Objective:To identify the nucleotide sequence of a novel HLA-DRB1*12: 106 allele. Methods:A blood donor who was joined into the database for platelet matching transfusion at the Blood Center of Zhejiang Province in 2023 was selected as the study subject. HLA genotyping was carried out through next-generation sequencing based on AllType NGS 11 locus, AllType FASTPlex NGS reagents, and Sanger sequencing method. The HLA genotype of the donor by Sanger sequencing and next generation sequencing were assigned by using uTYPE 7.3 and TypeStream Visual 3.0 software, respectively. This study was approved by Medical Ethics Committee of the Zhejiang Blood Center (Ethics No. Provincial Blood Center Ethics Review 2022 Research No. 001). Results:A novel HLA-DRB1*12 allele has been identified, and the full coding sequence has been submitted to the GenBank database (No. OR101190), and the length of submitted sequence was 801 bp, which was officially named as HLA-DRB1*12: 106 by the WHO Nomenclature Committee for Factors of the HLA System (submission No. HWS10066755). Compared with the sequence of the highest homology ( HLA-DRB1*12: 01: 01: 01 allele), a single nucleotide change was identified at position 344 T>G in the exon 2 of the HLA-DRB1*12: 106, which has resulted in replacement of Valine by Glycine at residue 86. The HLA genotype of the proband was determined as HLA-A*02: 01, 11: 01; -B*13: 02, 40: 01; -C*01: 02, 03: 03; -DRB1*07: 01, 12: 106; -DRB3*01: 01; -DRB4*01: 03; -DQA1*02: 01, 04: 01; -DQB1*02: 02, 04: 02; -DPA1*01: 03, 01: 03; -DPB1*02: 01: 02G, 04: 01: 01G. Conclusion:A novel HLA-DRB1 allele has been identified in the Chinese population. The mutated amino acid, located in the peptide binding region of the β chain, may affect the binding characteristics of antigen peptides.

OBJECTIVE: To identify the nucleotide sequence of a novel HLA-DRB1*12:106 allele. METHODS: A blood donor who was joined into the database for platelet matching transfusion at the Blood Center of Zhejiang Province in 2023 was selected as the study subject. HLA genotyping was carried out through next-generation sequencing based on AllType NGS 11 locus, AllType FASTPlex NGS reagents, and Sanger sequencing method. The HLA genotype of the donor by Sanger sequencing and next generation sequencing were assigned by using uTYPE 7.3 and TypeStream Visual 3.0 software, respectively. This study was approved by Medical Ethics Committee of the Zhejiang Blood Center (Ethics No. Provincial Blood Center Ethics Review 2022 Research No. 001). RESULTS: A novel HLA-DRB1*12 allele has been identified, and the full coding sequence has been submitted to the GenBank database (No. OR101190), and the length of submitted sequence was 801 bp, which was officially named as HLA-DRB1*12:106 by the WHO Nomenclature Committee for Factors of the HLA System (submission No. HWS10066755). Compared with the sequence of the highest homology (HLA-DRB1*12:01:01:01 allele), a single nucleotide change was identified at position 344 T>G in the exon 2 of the HLA-DRB1*12:106, which has resulted in replacement of Valine by Glycine at residue 86. The HLA genotype of the proband was determined as HLA-A*02:01, 11:01;-B*13:02, 40:01;-C*01:02, 03:03;-DRB1*07:01, 12:106;-DRB3*01:01;-DRB4*01:03;-DQA1*02:01,04:01;-DQB1*02:02,04:02;-DPA1*01:03,01:03; -- DPB1*02:01:02G,04:01:01G. CONCLUSION A novel HLA-DRB1 allele has been identified in the Chinese population. The mutated amino acid, located in the peptide binding region of the β chain, may affect the binding characteristics of antigen peptides.
Humans ; HLA-DRB1 Chains/genetics* ; Alleles ; Base Sequence ; Genotype ; Male ; High-Throughput Nucleotide Sequencing ; Blood Donors

[Objective] To resolve the ambiguities of HLA genotyping generated by next generation sequencing (NGS) using nanopore sequencing technology. [Methods] A total of 38 samples with ambiguous HLA genotyping by NGS in our laboratory were collected, and HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 loci in these samples were amplified using primers in the same commercial NGS HLA genotyping kit, then subjected to third-generation library construction, and sequenced on the nanopore sequencer. The sequencing data were converted into Fastq files and analyzed by software, and the genotypes of 11 HLA loci were obtained. The ambiguities were counted directly. [Results] The high-resolution genotyping at the second domain of 11 HLA loci of 38 samples using the third generation sequencing (TGS) were consistent with the results of the NGS method at a rate of 100%. The genotypes for the HLA-A, -B, -C, -DRB3, -DRB4, -DQA1 and -DPA1 loci by TGS were all only one result, and the discrimination rate for ambiguities of the HLA-A, -B, -C, and -DQA1 loci (all caused by the difficulty in phasing due to the short NGS read length) was 100%. Among the HLA-DRB1, -DRB5, -DQB1 and -DPB1 loci, the discrimination rate of TGS for the ambiguities caused by non-amplification of exon 1 was 0% and by the short NGS read length was 100%. [Conclusion] Nanopore technology was used to identify the ambiguities of 11 HLA loci in this study, and the ambiguities caused by the short read length disadvantage of the NGS method could be solved effectively and the accuracy of HLA genotyping would be improved.

Objective:To identify the nucleotide sequence of a novel HLA-DRB1*12: 106 allele. Methods:A blood donor who was joined into the database for platelet matching transfusion at the Blood Center of Zhejiang Province in 2023 was selected as the study subject. HLA genotyping was carried out through next-generation sequencing based on AllType NGS 11 locus, AllType FASTPlex NGS reagents, and Sanger sequencing method. The HLA genotype of the donor by Sanger sequencing and next generation sequencing were assigned by using uTYPE 7.3 and TypeStream Visual 3.0 software, respectively. This study was approved by Medical Ethics Committee of the Zhejiang Blood Center (Ethics No. Provincial Blood Center Ethics Review 2022 Research No. 001). Results:A novel HLA-DRB1*12 allele has been identified, and the full coding sequence has been submitted to the GenBank database (No. OR101190), and the length of submitted sequence was 801 bp, which was officially named as HLA-DRB1*12: 106 by the WHO Nomenclature Committee for Factors of the HLA System (submission No. HWS10066755). Compared with the sequence of the highest homology ( HLA-DRB1*12: 01: 01: 01 allele), a single nucleotide change was identified at position 344 T>G in the exon 2 of the HLA-DRB1*12: 106, which has resulted in replacement of Valine by Glycine at residue 86. The HLA genotype of the proband was determined as HLA-A*02: 01, 11: 01; -B*13: 02, 40: 01; -C*01: 02, 03: 03; -DRB1*07: 01, 12: 106; -DRB3*01: 01; -DRB4*01: 03; -DQA1*02: 01, 04: 01; -DQB1*02: 02, 04: 02; -DPA1*01: 03, 01: 03; -DPB1*02: 01: 02G, 04: 01: 01G. Conclusion:A novel HLA-DRB1 allele has been identified in the Chinese population. The mutated amino acid, located in the peptide binding region of the β chain, may affect the binding characteristics of antigen peptides.

Objective:To analyze the sequence of a novel HLA- DPB1 allele in an individual. Methods:A individual identified from the database of blood donors for matched platelet transfusion at the Blood Center of Zhejiang Province in May 2022 was selected as the study subject. HLA genotype of the individual was determined by next-generation sequencing (NGS) on an Ion Torrent S5 platform. The sequence of the HLA- DPB1 locus was also determined by NGS on an Illumina Miseq platform and third generation sequencing using Oxford Nanopore MinION. This study was approved by Medical Ethics Committee of the Blood Center of Zhejiang Province (Ethics No. 2021-001). Results:A novel HLA- DPB1*02 allele was identified in the specimen, for which the closest genotype was HLA- DPB1*02: new, 17: 01: 01G, with the variant located in exon 3. Meanwhile, the NGS also revealed a novel HLA- DPB1*17 allele, with the closest genotype being HLA- DPB1*02: 01: 02G, 17: new. Both the HLA- DPB1*17: 01: 01: 01 and novel HLA- DPB1*02 alleles were identified by third-generation sequencing. Compared with the HLA- DPB1*02: 01: 02: 01 allele, the novel allele had a G>A variation at position 369 in the exon 3, which did not result in amino acid change. Conclusion:A novel HLA- DPB1 allele has been identified and validated by both NGS and TGS, which has been named as HLA- DPB1*02: 01: 69 by the World Health Organization Committee on Nomenclature of Factors of the HLA System.

Objective:Assessing the accuracy of automated EasyNAT system for rapidly detecting par-affin-embedded tissue for the diagnosis of tuberculosis.Methods:A retrospective analysis was conducted on 134 patients,comprising 101 with confirmed tuberculosis and 33 without tuberculosis,treated at Beijing Chest Hospital,Capital Medical University,between 2018 and 2022.The clinical diagnostic results served as the standard for assessing the diagnostic performance of the EasyNAT system in comparison to quantita-tive real-time polymerase chain reaction(qPCR)for tuberculosis detection in paraffin-embedded tissues.The evaluation criteria included sensitivity,specificity,positive predictive value,negative predictive value,and accuracy rate.Results:Based on the clinical diagnostic results,the EasyNAT assay demonstrated a sensitivity of 87.1％(88/101,95％CI:79.2％-92.3％)and a specificity of 100.0％(33/33,95％CI:89.6％-100.0％).The positive predictive value,negative predictive value,and accuracy rate were 100％(88/88,95％CI:95.8％-100.0％),71.7％(33/46,95％CI:57.5％-82.7％),and 90.3％(121/134,95％CI:84.1％-94.2％),respectively.In comparison,the qPCR assay exhibited a sensi-tivity of 96.0％(90.3％-98.5％)and a specificity of 100.0％(89.6％-100.0％).The positive pre-dictive value,negative predictive value,and accuracy rate for qPCR were 100.0％(96.2％-100.0％),89.2％(75.3％-95.7％),and 97.0％(92.6％-98.8％).The Cohen's kappa value of 0.84 indicated substantial agreement between EasyNAT and qPCR.The detection rate of tuberculosis using this method was 86.4％(38/44,95％CI:73.3％-93.6％),while the detection rate for extrapulmonary tuberculo-sis was 87.7％(50/57,95％CI:76.8％-93.9％).In comparison,qPCR showed a detection rate of 97.7％(88.2％-99.6％)for pulmonary tuberculosis and 94.7％(85.6％-98.6％)for extrapulmonary tuberculosis.There was no statistically significant difference in the detection results between the method and qPCR for both pulmonary and extrapulmonary tuberculosis(P＞0.05).Importantly,the EasyNAT de-tection combined nucleic acid extraction,amplification,and analysis into one process.Compared with tra-ditional qPCR methods,manual operation time was reduced by 2 hours,leading to an overall reduction in total testing time by 3 hours.Conclusion:The EasyNAT nucleic acid rapid detection system can quickly,conveniently,and accurately detect Mycobacterium tuberculosis DNA in paraffin-embedded tissues,demon-strating significant clinical utility in the pathological diagnosis of tuberculosis.

【Objective】 To statistically analyze the characteristics of ambiguous results of HLA-DPB1 genotyping given by AllType NGS 11-loci sequencing reagent via two next generation sequencing platforms, i. e. Ion Torrent S5 and Illumina Miseq. 【Methods】 A total of 434 samples from patients or donors were genotyped for HLA-DPB1 locus using AllType NGS 11-loci sequencing reagent from One Lambda company; 336 samples of them were sequenced via the Ion Torrent S5 platform and other 98 samples were sequenced via the Illumina Miseq platform. All 434 samples were genotyped for HLA-DPB1 gene simultaneously using PCR-SSO flow fluorescent bead method. The ambiguous genotypes of HLA-DPB1^*13∶01∶01/107∶01 were distinguished by Sanger sequencing. The HLA-DPB1 genotype results by NGS method were assigned by TypeStream Visual professional software, and the ratio of ambiguous combination was calculated by direct count method. 【Results】 Ambiguous results were found in 357 out of 434 samples, accounting for 82.3% (357/434) when HLA-DPB1 allele was assigned to the third field using NGS method. Ambiguous results with 45 types were given in 275 out of 336 samples by the Ion Torrent S5 platform, accounting for 81.8% (275/336) and 82(with 27 types) out of 98 samples by the Illumina Miseq platform, accounting for 83.7% (82/98). All samples were re-genotyped for HLA-DPB1 gene by PCR-SSO, and none HLA-DPB1 allele had been missed by NGS. A total of 43 ambiguous alleles in HLA-DPB1^*13∶01∶01/107∶01 involving 41 samples were distinguished by Sanger sequencing; HLA-DPB1^*13∶01∶01 were detected in 25 (58.1%, 25/43) and HLA-DPB1^*107∶01 in 18 (41.9%, 18/43). 【Conclusion】 There were still a high proportion of HLA-DPB1 ambiguous combinations using the AllType NGS 11-loci sequencing reagent. Sequencing exon 1 of HLA-DPB1 gene by Sanger sequencing can resolve part of the ambiguous results in HLA-DPB1^*13∶01∶01/107∶01 alleles.

Objective:To explore the significance of enthesitis in psoriasis(PsO) and the value of high-frequency Doppler ultrasound (HDUS) in screening potential subclinical psoriatic arthritis(PsA) patients through enthesitis; Designing the semi-quantitative score of enthesitis explore the value of semi-quantitative score in evaluating the degree of joint structure damage and improving the curative effect in patients with subclinical PsA.Methods:Clinical and sonographic data were collected in 125 PsO patients and 50 osteoarthritis (OA) patients who attended hospital from January 2019 to March 2021, and healthy controls (HC) recruited during the same period. The prevalence of enthesitis of distal interphalangeal joint (DIP) of psoriasis patients, osteoarthritis patients and HC was compared. Meanwhile the semi-quantitative scoring system for ultrasound-guided enthesitis was designed, and the correlation between the severity of enthesitis and clinical score was analyzed. The correlation between psoriasis nail and enthesitis of extensor tendon of DIP finger was also analyzed.Results:The significant difference was found in the prevalence of enthesitis of DIP among PsO group, OA group and HC group (all P<0.001); There was a significant difference in the severity of enthesitis of DIP in PsO group before and after treatment ( P<0.001); In PsO group, the correlation between the severity of enthesitis of DIP and Psoriasis Area and Severity Index(PASI)score was moderate ( rs=0.538, P<0.001), and highly correlated with Nail Psoriasis Severity Index (NAPSI) scores( rs=0.877, P<0.001). There was a significant difference in the incidence of enthesitis between the patients with psoriasis nail and the patients without that ( P<0.001). The prevalance of enthesitis of nail disease free fingers was also found significantly different between PsO group and HC group( P<0.001). Conclusions:Enthesitis is a prominent characteristic of PsO.And the semi-quantitative scores designed in this research are in satisfied consistence with the clinical scores, and could be used to evaluate subclinical PsA structural damage.Furthermore, it is possible that the enthesitis of DIP is a relatively independent process from the nail disease.

OBJECTIVE: To detect loss of heterozygosity (LOH) at human leukocyte antigen (HLA) loci in a Chinese patient with leukemia after haploidentical hematopoietic stem cell transplantation. METHODS: HLA genotyping was carried out on peripheral blood, hair follicle and buccal swab samples derived from the patient after the transplantation as well as peripheral blood samples from his parents by using PCR-sequence specific oligonucleotide probe method and PCR-sequence based typing method. Short tandem repeat (STR) loci were detected by using a 23 site STR assay kit and a self-developed 6 STR loci assay for the HLA regions. RESULTS: After the transplantation, the HLA genotype of the peripheral blood sample of the patient was identical to his father. The patient was HLA-A*02:01,24:02, C*03:03,03:04, B*13:01,15:01, DRB1*08:03,12:02, DQB1*03:01,06:01 for his hair follicle specimen. However, homozygosity of the HLA loci was found in his buccal swab sample. Only the HLA-A*24:02-C*03:03-B*15:01-DRB1*08:03-DQB1*06:01 haplotype from his father's was present, while the HLA-A*02:01-C*03:04-B*13:01-DRB1*12:02-DQB1*03:01 haplotype from his mother was lost. After the transplantation, the alleles of the 23 STR sites in the patient's peripheral blood sample were consistent to his father, with no allelic loss detected in his buccal swab sample. However, at least 4 STR loci in the HLA region were lost in his buccal swab sample. CONCLUSION LOH at the HLA loci has been detected in the buccal swab sample of a patient with leukemia who received haploidentical hematopoietic stem cell transplantation.
HLA Antigens/genetics* ; HLA-A Antigens/genetics* ; Histocompatibility Antigens Class I/genetics* ; Humans ; Leukemia/genetics* ; Loss of Heterozygosity

Objective:To establish an artificial intelligence (AI)-assisted diagnostic system for lung cancer via deep transfer learning.Methods:The researchers collected 519 lung pathologic slides from 2016 to 2019, covering various lung tissues, including normal tissues, adenocarcinoma, squamous cell carcinoma and small cell carcinoma, from the Beijing Chest Hospital, the Capital Medical University. The slides were digitized by scanner, and 316 slides were used as training set and 203 as the internal test set. The researchers labeled all the training slides by pathologists and establish a semantic segmentation model based on DeepLab v3 with ResNet-50 to detect lung cancers at the pixel level. To perform transfer learning, the researchers utilized the gastric cancer detection model to initialize the deep neural network parameters. The lung cancer detection convolutional neural network was further trained by fine-tuning of the labeled data. The deep learning model was tested by 203 slides in the internal test set and 1 081 slides obtained from TCIA database, named as the external test set.Results:The model trained with transfer learning showed substantial accuracy advantage against the one trained from scratch for the internal test set [area under curve (AUC) 0.988 vs. 0.971, Kappa 0.852 vs. 0.832]. For the external test set, the transferred model achieved an AUC of 0.968 and Kappa of 0.828, indicating superior generalization ability. By studying the predictions made by the model, the researchers obtained deeper understandings of the deep learning model.Conclusions:The lung cancer histopathological diagnostic system achieves higher accuracy and superior generalization ability. With the development of histopathological AI, the transfer learning can effectively train diagnosis models and shorten the learning period, and improve the model performance.