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Biomedical subjects

A M Little

Publications and source records attributed to A M Little.

At least 37 records · Page 2Linked to original sources
PubMed1999

Aberrant splicing of intron 1 creates a novel null HLA-B*1501 allele.

A comparison of serological and DNA HLA class I typing data identified a serological "blank" HLA-B15 antigen in a volunteer donor on the bone marrow registry. Isoelectric focusing and Western blot analysis of a cell line established from this individual confirmed that the HLA-B15 antigen is not expressed at the cell surface. Nucleotide sequence analysis of the HLA-B*15 null allele revealed a 10-bp deletion near the 3' end of intron 1, when compared to the normal HLA-B*1501 sequence. All of the HLA-B*15 specific cDNA clones examined retained the intron 1 sequence. Reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis demonstrated that the HLA-B*15 mRNA molecule contained the intron 1 sequence, indicating an inability to efficiently splice out intron 1 from the mRNA transcript. The retention of the mutated intron 1 sequence in the mRNA causes a frameshift and premature termination of translation at the start of exon 2, explaining the HLA-B*1501 null phenotype. Our data predicts that the HLA-B*1501 null allele would express a small truncated protein containing the signal sequence fused to an ORF within intron 1 and terminating (out of frame) just within exon 2.

Alleles↗
PubMed1999

Sequence of HLA-A*6808.

A novel HLA-A*68 allele was identified by reference strand conformation analysis of a DNA sample studied for the International Cell Exchange program. Direct sequencing of HLA-A locus PCR products confirmed the presence of A*6602 and an A*68 allele which differed from A*68011 by two nucleotides at positions 538 and 539. The presence of this sequence motif in this allele which has been named A*6808 was verified by DNA sequencing of full-length A*6808 clones. No other coding differences between A*68011 and A*6808 were identified. The two nucleotide substitutions found in A*6808 effect an amino acid difference in the encoded protein at residue 156 within the peptide binding site which may evoke alloreactive immune response after HLA-A68 mismatched transplants.

Black People↗
PubMed1999

Characterization of the major susceptibility region for psoriasis at chromosome 6p21.3.

Psoriasis is a common inflammatory skin condition caused by genetic and environmental factors. Recent genome-wide linkage analyses have identified a locus encoding susceptibility to psoriasis and placed this gene in the 12 cM interval between markers D6S426 and D6S276 on chromosome 6p21.3. This is a broad region and encompasses the human major histocompatibility complex. We have sought to localize the susceptibility gene more precisely by exploiting the linkage, haplotype, and linkage disequilibrium information available through genotyping 118 affected sib pairs, their parents and other affected family members. A total of 14 highly polymorphic markers were genotyped, combining anonymous loci with the class I genes HLA-B and -C distributed across a genetic interval of approximately 14 cM including the entire major histocompatibility complex. Through the application of higher density mapping within the major histocompatibility complex, we identified those regions most commonly shared identical by descent in patients with psoriasis. Using the transmission-disequilibrium test, we found significant evidence of linkage and allelic association across an interval defined by the markers tn62 (p = 1.0 x 10(-7)), HLA-B (p = 4.0 x 10(-7)), and HLA-C (p = 2.7 x 10(-9)), a region encompassed within a 285 kb genomic DNA fragment. Hence these studies contribute to the refinement of the localization of a major psoriasis susceptibility gene and place the critical region near to HLA-C.

Adolescent↗
PubMed1999

Searching for an unrelated haemopoietic stem cell donor--a United Kingdom perspective.

The worldwide search for unrelated stem cell donors (now over 6 million) is one of the great success stories of international cooperation in the medical field. The initial search report from Bone Marrow Donors Worldwide estimates the chance of finding a suitably matched donor for a given patient. Registries whose donors are HLA-A, -B and -DR typed present the optimal prerequisite to identify a suitable donor for most patients. High-resolution matching HLA class I and class II alleles of the donor and recipient improves clinical outcome after unrelated donor transplantation. The clinical results of unrelated donor bone marrow transplantation are continually improving reflecting improvements in HLA matching, GvHD prophylaxis and transplantation in a favourable phase of disease. However, matching each HLA allele may or may not be critical for successful stem cell transplantation. Some degree of HLA mismatch ("permissible" mismatches) may be tolerated, especially in children. Cord blood banks provide a supplementary source of unrelated stem cell donors, in particular to patients from ethnic minority groups.

Family↗
PubMed1999

Polymorphism and evolution of HLA class I and II genes and molecules.

Genes in the HLA complex, the human major histocompatibility complex (MHC), encode polymorphic HLA class I and II molecules that help T lymphocytes recognise and respond to foreign antigens. Certain HLA class I allotypes also regulate the response of natural killer cells. HLA class I and II molecules with little or no polymorphism contribute a variety of functions to the immune response, as do class I molecules coded by genes outside of the HLA region. Knowledge of the organisation of HLA class I and II genes, of the nucleotide sequences of their alleles, and the three-dimensional structures of their protein products, has facilitated analysis of the evolution and polymorphism of HLA class I and II genes and molecules. In turn, these analyses have provided insight into the mechanisms and selective forces driving change in the HLA complex.

Genes, MHC Class I↗
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

Application of RSCA for the typing of HLA-DPB1.

We describe the application of RSCA, for the high resolution typing of alleles encoded at the HLA-DPB1 locus. RSCA differs from other sequence based typing methodologies in that the HLA type is assigned on the basis of differences in DNA conformation between different alleles. A total of 251 samples were typed in a blind study, of these 109 samples had been typed previously by conventional techniques. A comparison of the RSCA data with the historical typing results showed a concordance over 93%. Seven samples initially had discordant results, however, when these samples were typed by direct sequencing, the type assigned by RSCA was found to be correct in all but one case, indicating a concordance over 99%. RSCA has proved to be a simple reliable technique for the typing of the HLA-DPB1 locus, and is not limited by the ambiguous combinations of alleles determined in other conventional techniques.

Alleles↗
PubMed1998

Mutation detection and typing of polymorphic loci through double-strand conformation analysis.

Variations, such as nucleotide substitutions, deletions and insertions, within genes can affect the function of the gene product and in some cases be deleterious. Screening for known allelic variation is important for determining disease and gene associations. Techniques which target specific mutations such as restriction enzyme polymorphism and oligonucleotide probe or PCR primer reactivity are useful for the detection of specific mutations, but these techniques are not generally effective for the identification of new mutations. Approaches for measuring changes in DNA conformation have been developed, based on the principle that DNA fragments which differ in nucleotide composition exhibit different mobilities after separation by polyacrylamide gel electrophoresis (PAGE). Here we describe a conformation-based mutation detection system, double-strand conformation analysis (DSCA), which provides a simple means to detect genetic variants and to type complex polymorphic loci. We demonstrate the application of DSCA to detect genetic polymorphisms such as a single-nucleotide difference within DNA fragments of up to 979 base pairs in length. We present the application of DSCA in detecting four different mutations in the cystic fibrosis gene (CFTR) and 131 different alleles encoded by HLA class I genes.

DNA↗
PubMed1998

A high resolution HLA class I and class II matching method for bone marrow donor selection.

HLA matching in bone marrow transplantation has an important role in determining successful outcome. However HLA typing of both potential related and unrelated donors can be both time-consuming and laborious, and does not always resolve accurately the true level of histocompatibility. We have utilised a method, reference strand mediated conformation analysis (RSCA), which is technically simple and allows high resolution matching for all HLA loci, for the typing of 48 patients and their potential 120 donors. The results indicate that RSCA can detect many mismatches that are not routinely identified by conventional HLA typing methods. In addition, RSCA can be applied for the simultaneous analysis of multiple potential BM donor samples in order to quickly identify the best match for each patient.

Alleles↗
PubMed1998

Current methodologies of human leukocyte antigen typing utilized for bone marrow donor selection.

Matching for human leukocyte antigen (HLA) loci is the most crucial factor in the selection of bone marrow donors. The techniques utilized to assign HLA type have evolved significantly since the application of polymerase chain reaction-based methodologies. A significant challenge for DNA typing methodologies is to be able to maintain high resolution typing with the ever-increasing numbers of defined HLA alleles. To date, over 800 HLA class I and class II alleles have been identified by sequencing analysis, and this number shows no sign of reaching a plateau. It is only with high allelic level resolution typing analysis that an accurate definition of the role that individual HLA loci play in determining transplant outcome can be achieved, and this is a major goal for the future.

Bone Marrow Transplantation↗
PubMed1998

High resolution HLA class I typing by reference strand mediated conformation analysis (RSCA).

We describe the application of RSCA for the high resolution typing of alleles encoded within the highly polymorphic HLA class I system. RSCA differs from other sequence based typing methodologies in that the HLA type is assigned on the basis of differences in DNA conformation between different HLA alleles. HLA class I locus specific PCR was performed on the sample to be HLA typed and the PCR product mixed with a fluorescent labelled locus specific reference strand. DNA molecules were allowed to reanneal and the duplexes formed were separated by non-denaturing PAGE. Only duplexes which possess the fluorescent labelled reference strand were detected using an automated DNA sequencer. The mobilities of duplexes formed for 131 HLA class I alleles have been calculated and HLA type assigned. The use of a laser scanning system for accurate detection of migrating DNA, in combination with specific software for data analysis and correction of gel variablility, make RSCA sufficiently robust to detect alleles differing by as little as one nucleotide, resulting in a simple and automated technique for high resolution HLA typing.

Alleles↗
PubMed1997

Complementary strand analysis: a new approach for allelic separation in complex polyallelic genetic systems.

We describe a method, complementary strand analysis (CSA), for separating alleles potentially from any heterozygous genetic locus. Locus specific PCR is performed generating two allelic products. The antisense strands are isolated and hybridised with a sense reference strand to form a chimeric DNA duplex for each allele which is then separated by non-denaturing PAGE. We demonstrate the application of CSA for separation of highly polymorphic HLA-A, -B and -Cw alleles and characterisation of HLA identity in related bone marrow donors and patients. CSA is capable of resolving one nucleotide differences in a DNA fragment nearly as large as a kilobase in length.

Alleles↗
PubMed1997

Natural inactivation of a common HLA allele (A*2402) has occurred on at least three separate occasions.

HLA-A*2402 is common and widely distributed in human populations. Several individuals were identified who type genotypically for A*2402, but are serologically null for the HLA-A24 Ag. Sequencing and transfection of genomic DNA fragments containing null and wild-type A*2402 alleles, and the related A*2301 allele, revealed three different null alleles (A*2409N, A*2411N, and A*2402(low)), each of which differs from A*2402 by a single nucleotide change within the 6.7-kb sequence. The A*2301 and A*2402 sequences differ by no substitutions additional to those previously determined for the 1.1-kb cDNA. In exon 4, A*2409N has an in-frame stop codon, while A*2411N has a nucleotide insertion that alters the reading frame, causing premature termination. A*2402(low) has a nucleotide substitution near the splice acceptor site for intron 2 that impairs the production of correctly spliced mRNA. For A*2409N and A*2411N, mRNA is undetectable by Northern analysis, whereas A*2402(low) produces a low level of mRNA and a concomitant amount of normal A*2402 protein at the cell surface. The protein expressed from the A*2402(low) allele is sufficient to stimulate an alloreactive T cell response. On a background of unexpected sequence homogeneity, the single nucleotide changes in the A*2409N, A*2411, and A*2402(low) alleles have dramatic effects upon gene expression and are of likely importance for HLA matching in clinical transplantation. Segregation of at least three independently inactivated A*2402 alleles in human populations raises the possibility that loss of A*2402 may be the result of natural selection.

Alleles↗
PubMed1997

Episodic evolution and turnover of HLA-B in the indigenous human populations of the Americas.

Nucleotide sequences were determined for the HLA-A, B and C alleles of three populations of Amerindians: the Havasupai tribe from North America, and the Guarani and Kaingang tribes from South America. All 15 Havasupai alleles are found in Eastern Hemisphere populations, whereas the Guarani and Kaingang each have six alleles that appear to be present only in the Western Hemisphere. Nine of the "new" alleles come from HLA-B, one comes from HLA-A and one from HLA-C: ten appear to be the result of recombination and one the result of point substitution. Of the 14 Guarani alleles and 16 Kaingang alleles, only four are held in common. Despite their differences, the three tribes possess comparable numbers of HLA class I alleles, revealing a trend for "allele turnover", in which new alleles tends to supplant older alleles rather than supplement them. Although many new HLA-B alleles have been produced in Latin America, their net effect has been to differentiate populations, not to increase allele diversity within a population. From sequence comparisons, the Amerindian subset of HLA class I allotypes appears to cover the overall ranges of peptide binding specificity, natural killer-cell interactions, and CD8 interactions, that are found in all HLA class I. The recombinations that produced the new alleles of the Kaingang and Guarani class I are predicted to have modulated these functional properties rather than radically change them. Exchange of Bw4 and Bw6 motifs by recombination are noticeably absent in the events forming new alleles in America, whereas they have been the most common of recombinations elsewhere.

Alleles↗
PubMed1997

Factors influencing the outcome of bone marrow transplants using unrelated donors.

The use of unrelated donors for bone marrow transplantation (BMT) is associated with an increased morbidity and mortality when compared with HLA-identical donors, primarily due to an increased rate of graft-versus-host disease, but also to increased susceptibility to infections and graft failure. HLA matching for donors and recipients is the single most important factor influencing the outcome of BMT. However, unrelated donor selection generally relies on matching only for HLA-A, -B and -DR antigens without considering potential incompatibility for other HLA loci, such as HLA-C, -DQ and -DP. In addition, other factors that affect the outcome of BMT need to be taken into consideration in selecting the best unrelated donor. In this review, we will focus on the effects of HLA-associated factors in determining the result of a transplant procedure. We will also mention other relevant factors, drawing on our experience of laboratory studies performed at The Anthony Nolan Research Institute and clinical studies at the Hammersmith Hospital in London.

Bone Marrow Transplantation↗
PubMed1996

The presentation of a hepatitis C viral peptide by distinct major histocompatibility complex class I allotypes from two chimpanzee species.

A cytotoxic T lymphocyte (CTL) line, derived from the liver of a common chimpanzee (Pan troglodytes) with hepatitis C, specifically recognized a hepatitis C viral 9-mer peptide (KHP-DATYSR in single-letter amino acid code) bound by the major histocompatibility complex (MHC) class I molecule, Patr-A04. This same CTL line also recognized the identical peptide bound by a structurally different class I molecule, Papa-A06, derived from the separate chimpanzee species, Pan paniscus or pygmy chimpanzee. These class I allotypes differ by six amino acids but, in spite of the structural differences, share the same antigen-presenting function. This is the first observation of antigen presentation to a given T cell receptor by different MHC class I allotypes from separate species.

Animals↗