The distributions of HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 allele and haplotype at high-resolution level in Zhejiang Han population of China.

The distributions of HLA allele and haplotype are variable in different ethnic populations and the data for some populations have been published. However, the data on HLA-C and HLA-DQB1 loci and the haplotype of HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci at a high-resolution level are limited in Zhejiang Han population, China. In this study, the frequencies of the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci and haplotypes were analysed among 3,548 volunteers from the Zhejiang Han population using polymerase chain reaction sequencing-based typing method. Totals of 51 HLA-A, 97 HLA-B, 45 HLA-C, 53 HLA-DRB1 and 27 HLA-DQB1 alleles were observed. The top three frequent alleles of HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci were A*11:01 (23.83%), A*24:02 (17.16%), A*02:01 (11.36%); B*40:01 (14.08%), B*46:01 (12.20%), B*58:01 (8.50%); C*07:02 (18.25%), C*01:02:01G (18.15%), C*03:04 (9.88%); DRB1*09:01 (17.52%), DRB1*12:02 (10.57%), DRB1*15:01 (9.70%); DQB1*03:01 (22.63%), DQB1*03:03 (18.26%) and DQB1*06:01 (10.88%), respectively. A total of 141 HLA-A-C-B-DRB1-DQB1 haplotypes with a frequency of ≥0.1% were found and the haplotypes with frequency greater than 3% were A*02:07-C*01:02:01G-B*46:01-DRB1*09:01-DQB1*03:03 (4.20%), A*33:03-C*03:02-B*58:01-DRB1*03:01-DQB1*02:01 (4.15%), A*30:01-C*06:02-B*13:02-DRB1*07:01-DQB1*02:02 (3.20%). The likelihood ratios test for the linkage disequilibrium of two loci haplotypes was revealed that the majority of the pairwise associations were statistically significant. The data presented in this study will be useful for searching unrelated HLA-matched donor, planning donor registry and for anthropology studies in China.

Utility of next-generation sequencing methods to identify the novel HLA alleles in potential stem cell donors from Chinese Marrow Donor Program.

The human leucocyte antigen (HLA) is the most polymorphic region of the human genome. Compared with Sanger-sequencing-based typing (SBT) methods, next-generation sequencing (NGS) has significantly higher throughput and depth sequencing characteristics, having dramatic impacts on HLA typing in clinical settings. Here, we performed NGS technology with Ion Torrent S5 platform to evaluate the potential four novel HLA alleles detected in five donors from Chinese Marrow Donor Program (CMDP, Shaanxi Province) during routine Sanger SBT testing. We also predicted the highest estimated relative frequency novel allele-bearing haplotypes according to their phenotypes and HaploStats database. NGS assays, as it provided the phase-defined and complete sequencing information, undoubtedly increase novel allele identification which will greatly enrich HLA database and provide more information for donor selection.

A novel HLA allele, HLA-DRB1*13:204, detected in a Brazilian unrelated hematopoietic stem cell donor.

The HLA-DRB1*13:204 allele differs from HLA*13:64 by two nucleotide substitutions at positions 181 and 189 in the exon 2.

Two new HLA-B35 subtypes characterized in Spaniards: HLA-B*35:270 and HLA-B*35:273.

Two new HLA-B*35 alleles, B*35:270 and B*35:273, were characterized in the Spanish population.

A novel HLA allele, HLA-A*29:01:08, identified in a Brazilian individual.

HLA-A*29:01:08 differs from A*29:01:01:01 by a single synonymous substitution at position 519, codon 149 GCG→GCA in exon 3.

High density FTA plates serve as efficient long-term sample storage for HLA genotyping.

Storage of dried blood spots (DBS) on high-density FTA(®) plates could constitute an appealing alternative to frozen storage. However, it remains controversial whether DBS are suitable for high-resolution sequencing of human leukocyte antigen (HLA) alleles. Therefore, we extracted DNA from DBS that had been stored for up to 4 years, using six different methods. We identified those extraction methods that recovered sufficient high-quality DNA for reliable high-resolution HLA sequencing. Further, we confirmed that frozen whole blood samples that had been stored for several years can be transferred to filter paper without compromising HLA genotyping upon extraction. Concluding, DNA derived from high-density FTA(®) plates is suitable for high-resolution HLA sequencing, provided that appropriate extraction protocols are employed.

Three hundred and seventy-two novel HLA class II alleles identified in potential hematopoietic stem cell donors from Germany, the United States, and Poland.

We have characterized 372 novel human leukocyte antigen (HLA) class II alleles identified in newly registered stem cell donors, this includes 281 HLA-DRB1 alleles, 89 HLA-DQB1 alleles and 2 HLA-DPB1 alleles. Most novel alleles were single nucleotide variants when compared to their respective most homologous alleles. In 66.4% of all novel alleles non-synonymous nucleotide variations were identified, in 30.4% synonymous substitutions and in 3.2% nonsense mutations. Ninty-three (25.0%) novel alleles were found in several individuals; most often these were novel HLA-DRB1 alleles. Lastly, we underline the importance of recruiting ethnic minority donors in countries such as Germany and the United States, as novel alleles were frequently found among these groups.

Identification of 2127 new HLA class I alleles in potential stem cell donors from Germany, the United States and Poland.

We describe 2127 new human leukocyte antigen (HLA) class I alleles found in registered stem cell donors. These alleles represent 28.9% of the currently known class I alleles. Comparing new allele sequences to homologous sequences, we found 68.1% nonsynonymous nucleotide substitutions, 28.9% silent mutations and 3.0% nonsense mutations. Many substitutions occurred at positions that have not been known to be polymorphic before. A large number of HLA alleles and nucleotide variations underline the extreme diversity of the HLA system. Strikingly, 156 new alleles were found not only multiple times, but also in carriers of various parentage, suggesting that some new alleles are not necessarily rare. Moreover, new alleles were found especially often in minority donors. This emphasizes the benefits of specifically recruiting such groups of individuals.

Description and molecular modeling of four novel HLA-B alleles identified in Brazilian individuals.

Four novel HLA-B alleles, B*42:20, B*51:151, B*57:64 and B*58:42 were identified in Brazilian individuals.

A new HLA allele, HLA-B*08:122, described in an unrelated donor of Caucasian origin.

A new human leukocyte antigen-B allele was found in an unrelated Italian donor.

Characterization of a novel HLA-DRB1 allele: DRB1*11:153.

HLA-DRB1*11:153 differs from DRB1*11:131 by one nucleotide at position 286 where C > A, (codon 67 CTC>ATC), resulting in an amino acid substitution, 67L>67I.

The novel HLA-A*11:448 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-A*11:448 differs from HLA-A*11:01:01:01 by one nucleotide in exon 5.

Identification of a novel MICB allele, MICB*014:02, by cloning and sequencing.

The novel MICB*014:02 allele differs from MICB*014:01:01 by one nucleotide change in exon 2.

The novel HLA-C*01:255 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-C*01:255 differs from HLA-C*01:02:01:01 by one nucleotide in exon 2.

The novel HLA-C*17:69 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-C*17:69 differs from HLA-C*17:01:01:02 by one nucleotide in exon 4.

The novel HLA-B*51:389 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-B*51:389 differs from HLA-B*51:02:01:01 by one nucleotide in exon 2.

The novel HLA-C*03:651 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-C*03:651 differs from HLA-C*03:03:01:01 by one nucleotide in exon 4.

The novel HLA-B*40:555 allele identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-B*40:555 differs from HLA-B*40:01:02:01 by one nucleotide in exon 3.

Characterization of the novel HLA-B*14:121 allele by sequencing-based typing.

HLA-B*14:121 differs from HLA-B*14:01:01:01 by one nucleotide substitution in codon 319 in exon 6.

Characterization of the novel HLA-C*01:263 allele by sequencing-based typing.

HLA-C*01:263 differs from HLA-C*01:02:01:01 by one nucleotide substitution in codon 98 in exon 3.