[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.

HLA-B*07, HLA-DRB1*07, HLA-DRB1*12, and HLA-C*03:02 Strongly Associate With BMI: Data From 1.3 Million Healthy Chinese Adults.

Strong associations between HLA alleles and infectious and autoimmune diseases are well established. Although obesity is also associated with these diseases, the relationship between HLA and obesity has not been systematically investigated in a large cohort. In the current study, we analyzed the association of HLA alleles with BMI using data from 1.3 million healthy adult donors from the Chinese Marrow Donor Program (CMDP). We found 23 HLA alleles, including 12 low-resolution and 11 high-resolution alleles, were significantly associated with BMI after correction for multiple testing. Alleles associated with high BMI were enriched in haplotypes that were common in both Chinese and European populations, whereas the alleles associated with low BMI were enriched in haplotypes common only in Asians. Alleles B*07, DRB1*07, DRB1*12, and C*03:02 provided the strongest associations with BMI (P = 6.89 × 10-10, 1.32 × 10-9, 1.52 × 10-9, and 4.45 × 10-8, respectively), where B*07 and DRB1*07 also had evidence for sex-specific effects (Pheterogeneity = 0.0067 and 0.00058, respectively). These results, which identify associations between alleles of HLA-B, DRB1, and C with BMI in Chinese young adults, implicate a novel biological connection between HLA alleles and obesity.

Correlation Between HLA-A, B and DRB1 Alleles and Severe Fever with Thrombocytopenia Syndrome.

OBJECTIVE: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever caused by a tick-borne bunyavirus (SFTSV) in East Asian countries. The role of human leukocyte antigen (HLA) in resistance and susceptibility to SFTSV is not known. We investigated the correlation of HLA locus A, B and DRB1 alleles with the occurrence of SFTS. METHODS: A total of 84 confirmed SFTS patients (patient group) and 501 unrelated non-SFTS patients (healthy individuals as control group) from Shandong Province were genotyped by PCR-sequence specific oligonucleotide probe (PCR-SSOP) for HLA-A, B and DRB1 loci.Allele frequency was calculated and compared using χ2 test or the Fisher's exact test. A corrected P value was calculated with a bonferronis correction. Odds Ratio (OR) and 95% confidence intervals (CI) were calculated by Woolf's method. RESULTS: A total of 11 HLA-A, 23 HLA-B and 12 HLA-DRB1 alleles were identified in the patient group, whereas 15 HLA-A, 30 HLA-B and 13 HLA-DRB1 alleles were detected in the control group. The frequencies of A*30 and B*13 in the SFTS patient group were lower than that in the control group (P = 0.0341 and 0.0085, Pc = 0.5115 and 0.252). The ORs of A*30 and B*13 in the SFTS patient group were 0.54 and 0.49, respectively. The frequency of two-locus haplotype A*30-B*13 was lower in the patient group than in the control group(5.59% versus 12.27%, P = 0.037,OR = 0.41, 95%CI = 0.18-0.96) without significance(Pc>0.05). A*30-B*13-DRB1*07 and A*02-B*15-DRB1*04 had strong associations with SFTS resistance and susceptibility respectively (Pc = 0.0412 and 0.0001,OR = 0.43 and 5.07). CONCLUSION: The host HLA class I polymorphism might play an important role with the occurrence of SFTS. Negative associations were observed with HLA-A*30, HLA-B*13 and Haplotype A*30-B*13, although the associations were not statistically significant. A*30-B*13-DRB1*07 had negative correlation with the occurrence of SFTS; in contrast, haplotype A*02-B*15-DRB1*04 was positively correlated with SFTS.

High-Resolution Analyses of Human Leukocyte Antigens Allele and Haplotype Frequencies Based on 169,995 Volunteers from the China Bone Marrow Donor Registry Program.

Allogeneic hematopoietic stem cell transplantation is a widely used and effective therapy for hematopoietic malignant diseases and numerous other disorders. High-resolution human leukocyte antigen (HLA) haplotype frequency distributions not only facilitate individual donor searches but also determine the probability with which a particular patient can find HLA-matched donors in a registry. The frequencies of the HLA-A, -B, -C, -DRB1, and -DQB1 alleles and haplotypes were estimated among 169,995 Chinese volunteers using the sequencing-based typing (SBT) method. Totals of 191 HLA-A, 244 HLA-B, 146 HLA-C, 143 HLA-DRB1 and 47 HLA-DQB1 alleles were observed, which accounted for 6.98%, 7.06%, 6.46%, 9.11% and 7.91%, respectively, of the alleles in each locus in the world (IMGT 3.16 Release, Apr. 2014). Among the 100 most common haplotypes from the 169,995 individuals, nine distinct haplotypes displayed significant regionally specific distributions. Among these, three were predominant in the South China region (i.e., the 20th, 31st, and 81sthaplotypes), another three were predominant in the Southwest China region (i.e., the 68th, 79th, and 95th haplotypes), one was predominant in the South and Southwest China regions (the 18th haplotype), one was relatively common in the Northeast and North China regions (the 94th haplotype), and one was common in the Northeast, North and Northwest China (the 40th haplotype). In conclusion, this is the first to analyze high-resolution HLA diversities across the entire country of China, based on a detailed and complete data set that covered 31 provinces, autonomous regions, and municipalities. Specifically, we also evaluated the HLA matching probabilities within and between geographic regions and analyzed the regional differences in the HLA diversities in China. We believe that the data presented in this study might be useful for unrelated HLA-matched donor searches, donor registry planning, population genetic studies, and anthropogenesis studies.

[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*55:35].

OBJECTIVE: To identify a novel human leukocyte antigen (HLA) B allele and explore its family heritage. METHODS: A novel HLA allele was suspected upon routine HLA typing using a polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) assay. The sequence was confirmed with DNA sequencing and compared with its closest matching allele, B*55:02. The family was also investigated. RESULTS: An unusual reaction pattern was detected during routine HLA typing. The sequence was confirmed to be a novel HLA-B allele, which differed from the closest matching allele, B*55:02 in 7 nt positions in exon 2. Among the 7 mutations from 6 codons, there were two amino acids changes including 69Glu→Met and 70Glu→Ala. CONCLUSION: A novel HLA-B allele has been identified and officially named as B*55:35 by the WHO Nomenclature Committee for Factors of the HLA System (GenBank accession number FJ898284).

Human leukocyte antigen-C and killer cell immunoglobulin-like receptor gene polymorphisms among patients with syphilis in a Chinese Han population.

Syphilis is a sexually transmitted infection caused by the Treponema pallidum subspecies pallidum spirochete bacterium. The killer cell immunoglobulin-like receptors (KIR), interacting with human leukocyte antigens (HLA), regulate the activations of natural killer (NK) cells and certain T-cell subsets in response to microbe infection. The objective of this study was to explore whether KIR and HLA-C gene polymorphisms were associated with syphilis in a Chinese Han population. Polymerase chain reaction with sequence-specific primers (PCR-SSP) method was used to genotype KIR and HLA-C genes in 231 syphilis patients and 247 healthy controls. Framework genes KIR2DL4, KIR3DL2, KIR3DL3 and KIR3DP1 were present in all individuals. The frequencies of KIR2DS3 and KIR3DS1 were higher in syphilis patients than in healthy controls (p = 0.030 and p = 0.038, respectively), while the frequency of KIR2DS5 was higher in healthy controls than in syphilis patients (p = 0.015; OR = 0.575). The homozygote for HLA-C1 allele (HLA-C1C1) was more common in controls compared with syphilis patients (p = 0.030; OR = 0.667). The frequency of individuals with HLA-C1C1 and KIR2DL3 genotype was higher in control group relative to syphilis patient group (p = 0.018; OR = 0.647). These data indicated that KIR2DS3 and KIR3DS1 were more prevalent in syphilis patients than in controls, and that KIR2DS5, HLA-C1C1 and HLA-C1C1-KIR2DL3 were more prevalent in controls than in syphilis patients, respectively. These will require further investigation using functional studies.

[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.

[Identification of a novel HLA-A allele A*11:01:37 by sequence-based typing].

OBJECTIVE: To identify a novel HLA-A allele in a Chinese Han individual. METHODS: One mismatch was observed in HLA-A locus in HLA typing for CMDP donors using bi-allelic SBT kit. A confirmatory test for novel HLA allele was performed with mono-allelic SBT kit. RESULTS: The DNA sequence was confirmed to be a novel HLA-A allele. There was 1 nucleotide differed from the closest matching HLA-A*11:01:01 at position 393(G→A), which resulting a change from GGG to GGA at codon 107, led to a silent mutation, conserving the amino acid Gly. CONCLUSION: A novel HLA-A allele was confirmed and officially named HLA-A*11:01:37 (Genbank accession number, JN209962) by the WHO Nomenclature Committee for Factors of the HLA System in January 2012.

[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.