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Biomedical subjects

C Darke

Publications and source records attributed to C Darke.

At least 19 recordsLinked to original sources
PubMed2006

Non-B27 MHC associations of ankylosing spondylitis.

Ankylosing spondylitis (AS) has been associated with human leukocyte antigen (HLA)-B27 for over 30 years; however, the mechanism of action has remained elusive. Although many studies have reported associations between AS and other genes in the major histocompatibility complex (MHC) in AS, no conclusive results have emerged. To investigate the contribution of non-B27 MHC genes to AS, a large cohort of AS families and controls were B27 typed and genotyped across the region. Interrogation of the data identified a region of 270 kb, lying from 31 952 649 to 32 221 738 base pairs from the p-telomere of chromosome 6 and containing 23 genes, which is likely to include genes involved with susceptibility to AS.

Case-Control Studies↗
PubMed2006

A high-resolution polymerase chain reaction-sequence-specific primer HLA-B*27 typing set and its application in routine HLA-B27 testing.

We designed a set of 35 polymerase chain reaction sequence-specific primers (PCR-SSP) in 29 SSP mixtures to assign 29 HLA-B*27 4-digit level alleles (B*2701-B*2721 and B*2723-B*2730). This was used in conjunction with our 41 PCR-SSP primer mixture low-resolution HLA-B typing set to fully differentiate B*27 from all other HLA-B alleles. Successful typing set validation used 521 B*27 samples covering 13 (B*2701-B*2710 and B*2712, B*2717, B*2723) alleles. The distribution of B*27 alleles was determined in a random population of 4020 local blood donors and the use of PCR-SSP B*27 typing in our routine flow cytometry-based HLA-B27/B2708 typing strategy is described.

Alleles↗
PubMed2006

Identification of a novel HLA-A allele (A*1115) in the UK National External Quality Assessment Schemes for Histocompatibility and Immunogenetics' Educational Cell Exchange.

The novel allele, HLA-A*1115, was identified in an 'Educational Scheme' sample (ED03/03 - from a north-western European Caucasoid blood donor) distributed by the UK National External Quality Assessment Schemes for Histocompatibility and Immunogenetics. ED03/03 was typed by serology, the polymerase chain reaction using sequence-specific primers and sequence-based typing. A*1115 is most similar to A*110101 with a single mismatch (G to C) at constant position 565, leading to a conservative amino acid change from valine (GTG) to leucine (CTG) at codon 165 in the alpha(2) domain. This substitution has not been reported for any other HLA class I allele so far. The HLA-A*1115-bearing haplotype was B*350101; Cw*040101; DRB1*140101; DRB3*020201; DQA1*010401; DQB1*0503; DPA1*0103/07; DPB1*030101. Extensive serological typing indicated that this allele essentially encodes a 'normal' HLA-A11 specificity.

Alleles↗
PubMed2005

A typing system for the major histocompatibility complex class I chain related genes A and B using polymerase chain reaction with sequence-specific primers.

The Major Histocompatibility Complex (MHC) class I chain related (MIC) A and B genes are important additional loci within the MHC. We have developed a MICA and MICB typing system using the polymerase chain reaction with sequence-specific primers (PCR-SSP), which operates under the same conditions as our routine HLA-A, -B, and -C typing method. We designed 95 primers in 84 SSP mixtures for MICA and 39 primers in 29 mixtures for MICB. This detected and differentiated all 55 MICA and 19 MICB alleles (except MICA*00701 from MICA*026, MICA*00201 from MICA*020, and three MICB alleles, which are intronic variations). A computer program confirmed the MICA amplification reactivity of each SSP mixture and evaluated the typing set for MICA allele combination ambiguities. Seventy-six "reference" DNA samples were used for validation: 50 from International Histocompatibility Workshop B lymphoblastoid cell lines (IHW BCLs) and 26 MICA-typed samples from two laboratories. The reference material identified 28 out of the 55 MICA alleles and 13 of the 19 MICB alleles, and directly validated 62 of the 84 MICA and 20 of the 29 MICB SSP mixtures. Our genotyping agreed with 283 out of the 286 (98.95%) MICA and MICB reference laboratories' allele assignments or the consensus assignments. Two of the discrepancies remain unresolved, whereas one was probably due to a reference laboratory's failure to differentiate alleles differing in exon 5 of the MICA gene. A comparison of the MICA and MICB allele assignments between laboratories identified a "disagreement rate" of 19.4% for MICA alleles and 13.1% for MICB alleles. Accordingly, we have compiled "consensus" MICA and MICB genotypes for the 50 IHW BCLs tested, which have been confirmed by our typing. We also typed 166 random blood donors. Their MICA and MICB carriage and allele frequencies and HLA-B, MICA, MICB linkage disequilibrium parameters and haplotype frequencies largely concurred with other published data on United Kingdom subjects, further supporting the validity of our typing system. This PCR-SSP system is a simple, reliable and rapid technique for typing MICA and MICB alleles. It is easily updated as new alleles are identified but clearly requires a continuing validation review until all known MICA and MICB alleles have been identified.

DNA Primers↗
PubMed2004

Five-locus HLA typing of hematopoietic stem cell donor volunteers using PCR sequence specific primers.

We have developed a strategy for five-locus human leukocyte antigen (HLA) typing of hematopoeitic stem cell (HSC) donors using the polymerase chain reaction with sequence-specific primers (PCR-SSP). The PCR-SSP method is robust, reproducible, and accurate. New PCR-SSP mixtures can be added as required and all reactions are carried out under the same conditions, which can easily be applied to the typing of other loci, e.g., ABO blood groups. Initially, 127 PCR-SSP reactions were used to detect simultaneously HLA-A, -B, -C, -DRB1/3/4/5, and DQB1 alleles, differentiated generally to the level of the first two digits of the allele name, essentially equivalent to the serological split specificity. Approximately 40% of subjects were tested against a further 29 HLA-A, -B SSP mixtures to exclude rare alleles and unambiguously assign a two-digit HLA allele family. This gave an overall typing resolution equivalent to or greater than the split specificity level and covered all HLA-A, -B, -C, -DRBland DQB1 alleles listed in the WHO's Nomenclature for Factors of the HLA System, 2000. The Welsh Bone Marrow Donor Registry has used this strategy to HLA type over 35,000 HSC donors over 9 years. Comprehensive and accurate five-locus HLA typing allows confident and rapid identification of potential matched HSC donors for patients requiring stem cell transplantation generally without the need for typing additional loci. This allows resources to be focused directly on allele level typing of DRB1 and other loci. This strategy decreases overall donor work-up time, which is a major benefit to patients.

Alleles↗
PubMed2004

HLA-A*24020102L in the UK blood donor population.

The low-expression allele HLA-A*24020102L was identified on a likely haplotype bearing B*55, Cw*01 during the investigation of nine cases (four families and five unrelated subjects) of HLA-A*24 lacking the A24 specificity. A search was made of 70,007 HLA-A, HLA-B, and HLA-C DNA-based typed largely northwestern European Caucasoid blood donors on the British Bone Marrow Registry and Welsh Bone Marrow Donor Registry panels for phenotypes possessing A*24, B*55, Cw*01. Fifty three were found, and 12 of these were subsequently shown to possess A*24020102L by sequence-based typing or polymerase chain reaction with sequence-specific primers. This indicated that the likely A*24, B*55, Cw*01 haplotype has a frequency of 0.000378 in the UK and that approximately 22.6% of these will possess A*24020102L. Consequently, HLA-A*24020102L, B*55, Cw*01 is a principal A*24020102L-bearing haplotype, and the minimum carriage and gene frequencies of A*24020102L are 0.017% and 0.000086, respectively, in UK blood donors.

Blood Donors↗
PubMed2004

Human leucocyte antigen polymorphisms in nut-allergic patients in South Wales.

BACKGROUND: Peanuts and tree nuts are among the most common foods provoking severe allergic reactions including fatal anaphylaxis. However, little is known of the underlying genetic and immunological mechanisms involved. OBJECTIVE: Based on findings in other allergic diseases, we have investigated whether specific human leucocyte antigens (HLA) are associated with nut allergy. METHOD: Eighty-four patients presenting at the allergy clinic with symptoms of nut allergy were typed for the HLA Class I (HLA-A and B) and Class II (HLA-DRB1 and DQB1) loci by PCR using sequence-specific primers. Carriage frequencies were compared with 82 atopic non-nut-allergic subjects and 1798 random blood donors. RESULTS: The frequency of HLA-B(*)07 (28.57%) and DRB1(*)11 (15.48%) was increased in the nut-allergic patients compared to the atopic controls (12.20% and 3.66%, respectively) but not when compared to the blood donors (28.86% and 10.12%). DRB1(*)13 and DQB1(*)06 were both increased in frequency in the nut allergy patients over both the atopic and blood donor controls. However, none of these increased frequencies were significant when corrected for the number of comparisons undertaken. CONCLUSION: At HLA '2-digit resolution' and with undifferentiated patients with nut allergy, there are no major disturbances in the frequency of HLA-A, B, DRB1 or DQB1 types. However, the difference in frequency of HLA-DRB1(*)11 between the nut allergy patients and the atopic controls merits further investigation as this may represent an important phenotypic relationship.

Adolescent↗
PubMed2004

Provision of Epstein-Barr virus-transformed B-cell lines in a routine tissue typing laboratory: practicalities and applications.

A bank of Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (BCLs) has been established in this laboratory over the last 7 years. Novel, rare and unusual HLA phenotypes were highlighted during routine clinical testing and during typing of volunteer haematopoietic stem cell donors for the Welsh Bone Marrow Donor Registry. The BCLs are routinely used as laboratory reagents, as a source of DNA for reference purposes and for research work. Consenting donors are tested for hepatitis B surface antigens (HBsAg), human immunodeficiency virus (HIV) and hepatitis C virus (HCV) before their B-lymphocytes are transformed. Strict bio-identity checks are performed before and after transformation and after each BCL expansion. Each BCL is tested regularly for mycoplasma contamination. A total of 230 blood samples were transformed. One hundred and fifty-nine sterile samples produced 157 BCLs (98.7% success), while 71 non-sterile samples produced 50 BCLs (70.4% success), giving an overall success rate of 90.0%. Fifteen of the transformation failures have since been repeated successfully. Factors contributing to the high success rate and reasons for the 23 failed transformations are discussed. The successful development and use of pools of BCLs for HLA antibody screening by flow cytometry are described. Whilst certain training and health and safety issues require close attention, it is clear that EBV transformation of B lymphocytes and/or the use of BCLs is feasible in a routine tissue typing laboratory and that BCLs are a valuable resource. Careful adherence to the methods and procedures detailed here should virtually guarantee successful transformation (98.7%) from good quality, sterile whole blood samples.

B-Lymphocytes↗
PubMed2003

Immunogenetics of two new HLA alleles: A*0108 and B*4031.

Two new alleles, HLA-A*0108 and B*4031, were identified in north-western European Caucasoid subjects. A*0108 differed from A*010101 by a single substitution (C to T) at position 216 in exon 3, resulting in an amino acid difference of Arg to Trp at position 163. It was present on a haplotype with B*1501/60/70/71; Cw*0303; DRB1*1301; DRB3*0202; DQA1*0103; DQB1*0603 and its product reacted as a normal HLA-A1 specificity. B*4031 differed from B*4001 by two nucleotides in exon 3 (positions 20 (G to C) and 69 (A to G)) resulting in two amino acid differences (Arg to Ser at position 97 and Asn to Asp at position 114). It was found on a haplotype with HLA-A*03; Cw*0304; DRB1*0404/32; DRB4*0101/3/5; DQA1*03; DQB1*0302 and has the HLA-B60 specificity. Both alleles have frequencies of < 0.0002 in the largely north-western European Caucasoid blood donor population resident in Wales.

Gene Frequency↗
PubMed2003

Observations on the HLA-A2403 specificity.

We found HLA-A*2423 in a subject possessing the HLA-A2403 specificity. Consequently we delineated the A*24 alleles of 41 subjects previously assigned as 'A2403' by serology and/or 'low-resolution' PCR-SSP (allele group: A*2403/10/18/22/23/33) and found that 36 were A*2403, 3 were A*2410 and 2 were A*2433. The A*2410 and A*2433 subjects also typed as A2403. Several likely A*2410/23/33-bearing haplotypes were identified and the frequency of the A*2403/10/18/22/23/33 alleles was determined in 26,826 Welsh blood donors. We suggest that HLA-A9/A24 antibodies against epitope(s) involving 166D and 167G will fail to react with all A*2403/10/18/22/23/33 products. As it is this 'short' A9/A24 serological reactivity that defines HLA-A2403 all these products will be serologically designated as HLA-A2403.

HLA-A Antigens↗
PubMed2003

Immunogenetics of two new HLA-B alleles: B*4414 and B*5708.

Two new alleles, HLA-B*4414 and B*5708, were identified in north-western European Caucasoid blood donors. B*4414 differed from B*440201 by two nucleotide substitutions in exon 3 [positions 66 (T to C) and 69 (G to A)] producing two amino acid differences between the B*440201 and B*4414 specificities (tyrosine to histidine at codon 113 and aspartic acid to asparagine at codon 114). B*5708 differed from B*570101 by a single substitution (G to C) at position 247 in exon 2 causing an amino acid difference between B*570101 and B*5708 products of arginine to proline at codon 83. The likely haplotypes bearing these alleles were identified. Both alleles occurred once in approximately 25,000 random blood donors so both have a frequency of approximately 0.00002 (carriage frequency 0.004%). B*4414 and B*5708 specificities both gave 'short' serological reactivity for their expected specificities and Bw4. The likely reasons for this are discussed in relation to the epitopes of B44 and B57.

HLA-B Antigens↗
PubMed2003

Immunogenetics of the rare HLA-B allele B*4408.

The nucleotide sequence of exons 2 and 3 of B*4408 has been confirmed. This allele has now been found in five, apparently unrelated, north-western European Caucasoid subjects, residing in Wales, on HLA haplotypes bearing: A*0201, Cw*0501 with DRB1*0401, DQA1*03, DQB1*0301 in four instances and DRB1*0701, DQA1*0201, DQB1*0202 in one instance. The unique serological reactivity of 'HLA-B44BO' was established using up to nine B*4408 subjects and was easily identified using a variety of B44 and B62 antisera. The phenotype and gene frequencies of B*4408, in a sample of 40 473 subjects residing in Wales, were 0.01235% and 0.00006, respectively.

HLA-B Antigens↗
PubMed2003

An evaluation of Com-B27, a fluorescein-conjugated mouse anti-HLA-B27 reagent.

We evaluated the HLA-B27 typing reagent Com-B27, which is claimed to show minimal cross-reactivity with HLA-B7, for use in flow cytometry-based typing. This combination reagent consists of the fluorescein-conjugated HLA-B27 mouse monoclonal antibody ABC-m3 and a non-conjugated HLA-B7 monoclonal antibody that is claimed to block the reactivity of ABC-m3 to B7 without affecting its reactivity to B27. It reacted well with B27 (B*2702, B*2705) and B2708 [mean median channel fluorescence intensity (MCFI) 8.40] and weakly with B7 (including cells from homozygous B*07 donors), B42/B73 and B22/B37/B44 reference cells (mean MCFI 2.05, 3.09 and 1.10, respectively). It showed a uniform discrimination between B7 and B27/B2708 with no 'overlap' in MCFI values, which was seen with the standard ABC-m3 antibody. There was complete agreement when our standard three-antibody-based B27/B2708 flow cytometry assay and the Com-B27 reagent alone were used to independently assign B27/B2708 status to 651 random patients. Thus, the Com-B27 reagent provided improved discrimination between B7 and B27/B2708 over the ABC-m3 antibody, and its B27/B2708 assignments were comparable with our standard flow cytometry assay. However, for consistently reliable B27/B2708 typing we continue to recommend the use of a minimum of two B27 reagents in a protocol that includes DNA-based testing of 'equivocal' B27/B2708 assignments.

Animals↗
PubMed2003

A modified PCR-SSP method for the identification of ABO blood group antigens.

The ABO blood group antigens are carbohydrate molecules synthesized by the glycosyltransferases encoded by the ABO gene on chromosome 9. Kidney transplantation across the ABO barrier generally leads to rapid humoral graft rejection due to the presence of naturally occurring antibodies to the A and B antigens. We have developed a method for ABO typing our cadaveric organ donors by the polymerase chain reaction using sequence-specific primers (PCR-SSP). The method uses 12 primers in eight PCR mixtures and is performed under the same conditions as our routine HLA-A, B, C PCR-SSP typing. The PCR-SSP-based types of 166 regular blood donors and 148 cadaveric organ donors all showed total concordance with their serologically assigned ABO groups. Six individuals possessing the ABO A subgroups (A3, Ax and Aend) all typed as A1 by PCR-SSP, as expected. PCR-SSP is an appropriate method for ABO typing of cadaveric organ donors and, importantly, enables both ABO and HLA typing to be performed on the same DNA material.

ABO Blood-Group System↗
PubMed2003

Immunogenetics of a new HLA-B null allele, HLA-B*4423N.

The second example of an HLA-B*44 null allele (B*4423N) was identified by discrepancies between serological and polymerase chain reaction-sequence-specific primer (PCR-SSP) typing in two north-western European Caucasoid unrelated stem cell donor volunteers. HLA-B*4423N was identical to B*440201 except for a single nucleotide substitution at position 493 in exon 3, resulting in a premature stop codon at bases 493-495 (TAG rather than CAG at codon 141). As expected, comprehensive serological testing using 54 antisera, directed towards B44 or Bw4, failed to identify the HLA-B44 (Bw4) specificity. The B*4423N-bearing haplotype was identified as A*0201, Cw*0501, DRB1*0408, DRB4*01, DQA1*03, DQB1*0304 and the frequency of B*4423N estimated as 0.00006 (carriage frequency 0.0121%) in 16 533 subjects resident in Wales.

Europe↗