IntroductionPeripheral blood samples are widely used in HLA genotyping due to their easy accessibility and the high-quality DNA from nucleated leukocytes. However, in cases of disease relapse requiring a second transplantation, clinicians encounter significant challenges in performing HLA genotyping and interpreting complex results from patients who have undergone haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Furthermore, systematic studies investigating the impact of haplo-HSCT on recipients’ HLA genotypes across different tissues remain scarce. Therefore, this study aims to analyze HLA genotypes in various tissues of the recipient after haplo-HSCT.MethodsA total of 66 patients who received haplo-HSCT were enrolled, with peripheral blood, buccal swab and saliva samples collected for HLA genotyping. The results were compared with pre-HSCT HLA genotypes of the patients and their respective donors.ResultsThe majority of peripheral blood samples (55/57) exhibited the donor’s HLA genotype, whereas most buccal swabs (54/62) retained the patient’s pre-HSCT genotype. Among 20 salivary samples, 45% of patients retained their pre-HSCT genotype, while 30% exhibited the donor’s genotype. Notably, chimerism and HLA loss were detected in buccal swab and saliva cells of certain recipients. More strikingly, one patient’s buccal sample displayed complete donor HLA genotype replacement.ConclusionThese findings enhance our comprehension of the genetic effects of haplo-HSCT on the recipient’s different tissues and provide valuable insights for the rational selection of tissue samples and data interpretation in HLA genotyping for patients who underwent allogenic HSCT.

The novel allele HLA-DQB1*03:03:31 differs from DQB1*03:03:02:02 by one synonymous nucleotide substitution in exon 2.

OBJECTIVE:To investigate the missed detection of HLA-B allele in one case using AlltypeTM FastplexTM next generation sequencing (NGS), and evaluate the accuracy and limitations of NGS in HLA genotyping. METHODS:HLA routine detection was performed using PCR-sequence-specific oligonucleotide probe (SSOP) and AlltypeTM FastplexTM NGS. After discovering a sample from a patient with Glanzmann's thrombasthenia that had inconsistent HLA-B locus results, the sample was retested using PCR-sequence-based typing (SBT) and AlloSeq Tx17 hybrid capture NGS technology to verify the detection results of AlltypeTM FastplexTM NGS. RESULTS:The SSOP typing results showed that the patient's HLA-B locus was B*15:01, 40:01 , while the AlltypeTM FastplexTM NGS results showed that the patient was homozygous for B*40:01, 40:01 . The homozygous sample was retested with SBT and AlloSeq Tx17 hybrid capture NGS to confirm the NGS test results. The SBT result analysis showed that there were two possible combinations: B*15:01, 40:01 and B*15:07, 40:01, indicating that the SBT results were ambiguous. However, the AlloSeq Tx17 NGS results clearly showed B*15:01, 40:01 , which was consistent with the SSOP typing results, thus confirming that AlltypeTM FastplexTM NGS missed the B*15:01 allele at the HLA-B locus. CONCLUSION:The NGS method based on amplicon library construction has the limitation of missing detection of HLA alleles. Especially for the detected homozygous samples, multiple experimental methods should be used for verification to ensure the accuracy of HLA genotyping.

The new HLA-DQA1*03:02:07 allele differs from its most closely related allele DQA1*03:02:01:02 in exon 3.

The new HLA-DRB1*12:109 allele differs from its most closely related allele DRB1*12:02:01:04 in exon 1.

The novel HLA-DQA1*05:05:13:02 allele differs from its most closely related allele DQA1*05:05:01:01 in exon 4.

We report a confirmatory HLA-DQA1*05:82 allele detected in a Chinese individual.

HLA-DPB1*1763:01 differs from DPB1*02:01:64 by a single nucleotide substitution in codon-14 of exon 1.

Although the allele and haplotype frequencies of 11 HLA loci (HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1) have been reported in different populations, rare studies have simultaneously assessed the allele distributions of non-classical HLA class I genes (HLA-E/F/G/H) and MICA/MICB together with the 11 classical HLA loci, or further analysed the haplotype frequencies covering the 17 loci. The present study aims to investigate the allele diversity and haplotype frequencies of 17 HLA-related loci including HLA genes and MICA/MICB simultaneously using a hybrid capture (HC)-based NGS method. A total of 358 HLA alleles including 177 class I and 137 class II alleles, as well as 29 MICA and 15 MICB alleles were identified in this project. The most frequent alleles at each locus were A*11:01 (29.10%), B*40:01 (14.46%), C*01:02 (19.90%), DRB1*09:01 (15.61%), DQB1*03:01 (18.48%), DPB1*05:01 (40.13%), DQA1*01:02 (22.58%), DPA1*02:02 (55.27%), DRB3*02:02 (65.95%), DRB4*01:03 (95.20%), DRB5*01:01 (75.97%), E*01:03 (62.63%), F*01:01 (97.07%), G*01:01 (70.74%), H*01:01 (35.87%), MICA*010:01 (19.90%) and MICB*005:02 (57.53%), respectively. The haplotype frequencies for different combinations of HLA loci were estimated and linkage disequilibrium (LD) between alleles for all pairs of neighbouring loci were calculated. The most frequent haplotype covering 17 loci was F*01:01-G*01:01-H*01:01-A*02:07-E*01:03-C*01:02-B*46:01-MICA*010:01-MICB*005:02-DRB4*01:03-DRB1*09:01-DQA1*03:02-DQB1*03:03-DPA1*02:02-DPB1*05:01 with a frequency of 3.18%. This is the first study on allelic polymorphism, haplotype inference and LD covering 17 HLA-related loci simultaneously in the Shenzhen Chinese population. These results will extend our knowledge of the allelic diversity of the HLA complex and provide population genetics data for transplantation and HLA-associated disease studies.

OBJECTIVE:To confirm the sequence of a null allele HLA-C*08:127N produced by a base insertion. METHODS:PCR sequence-specific oligonucleotide probe (SSOP) and PCR sequence-based typing (SBT) were used for HLA routine detection, which discovered abnormal sequence maps of HLA-C in one acute myeloid leukemia patient. The sequence of the above loci was confirmed by next generation sequencing (NGS) technology. RESULTS:The SSOP typing result showed that HLA-C locus was C*03:04, C*08:01, while the sequence was suspected to be inserted or deleted in exon 3 by SBT, and finally confirmed by NGS as C*03:04, C*08:127N. CONCLUSION:When base insertion produces HLA null alleles, SBT analysis software cannot provide correct results, but NGS technology can more intuitively obtain accurate HLA typing results.

We report the confirmation and extension of the HLA-DQB1*05:305 allele detected in a Chinese individual.

[Objective] To analyze the HLA typing and STR loci chimerism in a patient with recurrent acute lymphoblastic leukemia after HLA-haploidentical hematopoietic stem cell transplantation. [Methods] HLA typing was performed on peripheral blood, buccal swabs and saliva samples after transplantation using PCR-sequence-specific oligonucleotide probes (PCR-SSOP) and next-generation sequencing (NGS). Additionally, STR analysis was conducted on these samples using a 21-locus STR assay kit to detect STR loci. [Results] The HLA typing and STR locus outcomes of the patient's peripheral blood and the second saliva sample post-transplantation were in full concordance with the test results of the donor (father), whereas the HLA typing and STR locus results derived from the buccal swabs and the first saliva sample indicated chimerism between the donor and the recipient. [Conclusion] In the follow-up and monitoring after transplantation, apart from focusing on peripheral blood samples, it is recommended to regularly monitor HLA typing and STR loci chimerism in patients' buccal swabs and saliva samples to comprehensively evaluate the transplantation effect and recurrence risk.

OBJECTIVE:To analyze and confirm the ambiguous results of HLA-DRB1 genotyping in one case. METHODS:HLA genotyping was performed on a sample of hematopoietic stem cell donor using Illumina MiSeq-based next-generation sequencing (NGS). The ambiguous results of HLA-DRB1 locus were further analyzed and confirmed through PacBio SMRT third-generation sequencing. RESULTS:The Illumina MiSeq-based NGS typing results suggested the presence of a new HLA-DRB1*11 allele (DRB1*11:NEW, 12:01) in the specimen, with a mismatch of G>A located in the 40th residue of exon 1 compared with the nearest allele DRB1*11:01:01:03. However, due to the long sequence of intron 1, this observed mutation site was so far away from the near heterozygous sites that no reads could cover this gap. Therefore, it was impossible to determine which consensus the mutation site was located in, and the NGS-based genotyping results were obtained from the random allocation by the software, which was ambiguous and unreliable. In order to confirm the results, the long-read third generation sequencing technology based on PacBio was applied to genotype the DRB1 locus. The results showed that the DRB1 typing was HLA-DRB1*11:01,12:10. E1-40A was actually located in the allele HLA-DRB1*12:XX , which was exactly matched with HLA-DRB1*12:10. CONCLUSION:For some new alleles suggested by NGS, especially the ambiguous ones that are far away from other heterozygous sites, it is necessary to analyze and confirm them by other methods such as the third-generation long-read sequencing technology to obtain reliable results.

The non-classical HLA-G*01:55 allele differs from G*01:01:12 at one position in exon 4.

HLA-A*31:01:53 differs from HLA-A*31:01:02:01 by one nucleotide change at nucleotide 900 in exon 5 from G to A.

OBJECTIVE:To delineate a deletional mutation of the HLA-B gene in a Chinese pedigree.METHODS:A female patient with acute myeloid leukemia who had visited Liuzhou People's Hospital in April 2022 was selected as the study subject. Routine human leukocyte antigen (HLA) was determined by using PCR-sequence specific oligonucleotide polymorphism (PCR-SSOP) and PCR-sequence-based typing (PCR-SBT) methods. Next generation sequencing (NGS) was used to validate the candidate variant in the HLA-B gene.RESULTS:The PCR-SBT and SSOP results for the HLA-B locus were inconsistent for the patient and her daughter. The SSOP results of the two individuals were HLA-B*35:01, 40:02 and HLA-B*35:01, 40:01, respectively. However, the PCR-SBT results has indicated a mismatch with the nearest HLA-B*35:01 at exon 4. NGS results showed that the HLA-B*35:01 had a 9 bp deletion in the intron 5. The patient's husband was HLA-B*40:01, 58:01, which was normal.CONCLUSION:The variant in intron 5 of the HLA-B gene in this pedigree has mapped to a primer-binding region for the SBT reagent, which has affected the accuracy of PCR-SBT results.

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.

HLA-A*02:1103 differs from HLA-A*02:01:01:01 by one nucleotide change at nucleotide 811 in exon 4 from G to A.

HLA‐A*30:211 differs from HLA‐A*30:01:01:01 by one nucleotide change at nucleotide 344 in exon 3 from G to C.

Compared with HLA-DRB1*09:01:02:05, the alleles HLA-DRB1*09:57 and HLA-DRB1*09:58 each show one nucleotide change, respectively.