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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.
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Humanos , Alelos , Reação em Cadeia da Polimerase , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Teste de Histocompatibilidade/métodos , TecnologiaRESUMO
【Objective】 To investigate the polymorphism of KIR2DL4 gene in northern Chinese Han population. 【Methods】 A total of 327 DNA samples were isolated by magnetic beads from unrelated individuals of northern Chinese Han population. The coding sequence (CDS) of KIR2DL4 were amplified using four pairs of KIR2DL4-specific PCR primers developed by our own KIR sequencing-based typing patent, and each exon of KIR2DL4 carried by the four PCR amplicons was then subjected to DNA Sanger sequencing in both directions. The genotype of each sample was assigned using the Assign 4.7 software. 【Results】 Among 327 individuals, 8 different kinds of KIR2DL4 alleles were detected, with observed frequencies ranked as KIR2DL4*00102 [77.1%(252/327)], *00501 [35.8%(117/327)], *011 [20.2%(66/327)], *00602 [12.5%(41/327)], *00801 [8.6%(28/327)], *00103 [4.9%(16/327)], *00503 [1.5%(5/327)] and *00504 [0.9%(3/327)]. In this study, the 10A type alleles which can encode a full membrane-bound receptor include 2DL4*00102, *00103, *00501, *00503, *00504 and *00602, whereas the 9A type alleles which produce truncated forms of receptor include 2DL4*00801 and *011. The observed frequencies for 10A and 9A type KIR2DL4 alleles were 97.6% (319/327) and 27.8% (91/327), respectively. The ratio of 10A to 9A type was 3.5: 1. The observed frequencies of KIR2DL4 alleles in northern Chinese Han population showed no significant difference compared with southern Chinese Han population (P>0.05). 【Conclusion】 The allelic diversity of KIR2DL4 elucidated in the present study can provide valuable data for KIR-associated disease studies and human evolution.
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【Objective】 To study the molecular basis of D variant and explore the molecular genetic mechanism of novel weak D alleles. 【Methods】 Blood samples were screened for D variants by serological method. The nucleotide sequences of coding region were amplified by PCR and sequenced directly, and RHD gene heterozygosity was detected. 【Results】 Weak D phenotype was confirmed by serological test, and two novel alleles were detected by DNA sequencing. The first was novel weak D 1102A allele, 1102G>A mutation in exon 8, resulting in a 368Glu>Arg substitution in two samples. The second was novel weak D 399C allele, carried a 399G>C mutation in exon 3, which led to a 133Lys>Asn substitution. 【Conclusion】 In this study, D variants were detected by sequence-based typing, and two new alleles were identified.
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BACKGROUND: The human leukocyte antigen (HLA) has undergone long-term evolution to form diverse polymorphisms. In recent years, due to the increase in the number of examinees and the rapid development of HLA typing technology, novel HLA alleles have been discovered constantly. OBJECTIVE: To analyze the full-length sequence and 18 point mutations of HLA-B gene in a leukemia patient and her family using the next-generation sequencing technology. METHODS: Polymerase chain reaction and sequence-specific oligonucleotide probes (PCR-SSOP) and polymerase chain reaction-sequence based typing (PCR-SBT) revealed abnormalities in the patient’s HLA-B. To identify the genotype, we sequenced the full length of the gene by next-generation sequencing technology and collected blood samples from the patient’s father, mother and two sisters for genetic analysis of HLA genes. RESULTS AND CONCLUSION: Both PCR-SSOP and PCR-SBT indicated that the HLA-B sample had no perfectly matched genotype. Further detection using the next-generation sequencing technology revealed that the novel allele had 18 base mutations in the exon, intron and 3’UTR compared to the most homologous allele B*15:09:01. Five exon base mutations were located in the exons 3 and 4, which were: 486G→C, 583T→C, 636T→C, 652A→G, 756C→T, resulting in changes in the five corresponding codons, including 171 tyrosine (Tyr) → histidine (His) and 194 isoleucine (Ile) → valine (Val). A pedigree survey found that the patient’s novel HLA B allele was inherited from her father. The novel allele sequence was submitted to the Genbank database (MG595995). A novel HLA-B allele was confirmed by the next-generation sequencing, which was officially named HLA-B*15:435 by the World Health Organization HLA Factor Nomenclature Committee in December 2017.
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Objective@#To explore the role of inhibitory KIR (iKIR) and its cognate HLA ligand in the occurrence and development of cervical cancer among ethnic Han Chinese and its potential mechanism.@*Methods@#Peripheral blood samples from 265 Han Chinese patients with cervical intraepithelial neoplasia (CIN)/cervical cancer and 200 ethnically matched healthy controls were collected. The results of KIR PCR-SSP, HLA PCR-rSSO and KIR3DL1 PCR-SBT, together with cervical cancer data from the TCGA database, were used to assess the association of iKIR genes, receptor-ligand gene combinations, iKIR transcription level in the tumor tissue and the KIR3DL1 alleles with the occurrence and development of cervical cancer.@*Results@#Among the four iKIR genes (KIR2DL1, 2DL2/3, 3DL1 and 3DL2), the frequencies of KIR3DL1 and KIR3DL1-HLA-Bw4 genes among controls were significantly higher than those of the cervical cancer group (96.5% vs. 87.0%, P = 0.018; 81.5% vs. 64.8%, P=0.009). The survival rate of cervical cancer patients with a high transcription level of KIR3DL1 in tumor tissues was significantly higher than those with a low/medium transcription level (P = 0.028). The frequency of strong-inhibitory and high-expression KIR3DL1*01502 allele among the healthy population was significantly higher than that of the cervical cancer group (76.0% vs. 59.3%, P = 0.015).@*Conclusion@#Combined KIR3DL1 and KIR3DL1-HLA-Bw4 can confer a protective effect against the development of cervical cancer, which may be attributed to the strong-inhibitory and high-expression allele of KIR3DL1*01502.
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Objective To do analysis of sequence and identify a novel HLA-A allele in Chinese hematopoietic stem cell do-nors.Methods A rare HLA-A allele was initially detected by Luminex PCR-SSO typing,then the sample was sequenced by sequence-based typing (SBT)and the group-specific sequencing primer (GSSP)to confirm the mutation allele and locus.Re-sults The sequence of the sample results showed that the allele compared with the highest homologous allele HLA-A*24∶02∶01∶01 was the difference in the exon 3 at position 544 G>A,resulting in an amino acid sequence of HLA-A*24∶02∶01∶01 at position 158 change Ala to Thr.Conclusion This allele is a new HLA-A allele and has been designated as HLA-A*24∶327 by the HLA Nomenclature Committee of World Health Organization (WHO).
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Objective To compare PCR-SBT to IMS-ELISA in the HLA-B27 detection in the ankylosing spondylitis (AS)pa-tients.Methods Simultaneously,PCR-SBT and IMS-ELISA were used to detect the HLA-B27 expression in peripheral blood samples which were suspected patients with AS from 120 cases.Chisquare test of paired design and the area under curve of receiver operating characteristics of SPSS17.0 software were used to evaluate the value of PCR-SBT and IMS-ELISA in HLA-B27 detection of AS patients.Results Among 120 cases of suspected patients with AS,the positive rates of HLA-B27 detected by PCR-SBT and IMS-ELISA were 45.83%(55/120)and 37.50% (45/120),respectively.There was statistical difference between the two methods in the HLA-B27 detection (χ2 =59.455,P =0.000).The sensibility and spe-cificity of PCR-SBT were 96.36% and 96.92%,respectively.While the sensibility and the specificity of IMS-ELISA were 69.09% and 89.23%,respectively.Area under the curve of two methods were 0.966 and 0.792,respectively.Conclusion In comparison with IMS-ELISA,the sensibility and the specificity of PCR-SBT in HLA-B27 detection were higher in AS diag-nosis,that is to say,PCR-SBT is better in HLA-B27 detection and AS diagnosis.
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Objective To investigate the correlation between human leukocyte antigens-A,-B,-DRB1 (HLA-A,-B,-DRB1) high resolution alleles and chronic renal failure (CRF) caused by immunoglobulin-a nephropathy (IgAN).Method The polymerase chain reaction-sequence-based typing (PCR-SBT) method was used to investigate the genotypes of HLA-A,-B and-DRB1 high-resolution alleles in 191 cases of CRF caused by IgAN (experimental group) and 503 healthy blood donors (control group).The alleles frequencies between two groups were compared and the association between CRF caused by IgAN and the polyrnorphism of HLA was analyzed.Result (1) There were 25 alleles at A locus,48 alleles at B locus and 32 alleles at DRB1 locus in experimental group.(2) The genetic frequency of HLAA * 2901 [Pc =0.033,OR =10.738,95％ CI (1.193,96.691)],HLA DRB1 * 1106 [Pc =0.0001,OR =0.969,95％ CI (0.944,0.994)],HLA-DRB1 * 1202[Pc =0.002,OR =1.859,95％ CI (1.259,2.745)],HLA-DRB1 * 1401 [Pc =0.021,OR =0.984,95％ CI (0.967,0.998)],HLA-DRB1 * 1602[Pc=0.015,OR=1.915,95％ CI (1.157,3.17)] in experimental group was higher than in control group (P＜0.05).Conclusion There is susceptibility association of HLA-A * 2901,HLA-DRB1 * 1106,HLA-DRE * 1202,HLA-DRB1 * 1401,HLA-DRB1 * 1602 with CRF caused by IgAN.It is concluded that there is a close genetic and immunological correlation between HLA alleles and the pathogenesis of CRF caused by IgAN.
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PURPOSE: To evaluate a multi-group-specific sequence-based typing (SBT) method for resolving ambiguous results from human leukocyte antigen (HLA) genotyping. MATERIALS AND METHODS: A total of 50 samples that showed ambiguous genotypes for at least two HLA loci from HLA-A, -B, -C and -DRB1 by the conventional SBT assay were evaluated using a new SBT test, the AVITA plus assay. The most likely HLA genotypes for the respective samples considering allele frequencies in Korean were concordant between the AVITA and conventional SBT assays. RESULTS: An average of 3.3 loci among the HLA-A, -B, -C and -DRB1 loci per sample gave results with two or more possible allele combinations with the conventional SBT, and 48 (96.0%) out of 50 showed reduced numbers of possible genotypes for at least one HLA locus with the AVITA. A total of 41, 43, 42, and 38 cases among the 50 samples showed ambiguous results for HLA-A, -B, -C, and -DRB1 typing by the conventional SBT, respectively. The average numbers of possible allele combinations for the respective four HLA loci were 8.2, 6.7, 5.9, and 3.2, and they were reduced to 1.5, 2.2, 4.4, and 1.8, respectively, by the AVITA. Ambiguity was resolved by the AVITA in 33 (80.5%), 31 (72.1%), 17 (40.5%) and 28 (73.7%) samples among the ambiguous cases from the conventional SBT for HLA-A, -B, -C, and -DRB1 typing, respectively. CONCLUSION: The multi-group-specific SBT method considerably reduced the number of ambiguous results, and thus may be useful for accurate HLA typing in clinical laboratories.
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Humanos , Povo Asiático/genética , Sequência de Bases , Frequência do Gene/genética , Genótipo , Antígenos HLA/genética , Teste de Histocompatibilidade , Reação em Cadeia da PolimeraseRESUMO
Using three Austrian case studies, the variegated applications of molecular typing in today's public health laboratories are discussed to help illustrate preventive management strategies relying on DNA subtyping. DNA macrorestriction analysis by pulsed field gel electrophoresis has become the gold standard for subtyping of food borne pathogens like listeria, salmonella, campylobacter and Bacillus cereus. Using a Salmonella Mbandaka outbreak from the year 2010 as example, it is shown how the comparison of patterns from human isolates, food isolates, animal isolates and feed isolates can allow to identify and confirm a source of disease. An epidemiological connection between the simultaneous occurrence of tuberculosis in cattle and deer with cases of human tuberculosis due to Mycobacterium caprae in 2010 was excluded using mycobacterial interspersed repetitive units variable-number tandem repeats subtyping. Also in 2010, multilocus sequence typing with nonselective housekeeping genes, the so-called sequence based typing protocol, was used to elucidate connections between an environmental source (a hospital drinking water system) and a case of legionellosis. During the last decades, molecular typing has evolved to become a routine tool in the daily work of public health laboratories. The challenge is now no longer to simply type microorganisms, but to type them in a way that allows for data exchange between public health laboratories all over the world.
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Humanos , Técnicas de Tipagem Bacteriana/métodos , Técnicas de Laboratório Clínico/métodos , Impressões Digitais de DNA , DNA Bacteriano/análise , Surtos de Doenças , Eletroforese em Gel de Campo Pulsado/métodos , Microbiologia de Alimentos , Laboratórios , Tipagem Molecular/métodos , Medicina Preventiva , Saúde PúblicaRESUMO
Objective To study the molecular polymorphism and the distribution of HLA-B27 subtypes in southern Chinese Han patients with Ankylosing Spondylitis and healthy controls.Methods A total of 46 samples form southern Chinese Han patients with AS and 80 non-related blood samples from healthy peripheral blood stem cell donors with B27-positive identified by rSSO Lumminex flow array assay were subjected to sequencing analysis of exon 2 ,3 and 4 of HLA-B gene by the sequence-based typing,the purified products of sequencing reaction were electrophoresed on ABI 3730 DNA sequencer and the designation of HLA-B27 allele was accomplished using the Assign3.5 software. The ambiguities and the detected "rare" alleles were confirmed using the PCR-SSP commercial kit. Results In the 46 B27-positive patients with the diagnosis of AS,four alleles,namely B2704,B2705,B2707 and B2724 were determined. The frequencies for these four alleles were 82.98%(39/47),12.77%(6/47),2.13%(1/47) and 2.13%(1/47),respectively. In the 80 B27-positive control individuals,seven B27 related alleles were identified. The frequency for the two dominant subtype B2704 and B2705 were 57.32%(47/82) and 26.83%(22/82),respectively. Both the B2706 and B2707 were observed 5 times with a frequency of 6.10%(5/82),three alleles B2703,B2715 and B2724 were detected only once with a frequency of 1.22%(1/82).Conclusion Our study shows that HLA-B2704 and B2705 were the predominant subtypes in normal healthy controls,however,B2704 was the predominant subtype for the AS group in southern Chinese Han patients.
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CG. And that was why SSP failed to determine the allele.Conclusion The difficulty in HLA genotyping by SSP resulted from the primers, which involved unknown sequence of Exon 1 at locus B in the studied sample.