33 results on '"DNA Probes, HLA genetics"'
Search Results
2. Resolution of HLA class I sequence-based typing ambiguities by group-specific sequencing primers.
- Author
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Lebedeva TV, Mastromarino SA, Lee E, Ohashi M, Alosco SM, and Yu N
- Subjects
- Alleles, Base Sequence, DNA Probes, HLA analysis, DNA Probes, HLA genetics, False Positive Reactions, Humans, Molecular Sequence Data, Retrospective Studies, Sequence Analysis, DNA methods, Sequence Homology, Software, Substrate Specificity genetics, Substrate Specificity immunology, DNA Primers genetics, Genes, MHC Class I genetics, Histocompatibility Testing methods, Sequence Analysis, DNA standards
- Abstract
The increasing demand for allele-level human leukocyte antigen (HLA) typing has led the sequence-based typing (SBT) to become the preferred method. In turn, the steady increase in the number of HLA alleles driven by the adoption of SBT as the ultimate typing method leads to the ever increasing number of cis/trans ambiguities. Over the last few years, additional sequencing with the commercially available group-specific sequencing primers (GSSPs) has replaced sequence-specific primer-polymerase chain reaction and group-specific amplification as the means of resolving cis/trans ambiguities in many laboratories. Here we summarize our 3-year experience in designing and utilizing GSSPs for resolution of HLA class I ambiguities. The panel of GSSPs used in our laboratory includes 14 primers for HLA-A, 18 for HLA-B, and 13 primers for HLA-C. The panel resolves 99.9% of all ambiguities., (© 2011 John Wiley & Sons A/S.)
- Published
- 2011
- Full Text
- View/download PDF
3. Improved KIR gene and HLA-C KIR ligand sequence-specific primer polymerase chain reaction genotyping using whole genome amplification.
- Author
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Chainonthee W, Böttcher G, Gagne K, Füssel M, Bignon JD, and Wassmuth R
- Subjects
- Alleles, Base Sequence, DNA Primers genetics, DNA Probes, HLA genetics, Genome-Wide Association Study, Genotype, Humans, Ligands, Polymorphism, Single Nucleotide, Receptors, KIR2DL1 genetics, Receptors, KIR3DL1 genetics, Receptors, KIR3DS1 genetics, HLA-C Antigens genetics, Polymerase Chain Reaction methods, Receptors, KIR genetics
- Abstract
Molecular analysis of genetic polymorphism for clinical or research purposes may be compromised by genomic DNA of limited quality and quantity. In this study, we have successfully tested the feasibility of using whole genome amplification (WGA) to allow genotyping for killer cell immunoglobulin-like receptor (KIR) genes and human leucocyte antigen (HLA)-C KIR ligand dimorphism on HLA-C. WGA was achieved by multiple displacement amplification (MDA) using bacteriophage phi29 polymerase. For KIR genotyping, a revised sequence-specific primer polymerase chain reaction protocol consisting of 23 primer pairs was used avoiding hitherto undetected cross-priming involving KIR2DL1, KIR2DS1, KIR3DL1 and KIR3DS1 alleles. Similarly, MDA-amplified genomic DNA was analyzed for the detection of the HLA-C KIR ligand groups C1 and C2, based on the amino acid K/N dimorphism in position 80.
- Published
- 2010
- Full Text
- View/download PDF
4. A one-step real-time PCR assay for detection of DQA1*05, DQB1*02 and DQB1*0302 to aid diagnosis of celiac disease.
- Author
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Reinton N, Helgheim A, Shegarfi H, and Moghaddam A
- Subjects
- Celiac Disease blood, Celiac Disease genetics, DNA chemistry, DNA genetics, DNA Primers chemistry, DNA Primers genetics, DNA Probes, HLA chemistry, DNA Probes, HLA genetics, HLA-DQ Antigens genetics, HLA-DQ Antigens immunology, HLA-DQ alpha-Chains, HLA-DQ beta-Chains, Humans, Sequence Analysis, DNA, Celiac Disease immunology, HLA-DQ Antigens blood, Polymerase Chain Reaction methods
- Abstract
Celiac disease is an autoimmune disorder that develops after dietary exposure of the small intestine to gluten peptides in cereals. Celiac disease has a strong genetic component associated with HLA-DQ2 and HLA-DQ8, and testing for absence of these genetic markers is useful when serological tests and biopsies are indeterminate, as it renders celiac disease highly unlikely. We have developed a new real-time PCR assay, using sequence-specific primers (PCR-SSP) and TaqMan probes, for detection of DQB1*05, DQB1*02 (coding for DQ2) and DQB1*0302 (coding for DQ8). PCR amplification and detection of DQ2 and DQ8 was accurately and unambiguously performed from genomic DNA isolated from cell lines and human DNA. Amplification was scored digitally, without laboratory manipulation of amplified PCR products and with a higher accuracy than PCR-SSP. This assay should increase accuracy and throughput, and reduce risks of contamination in laboratories where testing for HLA DQ2 and DQ8 is performed as part of diagnosis of celiac disease.
- Published
- 2006
- Full Text
- View/download PDF
5. Validation of sensitive human leukocyte antigen-sequence-specific primer and probe typing in forensic DNA examination.
- Author
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Ota M, Shimada K, Asamura H, Takayanagi K, Katsuyama Y, and Fukushima H
- Subjects
- Base Sequence, Blood Stains, Humans, Mouth Mucosa cytology, Polymerase Chain Reaction methods, Reproducibility of Results, DNA Primers genetics, DNA Probes, HLA genetics, Forensic Genetics methods, HLA-A Antigens genetics
- Abstract
Validation studies were carried out with the commercially available HLA typing kit using a PCR-SPP (sequence-specific primer and probe) technique. This technique has made it possible to type class I (HLA-A and -B) and class II (HLA-DRB1 and -DQB1) alleles at low-resolution level with total 10 ng of template DNA, in addition to amplify directly from various forms of blood samples without DNA isolation procedure. Experimental examinations with bloodstains smeared on cotton cloth that were a week to 3 months old, bloodstains on gauze stored for 18 years, and buccal cells revealed that this HLA-SPP typing kit is a sensitive and reliable method for forensic investigations.
- Published
- 2006
- Full Text
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6. Real-time PCR using fluorescent resonance emission transfer probes for HLA-B typing.
- Author
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Faner R, Casamitjana N, Coll J, Caro P, Pujol-Borrell R, Palou E, and Juan M
- Subjects
- DNA blood, DNA Probes, HLA chemistry, Genotype, Humans, Time Factors, DNA Probes, HLA genetics, Fluorescence Resonance Energy Transfer, Fluorescent Dyes chemistry, HLA-B Antigens genetics, Polymerase Chain Reaction methods
- Abstract
HLA genotyping by polymerase chain reaction (PCR) has some inherent labor-intensive and effort-demanding limitations. To overcome them, we have developed a real-time PCR with hybridization probes approach able to obtain a medium-low resolution HLA-B genotyping with fewer tubes and probes and with a shorter time requirement. Our strategy used 18 simultaneous reactions amplifying HLA-B alleles and an internal control. Monitorization of both amplifications in each tube is performed by the simultaneous application of two fluorescent resonance emission transfer probes: the first probe, different for each tube, is specific for the HLA-B locus and the second probe detects the control gene. A medium-low resolution (300 HLA-B allelic groups) typing is obtained for each sample by analyzing the melting curve patterns. Because some alleles may be determined without using the complete set of reactions, we present an alternative strategy: a first round of seven reactions and, according to the result, a second (or third) round of PCRs to solve the ambiguities. This method was validated in pretyped clinical samples and the results were completely concordant. Moreover, fewer ambiguous results were obtained. In summary, we present a new, faster, and more accurate method than currently used PCR techniques to type HLA-B alleles.
- Published
- 2006
- Full Text
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7. Identification of three novel HLA class I alleles: HLA-B*3928, HLA-B*400104 and HLA-B*4437.
- Author
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Velickovic ZM, Dodd R, Velickovic M, Hersee J, Le T, Taverniti A, Wallace R, and Dunckley H
- Subjects
- Amino Acid Substitution genetics, Base Sequence, Exons genetics, Humans, Molecular Sequence Data, Sequence Analysis, DNA, Alleles, DNA Probes, HLA genetics, Genetic Variation, HLA-B Antigens genetics
- Abstract
Three new human leukocyte antigen (HLA) class I alleles have been identified in the Tissue Typing Laboratory in Sydney, Australia. Sequence analysis of exon 2 and exon 3 of the HLA-B gene revealed the novel polymorphism. A silent substitution of C to T at nucleotide position 369 has been identified for the HLA-B*400104 allele when compared to the closest matched allele, HLA-B*400101. The HLA-B*3928 allele was identified with a nucleotide substitution of G to C at position 362 when compared to the closest matched allele, HLA-B*390101, resulting in an amino acid substitution of Arginine to Threonine. A nucleotide substitution of C to G at position 572 resulting in the amino acid change Serine to Tryptophan was identified in the new allele HLA-B*4437, when compared to the closest matched allele HLA-B*440301. Both amino acid substitutions implicate a different specificity and affinity of antigen binding for the alleles HLA-B*3928 and HLA-B*4437.
- Published
- 2004
- Full Text
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8. Identification of new HLA-C alleles by polymerase chain reaction-sequence specific oligonucleotide typing: Cw*0314 and Cw*1511*.
- Author
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Chapman G, Kennedy C, Greville WD, Dodd R, Hersee J, Taverniti A, Le T, Wallace R, Kennedy A, and Dunckley H
- Subjects
- Australia, Base Sequence, Biological Specimen Banks, Bone Marrow metabolism, Exons genetics, Fetal Blood metabolism, Humans, Molecular Sequence Data, Sequence Analysis, DNA, Alleles, DNA Probes, HLA genetics, Genetic Variation, HLA-C Antigens genetics
- Abstract
In this article, we report two new human leukocyte antigen-C (HLA-C) alleles, HLA-Cw*0314 and Cw*1511, which were identified during routine tissue typing of donors for the Australian Bone Marrow Donor Registry and Australian Cord Blood Bank. HLA-Cw*0314 shows six codon changes in exon 3 compared to Cw*030401 and shares some sequence homology with Cw*07 alleles. Cw*1511 has two nucleotide changes compared with Cw*150201 in exon 2, both resulting in amino acid changes in the protein sequence.
- Published
- 2004
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9. Ambiguities of human leukocyte antigen-B resolved by sequence-based typing of exons 1, 4, and 5.
- Author
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Swelsen WT, Voorter CE, and van den Berg-Loonen EM
- Subjects
- Base Sequence, DNA Probes, HLA genetics, Female, Humans, Leukocytes immunology, Male, Molecular Sequence Data, Exons, HLA-B Antigens genetics, Polymorphism, Genetic genetics, Sequence Analysis, DNA
- Abstract
The elucidation of the sequences of human leukocyte antigen-B (HLA-B)-exons 1 through 5 has led to an increase of ambiguities with alleles having identical exon 2 and 3 sequences, but differences in other exons. At the moment, 26 HLA-B alleles show such ambiguities which can be resolved by sequencing the exons in which the differences are located. Here we report a sequence-based typing (SBT) strategy for heterozygous sequencing of exons 1, 4, and 5, in addition to the previously described exons 2 and 3. The strategy was validated against a panel of 25 individuals, carrying HLA-B alleles from 33 different allele groups. Correct assignment of all HLA-B alleles was obtained for exons 1 through 5. In addition, the SBT protocol was used to resolve ambiguities in 50 individuals. The ambiguous combinations studied were B*0705/06, B*0801/19N, B*1512/19, B*180101/17N, B*270502/13/0504, B*350101/42/40N, B*390101/0103, B*400102/0101, B*440201/19N/27, and B*510101/11N/0105/30/32. In all cases, sequencing revealed the first allele to be present, except for three individuals with B*07. One of them typed B*0705; the other two were B*0706. The described SBT protocol for sequencing exons 1, 4, and 5 is a valuable tool for resolving ambiguities of HLA-B alleles with differences in these exons, as well as for studying the polymorphism of HLA-B outside exons 2 and 3.
- Published
- 2004
- Full Text
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10. Nonelectrophoretic method for high-throughput HLA-DRB1 group genotyping.
- Author
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Hampe J, Valentonyte R, Manaster C, Teuber M, Jenisch S, Entz P, Nagy M, and Schreiber S
- Subjects
- DNA Probes, HLA genetics, Electrophoresis, Genetic Testing, Genotype, HLA Antigens genetics, HLA-DRB1 Chains, Humans, User-Computer Interface, Gene Expression Profiling methods, HLA-DR Antigens genetics, Oligonucleotide Array Sequence Analysis methods, Polymerase Chain Reaction methods, Software
- Published
- 2004
- Full Text
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11. PCR-SSOP molecular typing of HLA-C alleles in an Iranian population.
- Author
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Buhler S, Sanchez-Mazas A, Zanone R, Djavad N, and Tiercy JM
- Subjects
- Genetics, Population, Humans, Iran, DNA Probes, HLA genetics, HLA-C Antigens genetics, Polymerase Chain Reaction methods, Polymorphism, Genetic
- Abstract
HLA-C alleles were characterized by a polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) hybridization protocol in a sample of 120 Iranians from Tehran. A total of 23 alleles were identified with the four most predominant--Cw*0401, Cw*0602, Cw*1202, and Cw*0701/06--accounting for almost 50% of HLA-C alleles. A comparison of HLA-C diversity among several populations indicates that Iranians stand at an intermediate genetic position between Europeans and Africans, an observation that may be related to their geographical location at a continental crossroads. The results also reveal a very high correlation between genetic and geographic distances on a global scale. A total of 30 HLA-C-DRB1 haplotypes were found in the Iranians, with the highest frequencies of 6.6% and 6.04 % being for Cw*0602-DRB1*0701 and Cw*1202-DRB1*1502, respectively.
- Published
- 2002
- Full Text
- View/download PDF
12. Use of quantitative ligation-mediated polymerase chain reaction to detect gene targeting by alkylating oligodeoxynucleotides.
- Author
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Gamper HB, Afonina I, Belousov E, Reed MW, and Podyminogin MA
- Subjects
- Alkylation, Chlorambucil chemistry, DNA chemistry, DNA Probes, HLA genetics, Humans, Rec A Recombinases genetics, Gene Targeting methods, Oligodeoxyribonucleotides chemistry, Polymerase Chain Reaction methods
- Published
- 2000
- Full Text
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13. Nomenclature for factors of the HLA system, update June 1999.
- Author
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Marsh SG
- Subjects
- DNA Probes, HLA genetics, Databases, Factual, HLA Antigens genetics, Humans, DNA Probes, HLA classification, Gene Library, HLA Antigens classification, Terminology as Topic
- Published
- 1999
- Full Text
- View/download PDF
14. Identification of DRB alleles in rhesus monkeys using polymerase chain reaction-sequence-specific primers (PCR-SSP) amplification.
- Author
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Lobashevsky A, Smith JP, Kasten-Jolly J, Horton H, Knapp L, Bontrop RE, Watkins D, and Thomas J
- Subjects
- Animals, Base Sequence, DNA Probes, HLA genetics, Haplotypes genetics, Macaca mulatta, Molecular Sequence Data, Pedigree, Sequence Alignment, Alleles, DNA Primers genetics, HLA-DR Antigens genetics, Polymerase Chain Reaction methods
- Abstract
Major histocompatibility complex (MHC) class In molecules play a vital role in the regulation of T-cell functions in the mammalian immune system. Two key features characterize the polymorphism of MHC haplotypes in humans and non-human primates: the existence of a large number of alleles, and the high degree of genetic diversity between those alleles. Rhesus monkeys and Chimpanzees have been extensively used as relevant models for human diseases and transplantation We have investigated DRB genes in 19 macaques, members of 3 families, using polymerase chain reaction with sequence-specific primers (PCR-SSP) and denaturing gradient gel electrophoresis (DGGE). After amplification PCR products were purified and subjected direct sequencing. Seven animals (Madison #1) were typed by DDGE also. We report that the DRB haplotypes defined by PCR-SSP exhibit a high degree of concordance with the data obtained by DGGE and direct sequening. Our data show prominent variability in the number of DRB1 alleles ranging from 1-4 per genotype within these families. This analysis demonstrated that most of the amplicons were identical to Mamu-DRB alleles that our PCR primers were to amplify. However, 98-99% similarity was noticed in the case of Mamu-DRB1*0303, Mamu-DRB6*0103 and Mamu-DRB*W201 alleles. The observed mismatches were located in non-polymorphic regions. Thus, family studies in rhesus macaques performed by molecular methods confirmed the multiplicity of Mamu-DRB1 alleles per haplotype and the existence of allelic associations published earlier. In addition, we propose 3 more DRB allele associations (haplotypes): Mamu-DRB1*04-DRB5*03; Mamu-DRB1*04-*DRB*W5; Mamu-DRB1*04*W2. The proposed medium-resolution PCR-SSP technique appears to be a highly reproducible and discriminatory typing method for detecting polymorphisms of DRB genes in rhesus monkeys.
- Published
- 1999
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15. Paternity assessment in rhesus macaques (Macaca mulatta): multilocus DNA fingerprinting and PCR marker typing.
- Author
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Nürnberg P, Sauermann U, Kayser M, Lanfer C, Manz E, Widdig A, Berard J, Bercovitch FB, Kessler M, Schmidtke J, and Krawczak M
- Subjects
- Animals, Cohort Studies, DNA blood, DNA Fingerprinting economics, DNA Fingerprinting methods, DNA Probes, HLA genetics, Female, Genetic Markers, Humans, Macaca mulatta blood, Male, Microsatellite Repeats, Paternity, Polymerase Chain Reaction veterinary, DNA Fingerprinting veterinary, Macaca mulatta genetics
- Abstract
Establishing kinship relations in primates using modern molecular genetic techniques has enhanced the ability to scrutinize a number of fundamental biological issues. We screened 51 human short tandem repeats (STRs) for cross-species PCR amplification in rhesus macaques (Macaca mulatta) and identified 11 polymorphic loci with heterozygosity rates of at least 0.6. These markers were used for paternity testing in three social groups (M, R, and S) of rhesus macaques from Cayo Santiago, Puerto Rico. Several consecutive birth cohorts were analyzed in which approximately 200 males were tested for paternity against more than 100 mother/ infant pairs. Despite a combined exclusion rate of more than 99.9% in all three groups, some cases could not be solved unequivocally with the STR markers and additional testing of the MHC-associated DQB1 polymorphism. A final decision became possible through multilocus DNA fingerprinting with one or more of the oligonucleotide probes (GATA)4, (CA)8, and (CAC)5. Paternity assessment by multilocus DNA analysis with probe (CAC)5 alone was found to have limitations in rhesus macaques as regards the number of potential sires which might be involved in a given case. Multilocus DNA fingerprinting requires large amounts of DNA, and the ensuing autoradiographic patterns present difficulties in comparisons across gels and even within the same gel across remote lanes. Computer-assisted image analysis was incapable of eliminating this problem. Therefore, a dual approach to DNA typing has been adopted, using STR markers to reduce the number of potential sires to a level where all remaining candidates can be tested by multilocus DNA fingerprinting on a single gel, preferably in lanes adjacent to the mother/infant pair.
- Published
- 1998
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16. Accurate typing of HLA-A antigens and analysis of serological deficiencies.
- Author
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Yu N, Ohashi M, Alosco S, Granja C, Salazar M, Hegland J, and Yunis E
- Subjects
- DNA genetics, Diagnostic Errors, Evaluation Studies as Topic, Genes, MHC Class I, Genotype, HLA-A Antigens genetics, Humans, Racial Groups genetics, Sensitivity and Specificity, DNA Probes, HLA genetics, HLA-A Antigens analysis, Histocompatibility Testing methods, Polymerase Chain Reaction methods, Serologic Tests
- Abstract
We are reporting the results of HLA-A typing by PCR-SSOP complemented by PCR-SSP of samples obtained from the National Marrow Donor Program (NMDP). These samples were a representative group from 2486 tested in duplicate by serology. A total of 390 samples gave HLA-A discrepant results. Comparing the molecular typing results of 238 samples (samples with available DNA) with the serological typing results, 54 homozygotes and 184 heterozygotes produced a total of 422 assignments by molecular methods. We found assignment discrepancies in 147/422 (35%) in laboratory 1 and 144/422 (34%) in laboratory 2 (a combined group of 4 NMDP laboratories; laboratory 1 is not included). The serological discrepancies found were of 3 categories: a) false negatives, b) incomplete typing (discrepancies due to the level of resolution within a cross-reactive or CREG group) and c) false positives. Major problems were identified using serology for typing HLA-A antigens: a) inability to identify all WHO-recognized specificities, more frequently in non-Caucasians or in HLA-A specificities known to be found more frequently in non-Caucasians for laboratory 1 and incorrect assignments of A19 specificities in laboratory 2, b) incorrect assignments in cells with poor viability and c) false-positive assignments in homozygotes. We propose a possible strategy to type HLA-A specificities with two steps: a) a minimum of serology for typing specificities for common CREG groups: A1, A2, A3, A11, A9, A10, A28, A19. However, a given laboratory can determine the level of serological assignments needed as a first step. And b) molecular methods to identify splits: A23, A24, A29, A30, A31, A32, A33, A34, A36, A66, A74 and A80. The technique described is useful for large-scale bone marrow donor typings for cells with poor viability, and for resolving ambiguous results including false-positive assignments of homozygous cells.
- Published
- 1997
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17. Comparison of typing results by serology and polymerase chain reaction with sequence-specific primers for HLA-Cw in 650 individuals.
- Author
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Mytilineos J, Christ U, Lempert M, and Opelz G
- Subjects
- Alleles, Blood Donors, Diagnostic Errors, Evaluation Studies as Topic, Gene Frequency, Genotype, HLA-C Antigens genetics, Humans, Kidney Transplantation immunology, Phenotype, Reagent Kits, Diagnostic, Sensitivity and Specificity, DNA Primers genetics, DNA Probes, HLA genetics, Genes, MHC Class I, HLA-C Antigens analysis, Histocompatibility Testing methods, Polymerase Chain Reaction methods, Serologic Tests
- Abstract
HLA-Cw typing by standard serological techniques is associated with a high frequency of blanks, and reliable typing reagents for several of the Cw specificities are scarce. We evaluated the PCR-SSP technique for Cw typing in 370 kidney transplant patients and 280 healthy blood donors. Serological typing of all individuals was performed in our laboratory from 1995 to 1997 using commercially available tissue-typing trays. Comparison between serological and PCR-SSP typing revealed a discrepancy rate of 33.6% (n= 94) in blood donors and 32.4%) (n=120) in kidney recipients. Incorrect antigen assignments occurred only rarely (3.6% of the blood donors and 3.2% of the kidney recipients). The vast majority of discrepancies were due to antigens that were not detected serologically. In 26 individuals no Cw antigen was detected by serological typing, whereas PCR-SSP showed 1 allele in 13 and 2 alleles in the other 13 cases. Another 269 individuals were typed serologically with one blank (presumably homozygous). Of these, only 108 were confirmed to be homozygous, whereas an additional Cw allele was found in the remaining 161 cases using the SSP technique. Most of the "missed" specificities (86.5%) were those for which serological reagents were not available (HLA-Cw*12-*17). The most commonly "missed" specificity was HLA-Cw*1203, which occurred in 13.9% of the healthy blood donors. These results indicate that serological HLA-Cw typing is insufficient for examining the clinical importance of HLA-Cw matching in transplantation. Future studies based on molecular typing should allow the proper investigation of HLA-Cw matching in kidney and bone marrow transplantation.
- Published
- 1997
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18. Error rate for HLA-B antigen assignment by serology: implications for proficiency testing and utilization of DNA-based typing methods.
- Author
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Bozón MV, Delgado JC, Selvakumar A, Clavijo OP, Salazar M, Ohashi M, Alosco SM, Russell J, Yu N, Dupont B, and Yunis EJ
- Subjects
- DNA Primers genetics, Evaluation Studies as Topic, Genotype, HLA-B Antigens genetics, Histocompatibility Testing statistics & numerical data, Humans, Racial Groups genetics, Reagent Kits, Diagnostic, Sensitivity and Specificity, DNA genetics, DNA Probes, HLA genetics, Diagnostic Errors, Genes, MHC Class I, HLA-B Antigens analysis, Histocompatibility Testing methods, Polymerase Chain Reaction methods, Sequence Analysis, DNA, Serologic Tests statistics & numerical data
- Abstract
Until recently, the majority of HLA class I typing has been performed by serology. Expensive commercial typing trays are frequently used for testing non-Caucasian subjects and new strategies using DNA-based methods have been adopted for improving clinical histocompatibility testing results and adapted as supplements in proficiency testing. A double-blind comparison of the typing of HLA-B specificities in 40 samples was carried out between serology and two polymerase chain reaction (PCR) methods, PCR amplification with sequence-specific primers (PCR-SSP) and PCR amplification and subsequent hybridization with sequence-specific oligonucleotide probes (PCR-SSOP). The results demonstrated 22.5% misassignments of HLA-B antigens by serology. There was complete concordance between the results obtained with the two PCR based typing methods. A second panel of 20 donor samples with incomplete or ambiguous serologic results was analyzed by PCR-SSP and SSOP Both PCR methods identified correctly the HLA-B antigens. Our results suggest that more accurate typing results can be achieved by complementing serologic testing with DNA-based typing techniques. The level of resolution for HLA-B antigen assignment can be obtained by this combination of serology and limited DNA-based typing is equivalent to the HLA-B specificities defined by the WHO-HLA Committee. This level of resolution cannot routinely be achieved in clinical histocompatibility testing or in proficiency testing using serologic reagents only.
- Published
- 1997
- Full Text
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19. Analysis of HLA-A and -B serologic typing of bone marrow registry donors using polymerase chain reaction with sequence-specific oligonucleotide probes and DNA sequencing.
- Author
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Sintasath DM, Bei M, Steiner N, Ng J, Alosco S, Hegland JD, and Hurley CK
- Subjects
- Diagnostic Errors, Evaluation Studies as Topic, Genes, MHC Class I, HLA-B Antigens genetics, Humans, Registries, Sensitivity and Specificity, Sequence Alignment, Sequence Homology, Serologic Tests, Bone Marrow Transplantation immunology, DNA Probes, HLA genetics, HLA-A Antigens analysis, HLA-B Antigens analysis, Histocompatibility Testing methods, Polymerase Chain Reaction, Sequence Analysis, DNA, Tissue Donors, Tissue and Organ Procurement methods
- Abstract
Unrelated volunteer donors (69) recruited by the National Marrow Donor Program were HLA typed by DNA-based methods for both the HLA-A and -B loci. Each donor had been previously typed by serology by at least two independent laboratories. Of the 69 samples, all serologic laboratories were in concordance for HLA-A in 62 typed samples and for HLA-B in 48 typed samples. Of the serologically concordant samples, 5 samples typed for HLA-A and 7 samples typed for HLA-B received DNA and serology types differing in their level of resolution. One sample typed for HLA-A and 3 samples typed for HLA-B by DNA methods gave different results from their serologic assignments. Of the samples exhibiting disparities among the different serologic typing laboratories, the DNA-defined types of 7 samples typed for HLA-A and 18 samples typed for HLA-B were consistent with at least one of the serologic assignments. The DNA types for the remaining 3 HLA-B typed samples did not agree with the serologic assignments and their alleles were subsequently sequenced. One of these sequences was a previously undefined allele, B*1537. Sharing of polymorphic sequences among HLA allelic products creates difficulties for consistent serologic assignments of some types complicating the process of identifying potential donors from bone marrow registries. Thus, the use of DNA-based typing techniques for characterization of donor class I types should allow a more consistent definition of types and should speed the donor selection process.
- Published
- 1997
- Full Text
- View/download PDF
20. Sequence of a new HLA-DR allele, DRB5*0106.
- Author
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Kervaire B and Tiercy JM
- Subjects
- Alleles, Amino Acid Sequence, Base Sequence, Bone Marrow Cells, DNA isolation & purification, DNA Probes, HLA genetics, Exons, Female, Histocompatibility Testing, Humans, Molecular Sequence Data, Polymerase Chain Reaction, Sequence Analysis, DNA, Switzerland, White People genetics, HLA-DR Antigens genetics
- Abstract
We report here the exon 2 sequence of a new HLA-DRB5 allele, DRB5*0106, that was identified in two volunteer bone marrow donors from the Swiss national registry. This new allele differs from DRB5*0101 by five amino acids at positions 67, 70, 71, 85 and 86. It is associated with DRB1*1501 and DQB1*0602. This unusual DRB1*1501-DRB5*0106 association increases the complexity of the DR2 group, although it appears to be very rare, at least in our population. HLA-DRB5*0106 can be readily detected upon DR generic oligotyping, provided the two probes that mark the major DRB5 subtypes, DRB5*0101 and DRB5*02, respectively, are included in the assay.
- Published
- 1997
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21. Bilateral simultaneous optic neuropathy in adults: clinical, imaging, serological, and genetic studies.
- Author
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Morrissey SP, Borruat FX, Miller DH, Moseley IF, Sweeney MG, Govan GG, Kelly MA, Francis DA, Harding AE, and McDonald WI
- Subjects
- Adolescent, Adult, Aged, Alleles, Brain physiopathology, DNA Probes, HLA genetics, DNA, Mitochondrial genetics, Female, Follow-Up Studies, Genome, Human, Haplotypes, Humans, Immunoglobulin G cerebrospinal fluid, Magnetic Resonance Imaging, Male, Middle Aged, Multiple Sclerosis diagnosis, Multiple Sclerosis genetics, Mutagenesis, Optic Atrophies, Hereditary diagnosis, Point Mutation genetics, Polymerase Chain Reaction, Functional Laterality, Optic Atrophies, Hereditary genetics, Optic Atrophies, Hereditary physiopathology
- Abstract
To elucidate the cause(s) of acute or subacute bilateral simultaneous optic neuropathy (BSON) in adult life, a follow up study of 23 patients was performed with clinical assessment, brain MRI, HLA typing, and mitochondrial DNA analysis. The results of CSF electrophoresis were available from previous investigations in 11 patients. At follow up, five (22%) had developed clinically definite multiple sclerosis, four (17%) had mitochondrial DNA point mutations indicating a diagnosis of Leber's hereditary optic neuropathy (LHON). The remaining 14 patients (61%) still had clinically isolated BSON a mean of 50 months after the onset of visual symptoms: three of 14 (21%) had multiple MRI white matter lesions compatible with multiple sclerosis, three of 14 (21%) had the multiple sclerosis associated HLA-DR15/DQw6 haplotype, and one of seven tested had CSF oligoclonal IgG bands; in total only five (36%) had one or more of these risk factors. The low frequency of risk factors for the development of multiple sclerosis in these 14 patients suggests that few will develop multiple sclerosis with more prolonged follow up. It is concluded that: (a) about 20% of cases of BSON without affected relatives are due to LHON; (b) multiple sclerosis develops after BSON in at least 20% of cases, but the long term conversion rate is likely to be considerably less than the rate of over 70% seen after an episode of acute unilateral optic neuritis in adult life.
- Published
- 1995
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22. Quantitation of residual white cells in filtered blood components by polymerase chain reaction amplification of HLA DQ-A DNA.
- Author
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Lee TH, Stromberg RR, Heitman J, Tran K, and Busch MP
- Subjects
- Blood Specimen Collection methods, DNA Probes, HLA genetics, Evaluation Studies as Topic, Gene Amplification, Humans, Polymerase Chain Reaction, Reproducibility of Results, Blood Component Removal methods, Cell Separation methods, DNA Probes, HLA analysis, HLA-A Antigens genetics, HLA-DQ Antigens genetics, Leukocytes cytology
- Abstract
Background: Over the past several years, blood filtration technology has improved dramatically, such that currently available experimental filters are capable of reducing white cells (WBCs) in blood components to less than 0.1 WBC per microL. These residual WBC concentrations are below the sensitivity of automated cell counters, as well as of large-volume (Nageotte) hemocytometers., Study Design and Methods: A quantitative polymerase chain reaction (PCR) amplification assay directed at HLA DQ-A DNA sequences has been developed for the enumeration of WBCs in filtered blood. To ensure quantitative recovery of WBCs at very low residual cell concentrations, a direct red cell lysis and WBC concentration protocol using 0.5 mL of filtered blood was perfected. Amplified product is detected by oligomer hybridization using 32P-labeled probes, with quantitation by image analysis of autoradiographic signals relative to a standardized dilution series processed in parallel., Results: Recovery of residual WBCs in filtrates was shown to be enhanced by the addition of xenogeneic WBCs or polystyrene beads, which served as "carrier" particles during red cell lysis and wash steps. A contribution of nuclear fragments in filtered blood to PCR signal in the range of 0.01 to 0.5 WBCs per microL was observed; a modified protocol was developed to minimize this effect. Parallel analysis of spiked dilution series and evaluations of 39 red cell components filtered through commercial filters indicated good correlation between PCR and standard Nageotte counts in the range of 0.1 to 10 WBCs per microL (r2 = 0.94); only PCR was able to detect residual WBCs in filtrates from prototype 6 log10 WBC-reduction filters., Conclusion: This assay should prove useful for the development and quality assurance of increasingly efficient WBC-reduction filters.
- Published
- 1994
- Full Text
- View/download PDF
23. HLA-DR polymorphism in a Senegalese Mandenka population: DNA oligotyping and population genetics of DRB1 specificities.
- Author
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Tiercy JM, Sanchez-Mazas A, Excoffier L, Shi-Isaac X, Jeannet M, Mach B, and Langaney A
- Subjects
- Base Sequence, HLA-DRB1 Chains, Heterozygote, Humans, Mathematics, Molecular Sequence Data, Polymerase Chain Reaction, Senegal, DNA Probes, HLA genetics, Gene Frequency genetics, Genes, MHC Class II, HLA-DR Antigens genetics, Histocompatibility Antigens Class II genetics, Polymorphism, Genetic genetics
- Abstract
HLA class II loci are useful markers in human population genetics, because they are extremely variable and because new molecular techniques allow large-scale analysis of DNA allele frequencies. Direct DNA typing by hybridization with sequence-specific oligonucleotide probes (HLA oligotyping) after enzymatic in vitro PCR amplification detects HLA allelic polymorphisms for all class II loci. A detailed HLA-DR oligotyping analysis of 191 individuals from a geographically, culturally, and genetically well-defined western African population, the Mandenkalu, reveals a high degree of polymorphism, with at least 24 alleles and a heterozygosity level of .884 for the DRB1 locus. The allele DRB1*1304, defined by DNA sequencing of the DRB1 first-domain exon, is the most frequent allele (27.1%). It accounts for an unusually high DR13 frequency, which is nevertheless within the neutral frequency range. The next most frequent specificities are DR11, DR3, and DR8. Among DRB3-encoded alleles, DR52b (DRB3*02) represents as much as 80.7% of all DR52 haplotypes. A survey of HLA-DR specificities in populations from different continents shows a significant positive correlation between genetic and geographic differentiation patterns. A homozygosity test for selective neutrality of DR specificities is not significant for the Mandenka population but is rejected for 20 of 24 populations. Observed high heterozygosity levels in tested populations are compatible with an overdominant model with a small selective advantage for heterozygotes.
- Published
- 1992
24. A method for using serum or plasma as a source of DNA for HLA typing.
- Author
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Martin M, Carrington M, and Mann D
- Subjects
- Base Sequence, Blood, DNA isolation & purification, DNA Probes, HLA genetics, Humans, Molecular Sequence Data, Oligonucleotides genetics, DNA blood, Genes, MHC Class II genetics, HLA-D Antigens genetics, Polymerase Chain Reaction methods
- Abstract
A simple method for obtaining DNA from serum and plasma is described. Using appropriate primer pairs the polymorphic segments of HLA class II genes were amplified by the polymerase chain reaction (PCR) from this DNA, and typed using allele-specific oligonucleotide hybridization. When compared with DNA obtained from peripheral blood lymphocytes, the efficiency of the PCR was only minimally compromised and could be augmented by increasing the number of amplification cycles and/or by the addition of glycerol to the reaction mixture. This method serves as a reasonable alternative when no other source of DNA is available.
- Published
- 1992
- Full Text
- View/download PDF
25. Extensive study of DRB, DQA, and DQB gene polymorphism in 23 DR2-positive, insulin-dependent diabetes mellitus patients.
- Author
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Zeliszewski D, Tiercy JM, Boitard C, Gu XF, Loche M, Krishnamoorthy R, Simonney N, Elion J, Bach JF, and Mach B
- Subjects
- DNA Probes, HLA genetics, HLA-DR1 Antigen genetics, Humans, Polymerase Chain Reaction, Recombination, Genetic genetics, Diabetes Mellitus, Type 1 genetics, HLA-DQ Antigens genetics, HLA-DR2 Antigen genetics, HLA-DR5 Antigen genetics, Polymorphism, Restriction Fragment Length
- Abstract
To gain insight into the HLA subregions involved in protection against insulin-dependent diabetes mellitus (IDDM) we investigated the polymorphism of HLA-DR and -DQ genes in 23 DR2 IDDM patients. Results show the following. (1) Fourteen patients (61%) possess the DRB1, DRB5, and DQB1 alleles found in DRw16/DQw5 healthy people. These data contrast with the 5% of DRw16 normally found in DR2 populations and are in agreement with former observations supporting that the DRw16 haplotype is not protective. (2) Nine DR2 patients, i.e., 39% versus 95% in published DR2 controls, possess the DRB alleles found in DRw15 unaffected people. Among them, six patients have also DQA1 and DQB1 alleles identical to those found in DRw15/DQw6 healthy individuals. These data confirm that the DRw15/DQw6 haplotype is protective but indicate that none of the DR or DQ alleles, alone or in association, confers an absolute protection. (3) Our most striking results concern the very high frequency of recombinant haplotypes among the DRw15 patients: 3 of 9. In these three patients recombinations led to the elimination of both DQB1 and DQA1 alleles usually associated with DRw15. This strongly suggests that the occurrence of IDDM in these DRw15 patients is due to the absence of the usual DQ product and thus reinforces the assumption that DQ rather than DR region is involved in the protection conferred by the DRw15/DQw6 haplotype. Finally, analysis of the non-DRw15 haplotypes in heterozygous patients showed that IDDM can occur in the absence of any DQ alpha beta heterodimer of susceptibility.
- Published
- 1992
- Full Text
- View/download PDF
26. A study of Italian pediatric celiac disease patients confirms that the primary HLA association is to the DQ(alpha 1*0501, beta 1*0201) heterodimer.
- Author
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Mazzilli MC, Ferrante P, Mariani P, Martone E, Petronzelli F, Triglione P, and Bonamico M
- Subjects
- Base Sequence, Celiac Disease genetics, Child, Child, Preschool, DNA Probes, HLA genetics, HLA-DQ Antigens immunology, HLA-DR Antigens genetics, HLA-DR Antigens immunology, Humans, Italy, Molecular Sequence Data, Oligodeoxyribonucleotides genetics, Polymerase Chain Reaction, Risk, Celiac Disease immunology, HLA-DQ Antigens genetics
- Abstract
Celiac disease (CD) has been recently reported to be primarily associated with the DQ(alpha 1*0501, beta 1*0201) heterodimer encoded in cis on DR3 haplotype and in trans in DR5,7 heterozygous individuals. The high incidence of DR5,7 heterozygotes, reflecting the high frequency of the DR5 allele in Italy, makes the analysis of the Italian CD patients critical. Polymerase chain reaction-amplified DNA from 50 CD patients and 50 controls, serologically typed for DR and DQw antigens, was hybridized with five DQA1-specific oligonucleotide probes detecting DQA1*0101 + 0102 + 0103, DQA1*0201, DQA1*0301 + 0302, DQA1*0401 + 0501 + 0601, and DQA1*0501 and a DQB1-sequence-specific oligonucleotide probe recognizing DQB1*0201 allele. As expected by the DR-DQ disequilibria, DQA1*0201 [62% in patients versus 26% in controls, relative risk (RR) = 5] and DQA1*0501 (96% versus 56%, RR = 19) show positive association with the disease. Of CD patients, 92% (50% DR3 and 42% DR5,7) compared to 18% of the controls carry both DQA1*0501 and DQB1*0201 alleles, so that the combination confers an RR of 52, higher than both the risks of the single alleles (DQA1*0501 RR = 19, DQB1*0201 RR = 30), confirming the primary role of the dimer in determining genetic predisposition to CD both in DR3 and in DR5,7 subjects.
- Published
- 1992
- Full Text
- View/download PDF
27. Polymorphism and distribution of HLA-DR2 alleles in Romanians.
- Author
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Lupu F, Reed E, McManus P, and Suciu-Foca N
- Subjects
- Alleles, Base Sequence, DNA Probes, HLA genetics, HLA-DRB1 Chains, HLA-DRB5 Chains, Humans, Molecular Sequence Data, Romania, White People genetics, Genes, MHC Class II, HLA-DR Antigens genetics, Histocompatibility Antigens Class II genetics, Polymorphism, Genetic genetics
- Published
- 1992
- Full Text
- View/download PDF
28. DPB1 polymorphism in rheumatoid arthritis: evidence of an association with allele DPB1 0401.
- Author
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Perdriger A, Semana G, Quillivic F, Chalès G, Chardevel F, Legrand E, Meadeb J, Fauchet R, and Pawlotsky Y
- Subjects
- Adult, Aged, DNA Probes, HLA genetics, Female, Gene Frequency, HLA-DP beta-Chains, Humans, Male, Middle Aged, Polymerase Chain Reaction, Risk, White People genetics, Arthritis, Rheumatoid genetics, Genes, MHC Class II, HLA-DP Antigens genetics, Polymorphism, Genetic genetics
- Abstract
HLA-DP polymorphism was examined in 71 rheumatoid arthritis patients and 148 controls, using dot-blot analysis with 14 synthetic oligonucleotide probes specific for the variable region of the DPB1 second exon. The DPB1 0401 allele was found to be significantly more frequent in RA patients than in controls (77.46% vs 55.40%, p less than 0.002, pc less than 0.03, relative risk value: 2.74). An association between DPB1 0401 and seropositivity for rhumatoid factors was also observed: 44 of the 55 seropositive RA patients were DPB1 0401 (p less than 0.001). Analyzing the HLA DPB1 alleles frequencies in 57 HLA-DR-typed RA patients did not show any linkage between the DPB1 0401 and the DR4 specificities. Furthermore, the DPB1 0401 homozygous frequency was increased in DR4-negative RA patients. Our findings suggest an independent role of the DPB1 0401 allele in the genetic susceptibility to RA.
- Published
- 1992
- Full Text
- View/download PDF
29. Strong linkage disequilibrium of HLA DPw11 with the HLA B44-DR7-DQw2 extended haplotype.
- Author
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Bignon JD, Cheneau ML, Herry P, Bonneville F, Cesbron A, and Muller JY
- Subjects
- DNA Probes, HLA genetics, Female, HLA-B44 Antigen, Haplotypes, Humans, Male, Polymerase Chain Reaction, White People genetics, Genes, MHC Class II, HLA-B Antigens genetics, HLA-DP Antigens genetics, HLA-DQ Antigens genetics, HLA-DR7 Antigen genetics, Linkage Disequilibrium genetics
- Published
- 1992
- Full Text
- View/download PDF
30. Oligotyping of Italian celiac patients with the 11th International Histocompatibility Workshop reagents.
- Author
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Ferrante P, Petronzelli F, Mariani P, Bonamico M, and Mazzilli MC
- Subjects
- Child, Child, Preschool, DNA Probes, HLA genetics, HLA-DQ alpha-Chains, HLA-DQ beta-Chains, HLA-DRB1 Chains, Humans, Italy, Polymerase Chain Reaction, Celiac Disease genetics, Genes, MHC Class II, HLA-DQ Antigens genetics, HLA-DR Antigens genetics, Histocompatibility Antigens Class II genetics
- Abstract
Oligotyping for HLA-DRB1, DQA1 and DQB1 specificities has been performed on PCR-amplified DNA from 55 Italian celiac children, living in Rome, and 50 blood donors. 52.6% of CD patients were DR3;DQA1*0501;DQB1*0201-positive versus 14% of controls (RR = 6.85) and 34.5% were DR5,7;DQA1*DQB1*0201-positive versus 2% of controls (RR = 25.86). 7 patients (12.7%) were negative for the DQA1*0501/B1*0201 dimer: 3 of them were DR4 (5.4%) and the others typed as DR1,5; 1,7; 5,7 and w6,7. No patient was negative for both DQA1*0501 and DQB1*0201 alleles.
- Published
- 1992
- Full Text
- View/download PDF
31. Analysis of HLA class II allogenotyping in Australian aborigines and Papua New Guinea populations.
- Author
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Trejaut J, Dunckley H, Sullivan J, Kennedy C, and Crane G
- Subjects
- Asian People genetics, Australia, Black People genetics, DNA Probes, HLA genetics, Gene Frequency, Genetic Linkage, Humans, Papua New Guinea, Polymorphism, Restriction Fragment Length, Genes, MHC Class II, HLA-DQ Antigens genetics, HLA-DR Antigens genetics, Native Hawaiian or Other Pacific Islander genetics, Racial Groups genetics, White People genetics
- Abstract
Human leukocyte antigen (HLA) class II allogenotyping has been applied to investigate the polymorphism of the DRB, DQB1, DQB2, DQA1, and DQA2 genes in Aborigines from the East Coast of Australia and in Melanesians from the Papua New Guinea North-East Coast and Highlands. Three new DR/DQ arrangements were observed, DRw14/DQB1-2b/DQA1-1a and DRw5Nauru/DQB1-3a/DQA1-2 (n Australian Aborigines), and DRw5Nauru/DQB1-1a/DQA1-1b (in Madang). DQA2 and DQB2 allogenotyping with TaqI and PstI digested genomic DNA revealed little polymorphism among the Papua New Guineans, with DQA2-Xa1 and DQB2-Xb1 the most common alleles in all the groups. However, the presence of DQA2-Xa2 in Papuans and Australian Aborigines reflects the degree of admixture with Caucasoids while the DQA2-Xa4 allele in Madang is probably a marker of Mongoloid origin.
- Published
- 1992
- Full Text
- View/download PDF
32. Polymorphism of HLA in the Romanian population.
- Author
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Reed E, Ho E, Lupu F, McManus P, Vasilescu R, Foca-Rodi A, and Suciu-Foca N
- Subjects
- Base Sequence, DNA Probes, HLA genetics, Gene Frequency, HLA Antigens genetics, Humans, Immunophenotyping, Linkage Disequilibrium genetics, Molecular Sequence Data, Polymerase Chain Reaction, Romania ethnology, United States, Genes, MHC Class I, Genes, MHC Class II, Polymorphism, Genetic genetics
- Abstract
We have investigated the HLA-class I and class II polymorphism in a population of 83 Romanians using conventional serology together with PCR amplification and oligonucleotide typing of HLA-class II genes. Romanians show a higher frequency of HLA-A11, B13, B18, B37, B39, B51 and DR2 than other European populations. HLA-DRB1*1501 and 1601 account for the high frequency of the serologic specificity DR2. In Romanians, HLA-DR2 is in linkage disequilibrium with HLA-B18 and HLA-Bw52 rather than with HLA-B7 as in the case in other Europeans. Unexpected HLA-DR2 haplotypes include HLA-DRB1*1502, DQA1*0102, DQB1*0601; HLA-DRB1*1602, DQA1*0102, DQB1*0502. Other unusual haplotypes include HLA-DRB1*0405, DQA1*03, DQB1*0302; HLA-DRB1*1305, DQA1*0103, DQB1*0603; and HLA-DRB1*1405, DQA1*0101, DQB1*05032. Analysis of the genetic distance between Romanians and other Europeans who have been studied serologically are consistent with the hypothesis that Romanians descend from Roman ancestors who colonized Dacia between the 1st century B.C. and 1st century A.D.
- Published
- 1992
- Full Text
- View/download PDF
33. Alloantisera can detect some of the variants of HLA DRw13 identified by oligotyping.
- Author
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Gebuhrer L, Lambert J, Labonne MP, Mollet I, Freidel AC, and Betuel H
- Subjects
- Amino Acid Sequence, DNA Probes, HLA genetics, HLA-DQ Antigens genetics, HLA-DQ Antigens immunology, HLA-DR Antigens genetics, HLA-DR Serological Subtypes, Haplotypes, Humans, Molecular Sequence Data, Polymerase Chain Reaction, White People genetics, Genes, MHC Class II, HLA-DR Antigens immunology, Immune Sera immunology, Immunophenotyping methods
- Abstract
Oligotyping has revealed a considerable polymorphism of HLA DRw13; so far, 5 alleles coded by DRB1 have been identified. An even greater number of haplotypes are apparent when considering the association of DRw13 with alleles coded by DRB3 and DQB1. Serology can now define some of these variants which had formerly escaped detection. We have tested by serology and by oligoprobes 66 genotyped Caucasoid cells comprising the known DR and DQ alleles. Among the 28 DRw6 cells, 10 different haplotype patterns were selected, of which 8 concern DRw13 and 2 DRw14. Four variants of DRw13 were represented: DRB1 *1301, *1302, *1303 and *1305 associated with usual and with uncommon alleles belonging to DRB3 and to DQB1. We have centered our analysis on 19 sera of the XIth Workshop defining DRw6. Two sera (639, 826) were noteworthy since they reacted with certain subtypes of DRw13 and with DRB1 *1102, *1103 only, the other DR specificities remaining negative.
- Published
- 1992
- Full Text
- View/download PDF
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