Using Next-Generation Sequencing Technology to Confirm the HLA Rare Alleles Detected by PCR-SSOP / 中国实验血液学杂志

OBJECTIVE@#To confirm the HLA genotypes of the samples including 4 cases of magnetic bead probe HLA genotyping result pattern abnormality and 3 cases of ambiguous result detected by PCR sequence-specific oligonudeotide probe (SSOP) method.@*METHODS@#All samples derived from HLA high-resolution typing laboratory were detected by PCR-SSOP. A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality and 3 samples of ambiguous result were further confirmed by PCR sequence-based typing (SBT) technology and next-generation sequencing (NGS) technology.@*RESULTS@#A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality were detected by PCR-SSOP method. The results of SBT and NGS showed that the HLA-A genotype of sample 1 did not match any known genotypes. NGS analysis revealed that the novel allele was different from the closest matching allele A*31:01:02:01at position 154 with G>A in exon 2, which resulting in one amino acid substitution at codon 28 from Valine to Methionine (p.Val28Met). The HLA-C genotype of sample 2 was C*03:119, 06:02, sample 3 was C*03:03, 07:137, and sample 4 was B*55:02, 55:12. A total of 3 samples with ambiguous result were initially detected by PCR-SSOP method. The re-examination results of SBT and NGS showed that the HLA-B genotype of sample 5 was B*15:58, 38:02, sample 6 was DRB1*04:05, 14:101, and sample 7 was DQB1*03:34, 05:02. Among them, alleles C*03:119, C*07:137 and DRB1*14:101 were not included in the Common and Well-documented Alleles (CWD) v2.4 of the Chinese Hematopoietic Stem Cell Donor Database.@*CONCLUSION@#The abnormal pattern of HLA genotyping results of magnetic probe by PCR-SSOP method suggests that it may be a rare allele or a novel allele, which needs to be verified by sequencing.

Association of hemorrhagic fever with renal syndrome and HLA-DRB allele polymorphisms in Han nationality, Hubei province / 中华流行病学杂志

Objective To understand the association of human leukocyte antigen (HLA)-DRB polymorphism and patients diagnosed as hemorrhagic fever with renal syndrome (HFRS). Methods HLA-DR allele polymorphism was detected by PCR-sequence specific primers (PCR-SSP). Hantavirus (HV) typed as Hantaan virus (HTNV) and Seoul virus (SEOV) in patients were detected by RT-heminested PCR. Results The gene frequency of DRB1*0401-0411, *1001 and *1101-1105 in HFRS case group were 3.1%, 2.2% and 15.7% respectively. Compared with control group, it was significant higher in HFRS case group (RR=13.87, 9.72 and 2.00 respectively with Chi-square value as 10.006,6.324 and 6.472 respectively, P<0.05). When compared with HFRS case group, the gene frequency of DRB1*1501-1502, DRB4 and DRB5 in control group were 11.0%, 19.0% and 16.9% respectively, markedly lower than in patients (RR=0.45, 0.58 and 0.23 respectively. Chi-square values were 6.138, 4.583 and 21.076 respectively, P<0.05). There was no significant difference in other HLA-DR gene frequencies. Mixed infection was found in Hubei, with HTNV slightly more than SEOV. Distinct hantaviruses could coexist in either different or the same geographic or ecological zores in Hubei province. Patients with HLA-DRB1*1101-1105 alleles were 81.8%(27/33) infected by HTNV and only 18.2% infected by SEOV, which had significant difference (P<0.05). Conclusion DRB1*0401-0411,*1001 and *1101-1105 were possibly associated with increased susceptibility to HV infection. On the other hand there was an inverse correlation among HFRS, DRB1*1501-1502, DRB4 and DRB5.

PCR sequence analysis of the 3'UTR of the SON gene of human and mammalian / 中国法医学杂志

Objective To study the specific sequences in the 3'untranslated region (UTR) of the SON gene of human beings and 14 kinds of mammalian. Method The specific sequences in the 3'untranslated region (UTR) of the SON gene of human beings and 14 kinds of mammalians were amplified and directly se-quenced. The sequence data of PCR products of human beings and mammalians were declared. Results The sequences of 3'UTR of SON gene of human beings differed from 14 kinds of mammalians. The sequences of samples came from different human individuals did not show any difference. Conclusion The 3'UTR of SON gene is a good DNA region for specie identification. Using this technology, DNA of human beings can be distinguished from 14 kinds of mammalians. This technology is very useful in of forensic science.