Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats.

BACKGROUND: Diseases caused by Toxoplasma gondii (T. gondii) have introduced serious threats to public health. There is an urgent need to develop a rapid detection method for T. gondii infection in cats, which are definitive hosts. Recombinant apical membrane antigen 1 (rAMA1) was produced in a prokaryotic expression system and used as the detection antigen. The aim of this study was to evaluate and optimize a reliable indirect enzyme-linked immunosorbent assay (iELISA) method based on rAMA1 for the detection of antibodies against T. gondii in cats. RESULTS: The rAMA1-iELISA method was developed and optimized by the chessboard titration method. There were no cross-reactions between T. gondii-positive cat serum and positive serum for other pathogens, indicating that rAMA1-iELISA could only detect T. gondii in most cases. The lowest detection limit of rAMA1-iELISA was 1:3200 (dilution of positive serum), and the CV of repeated tests within batches and between batches were confirmed to be less than 10%. The results of 247 cat serum samples detected by rAMA1-iELISA (kappa value = 0.622, p < 0.001) were in substantial agreement with commercial ELISA. The ROC curve analysis revealed the higher overall check accuracy of rAMA1-iELISA (sensitivity = 91.7%, specificity = 93.6%, AUC = 0.956, 95% CI 0.905 to 1.000) than GRA7-based iELISA (sensitivity = 91.7%, specificity = 85.5%, AUC = 0.936, 95% CI 0.892 to 0.980). Moreover, the positive rate of rAMA1-iELISA (6.5%, 16/247) was higher than that of GRA7-based iELISA (3.6%, 9/247) and that of commercial ELISA kit (4.9%, 12/247). CONCLUSION: The iELISA method with good specificity, sensitivity, and reproducibility was established and can be used for large-scale detection of T. gondii infection in clinical cat samples.

Preparation of highly specific monoclonal antibodies against SARS-CoV-2 nucleocapsid protein and the preliminary development of antigen detection test strips.

The coronavirus disease 2019 (COVID-19) is outbreaking all over the world. To help fight this disease, it is necessary to establish an effective and rapid detection method. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is involved in viral replication, assembly, and immune regulation and plays an important role in the viral life cycle. Moreover, the N protein also could be a diagnostic factor and potential drug target. Therefore, by synthesizing the N gene sequence of SARS-CoV-2, constructing the pET-28a (+)-N recombinant plasmid, we expressed the N protein in Escherichia coli and obtained 15 monoclonal antibody (mAbs) against SARS-CoV-2-N protein by the hybridomas and ascites, then an immunochromatographic test strip method detecting N antigen was established. In this study, we obtained 14 high-titer and high-specificity monoclonal antibodies, and the test strips exclusively react with the SARS-CoV-2-N protein and no cross-reactivity with other coronavirus and also recognize the recombinant N protein of Delta (B.1.617.2) variant. These mAbs can be used for the early and rapid diagnosis of SARS-CoV-2 infection through serological antigen.

Molecular basis and zygosity determination of D variants including identification of four novel alleles in Chinese individuals.

BACKGROUND: The frequency and molecular basis of the D variants have been reported in the Caucasian and African populations, but relatively little information was known in the Chinese population. Here, a study was investigated in Chinese persons with weak or discrepant D serologic typing. STUDY DESIGN AND METHODS: D variant was typed with a serologic method. The full coding regions of RHD of these variants were amplified with polymerase chain reaction and then directly sequenced. RHD zygosity test was performed using the hybrid Rhesus box technique and a multiplex ligation-dependent probe amplification (MLPA) assay was also used to analyze the variant alleles and RHD gene copy number. RESULTS: Twelve distinct RHD mutation alleles were found in 32 D variant individuals, with eight weak D and four partial D alleles. Weak D Type 15 and DVI Type 3 were the major weak D and partial D alleles in Zhejiang Han persons. Three novel weak D alleles (RHD weak D 95A, 779G, and 670G) and one new partial D allele (RHD130-132 del TCT) were identified. The results of RHD zygosity in three individuals disagreed between the RHD zygosity test and the MLPA assay. The most known variant alleles can be detected, but four novel alleles were missed using the RH-MLPA assay. CONCLUSION: The molecular basis and zygosity of D variants in Zhejiang Han persons were analyzed, and four novel RHD alleles were identified. These data extend the information of D variants and may help to improve the transfusion strategy of the D variants.

RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China.

Toxoplasma gondii, which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. T. gondii causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of toxoplasmosis. The current molecular diagnostic tools for T. gondii infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) assay to detect T. gondii. The lowest limit of detection of the assay was 31 copies/µL for the T. gondii B1 gene. In addition, we established a visual RPA-CRISPR/Cas12a lateral flow band assay (RPA-CRISPR/Cas12a-LFA) combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye, making the LFA assay results more accurate. The assay established in this study could identify T. gondii within 55 min with high accuracy and sensitivity, without cross-reaction with other tested parasites. The developed assay was validated by establishing a mouse model of toxoplasmosis. Finally, the developed assay was used to investigate the prevalence of T. gondii in stray cats and dogs in Zhejiang province, Eastern China. The positive rates of T. gondii infection in stray cats and dogs were 8.0% and 4.0%, respectively. In conclusion, the RPA-CRISPR/Cas12a-LFA is rapid, sensitive, and accurate for the early diagnosis of T. gondii, showing promise for on-site surveillance. IMPORTANCE: Toxoplasma gondii is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of T. gondii are distributed worldwide, and cats and dogs are common hosts of T. gondii. Therefore, rapid diagnosis of early T. gondii infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of T. gondii without false-negative results. Moreover, we detected the prevalence of T. gondii in stray cats and dogs in Zhejiang province, China. Our developed assay provides technical support for the early diagnosis of T. gondii and could be applied in prevalence surveys of T. gondii in stray dogs and cats.

Distribution of MICB diversity in the Zhejiang Han population: PCR sequence-based typing for exons 2-6 and identification of five novel MICB alleles.

The polymorphism of major histocompatibility complex class I chain-related gene B (MICB) and variations in MICB alleles in a variety of populations have been characterized using several genotyping approaches. In the present study, a novel polymerase chain reaction sequence-based typing (PCR-SBT) method was established for the genotyping of MICB exons 2-6, and the allelic frequency of MICB in the Zhejiang Han population was investigated. Among 400 unrelated healthy Han individuals from Zhejiang Province, China, a total of 20 MICB alleles were identified, of which MICB*005:02:01, MICB*002:01:01, and MICB*004:01:01 were the most predominant alleles, with frequencies of 0.57375, 0.1225, and 0.08375, respectively. Nine MICB alleles were detected on only one occasion, giving a frequency of 0.00125. Of the 118 distinct MICB ∼ HLA-B haplotypes identified, 42 showed significant linkage disequilibrium (P < 0.05). Haplotypes MICB*005:02:01 ∼ B*46:01, MICB*005:02:01 ∼ B*40:01, and MICB*008 ∼ B*58:01 were the most common haplotypes, with frequencies of 0.0978, 0.0761, and 0.0616, respectively. Five novel alleles, MICB*005:07, MICB*005:08, MICB*027, MICB*028, and MICB*029 were identified. Compared with the MICB*005:02:01 sequence, a G > A substitution was observed at nucleotide position 210 in MICB*005:07, and a 1,134 T > C substitution in MICB*005:08 and an 862 G > A substitution in MICB*027 were detected. In addition, it appears that MICB*028 probably arose from MICB*004:01:01 with an A to G substitution at position 1,147 in exon 6. MICB*029 had a G > T transversion at nucleotide position 730 in exon 4, compared with that of MICB*002:01:01. On the basis of the new PCR-SBT assay, these observed results demonstrated MICB allelic variations in the Zhejiang Han population.

The relationship of platelet yield, donor's characteristic and apheresis instruments in China.

Platelet yield was associated with donor's characteristic and property of apheresis instruments. Here, we have analyzed the relationship of platelet yield, physiologic parameters of donors for different apheresis instruments in China. Data were consecutively retrieved from plateletapheresis donors during March 1, 2007 and March 1, 2012. Three different apheresis instruments MCS+, Amicus, Trima system were used for plateletapheresis and defined as group 1, 2 and 3 respectively. Totally 77,091 Plateletapheresis donations were performed in this study. 17 donations were finally aborted because of vasovagal reaction with syncope. 5861, 37,036, 34,177 donations were performed in group 1, 2 and 3 respectively. Hct and platelet values before donations were similar, but platelet yield and collection rate were showed significantly difference (p<0.05) among the three groups. The values of platelet and Hct in the males before donations were higher than those in the females, and the platelet yield and collection rate were showed significantly difference between the male group and female group (p<0.05). The overall reaction rate was 1.56%. Most donors were chosen the group 2 (51.6%) for next donation, followed by group 3 (33%) and group 1 (15.4%). We concluded that the platelet yield and collection rate in the male group were higher than those in the female group and the efficiency of plateletapheresis was associated with the kind of apheresis instruments and donor's characteristic. These data will help to work out suitable apheresis protocol based on the Chinese donor's characteristic.

A dispermic chimera was identified in a healthy man with mixed field agglutination reaction in ABO blood grouping and mosaic 46, XY/46, XX karyotype.

BACKGROUND: Chimerism is the presence of two or more genetically distinct cell populations in one organism. Here, we reported the identification of dispermic chimerism in a 25-year-old male. METHODS: Blood grouping was performed with standard gel centrifugation test cards. ABO and HLA-A,-B,-C,-DRB1 and -DQB1 loci genotyping was determined with PCR sequence-based typing. A quantitative analysis of dual red cells populations was measured by flow cytometer. The karyotype was analyzed by G-banded chromosomes. Short tandem repeat (STR) analysis was performed on blood, buccal mucosal and hair shafts samples. RESULTS: A mixed-field agglutination with anti-B antibody was observed with gel centrifugation tests, which showed a double populations of O and B groups RBCs. Two groups RBCs were also observed by flow cytometer with nearly 90% O group cells and 10% B group cells. The normal O01,O02,B101 alleles were identified in DNA sample of the proband. STR analysis revealed three alleles for D8S1179,D3S1358,TH01,D13S317,D16S539,D2S1338,D19S433,TPOX and D18S51 loci. HLA-DRB1 and -DQB1 loci had three alleles and a karyotypic mosaic was found with 60% 46, XY and 40% 46, XX karyotype in the proband. In all studies, the third allele was attributable to a dual paternal contribution. CONCLUSION: A individual with dispermic chimerism was identified, which would generate by fertilization of an oocyte and the corresponding second polar body by two different sperms.

Serological characteristic and molecular basis of A2 subgroup in the Chinese population.

BACKGROUND: A2 phenotype is a common subgroup of blood group A, but the serological characteristic and genetics basis of A2 phenotype currently was rare reported in the Chinese Han population. Here, a large scale study of the serology and genetics of A2 and A2B phenotypes was performed. METHODS/MATERIALS: 11263 Chinese individuals with group A and AB phenotypes were determined for A2 antigen with the standard serological method. The full coding region of the ABO gene was sequenced in the individuals with A2 and A2B phenotypes. Some samples including each ABO genotypes were chosen for determining the activity of glycosyltransferase A (GTA) in plasma. RESULTS: 134 individuals were assigned as A2 and A2B phenotypes in 11263 individuals. There was imbalance in A2 and A2B phenotypes and the proportion of A2B among AB samples was significantly higher than that of A2 in group A samples. All samples of the A2 and A2B phenotypes were classified into A2-related allele group, A1-related allele group and the other group based on kind of the ABO genotype. Four novel A2-related alleles (A217, A218, A219, A220) were identified. The individuals with same genotype showed different agglutination strength with anti-A1 and anti-H on their RBCs. The plasma from individuals with A2-related allele had almost no GTA activity, while plasma from individuals with A1-related allele had some GTA activity. CONCLUSION: A2 and A2B phenotypes could derive from different genotypes and the serological characteristic may be heterogeneity in the Chinese Han population.

[A rare p phenotype caused by a 26-bp deletion in α 1,4-galactosyltransferase gene].

OBJECTIVE: To delineate serological features and genetic basis for a rare p phenotype of P1Pk blood group system found in a Chinese individual. METHODS: Serological assaying was carried out for a proband with unexpected antibody found in his serum using specific antibodies and panel cells. Coding regions and flanking introns of α 1,4-galactosyltransferase gene (A4GALT) associated with the p phenotype were screened with polymerase chain reaction and DNA sequencing. RESULTS: A rare p phenotype of the P1Pk blood group system has been identified with red blood cells from the proband, whose serum contained anti-Tja antibody which can agglutinate and hemolyze with other common red blood cells. Other members of the proband's family were all normal with P1 or P2 phenotype. DNA sequencing has identified in the proband a homozygous 26 bp deletion at position 972 to 997 of the A4GALT gene. The deletion has caused a shift of the reading frame, resulting in a variant polypeptide chain with additional 83 amino acid residues compared with the wild-type protein. Other family members were either heterozygous for above deletion or non-deleted. CONCLUSION: A 26 bp deletion at position 972 to 997 of the A4GALT gene has been identified in a Chinese individual with p phenotype.

Cord Blood Banking and Transplantation in China: A Ten Years Experience of a Single Public Bank.

BACKGROUND: Umbilical cord blood (UCB) has successfully used for transplantation to treat hematologic malignancies and genetic diseases. Herein, we describe the experience generated in a single public UCB bank at Zhejiang Province in China. METHODS: Good manufacturing practice and standard operating procedures were used to address donor selection as well as UCB collection, processing, and cryopreservation. Total nucleated cells (TNCs), cellular viability, CD34+ cells, and colony-forming units were determined, and infectious diseases screening test, sterility test, and HLA typing for UCB units were done. RESULTS: Only 18.51% of all collected UCB units met storage criteria, and 7,056 UCB units were cryopreserved in 10 years. The volume of UCB units was 95.0 ± 22.0 ml. The number of TNCs before and after processing was 13.32 ± 3.63 × 10(8) and 10.63 ± 2.80 × 10(8), respectively, and the recovery rate was 80.71 ± 11.26%. 0.4344 ± 0.1874% of the TNCs were CD34+ cells. The CFU-GM was 32.1 ± 28.0 colonies per 1 × 10(5) nucleated cells. Based mainly on HLA and nucleated cell content, 26 UCB units were released for transplantation. CONCLUSIONS: A public UCB bank was successfully established in China; collection and processing of UCB units should be optimized in order to gain maximum volume and cell count.

[Study on a novel mutation of B glycosyltransferase gene related with an ABx variant].

OBJECTIVE: To explore the molecular basis of an individual featuring an ABx variant of ABO blood group system. METHODS: Serological assays were used to characterize the erythrocyte phenotypes and salivary ABH secretors. All of the seven exons and flanking introns of ABO glycosyltransferase gene were amplified with polymerase chain reaction (PCR). And the products were sequenced bidirectionally following enzyme digestion. Exons 6 and 7 were also subcloned and analyzed for haplotypes of the ABO gene. RESULTS: Erythrocytes of the proband have expressed a strong A antigen and a weak B antigen, which was identified as a rare ABx variant in addition with other serological features. Nine heterozygous sites in exon 6 (297A/G) and exon 7 (467C/T, 526C/G, 657C/T, 703G/A, 796C/A, 803G/C, 808T/A, 930G/A) of the coding region of the ABO gene were identified. Based on haplotype analysis, one allele was determined as common A102, whilst another was consistent with B101 except for an 808T>A mutation which has resulted in replacement of phenylalanine with isoleucine at position 270 of glycosyltransferase B. CONCLUSION: The 808T>A mutation of the glycosyltransferase B gene may decrease the enzymatic activity and result in the Bx variant.

[Analysis of allele frequencies of HLA-DRB1*12:01:01G and HLA-DRB1*14:01:01G groups].

OBJECTIVE: To discriminate and analyze the relative frequencies of alleles in HLA-DRB1*12:01:01G(HLA-DRB1*12:01:01/12:06/12:10/12:17) and HLA-DRB1*14:01:01G (DRB1*14:01:01/14:54) groups and assess their associations with HLA-DRB3 and HLA-DQB1 loci. METHODS: A total of 115 DNA samples previously typed as HLA-DRB1*12:01:01G and 108 samples from HLA-DRB1*14:01:01G were selected. DNA sequences for exons 1 to 3 of the HLA-DRB1 locus were analyzed for HLA-DRB1*12:01:01G, and exons 2 to 3 were analyzed for HLA-DRB1*14:01:01G by polymerase chain reaction sequence-based typing (PCR-SBT). Genotyping of HLA-DRB3 and HLA-DQB1 were achieved by PCR-SBT. RESULTS: Among 115 samples previously typed as HLA-DRB1*12:01:01G, 101 (87.8%) were confirmed as HLA-DRB1*12:01:01 and 14 (12.2%) were HLA-DRB1*12:10, but HLA-DRB1*12:06 and HLA-DRB1*12:17 alleles were not identified. For 108 samples previously typed as HLA-DRB1*14:01:01G, all were typed as HLA-DRB1*14:54. HLA-DRB1*12:01:01 was linked with HLA-DRB3*01:01:02 and HLA-DQB1*03:01, while HLA-DRB1*12:10 was strongly linked with HLA-DRB3*02:02:01 and HLA-DQB1*03:01. HLA-DRB1*14:54 was strongly linked with HLA-DRB3*02:02:01 and two different HLA-DQB1*05:02, *05:03 alleles. CONCLUSION: HLA-DRB1*12:01:01 was more prevalent than HLA-DRB1*12:10 in the HLA-DRB1*12:01:01G group, and HLA-DRB1*14:54 was the dominant allele for HLA-DRB1*14:01:01G.

Antibody Response to SARS-CoV-2 in the First Batch of COVID-19 Patients in China by a Self-Developed Rapid IgM-IgG Test.

It has been over two years since the COVID-19 pandemic began and it is still an unprecedented global challenge. Here, we aim to characterize the antibody profile from a large batch of early COVID-19 cases in China, from January - March 2020. More than 1,000 serum samples from participants in Hubei and Zhejiang province were collected. A series of serum samples were also collected along the disease course from 70 patients in Shanghai and Chongqing for longitudinal analysis. The serologic assay (ALLtest) we developed was confirmed to have high sensitivity (92.58% - 97.55%) and high specificity (92.14% - 96.28%) for the detection of SARS-CoV-2 nucleocapsid-specific antibodies. Confirmed cases found in the Hubei Provincial Center for Disease Control and Prevention (HBCDC), showed a significantly (p = 0.0018) higher positive rate from the ALLtest than RNA test. Then, we further identified the disease course, age, sex, and symptoms that were correlating factors with our ALLtest results. In summary, we confirmed the high reliability of our ALLtest and its important role in COVID-19 diagnosis. The correlating factors we identified will require special attention during future clinical application.

[Study on polymorphism of membrane glycoprotein genes related to human platelet alloantigens].

OBJECTIVE: To investigate the polymorphisms of platelet membrane glycoprotein genes related to human platelet alloantigen (HPA)-1 to 17w. METHODS: The DNA segments of platelet membrane glycoprotein genes related to HPA-1 to 17w were amplified using author's designed primers. The amplification products were purified and directly sequenced to identify the HPA genotype and glycoprotein gene polymorphisms. RESULTS: Thirteen new single nucleotide polymorphisms (SNPs) and a micro-satellite sequence were found in the glycoprotein genes from the 112 random samples, in which two SNPs (1333G/A and 1960G/A) in ITGB3 gene result in two amino acid change (V419M and E628K) on glycoprotein GPIIIa. CONCLUSION: New variants in platelet membrane glycoprotein genes were identified, which may lead to structure change of platelet membrane glycoprotein and affect the accuracy of partial HPA genotyping method.

[Molecular mechanism of an individual with weaken B phenotype in ABO blood group].

OBJECTIVE: To elucidate the serological characteristics and molecular mechanism of a blood donor with weaken B antigen. METHODS: The ABO blood group antigens on red blood cells were identified by monoclonal antibodies, the ABO antibodies in serum were detected by standard A, B, O cells and the activity of the B glycosyltransferase was analyzed. The full-length sequence and 5'-untranslated region (5'-UTR) sequence of ABO gene were amplified by polymerase chain reaction (PCR) respectively and direct sequencing. The alternative splicing isoforms of ABO cDNA were obtained by reverse transcription-PCR(RT-PCR) and analyzed with cloning and sequencing techniques. The level of methylation of the CpG island in ABO gene promoter was analyzed by bisulfite sequencing method. RESULTS: The serological characteristic of the donor showed that the B antigen was decreased obviously without anti-B antibodies in serum and the B glycosyltransferase activity was decreased as well. The genotype of the donor was B101/O01 without any other mutations in the full-length coding sequences and splice receptor sites. The nucleotide characteristics of the 5'-UTR was consistent with B101/O01 and no any abnormity was identified in the promoter, enhancer and the negative regulatory sequence regions. The integrative cDNA transcript of ABO gene was obtained and no new splicing isoform was found. Compared with the normal B phenotype, a number of methylated CpG sites were found near the promoter of ABO gene in this sample. CONCLUSION: The methylation in the CpG island of ABO gene promoter region may cause weak expression of the B antigen.

[Recombination between human leukocyte antigen -A and -C loci within two Chinese Han families].

OBJECTIVE: To investigate the recombination events between human leukocyte antigen (HLA) loci within two families. METHODS: Identification of HLA-A, -C, -B, -DRB1 and -DQB1 loci was firstly carried out using polymerase chain reaction-sequence specific oligonucleotide. Then HLA high resolution typing was performed using polymerase chain reaction sequencing-based typing. The recombination between HLA loci was identified by family genetic analysis. The parentage possibility was analyzed by short tandom repeat technique. RESULTS: Recombination between the HLA-A and C loci was identified within two families. One individual inherited a paternal haplotype that was the result of a recombination event between the father's HLA-A and -C loci on his chromosomes. The other individual inherited a maternal haplotype that was the result of a recombination event between the mother's HLA-A and -C loci. The high parentage possibilities were obtained in the family members. CONCLUSION: The recombination events of HLA-A and -C have been found in two Chinese families, which may help further study on the mechanism of HLA recombination.

[Study on the expression stability of mutant alpha-1,2 fucosyltransferase gene 293C to T and 658C to T in eukaryotic cells].

OBJECTIVE: To establish the eukaryotic cell expression system for the alpha-1,2 fucosyltransferase gene FUT1 293C to T and 658C to T mutations and explore the mechanism of FUT1 mutations resulting in the reduced expression of H antigen. METHODS: Genomic DNAs were extracted from individuals with para-Bombay phenotype and full coding region of FUT1 was amplified. The amplification fragments were ligated with pcDNA3.1 plasmid to construct the recombinant expression vectors. The recombinant plasmids were transfected into the COS-7 cells using lipofectamine transfection reagent and stable expression screening was performed. FUT1 mRNA expression was determined by real-time quantitative PCR. The activity of enzyme was measured by high performance liquid chromatography. Expressed protein was identified by SDS-PAGE and Western blotting. RESULTS: pcDNA3.1-FUT1 wildtype, pcDNA3.1-FUT1 293C to T and pcDNA3.1-FUT1 658C to T recombinant vectors were constructed, respectively. COS-7 cells with stable expression of FUT1 were obtained through recombinant plasmid transfection and screening with G418. The FUT1 mRNA level of transfected cells with 293C to T and 658C to T recombinant vectors reached 97.10% and 104.74% of the wildtype FUT1 transfected cell. A specific protein band with about 46 000 was confirmed in the transfected cell lysates by SDS-PAGE electrophoresis and Western blotting with 6×His Tag antibody. The wildtype FUT1 transfected cell lysates can catalyze the enzymatic reaction, while the enzyme activity of cell lysates from 293C to T and 658C to T were abolished. CONCLUSION: The results suggested that the 293C to T and 658C to T mutations of FUT1 gene did not affect the RNA and protein expression levels, but the enzyme activity of cells with FUT1 mutations was significantly decreased which resulted in the reduced expressin of H antigen.

[Identification of a novel allele HLA-B*15:129 by polymerase chain reaction with allele group-specific primers].

OBJECTIVE: To analyze the sequence of the exons 2-4 of human leukocyte antigen (HLA) novel allele HLA-B*15:129. METHODS: DNA of the proband was extracted from whole blood by commercial DNA extraction kit. The amplification for HLA-B exons 2-4 was performed separately by polymerase chain reaction (PCR) with allele group specific primers. The PCR products were digested with enzymes and then directly sequenced for exons 2-4 of HLA-B locus in both directions. RESULTS: Sequencing results showed the HLA-B alleles of the proband included B*07:02 and a novel allele. The sequence of the novel allele has been submitted to GenBank (accession no. EF473219) and the allele has been officially named B*15:129 by the WHO Nomenclature Committee. Comparing with the HLA-B*15:01:01:01, the sequence of exons 2-4 of HLA-B*15:129 showed three nucleotide difference in exon 3 at positions 362 and 363 from GG to AT and positions 369 from C to T, which resulted in an amino acid change from Arg to Asn at codon 97. CONCLUSION: A novel HLA-B allele was identified and has been officially named B15:129 by the WHO Nomenclature Committee.

[Cloning and sequence analysis of 5'untranslated region of human ABO gene].

OBJECTIVE: To clone and investigate the polymorphism of the 5'-untranslated region (5'-UTR) of the human ABO gene, in order to provide the basis for exploring the transcriptional regulation of the human ABO histo-blood group genes. METHODS: ABO phenotypes of 30 unrelated healthy blood donors were determined by serological technique, their genotypes were analyzed by sequencing the exons 6 and 7 of the ABO gene. Nearly 5 kb of the 5'-UTR of ABO gene was obtained by PCR amplification and sequencing was performed bidirectionally. Haplotypes of samples with heterozygous sites in the 5'-UTR of ABO gene were separated and analyzed after cloning. RESULTS: Twenty polymorphic sites were identified in these samples where ABO genotypes were consistent with serological phenotypes. It included sixteen nucleotide sequence variations, one 8 bp deletion, one 6 bp deletion/insertion, one 36 bp insertion and one 43 bp repeats. Among them, 11 were novel polymorphic sites. Seven different haplotypes of 5'-UTR of ABO gene were defined to the cis/trans linkage of those mutations. CONCLUSION: There were polymorphisms in the 5'-UTR of ABO gene and the nucleotide sequences near the proximal promoter were conservative.

[Identification of an ABx09 phenotype of ABO subtype].

OBJECTIVE: To analyze the molecular basis for an individual with ABx09 phenotype of ABO subtype. METHODS: The ABO group antigens on red blood cells of the proband were identified by monoclonal antibodies, and the ABO antibody in serum was detected by standard A, B, O cells. The exons 1 to 7 of ABO gene were amplified by polymerase chain reaction (PCR) respectively and the PCR products were sequenced directly. The amplified products for exons 5 to 7 were also cloned by TOPO TA cloning sequencing kit to split the two alleles apart, selected colonies were sequenced bidirectionally for exons 5 to 7 of the ABO gene. The samples of the proband's parents were collected, then serological test of the blood group and sequence analysis for exons 6 and 7 of ABO gene were preformed. RESULTS: Both A and B antigens were detected on red blood cells of the proband and there was anti-B antibody in the serum. There was no G deletion at position 261, while 297AG in exon 6, 467CT, 526CG, 657CT, 703GA, 796CA, 803GC, 889GA and 930GA heterozygote in exon 7 were detected by direct DNA sequencing, which can be assigned for A102Bx09 genotype. After cloning and sequencing, two alleles A102 and Bx09 were obtained. The sequence of Bx09 had one nucleotide changes (G to A) at position 889 compared with that of B101, which resulted in an amino acid change of Glu to Lys at 297 position. The Bx09 in the proband was inherited from her mother by family investigation. CONCLUSION: G to A at nt889 of alpha-1,3 galactosyltransferasegene can result in Bx09 phenotype, with the presence of anti-B antibody in serum.