Determination for KIR genotype and allele copy number via real-time quantitative PCR method.

Killer cell immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) play crucial roles in regulating NK cell activity. Here, we report a real-time quantitative PCR (qPCR) to genotype all KIR genes and their copy numbers simultaneously. With 18 pairs of locus-specific primers, we identified KIR genes by Ct values and determined KIR copy number using the 2-∆Ct method. Haplotypes were assigned based on KIR gene copy numbers. The real-time qPCR results were consistent with the NGS method, except for one sample with KIR2DL5 discrepancy. qPCR is a multiplex method that can identify KIR copy number, which helps obtain a relatively accurate haplotype structure, facilitating increased KIR research in laboratories where NGS or other high-resolution methods are not available.

Prevalence and Residual Risk of HIV in Volunteer Blood Donors of Zhejiang Province, China, from 2018 to 2022.

Background: Blood safety levels have been significantly improved since the implementation of nucleic acid amplification technology (NAT) testing for blood donors. However, there remains a residual risk of transfusion transmission infections. This study aimed to evaluate the prevalence of HIV and its residual risk transmission among volunteer blood donors of Zhejiang Province, China, for five years after NAT implementation. Materials and Methods: All specimens and information were collected from voluntary unpaid donors at all blood services in Zhejiang Province, China, from January 2018 to December 2022. The HIV antibody or antigen and HIV RNA were detected using enzyme-linked immunosorbent assay and NAT, respectively. The HIV residual risk transmission was calculated using the incidence or window period model. Results: A total of 3,375,678 voluntary blood donors were detected, revealing an HIV prevalence of 9.92/100000. The HIV prevalence of blood donors in 12 blood services in Zhejiang Province was 6.11, 6.98, 7.45, 8.21, 8.36, 8.94, 9.04, 9.66, 9.73, 10.22, 11.80, and 12.47 per 100000 donors, without statistically significant difference observed among the services (p > 0.05). The HIV prevalence of males (15.49/100000) was significantly higher compared to females (1.95/100000; p < 0.05). There was an insignificant difference in HIV prevalence among blood donors of all different age groups (p > 0.05), but the HIV prevalence in the 26-35 age group and 18-25 age group was significantly higher compared to the 36-45 age group (p < 0.05). The difference in HIV prevalence between first-time blood donors (13.65/100,000) and repeat blood donors (6.78/100,000) was statistically significant (p < 0.05). From 2018 to 2022, the HIV residual risk in blood transfusion transmission was 0.266/100000. Conclusion: The prevalence of HIV among blood donors in Zhejiang Province, China, is associated with age, gender, and times of blood donation. The HIV residual risk in blood transfusion transmission remains low in the province, and increasing the rate of repeat blood donors is beneficial to improve blood safety.

HLA-A*02:06 allele may be susceptible to myelodysplastic syndrome in Zhejiang Han population, China.

The association between HLA loci and haematological malignancy has been reported in certain populations. However, there are limited data for HLA loci at a high-resolution level with haematological malignancy in China. In this study, a total of 1115 patients with haematological malignancies (including 490 AML, 410 acute lymphoblastic leukaemia (ALL), 122 myelodysplastic syndrome [MDS] and 93 non-Hodgkin's lymphoma [NHL]) and 1836 healthy individuals as a control group in the Han population of Zhejiang Province, China, were genotyped for HLA-A, HLA-C, HLA-B, HLA-DRB1 and HLA-DQB1 loci at high resolution. The possible association between HLA alleles and haplotypes and haematologic malignancy was analysed. The allele frequencies (AFs) of HLA-A*02:05, HLA-A*02:06, HLA-A*32:01, HLA-B*35:03, HLA-B*54:01, HLA-B*55:07, HLA-DRB1*04:05, HLA-DRB1*15:01, HLA-DQB1*04:01 and HLA-DQB1*06:02 in the MDS patients were much higher than those in the control group (P < 0.05), while the AFs of HLA-C*07:02, HLA-DRB1*03:01, HLA-DRB1*14:54, HLA-DQB1*02:01 and HLA-DQB1*05:03 were obviously lower than those in the control group (p < .05). Interestingly, the differences in these HLA alleles in patients with MDS were not significant after applying Bonferroni correction (Pc > .05), except for HLA-A*02:06 (Pc < .01). There were 13, 6 and 10 HLA alleles with uncorrected significant differences (p < .05) among patients with AML, ALL and NHL, respectively, compared with those in the control group, but the differences in these HLA alleles were not significant after correction (Pc > .05). Compared to those of the control group, there were some haplotypes over 1.00% frequency in patients with AML, MDS and NHL patients with uncorrected significant differences (p < .05). However, none of them showed a significant difference after correction as well (Pc > .05). The study reveals that HLA-A*02:06 may lead to susceptibility to MDS, but none of the HLA alleles were associated with AML, ALL or NHL after correction. These data will help to further understand the role of HLA loci in the pathogenesis of haematological malignancy in China.

[Study of the molecular characteristics of a Bweak phenotype due to a novel c.398T>C variant of the ABO gene].

OBJECTIVE: To explore the molecular mechanism for an individual with Bweak subtype. METHODS: Serological methods were used to identify the proband's phenotype. In vitro enzyme activity test was used to determine the activity of B-glycosyltransferase (GTB) in her serum. The genotype was determined by PCR amplification and direct sequencing of exons 5 to 7 and flanking sequences of the ABO gene. T-A cloning technology was used to isolate the haploids. The primary physical and chemical properties and secondary structure of the protein were analyzed with the ProtParam and PSIPRED software. Three software, including PolyPhen-2, SIFT, and PROVEAN, was used to analyze the effect of missense variant on the protein. RESULTS: Serological results showed that the proband's phenotype was Bweak subtype with anti-B antibodies presented in her serum. In vitro enzyme activity assay showed that the GTB activity of the subject was significantly reduced. Analysis of the haploid sequence revealed a c.398T>C missense variant on the B allele, which resulted in a novel B allele. The 398T>C variant has caused a p.Phe133S substitution at position 133 of the GTB protein. Based on bioinformatic analysis, the amino acid substitution had no obvious effect on the primary and secondary structure of the protein, but the thermodynamic energy of the variant protein has increased to 6.07 kcal/mol, which can severely reduce the protein stability. Meanwhile, bioinformatic analysis also predicted that the missense variant was harmful to the protein function. CONCLUSION: The weak expression of the Bweak subtype may be attributed to the novel allele of ABO*B.01-398C. Bioinformatic analysis is helpful for predicting the changes in protein structure and function.

The impact of nucleic acid testing to detect human immunodeficiency virus, hepatitis C virus, and hepatitis B virus yields from a single blood center in China with 10-years review.

BACKGROUND: Since 2010, the Blood Center of Zhejiang province, China, has conducted a pilot nucleic acid amplification testing (NAT) screening of blood donors for Hepatitis B virus (HBV), Hepatitis C virus (HCV), and Human immunodeficiency virus (HIV). This study aims to assess the results of NAT testing over 10 years to establish the effects and factors influencing NAT yields of HBV, HCV, and HIV. METHODS: Blood donations from seven different blood services were screened for HBV DNA, HCV RNA, and HIV RNA using 6 mini pools (6MP) or individual donation (ID)-NAT method between August 1, 2010, and December 31, 2019, at the NAT centralized screening center. We compared 3 transcription-mediated amplification (TMA) assays and 2 polymerase chain reaction (PCR) assays. Further, HBV, HCV, and HIV NAT yields were calculated and donor characteristics and prevalence of HBV NAT yields analyzed. Donors with HCV and HIV NAT yield were also followed up. RESULTS: 1916.31 per million donations were NAT screening positive overall. The NAT yields for HBV, HCV, HIV and non-discriminating reactive were 1062.90 per million, 0.97 per million, 1.45 per million, and 850.99 per million, respectively, which varied in the seven blood services and different years. HBV NAT yields were higher than those of HCV and HIV and varied across demographic groups. Risk factors included being male, old age, low education level, and first-time donors. We found no differences in NAT yields of HBV, HCV, and HIV between the 3 TMA and 2 PCR assays; nonetheless, statistically, significant differences were noted between the five assays. CONCLUSION: In summary, NAT screening in blood donations reduces the risk of transfusion-transmitted infections and shortens the window period for serological marker screening. Therefore, a sensitive NAT screening method, ID-NAT workflow, and recruitment of regular low-risk donors are critical for blood safety.

[Analysis of loss of heterozygosity at HLA loci in a patient with leukemia].

OBJECTIVE: To detect loss of heterozygosity (LOH) at human leukocyte antigen (HLA) loci in a Chinese patient with leukemia after haploidentical hematopoietic stem cell transplantation. METHODS: HLA genotyping was carried out on peripheral blood, hair follicle and buccal swab samples derived from the patient after the transplantation as well as peripheral blood samples from his parents by using PCR-sequence specific oligonucleotide probe method and PCR-sequence based typing method. Short tandem repeat (STR) loci were detected by using a 23 site STR assay kit and a self-developed 6 STR loci assay for the HLA regions. RESULTS: After the transplantation, the HLA genotype of the peripheral blood sample of the patient was identical to his father. The patient was HLA-A*02:01,24:02, C*03:03,03:04, B*13:01,15:01, DRB1*08:03,12:02, DQB1*03:01,06:01 for his hair follicle specimen. However, homozygosity of the HLA loci was found in his buccal swab sample. Only the HLA-A*24:02-C*03:03-B*15:01-DRB1*08:03-DQB1*06:01 haplotype from his father's was present, while the HLA-A*02:01-C*03:04-B*13:01-DRB1*12:02-DQB1*03:01 haplotype from his mother was lost. After the transplantation, the alleles of the 23 STR sites in the patient's peripheral blood sample were consistent to his father, with no allelic loss detected in his buccal swab sample. However, at least 4 STR loci in the HLA region were lost in his buccal swab sample. CONCLUSION: LOH at the HLA loci has been detected in the buccal swab sample of a patient with leukemia who received haploidentical hematopoietic stem cell transplantation.

Prevalence of Treponema Pallidum Antibody among Volunteer Blood Donors in China.

Background: Infection with syphilis is still a major public health problem. The precise data for syphilis seroprevalence in the populations will help to develop a strategy for prevention and treatment of it. However, the data for syphilis prevalence in continuous years among volunteer blood donors in China is rare. Methods: A retrospective study for Treponema pallidum (TP) antibody in blood donors was conducted from January 2010 to December 2019 at the Blood Center of Zhejiang Province, China. TP antibody was detected with two different reagents using enzyme-linked immunosorbent assay and the only sample which was reactive in the two reagents was defined as seropositive. Results: A total of 992,646 volunteer blood donors were analyzed and the positive rate of TP antibody in the blood donors was 0.43%. From 2010 to 2019, the positive rates of TP antibody were 0.53%, 0.51%, 0.51%, 0.43%, 0.36%, 0.18%, 0.11%, 0.12%, 0.11%, and 0.10%, respectively. The positive rates of TP antibody were significantly different among blood donor age group (p < 0.001), with the highest positive rate in 45-54-years-old group (0.93%). The positive rates of TP antibody in male and female blood donors were 0.44% and 0.41%, respectively. The positive rate was 0.57% among the first-time blood donors, which was significantly higher than that of the repeat blood donors (0.17%). The positive rate of TP antibody in blood donors decreased gradually with the increase of educational level. Conclusion: The syphilis seroprevalence is low in the blood donors of the Hangzhou area, and the positive rate of blood donors is associated with age, educational level, and times of blood donation. Increasing the number of repeat blood donations is helpful to improve blood safety.

The combinatorial diversity of KIR and HLA class I allotypes in Peninsular Malaysia.

Killer cell immunoglobulin-like receptors (KIRs) interact with polymorphic human leucocyte antigen (HLA) class I molecules, modulating natural killer (NK) cell functions and affecting both the susceptibility and outcome of immune-mediated diseases. The KIR locus is highly diverse in gene content, copy number and allelic polymorphism within individuals and across geographical populations. To analyse currently under-represented Asian and Pacific populations, we investigated the combinatorial diversity of KIR and HLA class I in 92 unrelated Malay and 75 Malaysian Chinese individuals from the Malay Peninsula. We identified substantial allelic and structural diversity of the KIR locus in both populations and characterized novel variations at each analysis level. The Malay population is more diverse than Malay Chinese, likely representing a unique history including admixture with immigrating populations spanning several thousand years. Characterizing the Malay population are KIR haplotypes with large structural variants present in 10% individuals, and KIR and HLA alleles previously identified in Austronesian populations. Despite the differences in ancestries, the proportion of HLA allotypes that serve as KIR ligands is similar in each population. The exception is a significantly reduced frequency of interactions of KIR2DL1 with C2+ HLA-C in the Malaysian Chinese group, caused by the low frequency of C2+ HLA. One likely implication is a greater protection from preeclampsia, a pregnancy disorder associated with KIR2DL1, which shows higher incidence in the Malay than in the Malaysian Chinese. This first complete, high-resolution, characterization of combinatorial diversity of KIR and HLA in Malaysians will form a valuable reference for future clinical and population studies.

Seroprevalence of human T-lymphotropic virus infection among blood donors in China: a first nationwide survey.

BACKGROUND: So far, the prevalence of human T-lymphotropic virus (HTLV) type 1 and 2 in some highly populated countries such as China is still unknown. In this study, a multi-center nationwide serological survey was designed and performed, to reveal the seroprevalence of HTLV infection among Chinese blood donors. RESULTS: Among 8,411,469 blood donors from 155 blood establishments, 435 were finally confirmed as HTLV carriers. The prevalence of HTLV infection in China varied in different provinces: Fujian had the highest prevalence of 36.240/100,000 (95% CI 31.990-41.050) and eleven provinces did not find HTLV-seropositive donors in the three years. no HTLV-2 infection was found. The overall prevalence of HTLV-1 in China decreased from 2016 to 2018. Female was identified as an independent risk factor of HTLV infection in China. Besides, seroconversion was observed in two of seven seroindeterminate donors 85 and 250 days after their last donation, respectively. CONCLUSIONS: The seroprevalence of HTLV infection in most areas of China among blood donors is quite low, but it varies significantly in different geographic areas. Screening anti-HTLV-1/2 antibody and follow-up of serointederminate donors are essential to ensure blood safety especially in areas where we have found HTLV infected donors.

Six splice site variations, three of them novel, in the ABO gene occurring in nine individuals with ABO subtypes.

BACKGROUND: Nucleotide mutations in the ABO gene may reduce the activity of glycosyltransferase, resulting in lower levels of A or B antigen expression in red blood cells. Six known splice sites have been identified according to the database of red cell immunogenetics and the blood group terminology of the International Society of Blood Transfusion. Here, we describe six distinct splice site variants in individuals with ABO subtypes. METHODS: The ABO phenotype was examined using a conventional serological method. A polymerase chain reaction sequence-based typing method was used to examine the whole coding sequence of the ABO gene. The ABO gene haplotypes were studied using allele-specific primer amplification or cloning technology. In silico analytic tools were used to assess the functional effect of splice site variations. RESULTS: Six distinct variants in the ABO gene splice sites were identified in nine individuals with ABO subtypes, including c.28 + 1_2delGT, c.28 + 5G > A, c.28 + 5G > C, c.155 + 5G > A, c.204-1G > A and c.374 + 5G > A. c.28 + 1_2delGT was detected in an Aw individual, while c.28 + 5G > A, c.28 + 5G > C, and c.204-1G > A were detected in Bel individuals. c.155 + 5G > A was detected in one B3 and two AB3 individuals, whereas c.374 + 5G > A was identified in two Ael individuals. Three novel splice site variants (c.28 + 1_2delGT, c.28 + 5G > A and c.28 + 5G > C) in the ABO gene were discovered, all of which resulted in low antigen expression. In silico analysis revealed that all variants had the potential to alter splice transcripts. CONCLUSIONS: Three novel splice site variations in the ABO gene were identified in Chinese individuals, resulting in decreased A or B antigen expression and the formation of ABO subtypes.

Mechanism evaluation for an amino acid substitution p.Y246C of B-glycosyltransferase enzyme with Bweak phenotype.

BACKGROUND: The amino acid substitutions caused by ABO gene variants are usually predicted to impact the glycosyltransferase function. Here, the effect of an amino acid substitution in the vicinity of the catalytic active region of the B-glycosyltransferase was explored in vitro and in silico study, which is important for further recognizing the ABO subgroup. METHODS: The ABO serological tests were performed by the routine methods. The ABO genotype was analyzed by polymerase chain reaction and sequenced bidirectionally. The haplotype of the variant allele was separated using single-strand amplification and sequencing with allele-specific primers. Stably expression cell lines with variant were constructed for study in vitro. 3D structure of the B-glycosyltransferase (GTB) variant was simulated by PyMOL software. The free energy change (ΔΔG) was calculated by FoldX. RESULTS: A variant c.737A > G was identified in a Chinese individual with Bweak phenotype, which led to an amino acid substitution p.Y246C in the vicinity of the catalytic active region of GTB enzyme. The stably expression cell lines with variant and wild type were successfully established and showed that the variant caused a decrease in protein levels and/or enzyme activity. The 3D structural of the GTB modelling found the amino acid substitution p.Y246C caused the hydrogen bond of the protein changes. Meanwhile, the free energy change (ΔΔG) value predicted the destabilizing effect on the variant GTB. DISCUSSION: The p.Y246C variant in the vicinity of the enzyme active centre reduced the antigen expression because of greatly destabilizing effect on the GTB variant.

Characteristic of HBV nucleic acid amplification testing yields from blood donors in China.

BACKGROUND: Nucleic acid amplification testing (NAT) for blood screening has been previously performed in some countries to determine NAT yields. The current study sought to explore the non-discriminating reactive NAT yields using individual-NAT (ID-NAT) and characteristics of HBV NAT yields through a 10-year retrospective analysis in Zhejiang, China. METHODS: Blood donations were analyzed using individual-NAT mode by the transcription-mediated amplification (TMA) method. Supplementary HBV serological tests were performed using chemiluminescent immunoassay, and HBV viral load assay was performed by real-time polymerase chain reaction. Follow-up studies were performed in partial donors with low HBV viral loads. RESULTS: Non-discriminating reactive NAT yields and HBV NAT yields varied in different years. The yields ranged from 853.73 per million to 2018.68 per million and 624.60 per million to 1669.50 per million, respectively. In the 476 NAT yields, 19 were probable window periods (WP), 33 probable occult hepatitis B virus infections (OBIs), 409 were confirmed OBIs and 15 were chronic HBV infections. ID-NAT results were categorized in four groups, and the findings showed that the levels of HBV DNA viral loads were different in the four different groups (χ2 = 275.02, p < 0.01). HBV viral load distribution was significantly different between anti-HBs positive and anti-HBc positive samples (χ2 = 49.429, p < 0.01). Notably, only 42.03% donors were NAT repeated positive in the 138 repeat donors' follow up tests. CONCLUSION: NAT screening of blood donations can reduce the risk of transfusion-transmitted HBV infections. Positive proportions of anti-HBs and anti-HBc are correlated with the HBV viral load level. However, low level of viral load donors pose risks in HBV NAT assays, and show fluctuating state for HBV viral load and leads to non-repeated NAT results during follow up studies.

The polymorphism of HLA-A, -C, -B, -DRB3/4/5, -DRB1, -DQB1 loci in Zhejiang Han population, China using NGS technology.

The distributions of HLA allele and haplotype are various in the populations. Currently, the data for HLA alleles and haplotypes at three fields resolution level in Chinese Han population is rare. Here, the HLA alleles and haplotypes of the 1734 cord blood samples from Zhejiang Han population, China were reported at three fields resolution. All samples were randomly collected from the Zhejiang Cord Blood Bank, China. HLA-A, -B, -C, -DRB1, -DQB1, -DRB3/4/5 loci was genotyped using next generation sequencing method. The genotypes of the samples were assigned using the HLA TypeStream Visual Software version 1.2.0. The frequency of alleles, haplotype estimation and linkage disequilibrium analysis were performed with the Arlequin software 3.5.2.2. It was found that the top three frequent alleles of HLA-A, -B, -C, -DRB1, -DQB1, -DRB3/4/5 loci were A*11:01:01 (25.81%), A*24:02:01 (16.70%), A*02:01:01 (10.61%); B*40:01:02 (15.97%), B*46:01:01 (11.48%), B*58:01:01 (7.96%); C*07:02:01 (19.03%), C*01:02:01 (17.65%), C*03:04:01 (10.41%); DRB1*09:01:02G (17.96%), DRB1*12:02:01 (9.57%), DRB1*08:03:02 (9.54%); DQB1*03:01:01G (21.05%), DQB1*03:03:02 (19.15%), DQB1*06:01:01G (12.08%); DRB4*01:03:01 (25.72%), DRB3*02:02:01 (20.27%), DRB5*01:01:01 (10.96%), respectively. A total of 1528 distinct A∼C∼B∼DRB3/4/5∼DRB1∼DQB1 haplotypes were estimated, and the top three most common haplotypes were A*33:03:01∼C*03:02:02∼B*58:01:01∼DRB3*02:02:01∼DRB1*03:01:01∼ DQB1*02:01:01 (4.02%), A*30:01:01∼C*06:02:01∼B*13:02:01∼DRB4*01:03:01∼ DRB1*07:01:01 ∼DQB1*02:02:01 (3.11%) and A*02:07:01∼C*01:02:01∼B*46:01:01 ∼DRB4*01:03:01∼DRB1*09:01:02G∼DQB1*03:03:02 (3.05%). Some alleles of different HLA loci were shown strong linkage disequilibrium. In conclusion, the data of allele and haplotype of HLA-A, -B, -C, -DRB1, -DQB1 and -DRB3/4/5 loci at three fields resolution level were obtained in Zhejiang Han population, thus contributing to analyze the HLA ploymorphism in the populations.

[Identification of a glycosyltransferase allele associated with Bw subtype and analysis of the protein structure].

OBJECTIVE: To explore the molecular basis for an individual with Bw subtype. METHODS: Routine serological reactions were used to determine the surface antigens of erythrocytes and antibodies in serum. PCR-sequence-based typing (PCR-SBT) was used to analyze the coding regions of the ABO gene and erythroid-specific regulatory element in its intron 1. Amplicons for exons 5 to 7 containing the variant site were subjected to TA cloning for the isolation of the haploid and verification of the sequence. The 3D structure of mutant protein was predicted with Pymol software. Changes of amino acid residues and structural stability were also analyzed. RESULTS: Serological assay showed that the individual had weakened B antigen and anti-B antibody in his serum. His genotype was determined as ABO*B.01/ABO*O.01.01. Sequencing of the entire coding region of the ABO gene identified an additional heterozygous c.734C/T variant. No variant was found in the erythroid-specific regulatory element of intron 1. Haploid cloning and isolation has obtained an ABO*O.01.01 allele and a ABO*B.01 allele containing a c.734T variant, which has led to substitution of Thr by Ile at position 245 in the functional center of glycosyltransferase. Based on the 3D structure of the protein, the residues binding with the mutation were unchanged, but the bonding distance between the hydrogens was changed with the amino acid substitution. Meanwhile, the connections with water molecules were increased. CONCLUSION: The c.734C>T variant of the GTB gene can lead to an amino acid substitution in the functional center of the enzyme, which in turn may affect the stability of glycosyltransferase B protein and reduceits enzymatic activity.

[Study of the distribution of KIR3DL2 alleles among ethnic Han Chinese from Zhejiang].

OBJECTIVE: To study the distribution of KIR3DL2 alleles among ethnic Han Chinese from Zhejiang. METHODS: Genomic DNA was extracted by using a magnetic bead method. The full sequence of the KIR3DL2 gene was amplified with four pairs by PCR primers. The coding regions of 208 unrelated ethnic Han Chinese blood donors were analyzed using a BigDye Terminator v3.1 Sequencing Kit. The genotypes were assigned based on the nucleotide polymorphism of the KIR3DL2 gene. RESULTS: Among the 208 samples, 133 were KIR3DL2 heterozygotes and 75 were homozygotes. Forty six KIR3DL2 genotypes were detected. Respectively, 70, 33 and 23 individuals were found to have a KIR3DL2*00201/KIR3DL2*00201, KIR3DL2*00201/KIR3DL2*00701, and KIR3DL2*00201/KIR3DL2*01001 genotype. Twenty-two KIR3DL2 alleles were discovered, and the frequencies of KIR3DL2*00201, KIR3DL2*00701 and KIR3DL2*01001 were 57.45%, 13.46% and 9.13%, respectively. CONCLUSION: The distribution of KIR3DL2 alleles among ethnic Han Chinese in Zhejiang has been determined and fits the criteria for genetic polymorphism.

[Molecular characterization of a recombination allele of ABO blood group].

OBJECTIVE: To analyze the molecular characteristics of a recombinant allele of the ABO blood group. METHODS: The ABO phenotype was determined with the tube method. The coding regions of the ABO and FUT1 genes were analyzed by PCR-sequence based typing. The ABO alleles of the proband were determined by allele-specific primer sequencing. The full sequences of the ABO gene of the proband and her mother were determined through next generation sequencing. RESULTS: The red blood cells of the proband did not agglutinate with anti-H, and the sequence of the FUT1 gene was homozygous for c.551_552delAG.The proband was thereby assigned as para-Bombay. Bi-directional sequencing also found that she was heterozygous for c.261G/del,467C>T,c.526C>G,c.657C>T,c.703G>A,c.796C>A,c.803G>C and c.930G>A of the coding regions of the ABO gene. Allele-specific primer sequencing also found her to carry a ABO*A1.02 allele and a recombinant allele from ABO*O.01.01 and ABO*B.01. The recombination site was located between nucleotide c.375-269 and c.526, and the allele was maternally derived. CONCLUSION: An recombinant allele of the ABO gene has been identified, which has originated from recombination of ABO*O.01.01 with the ABO*B.01 allele.

Multiple low-frequency and rare HLA-B allelic variants are associated with reduced risk in 1,105 nasopharyngeal carcinoma patients in Hunan province, southern China.

In our study, 1,105 cases of nasopharyngeal carcinoma (NPC) and 1,430 normal controls recruited from Hunan province, southern China were typed for human leukocyte antigen (HLA)-B locus by Sanger sequencing exons 2-4. Besides confirming the NPC association with HLA-B*46:01 allele, HLA-A*02:07-B*46:01 and HLA-A*33:03-B*58:01 haplotypes (all positive), and HLA-B*13 lineage (negative), all of which were relatively common, strong negative associations were observed for five low-frequency and rare alleles or lineages, including HLA-B*07, -B*27:04, -B*39, -B*51:02 and -B*55:02, with odds ratio (OR) ranging from 0.16 to 0.3 (all pcorrected < 0.05). These strong protective associations were independent of linkage disequilibrium (LD) between HLA-A and HLA-B loci. Further analysis indicated a single amino acid change from histidine to tyrosine at residue 171 is probably crucial for the mutant allele, HLA-B*51:02, to mediate resistance to NPC. A subset of NPC cases (n = 821) and normal controls (n = 1,035) were tested for antivirus capsid antigen immunoglobulin A (anti-VCA IgA), which differed drastically between the two groups [67.7% vs. 5.5%, OR (95% confidence interval) = 36 (26.55-48.81), p < 0.0001]. HLA-B allelic variation did not associate with seropositivity for anti-VCA IgA in either group. Results from our study show, more clearly than previously, the existence of a cluster of low-frequency and rare HLA-B variants conferring low, or very low risk to NPC, a phenomenon not observed in other ethnic groups. Our data shed new insights into genetic susceptibility to NPC in southern Chinese populations. Future independent studies are warranted to replicate the findings reported in our study.

A Novel c.796 A>C Mutation in the ABO*B.01 Allele Responsible for CisAB Phenotype.

BACKGROUND: Individuals with the CisAB phenotype are rare in the Chinese population. In the present study, we investigated the sequence of the ABO gene and family members of a newborn suspected to have the CisAB phenotype. METHODS: The ABO phenotype was detected using conventional serological tests. The full coding region of exons 1 to 7 of the ABO gene was amplified by polymerase chain reaction and was sequenced. The ABO haplotype was determined by the allele-specific primer sequencing method. RESULTS: The proband and his father and grandfather were assigned the CisAB phenotype according to the results of the serological tests and family investigation. A novel CisAB allele was identified in the proband and his father and grandfather, which has only one nucleotide difference at position 796 from A to C (c.796A>C) compared with the ABO*B.01 allele. CONCLUSION: A novel CisAB (c.796A>C mutation in ABO*B.01) allele is the first identified in the Chinese population.

Molecular Basis of ABO Variants Including Identification of 16 Novel ABO Subgroup Alleles in Chinese Han Population.

INTRODUCTION: The characteristic of ABO blood subgroup is crucial for elucidating the mechanisms of such variant phenotypes and offering useful information in blood transfusion. METHODS: In total, 211 ABO variants including part of available family members were investigated in this study. The phenotypes of these individuals were typed with serologic methods. The full coding regions of ABO gene and the erythroid cell-specific regulatory elements in intron 1 of them were amplified with polymerase chain reaction and then directly sequenced. The novel alleles were confirmed by cloning and sequencing. Phylogenetic tree was made using CLUSTAL W software. 3D structural analyses of the glycosyltransferases (GTs) with some typical mutations were performed by PyMOL software. RESULTS: Forty-eight distinctly rare ABO alleles were identified in 211 Chinese variant individuals, including 16 novel ABO alleles. All of the alleles were categorized as 5 groups: 16 ABO*A alleles, 23 ABO*B alleles, 4 ABO*BA alleles, 4 ABO*cisAB alleles, and 1 ABO*O alleles. ABO*A2.08 and ABO*BA.02 were the relatively predominant A and B subgroup alleles, respectively. According to the phylogenetic tree, 28 alleles (5 common alleles and 23 alleles identified in our laboratory) were classified into 3 major allelic lineages. The structural analysis of 3D homology modeling predicted reduced protein stability of the mutant GTs and may explain the reduced ABO antigen expression. CONCLUSIONS: The molecular basis of ABO variants was analyzed, and 16 novel ABO alleles were identified. The results extended the information of ABO variants and provided a basis for better transfusion strategies and helped to improve blood transfusion safety.

Novel method for simultaneously detecting HPA and HLA antibodies using Luminex microbeads.

BACKGROUND: Alloantibodies against human platelet antigens (HPAs) and human leukocyte antigen (HLA) are implicated in several immune-mediated platelet disorders. Detection of these antibodies is crucial in the diagnosis and management of these disorders. The aim of this study was to establish a novel method to simultaneously detect HPA-1, HPA-2, HPA-3, HPA-5 and HLA antibodies with Luminex microbeads technology. METHODS: Monoclonal antibodies specific for platelet glycoproteins and HLA class I molecules were separately coupled to the Luminex microbeads. We validated specificity of the Luminex platform using the following antibodies: anti-HPA-1a, anti-HPA-2b, anti-HPA-3a, anti-HPA-5a, and anti-HLA positive samples. Sensitivity was evaluated by a serial dilution (from neat to 1/1024) using the following antibodies: anti-HPA-1a, anti-HPA-3a standard sera, and anti-HPA-5a positive serum. Serum samples were collected from 36 neonatal alloimmune thrombocytopenia (NAIT) patients suspected of having HPA or HLA antibodies and 8 samples from ISBT platelet workshop were tested using the Luminex assay. RESULTS: The Luminex assay detected all antibodies tested from the known samples. The sensitivities of the Luminex assay detecting anti-HPA-1a, anti-HPA-3a, and anti-HPA-5a were 1:512, 1:64, and 1:128, respectively. The sensitivity of Luminex assay was higher than monoclonal antibody immobilization of platelet antigen method (MAIPA). No cross-reactivity was observed in the samples containing multi-platelet antibodies or mixture antibodies against HPA and HLA. The results of 44 samples with platelet disorders were consistent with those of the same samples processed with the MAIPA assay. CONCLUSION: Luminex microbeads coupled with monoclonal antibodies could be successfully used to detect HPA and HLA antibodies simultaneously, especially with high sensitivity in detecting HPA antibodies.