with B35 IEF variant form. Our study shows that the B35 antigen has a wide distribution of alleles, and that many more B35‐related alleles may yet to be uncovered.
A comprehensive approach for typing the alleles of the HLA‐B locus by automated sequencing
Authors: Petersdorf E. W. , Hansen J. A.
Published: December 2008

Abstract: We describe an approach for typing alleles of the HLA‐B locus by using automated sequencing technology. The exon 2 and exon 3 nucleotide sequence of each allele is determined directly from genomic DNA in two steps. In the first step, HLA‐B exon 2, intron 2 and exon 3 sequences are amplified with one or two primer pairs out of a panel of 5 primer pairs that describe all known HLA‐B alleles. In the second step, templates are sequenced in 5′ and 3′ orientations in a PCR assay that utilizes Taq polymerase to incorporate fluorescent dye‐labeled nucleotides into each new strand synthesized. Gel electrophoresis of the labeled products is performed in an automated DNA sequencer. The derived sequences are aligned against reference sequences and each nucleotide position is evaluated for homology to consensus sequence. Using this strategy, the HLA‐B allele sequence is directly ascertained with precision and efficiency. The automated sequencing strategy can be readily applied in the clinical laboratory as a practical tool for high resolution typing of HLA‐B alleles.
DQCAR microsatellite polymorphisms in three selected HLA class II‐associated diseases
Authors: Mignot E. , Kimura A. , Abbal M. , Thorsby E. , Lin X. , Voros A. , Macaubas C. , Bouissou F. , Sollid L. M. , Cambon‐Thomsen A. , Yasunaga S. , Grumet F. C.
Published: December 2008

Abstract: DQCAR is a very polymorphic CA repeat microsatellite located between the HLA DQA1 and DQB1 gene. Previous studies have shown that specific DQCAR alleles are in tight linkage disequilibrium with known HLA DR‐DQ haplotypes. Of special interest was the fact that haplotypes containing long CA repeat alleles (DQCAR > 111) were generally more polymorphic within and across ethnic groups. In these latter cases, several DQCAR alleles were found even in haplotypes containing the same flanking DQA1 and DQB1 alleles. In this work, three HLA class II associated diseases were studied using the DQCAR microsatellite. The aim of this study was to test if DQCAR typing could distinguish haplotypes with the same DRB1, DQA1 and DQB1 alleles in control and affected individuals. To do so, patients with selected HLA DR‐DQ susceptibility haplotypes were compared with HLA DR and DQ matched controls. This included: Norwegian subjects with Celiac disease and the HLA DRB1*0301, DQA1*05011, DQB1*02 haplotype; Japanese subjects with Type 1 (insulin‐dependent) Diabetes Mellitus and the HLA DRB1*0405, DQA 1*0302, DQB 1*0401 haplotype; and French patients with corticosensitive Idiopathic Nephrotic Syndrome and the HLA DRB 1*0701, DQA 1*0201, DQB1*0202 haplotype. These specific haplotypes were selected from our earlier work to include one haplotype bearing a short DQCAR allele (celiac disease and DR3, DQ2‐DQCAR99) and two haplotypes bearing long DQCAR alleles (Diabetes Mellitus and DR4, DQ4‐DQCAR 113 or 115 Idiopathic Nephrotic syndrome and DR7, DQ2‐DQCAR 111–121). Additional DQCAR diversity was found in both control and patients bearing haplotypes with long CA repeat alleles. The results indicate that DQCAR typing did not improve specificity in combination with high resolution DNA HLA typing as a marker for these three disorders.
Activation of extracellular signal‐regulated protein kinase (ERK/MAP kinase) following CD28 cross‐linking: activation in cells lacking p56lck
Authors: August A. , Dupont B.
Published: December 2008

Abstract: T lymphocytes require two signals for activation. Recognition of antigen/MHC complexes by the T cell receptor delivers the first signal, while a second signal, delivered by the cell surface receptors CD80 and/or CD86 binding to the T cell surface molecule CD28, has been shown to be effective for the initiation of effective T cell responses. While some of the cytoplasmic effector molecules involved in T cell receptor signaling is known, little is known regarding those involved in the co‐stimulation of T cells by CD28. Using the T cell leukemic cell line Jurkat as a model for T cell activation, we demonstrate that cross‐linking CD28 using monoclonal antibodies causes tyrosine phosphorylation and activation of MAP kinase/ERK. This activation was rapid, peaking at approximately 5 minutes post CD28 cross‐linking, and transient. Activation of MAP kinase/ERK occurred 3 fold less efficiently in a Jurkat line lacking functional p56lck (JCAM.1), and was almost undetectable in a line lacking CD45 (J45.01). These results suggest that CD28 cross‐linking can activate intracellular signaling pathways via several different tyrosine kinases. Thus CD28 signaling can activate src family kinases lck and fyn, as well as the Tec family kinase emt/ick. Activation of any one or a combination of these tyrosine kinases may be sufficient for the activation of MAPK following CD28 cross‐linking. Activation of MAPK has been shown to cause activation of AP‐I and other transcription factors via serine and/or threonine phosphorylation. This observation thus connects signaling via CD28 to activation of nuclear transcription factors required for the transcription of T cell genes including lymphokines such as IL‐2 and GM‐CSF.
Predictability of alloreactivity among unrelated individuals: role for HLA‐DPB1
Authors: Awdeh Z. L. , Alper C. A. , Fici D. A. , Ronco P. , Yunis E. J.
Published: December 2008

Abstract: We compared the mixed lymphocyte culture reaction (MLR‐I) among unrelated individuals who are carriers of the extended haplotype [HLA‐B8, SC01, DR3, DRB1*0301, DQB1*0201] on one chromosome and the generic specificity HLA‐DR4 on the second chromosome. Genomic DNA samples from the same individuals were also analyzed for HLA‐DRB1, DQB1 and DPB1 alleles by PCR and SSOPH typing and for DOB polymorphism by RFLP. HLA‐DRB1 alleles, in paired MLR responses between unrelated individuals indicated that matching of HLA‐DRB1 was a better predictor of non‐reactivity than identity in HLA‐DR generic types, (43% vs 22%). Moreover, 90% of the DRB1 matched pairs had nonreactive and weakly reactive MLR, whereas only 37% of DRB1 mismatched unrelated individuals gave weak or no reactions. Matching for HLA‐DRB1 and HLA‐DPB1 alleles eliminates a significant number of cell mixtures with MLR‐I reactivity. Furthermore, some DPB1 mismatches, but not all, do not seem to elicit MLR‐I reactivity.
Allelic frequencies of the HLA‐B17 antigen group: comparative analysis by serology, IEF and PCR‐SSOP typing
Authors: Levine J. E. , Yang S. Y.
Published: December 2008

Abstract: Current typing technology for class I HLA antigens uses serological and/or isoelectric focusing gel electrophoresis. DNA typing for the HLA class I antigens can accurately identify the class I genotype of individuals and cell lines. Here, we report correlation of DNA typing results with serological and IEF results for the B17 group. The B17 antigens are relatively common, being carried by almost 9% of Caucasians and 28% of blacks. In this study, five 10th International Histocompatibility Workshop cell lines carrying B17 and 106 individuals in 61 families carrying B17 were DNA typed for B17 using B17‐allele‐specific amplification and sequence specific oligonucleotide probe hybridization pattern analysis. 38 (55.07%) out of 69 unrelated haplotypes had B*5701, 23 (33.33%) had B*5801, 6 (8.70%) had B*5702, and 2 (2.90%) had B*5802. DNA typing results correlated well with serological and isoelectric focusing results. In general, there was high degree of agreement between all three methods, although heterozygosity for B17 poses a particular problem for serological and IEF methodology. Both B*5701 and B*5801 have the same electrophoretic mobility on IEF gel, corresponding to B17.2, B*5702 corresponds to B17.1, while B*5802 corresponds to B17.3.
HLA class II and HLA‐B27 oligotyping in two Siberian native population groups
Authors: Krylov M. , Erdesz S. , Alexeeva L. , Benevolenskaya L. , Arnett F. C. , Reveille J. D.
Published: December 2008

Abstract: It was the purpose of this study to better define the frequency of HLA‐B27 subtypes and HLA class II alleles among indigenous populations from the eastern tip of the Chukotka Peninsula of Siberia, Russia, which have higher frequencies of HLA‐B27 (40%) and spondyloarthropathies (2%) than Caucasian populations and test the hypothesis that these populations are more closely related to Orientals. Siberian Eskimos and Chukchi residing in four coastal villages on the Chukotka Peninsula inhabited by Siberian Eskimos and Chukchi people were examined using oligotyping of the polymerase‐chain reaction‐amplified second and third exons of the HLA‐B27 gene. HLA‐class II alleles (DRB1, DQA1 and DQB1) were similarly determined. Of 88 HLA‐B27 positive individuals from these villages, all had HLA‐B*2705, including the four patients with Reiter's syndrome and the five ankylosing spondylitis, except one Eskimo control who had HLA‐B*2702. None had HLA‐B*2704, a frequent subtype in Orientals. HLA‐class II typing in 70 Siberian Eskimos and 71 Siberian coastal Chukchi revealed HLA‐DRB1*0401, DRB1*0802, *0901 and *1402 to account for nearly all the DRB1 alleles found in this population, similar to what has been described in Eskimos in Alaska, but different from Chinese or native Americans in the U.S. The overwhelming majority of the individuals examined had HLA‐DQB1*0301, similar to what has been observed in Native Americans. The Siberian Eskimos differed from the coastal Chukchi only in the occurrence of HLA‐DRB1*0701, DQA1*0201, DQB1*0201 alleles, which occurred only in the former group. These data suggest that the Chukotka population is genetically more closely related to Caucasians and native Americans and less to other Oriental populations.
DNA typing of the HLA‐A gene: population study and identification of four new alleles in Japanese
Authors: Date Y. , Kimura A. , Kato H. , Sasazuki T.
Published: December 2008

With the use of polymerase chain reaction (PCR) and sequence‐specific oligonucleotide probe (SSOP), we established a DNA typing method of the HLA ‐ A locus. A pair of primers to amplify the highly polymorphic region of HLA‐A gene including exon 2 and exon 3 was designed and the amplified DNAs were hybridized with 91 types of 32P labeled SSOPs. This method allowed discrimination of all known HLA‐A alleles except for two combinations, A*0201 or A*0209 and A*0207 or A*0215N, which have identical sequences in exon 2 and exon 3. Another pair of primers was designed for amplification of exon 4 and the PCR products were hybridized with 5 SSOPs to distinguish A*0201 and A*0207 from A*0209 and A*0215N, respectively. In this study, 81 B‐lymphoblastoid cell lines (BLCL) homozygous for HLA and 553 unrelated healthy Japanese individuals were determined for their HLA‐A genotypes. Based on the genotyping results, frequency of HLA‐A alleles and linkage disequilibrium between HLA‐A and HLA‐B in the Japanese population were investigated. In addition, four new HLA‐A alleles were identified and their nucleotide sequences in exon 2 and exon 3 were determined to confirm the typing results.
Sequence analysis of the 2nd intron revealed common sequence motifs providing the means for a unique sequencing based typing protocol of the HLA‐A locus
Authors: Blasczyk R. , Wehling J. , Weber M. , Salama A.
Published: December 2008

We here present a sequencing strategy for the HLA‐A locus which is generally applicable for all HLA class I genes. The typing strategy is based on a group‐specific amplification according to the serologically defined antigens. The PCR products carry the typing‐relevant polymorphic regions of the 2nd and 3rd exon including the 2nd intron. The sequencing primers were designed to match conserved sequence motifs in the 2nd intron allowing a nested sequencing approach in 3′ and 5′ direction. These conserved regions were identified after sequence compilation of the 2nd intron of 143 clinical samples and 48 cell lines mostly from the 9th and 10th IHWC representing all serologically defined groups of alleles. This strategy allowed the use of only one 5′ and one 3′ sequencing primer regardless of the amplified allele. Therefore, it was possible to use dye terminator as well as dye primer sequencing chemistry. The amplification strategy allowed the separation of the haplotypes in almost all cases. Thus, an assignment of heterozygous positions requiring high sequencing quality was not necessary, allowing the application of Sequenase as well as TaqPolymerase as sequencing enzyme. Concerning the resolution of heterozygosity it is obvious that this approach is superior to a typing system using a single pair of generic primers followed by direct sequencing, since the latter technique is not capable of defining the cis/trans linkage of polymorphic sequences and, hence, cannot exclude the presence of unknown alleles.
HLA*A2 confers mortality risk for cardiovascular disease in Pimans
Authors: Williams R. C. , Hanson R. L. , Pettitt D. J. , Sievers M. L. , Nelson R. G. , Knowler W. C.
Published: December 2008

A sample of 1465 full heritage Piman Indians from Arizona were typed for the serological antigens of the HLA class I loci and then incorporated into a survival study that ended December 31, 1991. The total follow‐up time was 11,749 person‐years with an average of 8.0 years per person. During the study 298 persons died, 54 from cardiovascular disease (CVD). Allele HLA*A2 conferred a 4.94 fold rate for death from CVD (95% C.I. 1.91 – 12.77). When controlled for the potential confounding variables, cholesterol, mean blood pressure, smoking, body mass index, rheumatoid factor titer, and nephropathy, the mortality rate ratio (MRR) was 5.42 (95% C.I. 1.98 – 14.82). There was no statistically significant association of mortality with other HLA‐A or HLA‐B alleles, or for causes of death not related to cardiovascular disease.
The role of HLA antibodies in neonatal thrombocytopenia: a prospective study
Authors: King K. E. , Kao K. J. , Bray P. F. , Casella J. F. , Blakemore K. , Callan N. A. , Kennedy S. D. , Kickler T. S.
Published: December 2008

The role of HLA antibodies in neonatal alloimmune thrombocytopenia is controversial. We prospectively studied the sera of obstetric patients at delivery for HLA antibodies and correlated their presence with umbilical cord blood platelet counts. We studied 493 births at The Johns Hopkins Hospital comprising of 357 African American, 115 Caucasian, and 21 babies of other racial groups. One hundred and thirty nine mothers had HLA antibodies. Of these HLA alloimmunized mothers, only ten infants had platelet counts of 150,000/μL or less. Three hundred and eight mothers with no detectable antibodies gave birth to 27 infants with platelet counts of 150,000/μL or less. Yates corrected Chi square analysis showed no significant relationship between maternal HLA alloimmunization and baby platelet count (p=0.709). Only 8 of sixty cord sera from babies of HLA alloimmunized mothers were positive for HLA antibodies. The HLA cord blood antibody results were then correlated with the neonatal platelet counts. The Fisher's exact test showed no significant relationship between the presence of HLA antibodies in cord blood samples and neonatal platelet counts (p=0.232). Although one third (31%) of mothers have HLA antibodies, neonatal thrombocytopenia is rarely associated with this finding. However, HLA antibodies can cross the placenta, and in these unusual cases, may be associated with a higher risk of neonatal thrombocytopenia.
PCR‐RFLP‐based Mamu‐DQB1 typing of rhesus monkeys: characterization of two novel alleles
Authors: Sauermann U. , Arents A. , Hunsmann G.
Published: December 2008

Up to now 19 allelic sequences of the rhesus monkey DQB1 locus have been published. Referring to these sequences, we have developed a typing protocol for Mamu‐DQB1 alleles which was verified by additional cloning, sequence analysis and segregation studies. The protocol is based on the amplification of the second exon with only one specific primer pair followed by the digestion of the PCR products with up to 10 different restriction endonucleases. The alleles can be identified in homozyous and heterozygous combinations since most amplified second exon sequences give unique band patterns after digestion with at least one of the selected restriction endonucleases. By the use of this protocol we analyzed DNA‐samples from 182 rhesus monkeys. Among these samples two novel Mamu‐DQB1 alleles were detected, subsequently cloned and their nucleic sequence determined. Since we typed four complete breeding groups consisting of two generations we were able to identify several DQ haplotypes by segregation analysis using the previously developed typing protocol for DQA1.
Mechanisms of loss of HLA class I expression on colorectal tumor cells
Authors: Browning M. , Petronzelli F. , Bicknell D. , Krausa P. , Rowan A. , Tonks S. , Murray N. , Bodmer J. , Bodmer W.
Published: December 2008

For several years this laboratory has studied the expression of HLA class I on established colorectal tumor cell lines and on fresh tumors. We review here the mechanisms by which colorectal tumor cells may lose surface expression of HLA class I molecules. Several independent mechanisms have been identified, including loss or mutations in β2‐microglobulin genes, loss of HLA heavy chain genes, selective lack of expression of HLA alleles, and regulatory defects in HLA expression including loss of expression of the peptide transporters associated with antigen processing (TAP). The data suggest that colorectal tumor cells may evade tumor specific, HLA restricted immune attack by loss of HLA class I expression through a number of mechanisms.
Comparison of HLA‐A antigen typing by serology with two polymerase chain reaction based DNA typing methods: implications for proficiency testing
Authors: Bozón M. V. , Delgado J. C. , Turbay D. , Salazar M. , Granja C. B. , Alosco S. M. , Dupont B. , Yunis E. J.
Published: December 2008

Serology has been routinely used for class I HLA typing for the selection of donors for allotransplantation. However, serology is not adequate for the assignment of all class I specificities especially when testing non‐Caucasians subjects and it is necessary to adopt new strategies for routine testing. At the present time the extent of incorrect serologic HLA‐A assignments in clinical testing is not known. The polymerase chain reaction (PCR) based techniques have become useful standard clinical typing methods of HLA class II alleles but most laboratories still use serology for class I typing. In this report we have compared two PCR based techniques, PCR amplification with sequencespecific primers (PCR‐SSP) and PCR amplification and subsequent hybridization with sequence‐specific oligonucleotide probes (PCR‐SSOP), for the assignment of HLA‐A specificities in 56 blood samples from patients and families serologically typed for HLA‐A. This side‐by‐side comparison of PCR methods showed 100% correlation between them. However, serology showed 7.1% misassignments and, in an additional panel of 19 cells where serology produced equivocal results, the PCR‐SSP and SSOP methods identified the correct HLA‐A specificity. Our results emphasize the need to complement routine serologic testing of HLA specificities with a small number of primers designed to test HLA‐A34, A36, A43, A66, A74 and A80, that are not detected with high precision by serology. We concluded that the PCR‐SSP and ‐SSOP methods can be used in routine HLA‐A typing of patients and donors for transplantation with a greater precision than serology.
Linkage disequilibrium between HLA class II (DR, DQ, DP) and antigen processing (LMP, TAP, DM) genes of the major histocompatibility complex
Authors: Djilali‐Saiah I. , Benini V. , Daniel S. , Assan R. , Bach J.‐F. , Caillat‐Zucman S.
Published: December 2008

TAP, LMP and DM genes map within the major histocompatibility complex (MHC) class II region between the DQB1 and DPB1 loci, and are involved in the processing of peptides bound to HLA class I or class II molecules. In order to determine the various linkage disequilibria existing between these genes and HLA class II genes, we have analyzed TAP1, TAP2, LMP2, DMA, DMB, DRB1, DQA1, DQB1 and DPB1 polymorphisms in 162 unrelated healthy Caucasian individuals. Many positive or negative associations were observed between alleles at these loci, such as between DR/DQ and TAP2, DM or LMP, between DP and DMB, and between TAP2 and DM, TAP2 and LMP. Conversely, no linkage disequilibrium was detected between some closely related genes (DR/DQ and TAP1, TAP1 and TAP2, LMP2 and DM), in agreement with the existence of recombination hot spots in this region. Other weak linkage disequilibria are likely to exist in this region. These data allow to define some conserved MHC class II haplotypes including HLA class II and TAP, LMP and DM alleles. Furthermore, the knowledge of such linkage disequilibria is of outstanding importance in order to avoid misinterpretation of the data when studying MHC class II associations with autoimmune diseases.
Strategy for distinguishina new DQB1 allele (DQB1 *[0611) from the closely related DQB1 *0602 allele via sequence specific PCR or direct DNA sequencing
Authors: Williams T. M. , Bassinger S. , Moehlenkamp C. , Wu J. , Montoya G. D. , Griffith B. B. , McAuley J. D. , Goldman S. , Maurer D. H. , Troup G. M.
Published: December 2008

A novel DQ6 allele (DQB1*0611) was identified via direct DNA sequencing in an African‐American donor for bone marrow transplantation. The allele was not suspected on the basis of a sequence specific PCR assay which instead indicated the presence of DQB1*0602. DQB1*0602 and DQB1*0611 differ in exon 2 only at codon 9 resulting in a tyrosine substitution for phenylalanine. A modification of current DQB1 sequence specific PCR assays was devised which allows distinction between the closely related DQB1*0602 and DQB1*0611 alleles. Preliminary allele frequency studies suggest that DQB1*0611 is rare both in a non‐African American sample and in Americans of African descent carrying DR11, DQ6 haplotypes. The selection of various DQB1*0611 detection methods is discussed.
Genetic polymorphisms in the keratin‐like S gene within the human major histocompatibility complex and association analysis on the susceptibility to psoriasis vulgaris
Authors: Ishihara M. , Yamagata N. , Ohno S. , Naruse T. , Ando A. , Kawata H. , Ozawa A. , Ohkido M. , Mizuki N. , Shiina T. , Ando H. , Inoko H.
Published: December 2008

Psoriasis vulgaris is associated with the HLA‐Cw6 and Cw7 antigens. However, it has not yet been clarified if the HLA‐Cw6 and Cw7 genes themselves are the susceptible gene related to this disease or if it is some other non‐HLA gene in a linkage disequilibrium with these HLA‐C alleles. The S gene, recently identified in the HLA class I region 160 kb telomeric of HLA‐C, encodes a keratin‐like protein and is expressed specifically in the granular layer of the epidermis. Therefore, it is tempting to speculate that the S gene is one of the strong candidate genes responsible for the pathogenesis of psoriasis vulgaris. Direct sequencing of the first and second exon of the S gene after polymerase chain reaction (PCR) amplification has allowed the identification of two diallelic polymorphic sites in exon 1 and seven diallelic polymorphic sites in exon 2, three among which result in amino acid exchanges, a Ser‐Phe substitution at amino acid position 186, a Gly‐Val substitution at position 393 and a Ser‐Leu substitution at position 394. No significant difference in the dimorphic distributions of the S gene was observed between the patients with psoriasis vulgaris and healthy controls, suggesting that the susceptible gene for psoriasis is not the S gene itself.
A new murine lymphocytotoxic monoclonal antibody recognizing HLA‐A2, ‐A28 and ‐A9
Authors: Mizuno S. , Yako F. , Ohta H. , Kato T. , Wada K. , Uchigiri C. , Furuta M. , Ohya K. , Kurachi T. , Murase T. , Kamiya T. , Ozawa K.
Published: December 2008

Monoclonal antibodies recognizing polymorphic as well as monomorphic epitopes on HLA antigens are important tools for understanding the immuno‐biology of HLA molecules. We immunized BALB/c mice with a HLA‐A2 transfectant and screened for hybridomas which reacted with a HLA‐A2 trans‐fectant but not with a HLA‐B75 transfectant. After subcloning by limiting dilution four times, a hybridoma secreting a monoclonal antibody (mAb) (IgG 2a, kappa) designated 1–145 was established. 1–145 reacted with Epstein‐Barr virus transformed B lymphoblastoid cell lines (B cell lines) which expressed HLA‐A2, ‐A28, ‐A23 and ‐A24. The titer of 1–145 in culture supernatant against HLA‐A2 and ‐A28 antigens was similar and the titer against HLA‐A23 was lower. 1–145 reacted with cells expressing HLA‐A24 but the titer against HLA‐A24 antigens was even lower than that againt HLA‐A23 antigens. The HLA‐A24 antigens on the peripheral blood lymphocytes were not detected by 1–145 possibly due to the lower expression compared to the B cell lines. These differences of the titers were reflected to microlymphocytotoxic‐ity assay in which 1–145 culture supernatant lysed all PBLs expressing HLA‐A2.‐A28 and ‐A23 but did not lyse PBLs expressing HLA‐A24. Published deduced amino acid sequence data of HLA class I molecules indicate that Lys in position 127 may be critical for 1–145 binding.
Costimulation by CD28 sFv expressed on the tumor cell surface or as a soluble bispecific molecule targeted to the L6 carcinoma antigen
Authors: Hayden M. S. , Grosmaire L. S. , Norris N. A. , Gillil L. K. , Winberg G. , Tritschler D. , Tsu T. T. , Lihsley P. S. , Mittler R. S. , Senter P. D. , Fell H. P. , Ledbetter J. A.
Published: December 2008

Interaction of the CD80 (B7‐1) and CD86 (B7‐2) molecules on antigen presenting cells with the receptors CD28 and CTLA‐4 on T cells generates signals important in the regulation of immune responses. Because this receptor system involves multiple receptor‐ligand interactions, determining the function for individual receptors has been difficult. One approach is the use of antibodies and their derivatives with singular specificity as substitute ligands to explore the activities of these molecules. We have constructed recombinant mono‐and bi‐specific sFv molecules specific for the CD28 receptor that are capable of binding and generating costimulatory signals to activate T cells. We demonstrate that these soluble molecules are capable of higher levels of costimulation than soluble CD80Ig at equivalent concentrations. We also constructed artificial adhesion receptors on the cell surface using two different CD28‐specific sFvIgs fused to the CD80 cytoplasmic and transmembrane domains. In this report, we compared costimulation by a soluble bispecific (αCD28‐α6) single chain sFvIg fusion protein to that generated by L6 antigen positive (L6+) H3347 tumor cells transduced with cell surface expressed forms of aCD28 sFv's. We show that the bispecific protein can target potent CD28 costimulatory activity to L6+ tumor cells in vitro. We also show that transfection of the cell surface forms of the two different CD28 sFvIgs into H3347 tumor cells allows them to generate significant costimulatory signals to activated T cells. Finally, we demonstrate that tumor cell presentation of either the soluble bispecific or transduced cell surface sFv generate similar costimulatory effects resulting in T cell activation.
Molecular analysis of the DLA DR region
Authors: Wagner J. L. , Burnett R. C. , Storb R.
Published: December 2008

The dog is an important model for studying organ transplantation. In order to develop a DNA‐based typing system, a genomic analysis of the canine DR region of the MHC was undertaken. Southern analysis suggests the presence of two DRB genes in most dogs and all have one DRA gene. One dog out of 200 examined contains only one DRB gene. The DRA gene was mapped in one lambda phage clone. One DRB gene (DRBB1) was localized to two overlapping phage clones. Another DRB gene (DRBB2) was localized to two overlapping phage clones. Sequence analysis of the two DRB genes suggests that one gene is intact (DRBB1) and one gene is a pseudogene (DRBB2) because it lacks a splice acceptor signal for exon 3 and lacks exons 2 and exon 4. Intron 1 and 2 sequence data from DRBB1 allow amplification of the polymorphic exon 2 which, in turn, can serve as a basis for developing a typing system for DRB.
Distribution of the HLA‐A9 antigen family (A23, A24 and A2403)
Authors: Thomas M. , Street J. , Fusseil H. , Guttridge M. , Darke C.
Published: December 2008

Characterization of 50 local anti‐HLA‐A9 (A23 and/or A24) sera against the HLA‐A2403 antigen allowed the retrospective assignment of A2403 and the verification of A23 and A24 specificities in a panel of 9196 volunteer bone marrow donors. The six A2403 positive subjects identified were confirmed by PCR using four sequence‐specific primer mixtures. Population analysis showed the distribution and HLA‐A/B linkage disequilibrium of HLA‐A23 and A24 to be typical of a Northern European Caucasoid population. HLA‐A2403 has a phenotype frequency of 0.00065 (gene frequency ‐ 0.00033) and is in linkage disequilibrium with HLA‐B63 and possibly B35.
HLA‐A*02 subtype distribution in Caucasians from northern Italy: identification of A*0220
Authors: Fleischhauer K. , Zino E. , Mazzi B. , Severini G. M. , Benazzi E. , Bordignon C.
Published: December 2008

This study describes a comprehensive, easy to perform PCR‐SSOP typing approach suitable for complete genomic subtyping of HLA‐A*02. A single 1.6 kb PCR–amplificate spanning exons 2,3 and 4 of the HLA‐A*02 gene was used for hybridization with a panel of twenty‐four SSOPs. This allowed unequivocal assignment of all so far known HLA‐A2 subtypes, including A*0209 and A*0215N which differ for nucleotide substitutions in exon 4, without the need for two separate amplifications. Using this approach, HLA‐A*02 subtype distribution was analyzed in 218 samples from unrelated, healthy individuals from northern Italy enrolled in the Italian Bone Marrow Registry and typed as HLA‐A2 by serology or generic molecular analysis. As expected, A*0201 was found in the majority (92.6%) of samples. However, a significant number (6.8%) of individuals carried A*0205. Furthermore, A*0202, A*0208, A*0209 and A*0217, so far not described in Caucasians, were detected in a low number of samples (frequency ranging from 0.45% to 1.8%). Finally, a novel HLA‐A*02 subtype, A*0220, was detected in 0.9% of the samples. As confirmed by DNA sequencing of exons 2 and 3, this allele is identical to A*0201 except for a single nucleotide substitution in codon 66 which changes the predicted amino acid sequence form Lys to Asn. The findings of this study have implications for the selection of HLA‐A*02+ donors in unrelated bone marrow transplantation and of patients for specific immunotherapy with HLA‐A*02 restricted peptide vaccines.
Dermatitis herpetiformis and celiac disease are both primarily associated with the HLA‐DQ (α1*0501, (β1*02) or the HLA‐DQ (α1*03, (β1*0302) heterodimers
Authors: Spurkland A. , Ingvarsson G. , Falk E. S. , Knutsen I. , Sollid L. M. , Thorsby E.
Published: December 2008

HLA‐DRB1, ‐DQA1, and ‐DQB1 genomic typing of 50 patients with dermatitis herpetiformis and of 290 healthy blood donors was performed. Genes encoding the DQ (α1*0501, (β1*02) heterodimer were carried by 43 (86%) of the patients and 72 (25%) of the controls. Of die remaining seven patients six (12% of all the patients) carried genes encoding the DQ (α1*03, β1*0302) heterodimer. These HLA associations are very similar to those observed in patients with celiac disease. We thus conclude that dermatitis herpetiformis and celiac disease are associated to the very same HLA‐DQαβ heterodimers.
Brief communication HLA class II antigens DR4 and DQ8 are associated with allergy to hevein, a major allergen of Hevea latex
Authors: Rihs H.‐P. , Chen Z. , Cremer R. , Baur X.
Published: December 2008

In this study we investigated the relationship between HLA class II alleles and the IgE‐specific immune response to the 4.7 kDa polypeptide hevein of Hevea brasiliensis, a major latex allergen. 51 individuals with immediate‐type latex allergy and 90 controls were examined for the polymorphisms in exon 2 of HLA‐DRB1, 3, 4, 5 and DQB1 by sequence‐specific oligonucleotide probe typing. 35 (69%) out of 51 latex‐sensitized subjects showed positive hevein‐specific IgE values. Analysis of the HLA data among these 35 subjects revealed increased phenotype frequencies for DR4 (22/35, 63%) and DQ8 (18/35, 51%) when compared with those in the 16 hevein‐negative but latex‐positive subjects (DR4: 2/16, 13%, p=0.0009, pc=0.047; DQ8: 0/16, p=0.0003, pc=0.018) and with healthy controls (DR4: 22/90, 24%, p=0.00012, pc=0.013; DQ8: 16/89, 18%, p=0.0003, pc=0.036). Finally the DR4‐DQ8 haplotype frequency was significantly elevated in hevein‐positives when compared with hevein‐negatives (51% vs. 0, p=0.0003, pc=0.034) or controls (51% vs. 18%, p=0.0002, pc=0.045) The present data suggest DR4 and DQ8 to be operating jointly as susceptibility factor for the allergy to hevein.
HLA‐B27 polymorphism and worldwide susceptibility to ankylosing spondylitis
Authors: Gonzalez‐Roces S. , Alvarez M. V. , Gonzalez S. , Dieye A. , Makni H. , Woodfield D. G. , Housan L. , Konenkov V. , Abbadi M. C. , Grunnet N. , Coto E. , López‐Larrea C.
Published: December 2008

HLA‐B27 is strongly associated to ankylosing spondylitis (AS) and represents a family of eleven B27 alleles (B*2701–11). Our aim was to analyze the distribution of B27 subtypes by PCR/SSOP and genomic sequencing in a large group of populations (n=17). 711 B27‐positive samples from Caucasoid, Asian, African, Amerindian and Polynesian populations were selected to ascertain transracial gene mapping of the B27 subtypes. 476 of these were AS patients, chosen to investigate the contribution of B27 alleles to AS susceptibility. Some significant new findings have arisen from this study: 1) B*2705 was the predominant subtype in circumpolar and subarctic areas. B*2702 was found to be practically restricted to Caucasian populations, showing a higher frequency in Middle‐East (Jews) and North Africa (Arabs/Berbers) groups. 2) B*2703 appears associated with AS in Western Africans. This is of remarkable interest since it was suggested that B*2703 would be negatively disease‐associated. 3) Although B*2706 appears negatively associated with AS in Thais, we identified two patients from northern China carrying it. This may be a reflection of a disease heterogeneity and could indicate that more than one pathogenic agent can be involved in AS. B*2709 has been recently described as negatively associated with AS in Sardinians. The molecular changes His 114Asp (B*2706) and Asp 116His (B*2709) could modify the genetic susceptibility to AS.
Allele walking: a new and highly accurate approach to HLA‐DRB1 typing: Application to DRB1*04 alleles
Authors: Nieto A. , Tobes R. , Martin J. , Pareja E.
Published: December 2008

We have developed a typing method, which can be used even in small laboratories, to produce a highly accurate and reliable allele assignment in any homozygous or heterozygous situation. We have called the method allele walking (AW) and it consists of sequential rounds of PCR‐RFLP. After digestion, electrophoresis separates alleles positive for the mutation from the negative alleles; the cleaved fragment is then recovered from the gel and analyzed for mutations at another codon. In this way, AW is able to positively ascertain which mutations are in the same chromosome (cis‐linkage) and assigns alleles independently from each other. Artificial sites are created in the PCR step in order to positively detect substitutions not naturally recognized by any of the existing or convenient enzymes. We report the application of AW for typing the 22 DRB1*04 alleles. The first PCR‐RFLP round groups DRB1*04 alleles. Subsequently, the mutations at codons 86, 74, 71, 57 and 37 can be analyzed for the unambiguous assignment of the majority of the alleles. Additional polymorphisms at different codons can be assayed to resolve any undetermined alleles. The viability of all the restriction sites used as well as the feasibility of AW were successfully tested.
Striking diversity of DR15 haplotypes in Croatians
Authors: Grubic Z. , Zunec R. , Kastelan A.
Published: December 2008

Analysis of HLA class II polymorphism revealed DR2 as the most frequent antigen in Croatians (20, 9%). The observed DR2 subtypes and their frequencies were as follows: DRB1*1601 – 9.9%; 1501 – 8.5%; 1602 – 1.8%, 1502 – 1.4% and 1503 – 0.7%. Furthermore, analysis of probable DRB1‐DQA1‐DQB1 haplotype associations in unrelated individuals revealed a high diversity of DR15 haplotype associations. Now, to confirm this observation from unrelated individuals, family studies have been performed. Sixty‐eight families, with at least one parent and one offspring DR2 positive, have been studied. DRB1*1601‐DQA1*0102‐DQB1*0502 seems to be conserved haplotype in Croatians while DRB1*15 haplotypes showed extremely high diversity. Seven rare DR15 haplotypes are observed; six of these appear twice and one only once. Two haplotypes appear to be unique to Croatians: DRB1*1501‐DQA1*0102‐DQB1*0501 and DRB1* 1501‐DQA1*0102‐DQB1*0604. This confirmation of extremely high diversity of DR15 haplotypes in Croatians is a valuable tool for unrelated bone marrow transplantation and DR2 disease association studies.
Polymorphism of TAP1 and TAP2 in Japanese patients with rheumatoid arthritis
Authors: Takeuchi F. , Nakano K. , Matsuta K. , Takizawa K. , Nabeta H. , Kuwata S. , Ito K.
Published: December 2008

Contribution of polymorphism of transporter associated with antigen processing 1 and 2 (TAP1 and 2) alleles to pathogenesis of Japanese rheumatoid arthritis (RA) was studied in 92 RA patients by PCR‐RFLP. The allele frequency of TAP2A was slightly low (38.0%) and the frequencies of TAP2B and TAP2C were slightly high (39.7% and 17.9%) in RA, but these differences were not significant. These increases and decrease were due to the positive or negative associations with HLA‐DRB1*0405. It was very likely that slight differences in TAP2A, TAP2B and TA2C in RA were secondary phenomenon reflecting an increase in HLA‐DRB1*0405. The prevalence of TAP2E allele was low (3.3%, P<0.01, Pc=not significant) and not correlated with HLA‐DRB1*0405.
Five HLA‐B22 group alleles in Japanese
Authors: Bannal M. , Tokunaga K. , Tanaka H. , Lin L. , Kashiwase K. , Tokunaga K. , Juji T.
Published: December 2008

HLA‐B22‐group alleles in Japanese were identified using PCR‐single‐strand conformation polymorphism (SSCP) and sequence analyses. We analyzed genomic DNAs obtained from Japanese individuals positive for HLA‐B22 group antigens (HLA‐B54, B55, B56) including two locally proposed splits (B55.2, and B22N). In the SSCP analysis of both exons 2 and 3, we discriminated five different B22‐group alleles. Each allelic pattern corresponded to each serological split antigen. Direct sequencing analysis of exon 2 and exon 3 showed that alleles encoding B54, B55.1 and B56 antigens in Japanese are encoded by B*5401, B*5502 and B*5601, respectively, and those encoding B55.2 and B22N antigens are previously unidentified alleles, B*5504 and B*5603, respectively. Full‐length cDNA sequencing showed that the B*5603 sequence is identical to those of B*5501, B*5502, B*5601, and B*5602 in exons 1 to 2 except for a synonymous substitution at nucleotide position 165 in exon 2. On the other hand, the sequence of exons 3 to 7 was identical to those of some B15 and B46 alleles, suggesting that B*5603 was generated by a recombination event between one of the B55 and B56 alleles and one of the B15 and B46 alleles in intron 2. As for B*5504, the entire exon 1 to 7 sequence is identical to that of B*5502 except that the 5‐half of the exon 3 sequence is identical to those of some B7, B27, B40 and B48 alleles, suggesting that an event such as gene conversion, segmental exchange, or double recombination occurred in this region.
HLA‐DR 52‐ and 51‐associated DRB1 alleles in Kenya, East Africa
Authors: Dunand V. A. , Ng C.‐M. , Wade J. A. , Bwayo J. , Plummer F. A. , MacDonald K. S.
Published: December 2008

The genes of the major histocompatibility complex (MHC) are amongst the most polymorphic loci known in the human population. The population genetics of the MHC encoded HLA loci of sub‐Saharan Africa are of major interest because of their particular genetic diversity. Here we report on the HLA‐DR 52‐ and 51‐associated determinants of the DRB1 loci observed in 165 East African individuals studied in Nairobi, Kenya. The HLA‐DR typing was done by serologic and by molecular DNA techniques (PCR‐SSOP). The most frequent allele identified was DRB1*1101, followed by DRB1*1503 and DRB1*1302. Some unexpected alleles were repeatedly identified: DRB1*1108, DRB1*1316 and DRB1*1421. Most oftheDR 52‐and 51‐associated DRB1 alleles were correctly identified by serology as part of the DR3, DR5, DR6 and DR2 groups respectively. The HLA‐DRB1 profile reported here corroborates previous genetic and linguistic data supporting the concept that the Eastern African Black population is genetically distinct from other African Black populations. This has important implications in public health issues related to the genetic profile of a population (transplantation, vaccine design for example).