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D Middleton

Publications and source records attributed to D Middleton.

At least 55 records · Page 3Linked to original sources
PubMed2000

Development of a PCR-SSOP approach capable of defining the natural killer cell inhibitory receptor (KIR) gene sequence repertoires.

A molecular typing method based on polymerase chain reaction (PCR) amplification of three different target domains (immunoglobulin domains 1 and 3, and the transmembrane-cytoplasmic domain), followed by hybridisation with 26 digoxigenin-labelled sequence-specific oligonucleotide probes (SSOP) has been established for the polymorphic killer inhibitory receptor (KIR) genes. In addition to identifying the 12 KIR subfamilies, our PCR-SSOP typing approach could also distinguish the putative alleles, NKB1 and NKAT3, that comprise the KIR3DL1 subfamily. Ninety unrelated blood donors and 13 families (52 individuals), including both parents, were subjected to our KIR PCR-SSOP typing approach. All 12 KIR subfamilies, including a 2DS5 variant sequence, were present in the 90 individuals and displayed varied phenotype frequencies: 2DL1 (0.96), 2DL2 (0.31), 2DL3 (0.95), 2DS1 (0.56) 2DS2 (0.51), 2DS3 (0.27), 2DS4 (0.96), 2DS5v (0.35), 3DS1 (0.47), 3DL1 (0.96), 3DL2 (1.0) and 2DL4 (1.0). A total of 23 different KIR phenotypes were defined in this study, and 10 of these were only found on one occasion in one individual, indicating considerable diversity in the KIR phenotype profiles within the Irish population. Most individuals (93%) possessed the complement of inhibitory KIR specificities for the three well-defined HLA-B and -C ligands. An unusual probe pattern for 3DS1 was observed in 3 individuals indicating a variant 3DS1 gene sequence with changes at nucleotide positions 1185-1186, within the cytoplasmic domain. Sequencing analysis revealed a new single nucleotide polymorphism in exon 3 of 3DL1 NKB1(195, G-A) and a 22-bp deletion polymorphism in exon 5 of 2DS4 (nucleotides 777-798 deleted). A number of strong KIR associations were observed, namely 2DL1 with 2DL3, 2DS4 with 3DL1, 2DL2 with 2DS1/2DS2/2DS3, 2DS1 with 2DS3/2DS5v/3DS1, 2DS2 with 2DS3 and 2DS5v with 3DS1. Analysis of the KIR segregation observed in the 13 families confirmed these strong associations and permitted the definition of a number of partial KIR haplotypes, e.g. 2DL2-2DS1-2DS2-2DS3-3DL1. The segregation analysis concluded that at least 3 distinct gene loci encode 2DL1-4 and at least 4 gene loci encode the non-inhibitory KIR2DS1-2DS5. In the case of 3DL1-2 and 3DS1, our data suggests 3 gene loci, one for each subfamily.

Humans↗
PubMed2000

HLA class II typing of whole genome amplified mouth swab DNA.

Postal collection of mouth swabs provides a cheap and convenient means of DNA sampling but hitherto has not provided sufficient genetic material for HLA typing by polymerase chain reaction using sequence-specific primers (PCR-SSP). This study examined the feasibility of collecting mouth swabs from a test population by post, amplifying the DNA by whole genome amplification and genotyping for selected HLA class II alleles. We optimised a strategy for whole genome amplification or primer extension preamplification using a random 15 base pair primer which resulted in a 1,000-fold increase in DNA template. The amplified DNA was of sufficient quality for analysis of selected HLA Class II alleles by PCR-SSP and PCR using sequence-specific oligonucleotide probes. To test the reliability of our data, blood DNA from 30 individuals in 10 families, previously tested for all DRB1 alleles in a routine diagnostic laboratory, was then tested in our laboratory for DRB1 *03 and *04 following whole genome amplification. Further whole genome amplified product from another 10 families was tested for DRB1 *03, *04 in our laboratory and then tested for all DRB1 alleles in a routine diagnostic laboratory. One repeat typing was required to achieve 100% concordance between laboratories. Amplification of whole genome amplified DNA by PCR-SSP was then extended successfully to low-resolution HLA DRB1, DQA1, DQB1 and DPB1 typing. Mouth swab collection by post, followed by whole genome amplification of DNA provides an effective strategy for genetic analysis of large cohorts. We have optimised conditions for HLA class II typing on whole genome amplified DNA collected by mouth swab, but this method could potentially be applied to low concentrations of DNA from other sources.

Adult↗
PubMed2000

Genetic polymorphism of IL-12 p40 gene in immune-mediated disease.

Understanding of the genetic basis of autoimmune diseases is currently incomplete. Cytokine gene polymorphisms warrant consideration as factors explaining variation in the human immune and inflammatory responses and as candidate susceptibility genes for related pathological states. Interleukin 12 (IL-12) is a key regulator of the polarisation of immune responses to T helper 1 or 2 categories and plays a role in autoimmune and infectious diseases. Using a bioinformatic strategy, we aligned cDNA and expressed sequence tag sequences to identify putative polymorphic regions of the IL-12 p40 gene. Position 1188 in the 3' untranslated region (UTR) was polymorphic with the frequency of the common allele around 80% in healthy UK Caucasoids. PCR genotyping of multiple Caucasoid groups and an African group showed significant population variation. In a case-control design, the polymorphism was not associated with rheumatoid arthritis, Felty's syndrome or large granular lymphocyte syndrome with arthritis or multiple sclerosis. A nonsignificant increase in the B allele frequency was observed in the rare large granular lymphocyte syndrome without arthritis (odds ratio 2.02 95% CI 0.95-4.3). This new genetic marker could be useful in anthropological studies and should be investigated in other autoimmune, allergic, inflammatory and infectious diseases.

Alleles↗
PubMed2000

One thousand renal transplants at Belfast City Hospital: post-graft neoplasia 1968-1999, comparing azathioprine only with cyclosporin-based regimes in a single centre.

From 1968-1999, 868 recipients of 1,000 renal transplants were followed up for neoplasia. Altogether, 102 tumours were diagnosed in 94 patients (11.8% incidence). Eighty-seven occurred among 750 single and 15 occurred among 118 multiple graft recipients. Three of 11 patients with pre-existing tumour developed posttransplant neoplasia, either new or recurrent. The most frequently seen posttransplant neoplasms were squamous carcinoma of skin, basal cell carcinoma (BCC), posttransplant lymphoproliferative disease (PTLD) and gastro-intestinal (GI) cancer. Forty-one tumour-related deaths occurred (44% mortality). Patients on CSA (C) regimes had a greater cumulative incidence of tumour after transplantation than those on azathioprine and low-dose prednisolone alone (A regime) had--12.7% (34 of 268) vs. 4.5% (15 of 335) of those at risk up to 5 years (relative increased rate of incidence 2.4) with more early cases of PTLD. C-regime patients who developed neoplasia had been prescribed significantly higher CSA doses than tumour-free controls (4.5 vs. 3.4 mg/kg/day; p = 0.014). Patients who made a late conversion from the A to the C regime subsequently developed more neoplasms than nonconverted controls (25.7% vs. 12%), mainly due to early and often aggressive squamous carcinoma. Transplant survival figures were similar for both A- and C-regime groups. These findings suggest that current CSA doses are higher than are necessary for optimal graft survival and thus increase the risk of early neoplasia without any compensatory advantage. A dose reduction of CSA to less than 3.5 mg/kg/day in long-surviving, stable graft recipients should reduce tumour risk without imperilling function. Late conversion from the A to the C regime should be avoided where possible and CSA doses in this situation kept to a minimum.

Azathioprine↗
PubMed1999

Risk for Alzheimer's disease in older late-onset cases is associated with HLA-DRB1*03.

The allele frequency of the HLA-DRB1 gene was compared between groups of 48 clinically diagnosed elderly Alzheimer's disease (AD) cases and 44 pathologically confirmed elderly control cases. Specific primers were used to PCR amplify the highly polymorphic second exon of HLA-DRB1 using DNA extracted from blood samples or frozen brain tissue. The allele type was identified using sequence specific oligonucleotide probes. The results showed an increased frequency of DRB1*03 (P < 0.006) and decreased frequency of DRB1*09 (P < 0.049) in the AD cases compared with the controls. The results suggest that DRB1*03 is associated with an increased risk and DRB1*09 a possible decreased risk for the development of late-onset AD with first detectable clinical symptoms occurring at age 75 years or greater.

Age of Onset↗
PubMed1999

Characterization of kinetoplast DNA minicircles of an Indian isolate of Leishmania donovani.

Characterization of the kinetoplast DNA minicircles in a human pathogenic Indian isolate of Leishmania donovani has not been reported previously. Using inverse PCR, we constructed a library of PCR-amplified minicircle variable region from the kinetoplast DNA of this isolate. A combination of restriction enzyme digestion and nucleotide sequence analysis revealed five minicircle DNA sequence classes within the library, one of which was predominant, representing 75% of the kDNA network. Another distinct sequence class represented 15% of the minicircle network. Other minor sequence classes collectively constituted the remaining 10% of the network. Apart from generating basic information on the organisation and distribution of the different sequence classes within the minicircles, the DNA sequence analysis also revealed unique attributes to our minicircles. One was the surprising homology of our isolate (an Old World sp.) with distantly related New World Leishmania species. Secondly, open reading frames were also identified, indicating the possibility that these minicircles may have more than a structural role to play within the kinetoplast network.

Amino Acid Sequence↗
PubMed1999

A study of the HLA-DR region in clinical subgroups of multiple sclerosis and its influence on prognosis.

OBJECTIVE: To investigate the HLA-DR associations in relapsing-remitting/secondary progressive multiple sclerosis (RR/SPMS) and primary progressive MS (PPMS). The HLA-DR2 allele (or its split, HLA-DRB1*15) is felt to be a risk factor for MS, rather than a genetic marker for the population of origin. Some studies have indicated a different HLA-DR antigen profile in PPMS patients compared with those having an initially relapsing-remitting course, only those with relapsing disease showing an increase in HLA-DR2. Association of PPMS with DR4 has been suggested. Several DR alleles have also been felt to influence the prognosis in MS. METHODS: Genomic DNA was prepared from peripheral blood of 202 RR/SPMS patients identified in a population-based prevalence study, 102 PPMS patients identified throughout Northern Ireland and 398 normal controls (Nor) matched for the postcode areas of those identified in the prevalence study. Samples were typed for the HLA-DR antigens using polymerase chain reaction (PCR) technology and sequence specific oligonucleotide probes (SSOP). RESULTS: A high incidence of HLA-DRB1*15 was found in each MS group - PPMS (63.73%), RR/SPMS (66.83%) - compared with normals (32.41%), (PPMS vs. Nor, P<0.0001: RR/SPMS vs. Nor, P<0.0001). HLA-DRB1*04 occurred at a lower incidence in both MS groups compared with controls - RR/SPMS (22%), PPMS (30%), Nor (35%). Overall, highly significant differences existed across the full HLA-DR allele distribution (RR/SPMS vs. Nor, P<0.0001, df=12: PPMS vs. Nor, P=0.0007, df=12). No significant differences existed between PPMS and RR/SPMS (P=0.47, df=12), and the allele distributions in benign and aggressive MS were similar. CONCLUSIONS: These data suggest that in this population, HLA-DRB1*15 is indeed associated with PPMS and that PPMS has a HLA-DR profile distinct from the normal population but not from those with an initially relapsing-remitting course. No single allele is associated with either a good or poor prognosis.

Adult↗
PubMed1999

Frequency of HLA-A and B alleles in early and late-onset Alzheimer's disease.

The frequency of various allele types of the class I Major Histocompatibility Complex (MHC) genes HLA-A and HLA-B were compared between pathologically confirmed groups of late and early-onset Alzheimer's disease (AD) and a control group. DNA was extracted from frozen brain tissue and the highly polymorphic second and third exons of the HLA-A and HLA-B genes were independently PCR amplified using specific primers. Individual allele types were identified using sequence-specific oligonucleotide probes. The results showed that the main frequency differences occurred between the late-onset AD and the control group however none of these reached statistical significance.

Adult↗
PubMed1999

Genetic association studies of tumour necrosis factor alpha and beta and tumour necrosis factor receptor 1 and 2 polymorphisms across the clinical spectrum of multiple sclerosis.

Allelic association studies with microsatellite markers around the tumour-necrosis factor (TNF) genes have demonstrated significantly different allele distributions of TNF markers (a and b) between relapsing-remitting/secondary progressive multiple sclerosis (MS) (RR/SPMS) patients and normal controls. Considering the suspected genetic and immunological heterogeneity in MS, we tested this association in primary progressive MS (PPMS) patients. Elevated levels of serum soluble TNF receptors (sTNF-R) are reported in patients with gadolinium enhancing lesions, and animal models suggest a possible therapeutic role of sTNF-RI in MS. Thus we performed similar association studies using markers for the TNF-R genes. Gene association studies were carried out on 199-216 normal controls, 174 RR/SPMS patients and 102 PPMS patients using polymorphic dinucleotide repeat TNF markers (a, b and d), and separate markers for TNF-RI and TNF-RII. Forward primers were fluorescently labelled, polymerase chain reaction (PCR) products were analysed on a fluorescent fragment analyser, and Genescan 672 software was used for allele sizing. Samples were typed for HLA-DR antigens using PCR technology and sequence-specific oligonucleotide probes. TNFa marker allele distributions differed significantly between PPMS patients and controls (P = 0.028), largely attributable to an increase in the 118-bp TNFa allele in PPMS patients (P = 0.00024). Allele distributions were similar in PPMS and RR/SPMS patients (P = 0.91). Logistic regression analysis, however, indicated that these associations were not independent of that with HLA-DRB1*15. For the TNFb marker, the 127-bp allele showed association with both patient categories (PPMS vs. controls, P = 0.010; RR/SPMS vs. controls, P = 0.027), whilst the 128-bp allele occurred more frequently in controls (PPMS vs. controls, P = 0.036: RR/SPMS vs. controls, P = 0.0009). As with the TNFa 118 bp allele, the association with TNFb was not independent of the HLA association. No association occurred with the TNFd marker, and there were also no significant differences in allele frequencies between MS groups and controls regarding the marker for TNF-RI or TNF-RII. In Northern Irish patients the TNF contribution to MS genetic susceptibility is therefore similar across the clinical spectrum of the disease but is not independent of the association with HLA-DRB1*15.

Adult↗
PubMed1999

A special report: histocompatibility testing guidelines for hematopoietic stem cell transplantation using volunteer donors. Quality Assurance and Donor Registries Working Groups of the World Marrow Donor Association.

The World Marrow Donor Association has formulated guidelines for establishing the extent and quality of histocompatibility testing for unrelated donor registries, umbilical cord blood banks, and transplant centers involved in international exchange of hematopoietic stem cells for allogeneic transplantation. Registry and cord blood bank guidelines suggest that, at a minimum, initial HLA typing should be performed for three HLA loci, HLA-A, -B, and -DR, at low resolution/split antigen level. DNA-based testing methods should be utilized for HLA-DR typing. DNA-based testing for HLA-A and -B should replace serologic testing of new volunteer donors and cord blood units as robust protocols and reagents become available to the laboratories. Transplant center guidelines for typing of patient, family and to confirm the HLA types of potential unrelated donors should include, at the minimum, typing HLA-A, B, and -DR loci using primarily DNA-based testing methods at allele level resolution for DRB1 and low resolution/split antigen level for HLA-A and -B. It is strongly recommended that the typing of a patient and the selected donor be performed using the same set of reagents, methodology, and interpretation criteria with fresh tissue samples to ensure HLA identity. Guidelines for laboratory accreditation, approaches to quality assurance and quality control for HLA testing, and suggestions for the format of the HLA database of donor types are also outlined.

Bone Marrow Transplantation↗
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

A special report: histocompatibility testing guidelines for hematopoietic stem cell transplantation using volunteer donors.

The World Marrow Donor Association has formulated guidelines for establishing the extent and quality of histocompatibility testing for unrelated donor registries, umbilical cord blood banks, and transplant centers involved in international exchange of hematopoietic stem cells for allogeneic transplantation. Registry and cord blood bank guidelines suggest that, at a minimum, initial HLA typing should be performed for three HLA loci, HLA-A, -B, and -DR, at low resolution/split antigen level. DNA-based testing methods should be utilized for HLA-DR typing. DNA-based testing for HLA-A and -B should replace serologic testing of new volunteer donors and cord blood units as robust protocols and reagents become available to the laboratories. Transplant center guidelines for typing of patient, family and to confirm the HLA types of potential unrelated donors should include, at the minimum, typing HLA-A, B, and -DR loci using primarily DNA-based testing methods at allele level resolution for DRB1 and low resolution/ split antigen level for HLA-A and -B. It is strongly recommended that the typing of a patient and the selected donor be performed using the same set of reagents, methodology, and interpretation criteria with fresh tissue samples to ensure HLA identity. Guidelines for laboratory accreditation, approaches to quality assurance and quality control for HLA testing, and suggestions for the format of the HLA database of donor types are also outlined.

Hematopoietic Stem Cell Transplantation↗
PubMed1999

Molecular analysis of HLA class I alleles in the Mexican Seri Indians: implications for their origin.

The molecular analysis of HLA class I loci has demonstrated that, although, the genetic profile is restricted in Amerindians, several micropolymorphisms may be important in conferring a biological advantage. We analyzed the HLA-A and B genetic profile of Seris, a Mexican Indian tribe living in northwestern Mexico in the state of Sonora. There are presently only 619 individuals. Our study included 100 Seris belonging to nine families. HLA-A and -B loci typing was performed by polymerase chain reaction using an amplification refractory mutation system (PCR-ARMS) on a select group of samples; all of them were typed by polymerase chain reaction using sequence-specific oliogonuoleotide probes (PCR-SSOP) at a low-intermediate resolution level. The correlation between the techniques was 100%. Only five HLA-A alleles and seven HLA-B alleles were found. A*0201, A*68, A*31, A*24, B*3501, B*40, B*51, B*3512 and B*15 were present in over 5% of the individuals. B*27052 was detected in 2%. B27 is absent in any other Mexican Indian groups previously studied. The presence of B27 may be the result of a founder effect due to different waves of southward migrations. The B-locus is more diverse and the prevalent haplotypes were: A*0201-B*3501, A*0201-B*40, A*0201-B*3512, A*31-B*51, A*68-B*3501 and A*68-B*40. This genetic profile is different from the pattern of other Mexicans. The phylogenetic tree suggests that Seris are more closely related to the Warao Indians from Venezuela, who live in a similar ecosystem, and to some groups of Argentina, than they are to the Mexican Lacandones who live in the jungle. These data emphasize the relevance of the interaction between genes and environment.

Genetics, Population↗
PubMed1999

Allele resolution of HLA-A using oligonucleotide probes in a two-stage typing strategy.

High-resolution polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) typing methods for HLA-A identification have been established. The four systems, which operate independently of each other, are intended for use as secondary typing systems following HLA-A identification with a medium-resolution PCR-SSOP technique. The systems, all using digoxigenin-labelled probes, are based on group specific amplifications for resolution of: i) HLA-A*29 & -A*33; ii) HLA-A*24 & -A*30; and iii) HLA-A*26, -A*25, -A*11, -A*34, -A*66 and -A*68 alleles, respectively. The fourth system, for the detection of HLA-A*02 alleles, is a modification of a previously reported PCR-SSOP subtyping system. The methods have been applied to individuals from the local bone marrow registry and HLA-A allele frequencies for the Northern Ireland population have been established.

Alleles↗