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Biomedical subjects

M Bunce

Publications and source records attributed to M Bunce.

At least 55 records · Page 3Linked to original sources
PubMed1999

Molecular typing for HLA class I using ARMS-PCR: further developments following the 12th International Histocompatibility Workshop.

Molecular typing for HLA class I was introduced in the 12th International Histocompatibility Workshop. Following a pilot study using three methods, sequence specific oligotyping (SSO), reverse dot blot and amplification refractory mutation system (ARMS)-PCR, the ARMS-PCR method was selected for use. A great advantage of an ARMS-PCR method is that, unlike the other two methods, it can determine whether sequence motifs are in cis or in trans, as ARMS-PCR detects two cis located motifs per reaction using forward and reverse sequence specific primers. Resolution was designed to be low to medium level for HLA-A, -B and -C alleles. Two hundred and fifty class I kits and 83 HLA-A2 subtyping kits were distributed. The A2 subtyping kit used a two round nested PCR system to identify all of the A2 alleles known at the time. Typing results on control DNA samples distributed with both the kits showed a very satisfactory performance. Since the 12th Workshop, the kits have been developed with the addition of new primers and primer mixes to increase the resolution of the test.

DNA↗
PubMed1999

A DNA-based detection and screening system for identifying HLA class I expression variants by sequence-specific primers.

Molecular methods are now commonplace for HLA typing and they have replaced traditional serological methods in many histocompatibility laboratories. A consequence of reliance on molecular methods using primers or probes based on existing sequence information is that unsequenced or partially-sequenced null, or low expressed variants are not discriminated from expressed alleles. Failure to identify null alleles might have deleterious implications for allogeneic transplants. Expression variants may be classified into two categories: unique mutations and repeat mutations. For example, the alleles A*0303N, A*2409N, and B*1526N have apparently unique mutations. In contrast, repeat mutations may occur frequently at points where unusual nucleotide sequences make accurate DNA replication by DNA polymerases difficult. One example is between nucleotide positions 621-627, where HLA class I alleles may exhibit between three and seven consecutive cytosine residues. Incorrect insertion of an extra cytosine in this region is the cause of expression failure in A*2411N and A*0104N alleles. We hypothesise that insertion of an extra cytosine into the cytosine island between nucleotide positions 621-627 is likely to recur not only in other HLA-A alleles but also in HLA-B and even HLA-C alleles. We describe here a polymerase chain reaction using sequence-specific primers (PCR-SSP) system that can not only detect all previously-sequenced HLA class I expression variants but can also screen for mutations between positions 621-627 in HLA-A, B or C alleles which may give rise to potentially null alleles. Overall, in this study HLA class I expression variants were identified in 5 of 931 tested samples (0.53%).

Alleles↗
PubMed1999

Characterization of a novel HLA-A*24 allele containing an HLA-A*03 sequence motif.

HLA-A*2418 is described for the first time. It segregates in a Mendelian fashion. Serological analyses indicate that the new allele encodes epitopes from both HLA-A9 and -A3 specificities. Results from nucleotide sequencing analyses of polymerase chain reaction (PCR) amplification products derived from genomic and cDNA are compatible with those findings.

Alleles↗
PubMed1999

Variation in immunoregulatory genes determines the clinical phenotype of common variable immunodeficiency.

Variation in clinical phenotype is a hallmark of many complex diseases. The cause of this clinical heterogeneity is unknown, but it may be determined by genetic factors distinct from those conferring disease susceptibility. Common variable immunodeficiency (CVID) is a complex disease of unknown aetiology and diverse clinical manifestations. We have developed a unified polymerase chain reaction and sequence-specific primer (PCR-SSP) method to simultaneously genotype multiple polymorphisms under identical conditions, and have used this method to test the hypothesis that the clinical phenotype of CVID is determined by immunoregulatory gene polymorphism. Twenty-three polymorphisms in 13 genes were studied in 163 CVID patients. Vitamin D receptor and IL-6 alleles were associated with immunophenotypic abnormalities characteristic of more severe disease; and tumour necrosis factor and IL-10 alleles conferred susceptibility to the granulomatous form of CVID in an interacting fashion. These findings demonstrate that different clinical features of a disease may have unique pathogenetic abnormalities, determined by multiple interacting genetic factors. The ease of application of this efficient, robust genotyping technique to polymorphisms throughout the genome will make it a powerful tool in the investigation of the genetic basis of phenotypic variability in a wide variety of diseases.

Abatacept↗
PubMed1999

HLA-A, -B, -C, -DRB1, DRB3, DRB4, DRB5 and DQB1 polymorphism detected by PCR-SSP in a semi-urban HIV-positive Ugandan population.

PCR-SSP was used to HLA-type a cohort of Ugandan HIV-positive individuals. The results represent a more comprehensive description of HLA in an African population than previously described and are in concordance with data from a general Black population. Substantial differences exist between this population and Caucasoid populations in which immunological responses to HIV have been investigated; this emphasises that the main HLA-restrictive elements for HIV-specific cytotoxic T lymphocytes will most likely be different for each population.

Base Sequence↗
PubMed1999

Molecular typing for the MHC with PCR-SSP.

Sequence-specific amplification (SSP) is simply a form of polymerase chain reaction (PCR) which involves designing one or both primers so that they will or will not allow amplification (the 3'-mismatch principle). Its origins are probably legion, i.e. many people probably thought of it at the same time. For example, in 1988 a group from Guy's Hospital, London, described a form of SSP for HLA-DR4 detection and in the same year a group from Upjohn described its use at the American Society of Histocompatibility and Immunogenetics (ASHI). Both are published in abstract form (British Society of Rheumatology and ASHI). The 3'-mismatch principle can be used to identify virtually any single nucleotide point mutation (SNP) within one or two PCR-SSP reactions and the first peer-reviewed statements of this came in 1989 (1, 2). Thus, although the use of SSP probably began around 1990, it was 5 years before its popularity erupted, mainly due to the work of Olerup & Zetterquist (3, 4), who defined its potential for solid organ transplantation. It is now the method of choice for high resolution HLA typing in many laboratories. In addition, over a thousand applications for genes outside the MHC are in the literature.

Histocompatibility Testing↗
PubMed1998

Molecular typing shows a high level of HLA class I incompatibility in serologically well matched donor/patient pairs: implications for unrelated bone marrow donor selection.

In comparison with HLA-matched sibling bone marrow transplants, unrelated donor transplants are associated with increased graft-versus-host disease and graft failure. This is likely in part due to HLA incompatibilities not identified by current matching strategies. High resolution DNA-based typing methods for HLA class II loci have improved donor selection and treatment outcome in unrelated donor bone marrow transplantation. By using DNA-based typing methods for HLA-A and -B on a cohort of 100 potential bone marrow donor/patient pairs, we find that serological typing for HLA class I is limited in its ability to identify incompatibilities in unrelated pairs. Furthermore, the incompatibilities identified are associated with the presence at high frequency of alloreactive cytotoxic T-lymphocyte precursors. DNA typing also indicates that HLA-C mismatches are common in HLA-A and -B serologically matched pairs. Such mismatches appear to be significantly less immunogenic with respect to cytotoxic T-lymphocyte recognition, but are expected to influence natural killer cell activity. Thus, improved resolution of HLA class I shows many previously undisclosed mismatches that appear to be immunologically functional. Use of high resolution typing methods in routine matching is expected to improve unrelated donor selection and transplant outcome.

Base Pair Mismatch↗
PubMed1998

Detection of microchimerism after allogeneic blood transfusion using nested polymerase chain reaction amplification with sequence-specific primers (PCR-SSP): a cautionary tale.

In bone marrow transplantation, the detection of chimerism is an important adjunct to the repertoire of tests available for determining acceptance of the graft. In solid organ transplantation, there is currently intense interest in the role that chimerism plays in both short- and long-term host reactivity to the graft. Allogeneic blood transfusion has been associated with a subtle immunosuppressive effect in renal transplantation and chimerism is implicated as a possible mechanism for this effect. To assess the survival of allogeneic cells after blood transfusion or transplantation, we have developed a technique based on molecular typing for HLA class II alleles, which enables the detection of donor-derived cells in patients receiving blood transfusions. While developing this technology, we investigated why we and others observe false amplification. Sequencing of false products has shown that they arise from amplification of both pseudogenes and non-pseudogenes present in the DNA under test. Elucidation of this phenomenon allows the amplification of these false products to be predicted in any given combination and hence avoided by the judicious selection of primers. Validation has been achieved by following donor alleles after transfusion of blood containing defined numbers of leukocytes expressing selected mismatched antigens.

Blood Donors↗
PubMed1998

HLA-Cw*0602 and HIV-associated psoriasis.

The aetiopathogenesis of psoriasis is unknown, but genetic and environmental factors may be involved. Psoriasis may not be one disease but a cutaneous inflammatory reaction pattern consequent upon several different independent or related stimuli in susceptible individuals. There are controversial issues regarding the immunological basis of psoriasis and the role of CD4 vs. CD8 T lymphocytes. Psoriasis has been associated with HLA-Cw6 and Cw7 by serology and specifically with HLA-Cw*0602 by polymerase chain reaction (PCR) typing. Psoriasis is probably no more common in HIV infection than in the general population; however, it may appear for the first time or pre-existing psoriasis may worsen and be difficult to treat in HIV disease. We have investigated the prevalence of HLA-C alleles, in the specific clinical context of HIV infection complicated by type 1 psoriasis, in a case control study of 14 men with HIV disease and type 1 psoriasis and 147 HIV-infected patients without psoriasis. Typing was performed using PCR with sequence-specific amplification primers. Eleven of 14 patients (79%) with psoriasis carried the HLA-Cw*0602 allele compared with 24.5% of those without psoriasis (odds ratio = 11.31; 95% confidence limits 2. 73 to 65.36; P = 0.0001). Two patients without the HLA-Cw*0602 allele carried instead the closely related Cw*0401/3 allele. The results confirm the previously reported association between the HLA-Cw*0602 allele and type 1 psoriasis, and suggest that the association with HLA-Cw*0602 is stronger in HIV-associated psoriasis although this trend needs to be supported by a larger sample. The immunodysregulation resulting from HIV infection may trigger psoriasis in those genetically predisposed by the Cw*0602 allele. As CD8 T cells recognize antigens in the context of class I major histocompatibility complex, the identification of an HLA class I association in HIV-associated psoriasis strengthens the argument for an important role for CD8 + T lymphocytes in the immunopathogenesis of psoriasis. Investigations of the pathogenesis of psoriasis should take account of clinical and other subtypes already identified.

Adult↗
PubMed1998

The PCR-SSP Manager computer program: a tool for maintaining sequence alignments and automatically updating the specificities of PCR-SSP primers and primer mixes.

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

Antisense Elements (Genetics)↗
PubMed1998

Comprehensive HLA-DP typing using polymerase chain reaction with sequence-specific primers and 95 sequence-specific primer mixes.

HLA-DP is the third of the class II molecules. Its role is antigen presentation, and it has been suggested to play a part in the susceptibility to certain diseases such as berylliosis, sarcoidosis and juvenile chronic arthritis. The standard typing method is SSO typing, although other methods have been used. Probably the best is sequence-based typing, but this is time-consuming and requires expensive equipment. We describe a method for comprehensive HLA-DPB1 and HLA-DPA1 typing using sequence-specific primers. This method has the advantages that it is rapid - typing a single DNA sample takes under 3 hours - and does not require any special equipment or reagents. The method has been shown to be highly accurate by typing 60 cell line DNA samples in which there was 100% agreement between the types obtained and the published information. Similarly typing of 20 DNA samples previously typed by sequence-based typing gave 100% concordance. We used the method to type DNA samples from 102 UK Caucasoid kidney donors. The allele frequencies agree with previously published data. Linkage disequilibria between HLA-DPB1, HLA-DPA1 and the other class II antigens have been investigated. Strong linkage disequilibria exist between certain HLA-DPB1 and HLA-DPA1 alleles. This is unsurprising in view of their proximity on the chromosome. More unexpectedly, the data also suggest that genes further away along the chromosome are in linkage disequilibrium with HLA-DP, forming extended haplotypes.

DNA Primers↗
PubMed1998

A new pair of HLA-C alleles, Cw*12042 and Cw*1203, differing at the KIR-related dimorphism of codons 77-80.

A previously unknown HLA-C variant of the Cw*12 group was identified by PCR-SSP from genomic DNA of cell NDS-JD. Molecular cloning and nucleotide sequence analysis permitted the characterization of the complete coding region of this new allele, Cw*12042. The new variant differs from the recently reported Cw*12041 by two silent changes at exons 2 and 3, and from Cw*1203 by coding changes at codons 77 and 80. Cw*1203 (Ser-Asn) and Cw*12042 (Asn-Lys) constitute the second known example of HLA-C alleles only differing at the KIR-related dimorphism of residues 77-80. The new allele is associated in cell NDS-JD with the haplotype HLA-A*2403, Cw*12042, B*51, DRB1*1502, DRB5*0102, DQB1*0601, possibly related from the evolutionary aspect to the ancestral haplotype A*2402, Cw*1202, B*5201, DRB1*1502, DRB5*0102, DQB1*0601.

Alleles↗
PubMed1998

On the nature of "HLA-B42" alloantibodies. Specific reagents for HLA-Cw*17?

An almost complete and bidirectional association exists between HLA-Cw*17 and the HLA-B antigens B41 and B42. Serological and molecular analysis of an individual in which HLA-B*4101 was identified in the absence of Cw*17 provides experimental evidence to prove a previously proposed hypothesis predicting that alloantisera classified as "B41+B42" are instead specific reagents for HLA-Cw*17.

Amino Acid Sequence↗
PubMed1998

Allele-specific HLA-B*15 typing by PCR-SSP and its application to four distinct ethnic populations.

We present a set of primer mixes for the allele-specific typing of the HLA-B*15 group by PCR-SSP. The set comprises 46 primer mixes which are designed to unequivocally resolve all but two of the 666 possible combinations of the B*15 alleles, B*1501-37 (B*1536 sequence unavailable). A core subset of 34 of the 46 mixes can be used alone to give a high resolution B*15 typing set. This allows for the identification of each B*15 allele when present as the only B*15 allele and the majority of the possible B*15 homozygotic combinations. The method was validated using reference DNA samples and the B*15 allele frequency in 4 distinct ethnic populations was investigated. The results show that these populations contain predominantly mutually exclusive sets of B*15 alleles.

Alleles↗
PubMed1998

A rapid method of haplotyping HFE mutations and linkage disequilibrium in a Caucasoid population.

BACKGROUND: HFE mutations are associated with hereditary haemochromatosis. However, a simple method capable of demonstrating the cis/trans arrangement of alleles is lacking, and linkage disequilibrium between HFE alleles and classic HLA loci is unknown. These are important issues as the pathogenic role of the mutations is not known. AIMS: To develop a simple method of genotyping HFE mutations suitable for clinical use in addition to large disease studies. PATIENTS: A total of 330 Caucasoid cadaveric organ donor controls were examined. Ten individuals previously HLA-H genotyped by polymerase chain reaction using restriction fragment length polymorphism (PCR-RFLP) were also examined to validate the method. METHODS: A simple polymerase chain reaction using sequence specific primers (PCR-SSP) capable of haplotyping the mutations was developed. HFE allele and haplotype frequencies and linkage disequilibrium with eight HLA class I and II loci were examined in the control population. RESULTS: 27% and 19.7% of patients were positive for the 63D and 282Y alleles, respectively. No chromosome carried both 63D and 282Y. Linkage disequilibrium between 282Y and HLA-A*03 was confirmed, but was not straightforward: some A*03-associated alleles (DRB1*15, DQB1*06), but not all (B*07, Cw*0702), were associated with 282Y. CONCLUSIONS: Linkage disequilibrium data suggest that an HLA-B*07 containing haplotype contains an element affording protection from haemochromatosis and may suggest the timing of the founder 282Y mutation.

Alleles↗
PubMed1997

HLA-C genes and susceptibility to type 1 autoimmune hepatitis.

Susceptibility to autoimmune hepatitis (AIH) is associated with the HLA A1-B8-DR3 haplotype, DR4 antigen, and, more specifically, the HLA DRB3*0101, DRB1*0301, and DRB1*0401 alleles. Few investigators, however, have examined the HLA C locus in AIH, which warrants detailed study in view of its recently described roles in immunoregulation. Eighty-seven adult, white patients with well-characterized type 1 AIH and 100 controls were studied. HLA C and HLA DRB1 alleles were assigned by polymerase chain reaction (PCR)-based genotyping. HLA A and B antigens were determined by standard microlymphocytotoxicity assay. Extended haplotypes were constructed according to known patterns of linkage disequilibrium. Only one HLA C locus allele, Cw*0701, which was present in 54% of patients versus 34% of controls (P = .006; relative risk [RR] = 1.54) was associated with AIH. The overall increase in the frequency of the Cw*07 gene (70.1% of patients vs. 54% of controls; P = .024; RR = 1.3) was due entirely to inheritance of the Cw*0701 allele rather than the other Cw*07 alleles, Cw*0702, *0703, and *0704. The RR for Cw*0701 (RR = 1.54) is greater than that for HLA A1 (RR = 1.33) and DRB1*0301 (RR = 1.49), but less than that for HLA-B8 (RR = 1.75). The present findings suggest that the gene or genes conferring susceptibility to AIH lie in the region centromeric to the HLA A locus between HLA C and DRB1. Although linkage disequilibrium with both B8 and DRB1*0301 may account for our finding of an increased frequency of Cw*0701, it is also possible that this allele contributes to disease susceptibility, perhaps by interaction with natural killer cells or cytotoxic T lymphocytes.

Adult↗
PubMed1997

Cw*1701 defines a divergent african HLA-C allelic lineage.

The complete sequence of a new HLA-C allele, Cw*1701, was determined from a South African Zulu individual. Unique features that distinguish Cw*1701 from other HLA-C alleles include multiple point substitutions and an 18 nucleotide insertion in exon 5, which encodes the transmembrane domain. In a phylogenetic analysis of HLA-C sequences, Cw*1701 forms a third, distinct allelic lineage. A comparison of the transmembrane domain of Cw*1701 with other HLA-B and -C alleles reveals that duplications and deletions have been common in the evolution of these loci. A polymerase chain reaction based typing method was used to determine the distribution of this unusual allele in human populations. In contrast to the other two lineages of HLA-C alleles, the Cw*17 lineage is found at high frequencies only in populations of African descent. In addition, the HLA-B/Cw*17 haplotype diversity is higher in Africa.

Africa↗