Identification of the novel allele HLA-A*02:1144 in a Chinese platelet donor.

The novel HLA-A*02:1144 allele differs from HLA-A*02:03:01:01 by 3 nucleotides in exon 7.

Genomic full-length sequence of the HLA-B*40:86 allele identified in a Chinese individual.

HLA-B*40:86 differs from B*40:06:01:03 by a single nucleotide exchange in exon 3.

[The Polymorphism Analysis of HLA Class II Alleles Based on Next-Generation Sequencing and Prevention Strategy for Allele Dropout].

OBJECTIVE: To investigate the accuracy of next-generation sequencing technology (NGS) in detecting the polymorphisms of HLA-DRB1, DQB1, DQA1, DRB3, DRB4, DRB5, DPA1 and DPB1 alleles in randomly-selected unrelated healthy individuals from Shenzhen Han population, investigate the potential reason for HLA-DRB1 allele dropout in routine NGS, and establish an internal quality control system. METHODS: NGS-based HLA class II genotyping was performed on 1 012 samples using the MiSeqDxTM platform. The suspected missed alleles indicated by the quality control software and HLA-DRB1 homozygotes were confirmed by PCR-SSOP or PCR-SBT methods. RESULTS: A total of 139 alleles were detected, including HLA-DRB1(45), DRB3(7), DRB4(5), DRB5(7), DQA1(17), DQB1(21), DPA1(10) and DPB1(27). HLA-DRB1*09:01(17.09%),15:01(10.72%); DRB3*02:02(25.99%),03:01(10.18%); DRB4*01:03(36.46%); DRB5*01:01(15.42%); DQA1*01:02(20.01%),03:02(17.19%); DQB1*03:01(19.47%),03:03(17.98%), 05:02(11.66%), 06:01(10.67%); DPA1*02:02(54.45%), 01:03(31.18%) and DPB1*05:01(39.13%), 02:01(16.90%) alleles were the most common alleles in Shenzhen Han population (frequencies >10%). There was no statistical difference between the gene frequencies of HLA-DRB1 and DQB1 loci in our study. The HLA Common and Well-Documented Alleles in China (CWD2.4) (χ2=12.68, P >0.05). 94 cases of HLA-DRB1 homozygous samples detected by NGS were retested by PCR-SSOP or SBT method, and one case of allele dropout at HLA-DRB1 locus was found. SBT method confirmed that the allele of DRB1*04:03 was missed. The laboratory internal quality control system was established. Two cases of new alleles were detected and named by WHO Nomenclature Committee for Factors of the HLA System. CONCLUSION: The HLA genotyping results based on NGS showed a significantly lower ambiguity rate. The HLA class II alleles exhibit genetic polymorphism in the Han population of unrelated healthy individuals in Shenzhen. The independent method based on NGS in clinical histocompatibility testing has limitations and requires internal quality control strategies to avoid allele-dropout events.

Full-length sequence of the novel allele, HLA-B*58:01:40, identified in a Chinese individual.

HLA-B*58:01:40 differs from HLA-B*58:01:01 by a single nucleotide change in exon 3, 507 C- > T (codon 145.3 CGC- > CGT).

[Establish a Graded Method to Avoid HLA Class I Antibodies Corresponding Antigen and Combining HLAMatchmaker Application in Improving the CCI Value after Platelet Transfusion for Patients with IPTR].

OBJECTIVE: To establish a graded method to avoid mean fluorescence intensity (MFI) threshold of HLA Class I antibodies corresponding antigen, and the HLAMatchmaker program has been used to select the minimum mismatch value of donor-patient epitopes. Evaluate the application value of combining both methods in selecting HLA compatible platelets (PTL) for patients with immune platelet transfusion failure (IPTR) in improving platelet the corrected count increment (CCI). METHODS: A total 7 807 PLT cross-matching compatible were performed by the solid-phase red cell adherence (SPRCA) method for 51 IPTR patients. The Luminex single antigen flow cytometry was used to detect HLA Class I antibodies in patients, and detected the MFI value for different specificity antigens of HLA Class I antibodies, was graded into strong positive group (MFI>4 000, level 1), medium positive group (1 000< MFI≤4 000, 2), weak positive group (500< MFI≤1 000, 3), and one negative control group (MFI≤500). The results of 7 807 SPRCA their negative/positive reaction wells were enrolled and statistically analyzed in different grades and the four groups, the statistical differences between the four groups were compared. Multiple applications for the select HLA Class I compatible donor events were made for patients in two cases, and HLAMatchmaker program was used to calculate the number of HLA Class I epitopes mismatches between the donors and patients. The donor with the minimum number of epitopes mismatches was selected, while avoiding the corresponding antigens of HLA Class I antibodies in levels 1 and 2, the provision of HLA compatible platelets for IPTR. After the transfusions, the CCI value of the platelet transfusion efficacy evaluation index was calculated, and the clinical evaluation of the transfusion effect was obtained through statistical analysis. RESULTS: There were statistically significant differences in the positive results of SPRCA immunoassay among the strong positive group, medium positive group, and weak positive group of 51 IPTR patients with different specific of HLA -I class antibodies and corresponding antigens(all P <0.001). The positive results showed a range from high to low, with strong positive group>medium positive group>weak positive group. There were a statistical difference among between the strongly positive or moderately positive groups and the negative control group(P <0.001). There was no statistical difference between the weakly positive group and the negative control group(P >0.05). The strong positive group was set as the corresponding specific HLA Class I site corresponding antigen grade 1 avoidance threshold, the medium positive group as the grade 2 avoidance thresholds, and the weak positive group as the grade 3 avoidance threshold. In the case of donor platelet shortage, it is not necessary to avoid the weak positive group. Avoiding the strategy of donor antigens and HLAMatchmaker program scores ≤7 corresponding to HLA Class I antibodies of levels 1 and 2, with CCI values>4.5×109/L within 24 hours, can obtain effective clinical platelet transfusion conclusions. CONCLUSION: When selecting HLA Class I compatible donors for IPTR patients, the grading avoids HLA Class I antibodies corresponding to donor antigens, and the donor selection strategy with the minimum scores of HLAMatchmaker program is comprehensively selected. The negative result confirmed by platelet cross-matching experiments has certain practical application value for improving platelet count in IPTR patients.

Discovery of the novel HLA-A*11:452N allele by next-generation sequencing in a Chinese individual.

HLA-A*11:452N differs from A*11:01:01:01 by a single nucleotide exchange in exon 1.

Genomic sequence of the novel HLA-A*26:206:02N allele, identified in a patient with hepatitis B infection.

HLA-A*26:206:02N differs from A*26:01:01:01 by a single nucleotide exchange in exon 3.

[Using Next-Generation Sequencing Technology to Confirm the HLA Rare Alleles Detected by PCR-SSOP].

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.

A novel HLA-C null allele, HLA-C*08:236N, identified by next-generation sequencing in a Chinese individual.

HLA-C*08:236N differs from C*08:01:01 by a single nucleotide exchange in exon 5 at position 1991.

Discovery of the HLA-C*08:99 allele in a Chinese individual.

HLA-C*08:99 differs by one non-synonymous nucleotide from C*08:01:01 in exon 5, codon 288 GTT>ATT.

A novel HLA-A null allele, HLA-A*31:188N, identified by next-generation sequencing in a Chinese individual.

The HLA-A*31:188N allele differs from A*31:01:02:01 by a single nucleotide deletion in exon 3.

Identification of the novel HLA-DRB3*02:02:19 allele.

The HLA-DRB3*02:02:19 allele differs from DRB3*02:02:01:02 by a single nucleotide change in exon 2.

Identification of the HLA-DPA1*02:33 allele by next-generation sequencing in a Chinese individual.

The HLA-DPA1*02:33 allele differs from DPA1*02:02:02:04 by two nucleotide change in exon 4.

Identification of HLA-DPB1*1104:01 by next-generation sequencing in a Chinese individual.

HLA-DPB1*1104:01 differs from HLA-DPB1*540:01 by a single nucleotide change in exon 2.

Identification of HLA-DQB1*03:222 by cloning and sequencing in a Chinese individual.

HLA-DQB1*03:222 differs from HLA-DQB1*03:03:02:01 by a single nucleotide change in exon 3.

Identification of HLA-A*24:02:78 by next-generation sequencing in a Chinese Han individual.

HLA-A*24:02:78 differs from HLA-A*24:02:01:01 in exon 3 by a single nucleotide.

[A study of HLA-DPA1 and DPB1 matching status for unrelated donor-recipient pairs matched at allele level for HLA-A, -B, -C, -DRB1 and -DQB1 loci].

OBJECTIVE: To analyze the status of HLA-DPA1 and DPB1 matching for unrelated donor-recipient pairs matched at high-resolution allele level for HLA-A, B, C, DRB1 and DQB1 loci. METHODS: A total of 76 unrelated donor-recipient pairs matching at allele level for HLA-A, B, C, DRB1 and DQB1 loci were subjected to HLA-DPA1 and DPB1 sequence-based typing (SBT). HLA-DPA1and DPB1 matching status at high-resolution allelic level was also analyzed. RESULTS: The allelic identity ratio for single HLA-DPA1 and DPB1 were 17.1% and 9.2%, respectively. HLA-DPA1 and DPB1 allelic identity ratio were both very low. The majority of unrelated donor-recipient pairs (73.7%) had an incompatibility at 1 HLA-DPA1 allele, 9.2% of pairs had an incompatibility at 2 DPA1 alleles. As for the high-polymorphic HLA-DPB1 gene, 57.9% of studied donor-recipient pairs had an incompatibility at 1 HLA-DPB1 allele, almost 1/3 (32.9%) of them were completely incompatible. When HLA-DPA1 and DPB1 genes were analyzed together, the donor-recipient pairs matched at 2/4 was the most common (51.4%), 4/4 allelic complete matched pairs accounted for 5.6%, and 0/4 matched pairs accounted for 8.3%. CONCLUSION: Our results indicated that the ratio of HLA-DPA1 and DPB1 complete match in the unrelated donor-recipient pairs matching at allelic level for HLA-A, B, C, DRB1 and DQB1 loci were very low. The effect of HLA-DPA1 and DPB1 matching status on clinical unrelated stem cell transplantation still needs to be elucidated.

Decision support system for the response to infectious disease emergencies based on WebGIS and mobile services in China.

BACKGROUND: For years, emerging infectious diseases have appeared worldwide and threatened the health of people. The emergence and spread of an infectious-disease outbreak are usually unforeseen, and have the features of suddenness and uncertainty. Timely understanding of basic information in the field, and the collection and analysis of epidemiological information, is helpful in making rapid decisions and responding to an infectious-disease emergency. Therefore, it is necessary to have an unobstructed channel and convenient tool for the collection and analysis of epidemiologic information in the field. METHODOLOGY/PRINCIPAL FINDINGS: Baseline information for each county in mainland China was collected and a database was established by geo-coding information on a digital map of county boundaries throughout the country. Google Maps was used to display geographic information and to conduct calculations related to maps, and the 3G wireless network was used to transmit information collected in the field to the server. This study established a decision support system for the response to infectious-disease emergencies based on WebGIS and mobile services (DSSRIDE). The DSSRIDE provides functions including data collection, communication and analyses in real time, epidemiological detection, the provision of customized epidemiological questionnaires and guides for handling infectious disease emergencies, and the querying of professional knowledge in the field. These functions of the DSSRIDE could be helpful for epidemiological investigations in the field and the handling of infectious-disease emergencies. CONCLUSIONS/SIGNIFICANCE: The DSSRIDE provides a geographic information platform based on the Google Maps application programming interface to display information of infectious disease emergencies, and transfers information between workers in the field and decision makers through wireless transmission based on personal computers, mobile phones and personal digital assistants. After a 2-year practice and application in infectious disease emergencies, the DSSRIDE is becoming a useful platform and is a useful tool for investigations in the field carried out by response sections and individuals. The system is suitable for use in developing countries and low-income districts.

[Establishment of a large-scale bi-directional sequencing and genotyping platform for MICA gene exons 2 to 4].

OBJECTIVE: To establish a stable and large-scale bi-directional sequencing platform for genotyping MICA gene exons 2 to 4, and to analyze single nucleotide polymorphisms(SNP) of the region. METHODS: Primers for particular alleles of MICA gene exons 2 to 5 were designed. Optimal conditions for PCR amplification and sequencing reaction were explored. A commercialized one-way sequencing kit for MICA allele was used as a parallel control. Four samples carrying a MICA *010 allele were subjected to cloning and haplotype sequencing. RESULTS: Results of MICA allele typing of 100 samples for a parallel control group were confirmed by the establish method. Twenty-two SNP in MICA gene exons 2 to 4 were detected in Chinese population. Two novel allelic sequences were accepted by GenBank and IMGT/HLA database and officially named as MICA*065 and MICA*066 by the WHO Nomenclature Committee. A novel SNP in MICA gene intron 3 was discovered, with allelic sequence submitted to GenBank and IMGT/HLA database. CONCLUSION: The bi-directional sequencing genotyping platform may be applied for large-scale study of MICA allelic polymorphisms, tissue typing, organ transplantation and disease research.

[Study on HLA nucleotide sequence matching in epitope positions among recipient-donor pairs for allogenic hematopoietic stem-cell transplantation].

OBJECTIVE: To analyze the human leukocyte antigens(HLA)-A, -B, -Cw, -DRB1 and DQB1 nucleotide sequences between patients waiting for allogenic hematopoietic stem-cell transplantation (HSCT) and donors in Chinese population, and to establish strategy for maximizing optimal donor selection. METHODS: HLA high-resolution typing in a total of 537 recipient-donor pairs was determined by sequence based typing (SBT) method. The nucleotide BLAST tool was used to compare the nucleotide sequences among recipient-donor pairs. RESULTS: Only 16.20% (88/537) of recipient-donor pairs were found to fully match for nucleotide sequences of all HLA-A,-B,-Cw, -DRB1 and -DQB1 loci. Mismatch rate in single locus were 8.38% in HLA-A, 0.74% in HLA-B, 12.29% in HLA-C, 2.42% in HLA-DRB1, and 2.79% in HLA-DQB1, respectively. Mismatch rate in two or multiple HLA loci was 42.65%. Nonpermissive allele mismatch combinations (A 02:01-A 02:06, A 02:06-A 02:07, Cw 03:04-Cw 15:02, Cw 03:03-Cw 04:01, Cw 03:04-Cw 14:02, Cw 03:03-Cw 08:01, DRB1 04:03:01-DRB1 04:05) were detected in single mismatch HLA locus of recipient-donor pairs, mismatches of B 07:05:01-B 07:06, Cw 07:01:01-Cw 07:06 combinations outside of epitope positions were detected in two recipient-donor pairs. CONCLUSION: Our data suggested that attention should be paid in comparing nucleotide sequences between recipient and donor, and in distinguishing nucleotide sequence mismatches within and outside of the epitope positions. These results could serve as guidelines for donor selection.