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M Bunce

Publications and source records attributed to M Bunce.

85 records · Page 5Linked to original sources
PubMed1995

HLA typing for DR3 and DR4 using artificial restriction fragment length polymorphism PCR from archival DNA.

AIM: To develop polymerase chain reaction based artificial restriction fragment length polymorphism (artificial RFLP PCR) assays for DR3 and DR4 alleles of the multiallelic DRB1 locus and to apply them to paraffin wax embedded archival material. METHODS: Sixty five samples from DRB1 typed cell lines were analysed using the artificial RFLP PCR method to determine the specificity and sensitivity of the system. RESULTS: The artificial RFLP PCR method for typing the DRB1 locus showed 100% accuracy in the 65 samples previously typed using allele specific PCR and serology. The samples included 18 combinations of alleles that included DR3, 18 that included DR4, four that were DR3/DR4 heterozygotes, and 10 samples that were neither DR3 nor DR4. Typing of 10 paraffin wax embedded samples using artificial RFLP PCR was in complete agreement with previous typing at the DRB1 locus. CONCLUSION: The application of artificial RFLP PCR for the analysis of multiallelic loci, such as those of the HLA system, in archival DNA samples has been achieved. Artificial RFLP PCR is a robust, easily implemented, non-isotopic system and may be useful for large retrospective studies.

Alleles↗
PubMed1994

Molecular characterization of a novel, serologically detectable, HLA-C allele: Cw*1602.

Cw*1602, a novel HLA-C allele belonging to the newly assigned Cw*16 group, has been cloned and sequenced from a Spanish Caucasoid cell expressing a "Cw6.2" phenotype. Some of the polymorphic substitutions of the new allele, and linkage disequilibrium to B51, had been predicted on the basis of previously published studies. The primary structure of Cw*1602 is in agreement with its serologic reactivity and, in comparison with that of Cw*1601, underlines the dimorphism of HLA-C molecules at residues 77 and 80 of the alpha 1-domain alpha helix.

Alleles↗
PubMed1994

Rapid DNA typing for HLA-C using sequence-specific primers (PCR-SSP): identification of serological and non-serologically defined HLA-C alleles including several new alleles.

Detection of HLA-C antigens by complement mediated cytotoxicity using human alloantisera is often difficult. Between 20 to 40% of individuals in every race have undetectable HLA-C locus antigens and 9 out of the 29 sequenced HLA-C alleles so far published encode serologically undetected antigens. In addition, HLA-C molecules are expressed at the cell surface at about 10% of the levels of HLA-A and HLA-B. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable and rapid method for typing HLA-DR, HLA-DQA and HLA-DQB genes. PCR-SSP takes two hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers which will positively identify the HLA-C alleles corresponding to the serologically defined series HLA-Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7 and Cw8. The serologically undetectable alleles have also been detected in groups according to sequence homology. In addition, three new unsequenced variants have been identified. DNA samples from 56 International Histocompatibility Workshop reference cell lines and 103 control individuals have been typed by the HLA-C PCR-SSP technique. 4/56 cell line types and 11/103 normal control individuals types were discrepant with the reported serological types. All combinations of serologically detectable and most of the serologically blank HLA-C antigens can be readily identified. DNA typing for HLA-Cw by PCR-SSP can take as little as 130 minutes from start to finish, including DNA preparation.

Alleles↗
PubMed1994

HLA-B15: a widespread and diverse family of HLA-B alleles.

HLA-B15 embraces a multiplicity of antigenic specificities which vary in their distribution amongst human populations. To correlate B15 molecular structure with the serological picture we have sequenced alleles encoding the various subspecificities of the B15 antigen: B62, B63, B75, B76 and B77, and a number of "variants" of these antigens including the 8w66 split of B63. HLA-B63 (B*1517) and 8w66 (B*1516) heavy chains have sequence identity to B17 in the alpha 1 helix correlating with the antigenic crossreactivity of these molecules. HLA-B77(B*1513) and B75 (B*1502) heavy chains differ solely in segments determining the Bw4 and Bw6 public epitopes, consistent with the serological description of the B77 and B75 antigens. One allele encoding the B76 antigen (B*1512) appears to be the product of gene conversion between the HLA-A and -B loci and differs from B*1501 in codons 166 and 167. In contrast, a second allele encoding the B76 antigen (B*1514) differs from B*1501 by an unrelated substitution in codon 167 which confers similarily with B45, an antigen crossreactive with B76. A third allele encoding B76, B*1519, differs from B*1512 by a unique point substitution in exon 4. Three alleles encoding variant B15 and B62 antigens (B*1508, B*1511 and B*1515) differ from B*1501 by localized clusters of substitutions that probably result from interallelic conversion. The B15 sequences described in this paper, in combination with those previously determined, define a family of 22 alleles, including those encoding the B46 and B70 antigens. Within this family the patterns of allelic substitution are analogous to those of other HLA-A and -B families, in that pairwise differences almost always involve functional positions of the antigen recognition site and recombination is the major agent of diversification.

Alleles↗
PubMed1993

Rapid HLA-DQB typing by eight polymerase chain reaction amplifications with sequence-specific primers (PCR-SSP).

Molecular genotyping of HLA class II genes using group-specific DNA amplification by the PCR followed by probing with (PCR-SSO) probes is too time consuming for the typing of cadaveric organ donors. Recently, amplification of DNA using PCR-SSP has proved a reliable and rapid method for typing HLA-DRB1 genes. PCR-SSP takes 2 hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers that in eight PCR reactions will positively identify the HLA-DQB1 alleles corresponding to the serologically defined series HLA-DQ2, DQ4, DQ5, DQ6, DQ7, DQ8, and DQ9. Presently, 30 homozygous cell lines and 138 individuals have been typed by the DQB1 PCR-SSP technique and compared with a combination of serology and RFLP with 100% concordance. No false-negative or false-positive amplifications were recorded. All combinations of DQB1 can be readily identified. DQB1 PCR-SSP can take as little as 130 minutes from start to finish, including DNA preparation.

Alleles↗
PubMed1993

Clinical and socioeconomic benefits of serological HLA-DR matching for renal transplantation over three eras of immunosuppression regimens at a single unit.

The efficacy of HLA-DR matching in cadaveric renal transplantation is controversial in the cyclosporine (CsA) era. Reports have questioned both the reliability of serological HLA-DR typing as well as the benefit of matching in terms of improved graft survival. Analysis of 1,000 consecutive cadaver donor transplants performed at Oxford between 1975 and 1992 has shown that with improved immunosuppressive regimens and increased transplant success there has been a steadily diminishing influence of HLA-DR matching measured in terms of first graft outcome. For patients treated with azathioprine and prednisolone (n = 278) overall one-year first graft survival was 65%, but there was a 20% improvement associated with HLA-DR matching which has been maintained for up to 15 years. With the introduction of CsA, used either alone or in conjunction with low dose steroids (n = 96), one-year first graft survival was 69% and the difference between HLA-DR-matched and -mismatched transplants was 14%. Our current maintenance immunosuppressive protocol is triple therapy (N = 425) with an 81% one-year first graft survival for both matched and mismatched transplants. However, we do continue to find a marked correlation between HLA-DR matching and clinical course. HLA-DR-mismatched patients suffer more rejection episodes, spend a longer time in the hospital, and have higher creatinine levels at 3 months. This costs, on average, an extra 1,500 pounds for each mismatched transplant. The effect is most apparent in unsensitized males. For cadaveric regrafts, one-year graft survival for patients on triple therapy is 80% (n = 116) which does not differ from first graft survival rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Graft Rejection↗
PubMed1992

Interethnic genetic differentiation in Africa: HLA class I antigens in The Gambia.

A total of 752 individuals from The Gambia, west Africa who are representative of the major ethnic groups in the capital, Banjul, were serologically typed for HLA-A, -B, and -C antigens. Although all were typically "African" in their antigenic profiles, some marked frequency differences were found between the ethnic groups. Genetic distance comparisons with several other African populations showed that, although these west African populations clustered closely together, the positions of the various ethnic groups in The Gambia were consistent with historical and linguistic evidence of their affinities with one another and with other African populations. Despite the potential confounding effects both of selection by infectious diseases and of genetic drift caused by local differences in population structure, HLA frequencies appear to be of value in measuring inter- and intraregional population affinities in sub-Saharan Africa.

Africa↗
PubMed1991

Sequence analysis of HLA-Bw53, a common West African allele, suggests an origin by gene conversion of HLA-B35.

In the West African population of the Gambia the class I antigen HLA-Bw53 is found at high frequency. We used the polymerase chain reaction to amplify cDNA from an individual homozygous for this allele and determined the nucleotide sequence of the polymorphic alpha 1 and alpha 2 domains. The HLA-Bw53 sequence is identical to HLA-B35 except for a short sequence at the 3' end of exon 2 (encoding the alpha 1 domain) which specifies a Bw4 rather than a Bw6 motif. This suggests an origin for HLA-Bw53 involving a gene conversion of HLA-B35 by an allele containing this Bw4 sequence. The alpha 2 domain shared by HLA-Bw53, -B35, and -Bw58 is particularly common in sub-Saharan Africans.

Adult↗
PubMed1990

The production of a human monoclonal antibody defining a split of HLA-DRw13 (DRw13b).

By use of the heterohybridoma technique we have produced a human monoclonal antibody (NDS40) which detects a split of HLA-DRw13 (DRw13b) which is in linkage with HLA-DQw1. In addition, the antibody reacts with cells positive for HLA-DRw8 and DRw11, but does not react with the commonly found split of DRw13 (DRw13a) which is associated with DQw1. NDS40 is cytotoxic and is of the lambda IgM class.

Antibodies, Monoclonal↗
PubMed1985

Ulcerative colitis and HLA phenotype.

The distribution of HLA A, B, C, DR antigens was investigated in a British population with ulcerative colitis. Fifty six patients were typed for HLA, A, B, C and 46 additionally for DR. No association was found between the HLA phenotype and the presence or absence of ulcerative colitis. Serum from 52 patients was tested for the presence of the anticolon antibody. There was no relation between the presence of the antibody and the HLA phenotype. Finally, no correlation was found between the HLA phenotypes, the age of onset of the disease, the extent and the clinical course.

Adolescent↗