摘要:

Abstract:We describe an approach for typing alleles of the HLA‐B locus by using automated sequencing technology. The exon 2 and exon 3 nucleotide sequence of each allele is determined directly from genomic DNA in two steps. In the first step, HLA‐B exon 2, intron 2 and exon 3 sequences are amplified with one or two primer pairs out of a panel of 5 primer pairs that describe all known HLA‐B alleles. In the second step, templates are sequenced in 5′ and 3′ orientations in a PCR assay that utilizes Taq polymerase to incorporate fluorescent dye‐labeled nucleotides into each new strand synthesized. Gel electrophoresis of the labeled products is performed in an automated DNA sequencer. The derived sequences are aligned against reference sequences and each nucleotide position is evaluated for homology to consensus sequence. Using this strategy, the HLA‐B allele sequence is directly ascertained with precision and efficiency. The automated sequencing strategy can be readily applied in the clinical laboratory as a practical tool for high resolution typing of

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02482.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR‐based HLA‐A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA‐A locus. The method is based on a sequence‐specific amplification identifying the serologically defined HLA‐A specificities. The PCR products generated by these group‐specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single‐strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1‐A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1‐A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid‐phase sequencing or SSCP analysis of the group‐specific PCR fragments allowed complete subtyping of the HLA‐A locus. This strategy can identify all 48 HLA‐A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA‐A locus. Only 4 out of these 1128 allele combi

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02483.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:In HLA Class II genes, polymorphism is mainly located in the second exon. Most DNA based typing methods are confined to the identification of specific sequence motifs in the second exon. In contrast, Sequencing Based Typing (SBT) elucidates the entire exon 2 sequence for typing. Comparison of the obtained exon 2 sequence with an allele sequence library results in allele assignment. We tested the applicability of SBT using a protocol for amplification followed by solid phase Taq‐cycle sequencing for HLA‐DPB1 typing. A panel of 32 samples were typed by SBT at five test sites which are participating in the Sequencing Based Typing component of the 12th International Histocompatibility Workshop. The panel represents the existing polymorphism at all known polymorphic positions of exon 2, both in homozygous and heterozygous combinations. In this multicenter study we focused on the reliability of analyzing heterozygous sequences for HLA typing. A multi‐sequence analysis approach, Polall, was developed to evaluate sequences obtained. The assignment of homozygosity and heterozygosity was validated by cluster analysis of chromatographic data of all sequences. Sequence characteristics were examined and considered for appropriate assignment. Differences in sequence characteristics that occurred between the test sites are considered in detail. The evaluation of data of 5 test sites reveals that Taq‐cycle sequencing can reliably be performed for HLA

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02484.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The Jewish people comprise two major groups, one encompassing the Jews of Ashkenazi (Central and Eastern European) origin and the other including those of Sephardic (Middle Eastern and North African) descent. To the latter belong the Jews of Moroccan stock, who form the largest Jewish subgroup among the non‐Ashkenazi population living in Israel. As the members of each of these groups differ in physiognomy and life style, it was of interest to investigate whether these differences are also reflected in their respective HLA compositions. To this end, 132 subjects of Ashkenazi and 113 individuals of Moroccan origin residing in Israel were tested and the results compared with data for other populations made available by the 11th International Histocompatibility Workshop. Comparison between their HLA profiles and those of non‐Jews revealed that the Jewish groups in some aspects resembled one another but in others showed disparities. The dissimilarities between the various groups are expressed in terms of gene and haplotype frequencies, as well as in HLA‐disease associations (as for example rheumatoid arthritis, erosive lichen planus, primary Sjögren's syndrome, pemphigus vulgaris). However, both Jewish groups shared some unique features with respect to HLA class II allelic frequencies, pointing to a common an

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02485.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The genetic polymorphism of the HLA class II loci was investigated in a Manchu population resident in the northern part of China and compared with those of other Asian populations including Japanese and Han. In 8 DQA1 alleles, the most frequent allele was DQA1*03 with the gene frequency of 25.5%. Of 15 DQB1 alleles tested, 11 were observed and the most common allele was DQB1*0301 with the gene frequency of 24.5%. Among 19 DPB1 alleles, 11 were detected and DPB1*0501 (43.8%) was the most frequent allele as observed in other Asian populations such as Japanese, Chinese and Korean. Of 43 DRB1 alleles tested, 21 were detected and DRB1*0901 (14.0%), *1501 (11.0%), *1201 (11.0%), *07 (9.0%) and *1401 (9.0%) were highly predominant and account for the high frequencies of DR9, DR2, DR5, DR7 and DR6. In the DRB3 gene (DR52), DRB3*0202 (18.0%) was the most frequent. With respect to the DRB4 gene (DR53), the gene frequency of DRB4*0101 was 35.0%. Of 3 DRB5 alleles detected, DRB5*0101 (11.0%) was highly predominant. Comparison of HLA class II allele frequencies in Manchu with those in Japanese and Han Chinese populations (South&North) detected some significant differences and genetic divergence between these Oriental populations. The dendrogram constructed by the neighbor‐joining (NJ) method based on the allele frequencies of DQA1, DQB1, DPB1 and DRB1 of 10 representative populations over the world suggested that Manchu is the closest, but at the same genetic distance to both Northern and Southern Han Chines

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02486.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:HLA class I and class II antigens circulate in serum as soluble molecules. Increased concentrations of soluble HLA class I molecules have been demonstrated in viral diseases, in rejection episodes following organ transplantation and in graft versus host disease. To explore the possibility of a variation of the serum concentrations of soluble HLA class II molecules in the same pathologic conditions we developed a double determinant immune assay that detects whole soluble HLA‐DR molecules (sHLA‐DR). The mean level of sHLA‐DR antigens in sera from 23 healthy individuals was 0.64±0.72 μg/ml. Elevated serum concentrations of sHLA‐DR molecules were detected in sera from HIV infected patients in CDC2/3 and in CDC4C1 stages (2.0±1.7m̀g/ml and 4.6±1.7m̀g/ml, respectively), in sera from patients affected by acute rejection after liver transplantation (5.3±3.7 μg/ml) and in sera from patients affected by severe acute graft versus host disease following bone marrow transplantation (8.8±3.1 μg/ml). The increase of sHLA‐DR molecules in these sera significantly correlated with the elevation of soluble HLA class I antigens (P=0.0004). The reported data suggest that both soluble HLA class I and class II molecules serum levels increase during viral infections and strong immune reactions and could suggest the involvement of these molecules

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02487.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02488.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02489.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02490.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1995.tb02491.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY