摘要:

Abstract:We have identified locus‐specific sequences in the first and third introns flanking the polymorphic second and third exons of HLA class I genes. PCR primers derived from these conserved sequences produced DNA fragments of the expected sizes for each of the HLA‐A, ‐B, and ‐C loci in the amplification of genomic DNA. PCR products generated using each of the locus‐specific sets of primers displayed exquisite locus specificity, as assessed by hybridization with oligonucleotide probes specific for ten classical and non‐classical HLA class I genes. Amplification with these primer sets was effective and specific for the HLA alleles tested under the given PCR conditions. When hybridized with oligonucleotides derived from shared polymorphic sequence motifs, reaction patterns of PCR products from each locus were precisely as expected from published or database sequences. Chemiluminescent signals generated from digoxygenin‐ddUTP‐labeled probes were even for all samples and as strong as those obtained in MHC class II typing. These locus‐specific primer sets derived from intron sequences provide an effective means to amplify genomic DNA which will facilitate PCR‐based HLA class I typing methods. This will also allow HLA class I typing to be conducted with greater precision, at lower cost, and faster than previously described class I t

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02408.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Traditional methods of serological typing have largely used antisera of Caucasoid origin, which can overlook HLA heterogeneity in non‐Caucasoid populations. Therefore, we have used molecular techniques to evaluate potential polymorphism in HLA class I molecules of Aborigines from the central desert and northern coast of Australia. The DNA sequence of common Aboriginal HLA‐A and B antigens were compared with serological reaction patterns which suggested new polymorphisms. Although serological data indicated that long and short variants of A34 may exist, regardless of the serological pattern, all individuals carried the A*3401 allele. Therefore, the variation in A34 reaction pattern observed serologically was not attributable to primary sequence variation in the HLA A*3401 allele. Similarly, there was no detectable polymorphism in the sequences of selected HLA‐B alleles, even though some of these alleles showed unusual serological reaction patterns. However, a new allele of B15 (B*1521) was detected in two individuals carrying this serotype. The cells from both of these individuals showed ambiguous reaction patterns with monospecific B62 and B75 sera. cDNA sequencing of the HLA B15 gene from these cells revealed a B15 allele that differed from B*1502 by a single nucleotide change. This change occurred at position 272, resulting in a C to G substitution at residue 67 in the consensus B15 cDNA sequence. Hence, the Australian Aborigines as an ethnic group show very little primary sequence polymorphism within the class I loci, consistent with the results obtained from previous serological st

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02409.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The ST‐16 antigenic specificity of the HLA‐B locus is defined as a B39 variant of Mexican‐Americans. Nucleotide sequencing of cDNA shows the ST‐16 allele (B*3905) differs from B*39011 by a single substitution that substitutes tyrosine for aspartic acid at position 74 of the mature class I heavy chain. The complete coding region sequence for the common caucasoid allele encoding the B38 antigen has been determined. This B*3801 allele differs from B*3802 at two nucleotide substitutions within the Bw4 sequence motif. B*3801 and B*3802 may have been derived independently from B*39011 by conversion events with B alleles donating distinctive Bw4 motifs. A novel allele B*39022 derived from a Colombian Indian differs from the B*39021 allele of Japanese origin at two widely separated silent substitutions. Comparison of sequences for the known B16 alleles suggest that B*39021 and B*39022 were independently derived by recombination from B*39013 and B*39011 respe

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02410.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:We have established a DNA typing system for the HLA‐B5 serologically cross‐reactive group (CREG) by means of a two‐step PCR amplification with nested sequence‐specific primers (nPCR‐SSP). The present study provides a low resolution definition of the HLA‐B5 CREG, i.e. identifying polymorphism equivalent to serology. Two different primer combinations allow group‐specific amplification of all HLA‐B5 CREG alleles and other related HLA class I alleles from genomic DNA. The amplified DNA is subjected to a second amplification step using eleven nested primer pairs. This assay permits the detection of the HLA‐B5 CREG specificities B35, B51, B52, B53, and B7801 in all homozygous and heterozygous combinations. Sensitivity and specificity as judged by a blind quality control study investigating a reference panel (n=50) is 100%. Extension of this approach should allow rapid DNA typing of all serologically defined HLA‐B specif

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02411.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:In this study, serological HLA‐DR and ‐DQ typing results were compared to typing results obtained with sequence‐specific primers in the polymerase chain reaction (PCR‐SSP). HLA‐DR typing was performed on a random Caucasian population consisting of 31 patients and 73 healthy individuals. Considering HLA‐DR1‐10, differences in typing results were found in 3 out of 73 healthy individuals and 8 out of 31 patients. When HLA‐DR1‐16 alleles were taken into account, differences in typing results were found in 11 out of 31 patients and 14 out of 73 healthy individuals. Typing results of PCR‐SSP, different from that of serology, were all confirmed by sequencing‐based typing of HLA‐DRB1 alleles. HLA‐DQ 1–3 typings were performed on 40 individuals consisting of 17 patients and 23 healthy individuals. Differences in typing results were found in 5 out of 17 patients and 1 out of 23 healthy individuals. From the results of this study it can be concluded that serology is a reliable technique, when restricted to identification of HLA‐DR1‐10 and HLA‐DQ1‐3 antigens in healthy individuals. By PCR‐SSP, however, reliable HLA‐DR1‐16 and ‐DQ1‐3 typings can be obta

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02412.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:An HLA‐DRB1 typing procedure by means of sequence‐specific primer (SSP) amplification was developed for 65 different DRB1 subtypes. Subtyping is achieved by the performance of two subsequent PCR assays (PCR‐1 assay and PCR‐2 assay) using a limited number of reactions. The PCR‐1 assay determined low‐resolution HLA‐DRB1 typing, i.e. the serologically defined specificities DR1, 2, 3, 4, 11, 12, 6, 7, 8, 9 and 10. The second exon of the DRB1 gene is amplified also in this PCR‐1 assay. High‐resolution subtyping for positively identified alleles was performed in the PCR‐2 assay with the exon‐2 product from PCR‐1 assay as DNA template. PCR reactions were carried out using unpurified primers in reaction volumes of 20 μ1 and 100 ng of chromosomal DNA. After 3 hours, the results of the PCR‐1 assay were analyzed and subsequently subtyping results in the PCR‐2 assay were obtained in another 1.5 hours. A total of 249 DNA samples was typed by this method. No false positive nor false negative results were obtained in DRB1 typing of 32 homozygous cell lines, 56 serologically well‐defined panel cells and 125 unrelated individuals. Segregation of the amplification patterns was investigated in 36 members of 7 two‐generation families. DRB1 subtyping revealed codominant Mendelian segregation for all subtypes investigated. In conclusion, LR‐HR‐PCR‐SSP typing is a fast and reliable typing technique for routine DNA typing purposes which gives complete DRB1 subtyping within 4.5 h. Besides low‐resolution DRB typing, also high‐resolution DRB subtyping for prospective HLA‐DR matching in cadaveric rena

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02413.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Sixty‐five living related kidney transplant pairs were analyzed for matching at HLA class II loci by the polymerase chain reaction (PCR)‐ sequence specific oligonucleotide probe (SSOP) method. The retrospective HLA matching study revealed that there were many early graft loss cases despite the DQB compatibility, contrary to our expectation. There were 54 DRB1 one‐mismatched cases, in which 7 of the 11 (64%) DQB zero‐mismatched cases had lost their grafts, while the graft loss cases were only 10 of the 43 (23%) DQB one‐mismatched pairs (P value=0.0006). The DQB matching of these cases was studied in detail, because the PCR‐SSOP methods are based on the detection of sequence polymorphisms in a relatively narrow range, i.e., recognized sequences by SSOPs. The PCR ‐ heteroduplex ‐ polymorphism analysis method was developed to analyze the polymorphism in exon 2 of the DQB1 gene. However, all the pairs proved to be compatible for the DQB, demonstrating that the DQB compatibility was associated with a harmful influence on the graft outcome. These observations suggested that the DQB1 incompatibility might exert the low responsiveness to HLA haplo‐identical allog

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02414.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:An HLA‐DPA1 sequencing‐based typing (SBT) system has been developed to identify DPA1 alleles. Up to now eight DPA1 alleles have been defined. Six can be discriminated based upon exon 2 polymorphism. The three subtypes of DPA1*01: DPA1*0101, DPA1*0102 and DPA1*0103, have identical exon 2 sequences but show differences in exon 4. Exon 4 sequences were known for only the three DPA1*01 subtypes and for DPA1*0201. We now present additional sequence information for exon 4 and the unknown segments at the 3′ end of exon 2. Additionally with the use of this sequencing technique it is also possible to identify previously unidentified polymorphism. We have studied the exon 2 and exon 4 polymorphism of DPA1 in 40 samples which include all known DPA1 alleles. A new allele, DPA1*01 new, was identified which differs by one nucleotide in exon 2 from DPA1*0103, resulting in an aspartic acid at codon 28. The DPA1*01 subtypes DPA1*0101 and DPA1*0102 could not be confirmed in samples which previously were used to define these subtypes, and consequently they do not exist. The exon 4 sequence of DPA1*0201 is corrected based on sequence data of DAUDI, the cell line in which DPA1*0202 was originally defined. The exon 4 regions of the remaining four alleles were resolved: the exon 4 regions of the alleles DPA1*02021 and DPA1*02022 were found to be identical to the — corrected — DPA1*0201 whereas the exon 4 region of DPA1*0301 differs by one nucleotide compared to DPA1*0103. The DPA1*0401 exon 4 region differs by one nucleotide compared to the corrected DPA1*0201. As is found in other class II genes, all exons of DPA1 show some polymorphism, but the polymorphism in exon 2 is sufficient to identify the differen

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02415.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The polymorphism of HLA clas II genes (HLA‐DRB, DQB, DPB) was investigated in 101 Tunisians using polymerase chain reaction (PCR) amplification and reverse dot blot (RDB) hybridization. Allele and haplotype frequencies, as well as DRB1‐DQB1 linkage disequilibria, were calculated. A total of 26 DRB1 alleles were detected and the most prevalent variant was DRB1*0301 with an allelic frequency at 21.87%. In the DR1 group, DRB1*0102 was most frequent than DRB1*0101. In the DR4 group, DRB1*0403 was the most common allele and was associated with DQB1*0402. Interestingly this DRB 1‐DQB1 association has not been observed in other populations. With regard to the DR8 group, DRB1*0804 was the unique variant detected, whereas with the DR13 specificity, the most common variant was DRB1*1303 in Algerians also. Although the DQB1 polymorphism analysis showed an allelic distribution very close to that observed in caucasoids, many DRB1‐DQB1 associations which have not been reported in studies of other populations, were described. Finally at the DPB1 locus DPB1*1701 and *1301 allele frequencies distinguish clearly this Tunisian sample from a French caucasoïd panel of 83 subjects. In conclusion, a specific distribution of HLA components in terms of gene and haplotype frequencies characterizises this Tunisian population. This specific pattern may reflect the great ethnic diversity of this community. All these informations may be helpful in the future for HLA and disease association

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02416.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:To avoid the well‐known shortcomings of phenotyping granulocytes for the NA antigens using NA‐specific human sera, a DNA‐based method to determine the NA genotype was developed. Genomic DNA was isolated from blood cells or serum, amplified by polymerase chain reaction (PCR), immobilized on nylon membrane and genotyped using digoxigenin‐labeled, sequence‐specific oligonucleotides (SSO). The genotyping results of whole blood samples from 54 and of serum from 20 individuals correlated perfectly with our phenotyping using the antigen capture assay MAIGA. In three cases with the phenotype “NA‐null” no hybridization of the NA‐specific oligonucleotides occurred. These data show that SSO is a reliable method for NA genotyping especially if only small volumes of blood or even only serum pro

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02417.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY