Complete subtyping of the HLA-A locus by sequence-specific amplification followed by direct sequencing or single-strand conformation polymorphism analysis

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 combinations remained unresolved.     

Neurological Manifestations of COVID-19 Feature T Cell Exhaustion and Dedifferentiated Monocytes in Cerebrospinal Fluid

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Prevalence and genotypic distribution of TT virus in Athens, Greece

The prevalence of TT virus (TTV) infection in various population groups from Athens, Greece, was assessed by the polymerase chain reaction (PCR) using two primer sets from distinct regions of the genome: the conventional set derived from the open reading frame-1 (ORF-1) and the new, highly sensitive set targeting the region that includes the TATA signal localized upstream of ORF-2. Based on both primer sets, TTV DNA was detected in 42/50 (84.0%) healthy individuals, 42/50 (84.0%) chronic hepatitis C patients, 31/39 (79.5%) acute non-A-E hepatitis patients (group I), 14/16 (87.5%) renal failure patients with acute non-A-E hepatitis (group II), 47/50 (94.0%) intravenous drug users (IVDU), 36/50 (72.0%) hemophiliacs, and 21/31 (67.7%) hemodialysis patients. The presence of TTV was not associated with any particular risk group, and no differences were observed in relation to demographic, biochemical and virological characteristics between TTV DNA-positive and -negative patients. TTV did not seem to have a profound effect on the course of chronic C or acute non-A-E hepatitis either. Phylogenetic analysis revealed that TTV strains circulating in the greater metropolitan area of Athens belong not only to the G1 and G2 genotypes that are encountered worldwide, but also to G3 and to G5 that are found mainly in Europe and Asia, respectively. Further studies will shed light on the role of this highly prevalent virus.     

Direct sequencing of SSP-PCR-amplified cDNA to identify new alleles in the DR52-associated DRB1 group: identification of DRB1*1115, DRB1*1117 and DRB1*1319

Abstract: Low and high resolution sequence specific oligonucleotide probe hybridization patterns were used to design an approach to direct sequencing of allele specific amplified cDNA. Several PCR amplifications were used to derive overlapping sequence fragments to define complete first domain sequences for a single allele. This method has been used to characterize three new DRB1 alleles in the DR52 family, DRB1*1115, DRB1* 1117, and DRB1*1319. All three alleles carry polymorphisms previously observed in other DRB alleles and underscore the importance of utilizing a directed sequencing approach for obtaining unambiguous typing results in matching for bone marrow transplantation between unrelated donor and recipient.     

A bioinformatics pipeline for high-throughput microbial multilocus sequence typing (MLST) analyses

Multilocus sequence typing (MLST) analysis for semi-routine applications is hindered by the downstream, manually intensive steps of processing the raw sequence data files. This report describes the development of an MLST pipeline that automates DNA sequence editing and analysis in order to significantly reduce the time required for processing data. Validation using a pneumococcal dataset revealed complete agreement between the results generated by manual and automated workflows. The MLST pipeline was developed for both double-strand and single-strand sequencing.     

New HLA-A*11 allele,A*1112, identified by sequence-based typing

In this report, we describe the identification of HLA-A*1112, a novel HLA-A*11 allele found in two Italian families. The new allele was detected during routine HLA typing by a polymerase chain reaction sequence-specific primer and was confirmed by high-resolution sequencing-based typing. The nucleotide sequences of HLA-A*1112 exons 2 and 3 are identical to HLA-A*11011 except for a single nucleotide substitution in codon 90 (GAC-->GCC).     

Molecular characterization and determination of the coding capacity of the genome of equine herpesvirus type 2 between the genome coordinates 0.235 and 0.258 (theEcoRI DNA fragment N; 4.2 kbp)

The complete DNA nucleotide sequence of theEcoRI DNA fragment N (0.235 to 0.258 viral map units) of equine herpes virus type 2 (EHV-2) strain T400/3 was determined. This DNA fragment comprises 4237 bp with a base composition of 55.23% G+C and 44.77% A+T. Nineteen open reading frames (ORFs) of 50-287 amino acid (aa) residues were detected. ORF number 10 is located between the nucleotide position 2220 and 2756 coding for a protein of 179 amino acid residues. This protein shows significant homology to the cytokine synthesis inhibitory factor (CSIF; interleukin 10) of human (76.4%) and mouse (68.5%), and to the Epstein-Barr virus (EBV) protein BCRF1 (70.6%). The existence of an interleukin 10 (IL-10) analogous gene within the genome of the EHV-2 was confirmed by screening the genome of nine EHV-2 strains using specific oligonucleotide primers corresponding to the 5 and 3 region of this particular gene by polymerase chain reaction. In all experiments an 870 bp DNA product was amplified. The specifity of the amplified DNA fragments obtained from individual EHV-2 strains was confirmed by DNA-DNA hybridization experiments. The DNA sequence analysis of the amplified DNA products of the EHV-2 strain LK was carried out. This analysis revealed the identity of the corresponding IL-10 gene (540 bp) of this strain to the IL-10 gene of EHV-2 strain T400/3. The presented data indicate that the EHV-2 genome harbors a viral interleukin 10-like gene. This is further evidence that the IL-10 gene can be present in the genomes of members of the Herpesviridae family.     

Five newly identified HLA alleles: A*0310, A*2907, B*4435, Cw*0206, Cw*0506, and confirmation of A*3106, B*3924, Cw*0314, DRB1*0322, and DRB1*1433 alleles

A number of HLA alleles have been newly identified. This concerns HLA-A*0310, A*2907, B*4435, Cw*0206, Cw*0506, of which Cw*0206 was found in three unrelated individuals, all B*4002 positive. Some other alleles are also presented but confirm earlier detected sequences: A*3106, Cw*0314, DRB1*0322, and DRB1*1433. Moreover, we identified B*3924 in a bone marrow transplant recipient and in five of six unrelated stem cell donors, selected for this patient. In all cases, B*3924 was found on a haplotype combining A*0201, B*3924, Cw*0701, and DRB1*1303. The observation of this extended haplotype is of importance for the selection for stem cell transplantation. Cells expressing B*3924 and B*4435 were typed by serology as B39 and B44, respectively. Cells expressing HLA-A*0310 do not express A3 but type as A-Blank.     

Novel germline BRCA1 and BRCA2 mutations in breast and breast/ovarian cancer families from the Czech Republic

Germline mutations in breast cancer susceptibility genes, BRCA1 and BRCA2, are responsible for a substantial proportion of high‐risk breast and breast/ovarian cancer families. To characterize the spectrum of BRCA1 and BRCA2 mutations, we screened Czech families with breast/ovarian cancer using the non‐radioactive protein truncation test, heteroduplex analysis and direct sequencing. In a group of 100 high‐risk breast and breast/ovarian cancer families, four novel frame shift mutations were identified in BRCA1 and BRCA2 genes. In BRCA1, two novel frame shift mutations were identified as 3761‐3762delGA and 2616‐2617ins10; in BRCA2, two novel frame shift mutations were identified as 5073‐5074delCT and 6866delC. Furthermore, a novel missense substitution M18K in BRCA1 gene in a breast/ovarian cancer family was identified which lies adjacent just upstream of the most highly conserved C3HC4 RING zinc finger motif. To examine the tertiary structure of the RING zinc finger domain and possible effects of M18K substitution on its stability, we used threading techniques according to the crystal structure of RAG1 dimerization domain of the DNA‐binding protein. © 2000 Wiley‐Liss, Inc.     

Identification of HLA-C alleles using PCR—single-strand-conformation polymorphism and direct sequencing

Alleles encoding HLA-C antigens in Japanese were identified by polymerase chain reaction followed by single strand conformation polymorphism (PCR-SSCP) and nucleotide sequencing analyses. The results showed that at least sixteen different alleles code for eight serologically detectable antigen groups and undetectable blanks. Cwl was mainly encoded by Cw*0102, whereas two split antigens of Cw3, Cw9 and Cw10, were encoded by Cw*0303 and Cw*0304, respectively. Cw4 and Cw6 were encoded by Cw*0401 and Cw*0602, respectively. Seven alleles, Cw*0801, Cw*0803, Cw*1202, Cw*1203, Cw*1402, Cw*1403 and Cw*1502, were found to encode serological HLA-C "blanks" in Japanese. Moreover, errors in the published nucleotide sequences of Cw*0501 and Cw*1201 were corrected. Twenty-one HLA-C alleles were distinguished from each other by means of group-specific PCR amplification followed by the SSCP method developed in the present study. The system using genomic DNAs can be used effectively for identification of new HLA-C alleles.