[Characterization of the novel HLA-A*24:191 allele and analysis of its MHC molecular modeling structure].

OBJECTIVE: To characterize a novel HLA allele, A*24:191, its DNA sequence, MHC modeling structure, and the possible influence of the amino-acid residue variations on the molecule. METHODS: The HLA sequence was determined by Luminex PCR-SSO and PCR-SBT. Its MHC molecular structure and the possible effects of the amino-acid residue variations were modeled and analyzed with Phyre2, RCSB PDB and HistoCheck software. RESULTS: The PCR-SBT revealed the novel A*24:191 differs from A*24:02 in exon 2 at position 256, 265, 270 with G>C, G>C, A>T. The MHC molecular structure prediction showed that, compared with A*24:02, the 62nd residue of A*24:191 changed from the acidic E to a neutral Q, both with the side chain extending outside the α helix pointing forward the groove, (Risler's score, R=2), the 65th changed from the smaller neutral G extending inside the helix to a basic R with a long-chain extending upward outside the helix (R=52), and the 66th changed from the basic K to a neutral N both with a long side chain extending inside the groove (R=31). The above residues are located on the α helix of the α 1 domain which constituting the side wall of the peptide-binding groove. The DSS Score=3.85. From the surface image of the molecule, it can be clearly seen that the variations of the properties, sizes and configurations of the residues caused significant changes in the shape of the surface structure of the α helix. CONCLUSION: It suggested that the residue variations are likely to change the peptide binding properties as well as the TCR and antibody binding characteristics of the molecule.

[Identification of a rare platelet-specific antigen HPA-10bw allele among ethnic Han Chinese population in Shandong].

OBJECTIVE: To study the polymorphism of human platelet antigen (HPA) system 10 among ethnic Han Chinese from Shandong, China so as to supplement the data of platelet donor bank in the region. METHODS: Peripheral blood samples of platelet donors from the region were genotyped for HPA-10 alleles by PCR-sequence specific primer (PCR-SSP) and direct sequencing. RESULTS: Among 1401 donors, a rare heterozygote carrier of HPA-10w (a+b+) was identified, which gave an allelic frequency of approximately 0.035%. CONCLUSION: The detection of rare HPA-10bw antigen allele among ethnic Han Chinese from Shandong is useful for the diagnosis and prevention of neonatal alloimmune thrombocytopenia and post-transfusion purpura in the region.

[Analysis on Polymorphism of Platelet Antigen Gene in Shandong Han Population].

OBJECTIVE: To study the Polymorphism of the human platelet antigen(HPA) gene 1-17 and human leukocyte antigen(HLA) gene-A and B locus in Shandong Han population. METHODS: A total of 962 samples from routine voluntary platelet donors were genotyped for HPA1-17 system and HLA-A site, B by PCR-SSP and PCR-SSOP respectively．Gene frequencies were calculated by counting. HPA1-17 and HLA genotype combinations were analyzed by Arelequin 3.5. RESULTS: The gene frequencies of HPA-la, -1b, HPA-2a, -2b, HPA-3a, -3b, HPA-4a, -4b, HPA-5a, -5b, HPA-6a, -6b, HPA-15a, -15b were 0.9918, 0.0082, 0.9419, 0.0592, 0.5841, 0.4174, 0.9969, 0.0031, 0.9892, 0.0108, 0.9835, 0.0175,0.5488 and 0.4512, respectively． The most common HPA genotype combination was HPA-(1, 2, 4, 5, 6, 7-14, 16, 17) aa-3ab-15ab (0.2048). Moreover, HLA-A*2(0.3094) and HLA-B*13(0.1513) showed the highest frequency in their respective locus. The most common HLA genotype combination was HLA-A*2-B*13(0.1397) . CONCLUSION: Distributions of HPA and HLA show high polymorphism in Shandong Han population. The ethnic and territorial difference of HPA distribution is also confirmed. It is imperative to establish local genetic database of volunteer platelet donors.

[Sequence analysis and 3D molecular structure simulation of a novel HLA allele B*51:159].

OBJECTIVE: To identify a novel human leukocyte antigen (HLA) B allele in a Chinese Han individual and construct its three-dimensional structure. METHODS: The initial HLA genotyping was performed by PCR-sequence-based typing (PCR-SBT). The ambiguous allele was confirmed with single-strand DNA sequencing. The DNA sequence was analyzed to identify the difference between the novel allele and its closest matching allele. Finally, the three-dimensional molecular structure of the novel allele was constructed using a Swiss-Model. RESULTS: One allele of the subject at the HLA-B locus was B*44:03:01, whilst the other was a novel allele which differed from the closest matching allele B*51:01:01:01 by nucleotide (nt) 329 A to C in exon 2, resulting in an amino acid change at codon 86 (p.Asn86Thr). CONCLUSION: A novel HLA-B allele has been identified and officially named as HLA-B*51:159 by the WHO Nomenclature Committee for Factors of the HLA System. The three-dimensional structure of B*51:159 was simulated.

[Identification of 3 novel HLA-A alleles A*24:224, A*24:225 and A*24:257 by sequence-based typing].

OBJECTIVE: To verify 3 novel HLA-A alleles A*24:224, A*24:225 and A*24:257 identified in Chinese Han individuals. METHODS: No full matched results were obtained at HLA-A locus in HLA typing for China Marrow Donor Program (CMDP) using bi-allelic Sequence-Based Typing (SBT). The novel HLA alleles were identified with allele-specific amplification SBT. RESULTS: All of the three probands had a novel nucleotide sequence at HLA-A locus. All of the 3 new sequences are most close to HLA-A*24:02:01:01 except for 1 or 2 nucleotide substitution in exon 2, which resulted in different changes in corresponding codons and encoded amino acids. CONCLUSION: Three novel HLA-A alleles were confirmed and officially named as HLA-A*24:224, HLA-A*24:225 and HLA-A*24:257 under the GenBank accession numbers JQ899198, JQ924283 and HG003642 by the WHO Nomenclature Committee for Factors of the HLA System in November 2012 and November 2013, respectively.

[Confirmation of 17 rare HLA alleles and prediction of their haplotypes].

OBJECTIVE: To confirm 17 rare HLA alleles detected during routine HLA typing and deduce their haplotypes. METHODS: Bi-allelic sequence-based typing and Luminex DNA PCR-SSOP assay were applied for the initial or repeat HLA typing, respectively. The rare HLA alleles were confirmed with mono-allelic sequence-based typing. Predicted haplotypes of the rare alleles were inferred based on the frequencies of HLA alleles and haplotypes in Han population. RESULTS: The authenticity of the total 17 rare HLA alleles was proven, and 18 predicted haplotypes associated with the rare alleles were recognized. A*11:12 and DRB1*13:19 were detected twice among unrelated individuals. CONCLUSION: Study of rare HLA alleles and predicted haplotype can provide useful information for donor searching and transplantation, and enrich polymorphisms of HLA in this population.

In vitro properties of apheresis platelet during extended storage in plasma treated with anandamide.

BACKGROUND: In China apheresis platelets (PLTs) are stored in plasma for only 5 days, resulting in PLT inventory pressures. Anandamide (ANA) was reported to be a potential agent to inhibit PLT apoptosis. The aim of this study was to evaluate the characteristics of extended storage PLTs in plasma treated with ANA in vitro. METHODS: Apheresis PLTs (n = 20) were prepared in plasma treated with ANA, and stored at 22 °C for up to 11 days. On day 1, 3, 5, 7, 9 and 11, PLTs were tested for PLT count, mean PLT volume (MPV), PLT distribution width (PDW), pH, pCO(2), pO(2), hypotonic shock response (HSR), phosphatidylserine (PS) exposure and soluble P-selectin content. RESULTS: PLTs stored in plasma with/without ANA didn't show significant differences during the first 5 days of storage. From the 7(th) day on, PLTs stored in plasma with ANA displayed significantly lower PS expression, soluble P-selectin content and higher HSR scores than those stored in plasma without ANA (P <0.05), respectively. CONCLUSION: The extended storage of PLTs in plasma treated with 0.5 µmol/l ANA showed better characteristics of the PLTs, compared with the control group, which was suggested to potentially alleviate the PLT storage lesion.

[Identification of a novel HLA allele, HLA-DRB1*03:80, by sequencing-based typing].

This study was aimed to identify a novel HLA-DRB1 allele from a Chinese potential hemopoietic stem cell donor of Northeast China. A rare HLA-DRB1 allele was initially detected by Luminex PCR-SSO typing, then the sample was sequenced by sequence-based typing (SBT) and the alignments of sample's alleles was identified by single allele-specific sequencing strategy. The results revealed the existence of a new allele which differs from the closest matching allele DRB1*03:06 by a single nucleotide substitution at position 239, where C→G in exon 2, resulting in an amino acid exchange from Thr to Arg at codon 51. It is concluded that a novel allele has been confirmed and its name DRB1*03:80 is officially assigned by the WHO Nomenclature Committee in February 2012.

[Effects of N-Arachidonoylethanolamine on the quality of platelets stored in M-sol platelet preservative solution in vitro].

This study was purposed to investigate the effects of N-Arachidonoylethanolamine (ANA) on the quality of platelets (Plt) stored in Plt M-sol preservative solution at 22 ± 2°C. Samples taken from collecting apheresis Plt by the Amicus instrument and splited into two equal parts were stored in Plt M-sol preservative solution on a shaker at 22 ± 2°C. Different working concentrations of ANA (from 0.1 to 50 µmol/L) were then added into one part of stored Plt as the experimental group, the other without ANA was used as the control group. The viability of Plts stored at 22 ± 2°C for 7 days was evaluated by MTT colorimetric assay. The most effective concentration of ANA was selected and added to the subsequent experimental group. Plt count (BPC), mean Plt volume (MPV), Plt distribution width (PDW), phosphatidyl serine (PS) and soluble P-selectin were detected on the 1(st), 5(th), 7(th), 9(th) and 11(th) day of storage. The results showed that the most effective working concentration of ANA was 0.5 µmol/L, which showed significant increasing Plt viability (91.23 ± 5.44%) compared to the control group (62.54 ± 4.79%). Thus, ANA concentration at 0.5 µmol/L was choose to perform subsequent experiments. During 11 days of storage, the BPC, MPV and PDW were not changed significantly between the experimental group and control group, although there was decreasing trend in the BPC and increasing trends in MPV and PDW in the two groups. The rate of Plt PS positive was enhanced during the storage period: the rate of PS positive in experimental group increased from 7.69 ± 1.82% to 10.74 ± 1.78% while it in control group increased from 11.21 ± 2.03% to 15.37 ± 1.95%, with significant differences between the two groups (P < 0.05) on the 9(th) and 11(th) day of storage, respectively. Soluble P-selectin contents in experimental group on the 9(th) and 11(th) day of storage were 30.19 ± 2.03 ng/ml and 34.52 ± 2.64 ng/mL, respectively, while those in control group were 39.18 ± 2.66 ng/ml and 43.23 ± 2.58 ng/ml, respectively, with significant differences between the two groups (P < 0.05). It is concluded that the extended storage of Plt in M-sol treated with low concentration ANA can potentially alleviate Plt storage lesions.

[Identification of a novel HLA allele, HLA-B*35:03:07, by sequencing-based typing].

This study was purposed to analyze and identify a novel HLA allele in Chinese population. A new HLA-B allele which is closely related to HLA-B*35:03:01 was initially detected by PCR-SSOP, then DNA sequencing was performed to identify the difference between the novel allele and HLA-B*35:03:01 allele. The result showed that the sequence of the new allele was different from all other known sequence. It differs from the closest matching HLA-B*35:03:01 by a single substitution at position 387 C→G in exon 3, no resulting in amino acid change. It is concluded that this allele is a novel one and has been officially named B*35:03:07 by the WHO Nomenclature Committee.

[Analysis of HLA-A, B, and DRB1 polymorphism and genetic distance in Han population living in Yantai and Weihai regions of Shandong province].

OBJECTIVE: To investigate allelic and haplotypic polymorphisms of human leukocyte antigen(HLA) genes at A, B and DRB1 loci in Yantai and Weihai Han population and analyze the genetic relationship between Yantai, Weihai Han population and other populations. METHODS: A total of 4062 unrelated Han ethnic individual from Yantai and Weihai regions were genotyped by polymerase chain reaction-sequence specific olignucleotide probe(PCR-SSOP) for HLA-A, B and DRB1 loci. Allelic and haplotypic frequencies were estimated by maximum likelihood estimation method using Arlequin 3.5 software. Genetic distances were computed, and phylogenetic tree was constructed using Mega5.0 software. RESULTS: Respectively 18, 33 and 13 alleles were observed at HLA-A, B and DRB1 loci. The most frequent alleles were HLA-A*02(0.2935), HLA-B*15(0.1485) and HLA-DRB1*15(0.1621). And the most common three loci haplotype was A*30-B*13-DRB1*07(0.0649). A*33-B*58, A*66-DRB1*13 and B*08-DRB1*03 showed the strongest linkage disequilibrium. Yantai and Weihai Han population has the shortest genetic distance with Jilin Han population (0.0034). CONCLUSION: The HLA-A, B and DRB1 loci are highly polymorphic in Han population from Yantai and Weihai, and this population has closest relationship with Han population from Jilin province.

[HLA-A, B and DRB1 polymorphism at high-resolution in Han population from southern area of Shandong province in China].

This study was aimed to analyze the polymorphism of HLA-A, B, DRB1 alleles at high-resolution level in Han population from southern area of Shandong province in China. 688 randomly selected, unrelated and healthy individual from southern area of Shandong province were genotyped for HLA-A, -B and HLA-DRB1 loci by PCR-SBT. Then, allelic and haplotypic distributions of HLA-A, B and DRB1 were estimated by maximum likelihood estimation method using Arlequin 3.0. The results indicated that a total of 31 HLA-A, 63 HLA-B and 39 HLA-DRB1 alleles were identified in Han Population from southern area of Shandong province. Six HLA-A alleles were found with a frequency greater than 0.05 (A*24:02, *30:01, *11:01, *02:01, *33:03 and *02:06), with a cumulative frequency of 0.7223. For HLA-B locus, there were also six alleles which had a frequency higher than 5% (B*1302, *4403, *5101, B*4601, *1501 and *5801), representing 0.4432 of the all alleles in the population. And four HLA-DRB1 alleles were defined as predominant (DRB1*0701, *1501, *0901and *0803), accounting for 0.5453 of the defined alleles. The most common three-loci haplotype was A*30:01-B*13:02-DRB1*07:01 (0.1151) and the most frequent two-loci haplotype were A*30:01-B*13:02 (0.1303), A*30:01-DRB1*07:01 (0.1157) and B*13:02-DRB1*07:01 (0.1307). It is concluded that the allelic and haplotypic diversities of HLA-A, -B and HLA-DRB1 at high-resolution in Han population from southern area of Shandong province in China provide useful information for HLA matching in transplantation and diseases-associated study in this population.

[Identification of a novel HLA allele HLA-B*40:96].

OBJECTIVE: To identify a novel human leukocyte antigen (HLA) allele in Chinese and investigate its inheritance in the family. METHODS: Exceptional reaction pattern was detected in HLA-B locus in HLA typing using Luminex DNA polymerase chain reaction with sequence specific oligonucleotide probe hybridization (PCR-SSOP) assay. A confirmatory test for the novel HLA allele was performed by DNA sequencing based typing of the proband's family. RESULTS: The DNA sequence was confirmed to be a novel HLA B allele. There were 7 nucleotides which differed from the closest matching HLA B*40:06:01 at positions 302(G to A), 309(G to C), 311(A to C), 313(C to G), 314(T to C), 317(G to T), and 319(G to C) in exon 2, which resulted in 5 amino acid changes at codon 101 (Ser to Asn), 104 (Asn to Thr), 105 (Leu to Ala), 106 (Arg to Leu), and 107 (Gly to Arg), respectively. Family investigation indicated that the novel allele was transmitted from the proband's father. CONCLUSION: A novel HLA B allele was identified and officially named as HLA-B*40:96 (GenBank accession No. FJ374890) by the WHO Nomenclature Committee for Factors of the HLA System.

[Identification of a novel allele HLA-DRB1*1219].

OBJECTIVE: To identify a novel HLA DRB1 allele in a Chinese leukemia family. METHODS: A new HLA-DRB1 allele was initially detected by polymerase chain reaction-sequence specific primer and unusual reaction pattern by Luminex RSSO, then DNA sequencing was performed to identify the sequence of the novel allele. RESULTS: The DNA sequencing revealed the presence of the new allele which differs from the closest matching HLA-DRB1*120201 by a single nucleotide substitution at position (341 C > T in exon 2), resulting in an amino acid change from Ala to Val at coden 85. CONCLUSION: A novel allele was confirmed by DNA sequencing and has been designated HLA-DRB1*1219 by the WHO Nomenclature Committee.