The aim of this study was to clone human RHD gene and to investigate its expression in transduced K562 cells. Total RNA was extracted from reticulocyte of cord blood. RHD and RHCE genes were amplified using RT-PCR method. The amplified products were cloned into pGEM-T plasmid by TA ligation and several clones were screened by direct sequencing method in order to obtain the RHD gene. RHD gene was subcloned into pcDNA3.1(-) expression vector, then the recombined plasmids were transduced into K562 cells with superfect transfection reagent kit. Finally transcription and expression of RHD gene in K562 cells were detected. The result showed that RHD gene has been cloned sucessfully, the inserted sequence and direction of RHD cDNA in its recombined pcDNA3.1(-) vector were identified using enzyme cutting and sequencing method. After transduced with recombined pcDNA3.1(-) vector, K562 cells could transcribe RHD mRNA in its cytoplasm and express RhD antigen on its membrane surface. In conclusion, RhD antigen can expressed in K562 cells with RHD cDNA transduction, and the expression system in vitro may be helpful to further investigate the molecular basis of RhD variants.
Base Sequence ; Cell Membrane ; metabolism ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; Molecular Sequence Data ; RNA, Messenger ; biosynthesis ; genetics ; Rh-Hr Blood-Group System ; biosynthesis ; genetics ; Sequence Analysis, DNA ; Transfection

OBJECTIVETo analyze the influence of benign prostatic hyperplasia (BPH) drugs on incidence and pathology grading of prostate cancer in China.

METHODSRetrospectively investigated the history of drug treatment in 1029 cases of BPH in patients from February 1998 to December 2004. According to the history of drug use, the patients were divided into 4 groups: finasteride group, alpha-receptor inhibitor group, finasteride and alpha-receptor inhibitor combination group and control group (untreated group). We gathered pathology sections of patients in all groups, and gave Gleason Score to each. The difference of incidence and pathology grading of prostate cancer were analyzed by Stata 7.0.

RESULTSThe incidence of prostate cancer in the population of our study was 13.5%; The incidence in finasteride group, alpha-receptor inhibitor group, combination group and control group was 9.8%, 16.0%, 10.3% and 18.6%, respectively. There was significant difference between the two groups with the use of finasteride and the two groups without it (P < 0.05). In our study, the ratio of middle or high level pathology grading (Gleason ≥ 7) in prostate cancer patients was 58.3%, the ratio of middle or high level pathology grading prostate cancer patients in the four groups was 71.4%, 59.6%, 67.7% and 40.0%, respectively. In the comparison of composition ratio of middle or high level prostate cancer, there was significant difference between the two groups with the use of finasteride and the two groups without it (P < 0.05).

CONCLUSIONSFinasteride can lower the risk of prostate cancer, but increase the pathology grade of the prostate cancer which has occurred in the same time. The alpha-receptor inhibitor does not have the same effect.

Adrenergic alpha-Antagonists ; therapeutic use ; Aged ; Aged, 80 and over ; Finasteride ; therapeutic use ; Humans ; Incidence ; Male ; Middle Aged ; Prostatic Hyperplasia ; drug therapy ; Prostatic Neoplasms ; epidemiology ; pathology ; Retrospective Studies

To investigate the molecular genetics basis for one para-Bombay phenotype, the red blood cell phenotype of the proband was characterized by standard serological techniques. Exon 6 and 7 of ABO gene, the entire coding region of FUT1 gene and FUT2 gene were amplified by polymerase chain reaction from genomic DNA of the proband respectively. The PCR products were purified by agarose gels and directly sequenced. The PCR-SSP and genescan were performed to confirm the mutations detected by sequencing. The results showed that the proband ABO genotype was A(102)A(102). Two heterozygous mutations of FUT1 gene, an A to G transition at position 682 and AG deletion at position 547-552 were detected in the proband. A682G could cause transition of Met-->Val at amino acid position 228, AG deletion at position 547-552 caused a reading frame shift and a premature stop codon. The FUT2 genotype was heterozygous for a functional allele Se(357) and a weakly functional allele Se(357), 385 (T/T homozygous at position 357 and A/T heterozygous at 385 position). It is concluded that the compound heterozygous mutation--a novel A682G missense mutation and a 547-552 del AG is the molecular mechanism of this para-Bombay phenotype.
ABO Blood-Group System ; genetics ; China ; DNA Mutational Analysis ; Female ; Fucosyltransferases ; genetics ; Genotype ; Humans ; Male ; Mutation ; Mutation, Missense ; Pedigree ; Phenotype ; Sequence Deletion

OBJECTIVETo probe into the molecular genetics basis for para-Bombay phenotype.

METHODSRed blood cell phenotype of the proband was characterized by serological techniques. Exons 6 and 7 of ABO gene, the entire coding region of alpha(1,2) fucosyltransferase (FUT1) gene and FUT2 gene were amplified by polymerase chain reaction (PCR) from genomic DNA of the proband respectively. The PCR products were excised and purified from agarose gels and were directly sequenced.

RESULTSAG at 547-552 deletion homozygous allele was found in the proband, which caused a reading frame shift and a premature stop codon. Parents of proband were heterozygous carriers.

CONCLUSIONTwo base deletion at position 547-552 of alpha (1,2) fucosyltransferase gene may cause para-Bombay phenotype.

ABO Blood-Group System ; genetics ; Fucosyltransferases ; genetics ; Humans ; Male ; Mutation ; Phenotype

To analyse the reason for one case of hemolytic transfusion reaction, antibodies in a patient's serum were identified using panel cells and Le (a-b-) phenotype cells, patient phenotype was identified by using anti-Le(a) and anti-Le(b) blood grouping reagents and the entire coding region of FUT3 gene was amplified by PCR and sequenced directly. The results showed that both IgM anti-Le(a) and anti-Le(b) antibodies were detected in patient's serum. Red cells was typed as Le (a-b-) phenotype and the FUT3 genotype was homozygote for non-functional le(59, 508) alleles. In conclusion, anti-Le(b) antibody can result in hemolytic transfusion reaction, FUT3 gene is homozygous for le(59, 508) allele resulting in Le (a-b-) phenotype.
Adult ; Antibodies ; adverse effects ; immunology ; Blood Grouping and Crossmatching ; Female ; Fucosyltransferases ; genetics ; Genotype ; Hematologic Diseases ; Humans ; Lewis Blood-Group System ; immunology ; Serology ; Transfusion Reaction

This study was purposed to investigate the molecular basis for RhD negative phenotype in Yiwu population in Zhejiang Province of China. The RhD negative samples were screened by saline agglutination test in blood donors. Some real RhD negative and RhDel phenotypes were identified using anti-human globulin test and absorbtion elution test. Ten exons of RHD gene in these samples were amplified by PCR-SSP, and positive exons were DNA sequenced. The results indicated that 30 real RhD negative and 8 RhDel phenotypes were identified in 38 initial RhD negative samples. Ten exons were complete negative in 28 real RhD negative samples and only exon 1, 2 and 10 were positive in 2 real RhD negative samples amplified by PCR. All 10 exons in 8 RbDel samples were positive and a DNA variant (1227G > A) was found in 8 RhDel samples. It is concluded that all exons are absence in most real RhD negative phenotypes, and the partial exons absence is also found in some real negative phenotypes among Yiwu population in Zhejiang province of China. The G to A mutation at position 1227 is found in all RhDel phenotypes.
Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Exons ; Genotype ; Humans ; Molecular Sequence Data ; Phenotype ; Polymorphism, Genetic ; Rh-Hr Blood-Group System ; genetics ; immunology

OBJECTIVETo explore the molecular basis of an individual featuring an ABx variant of ABO blood group system.

METHODSSerological 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.

RESULTSErythrocytes 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.

CONCLUSIONThe 808T>A mutation of the glycosyltransferase B gene may decrease the enzymatic activity and result in the Bx variant.

ABO Blood-Group System ; genetics ; Adult ; Exons ; Female ; Glycosyltransferases ; genetics ; Haplotypes ; Humans ; Mutation

This study was aimed to establish the real-time fluorescent quantitative PCR (RT-qPCR) with erythrocyte Kidd blood group gene for detecting the hematopoietic chimera and to investigate the feasibility of this method. The TaqMan MGB probes and special primers were designed on basis of difference of erythrocyte Kidd blood group alleles, the hematopoietic chimerism was detected by RT-qPCR, the DNA chimerism was simulated by means of dilution of multiple proportions, and the sensitivity analysis was performed. The results showed that the RT-qPCR with erythrocyte Kidd blood group gene could effectively distinguish JK*A and JK*B alleles. There was no significant difference between the theoretic value and the practical measured value by this method (P > 0.05). As 156 donor's cells could be discriminated from 10(4) chimeric cells, this method may effectively detect donor's cells with correlation coefficient 0.998. It is concluded that the established RT-qPCR with erythrocyte Kidd blood group gene shows the feasibility for quantitative detection of hematopoietic chimera, and may be used to quantitatively detect chimera in a certain range.
Chimera ; Erythrocytes ; Humans ; Kidd Blood-Group System ; genetics ; Real-Time Polymerase Chain Reaction

OBJECTIVETo investigate the application of flow cytometry in the differential diagnosis of lymphoma/leukemia with aberrant antigen expression.

METHODSThe results of flow cytometry of 30 lymphoma/leukemia cases with aberrant antigen expression, of which 3 cases being lymphomas, 8 B-cell leukemia, 1 T-cell leukemia, 17 acute non-lymphoid leukemia and 1 acute non-lymphoid leukemia involving lymph nodes were analyzed. Immunohistochemistry (EnVision) for CD79a, CD3 and MPO was performed on all cases.

RESULTSEleven cases of B-cell lymphoma/leukemia were cytoplasmic CD79a (cCD79a)-positive, cytoplasmic CD3 (cCD3epsilon) and cytoplasmic MPO (cMPO)-negative. Five of these cases were positive for CD5 and 2 for CD5, 1 or 2 for myeloid marker(s). The T-cell leukemia cases were cCD3epsilon-positive, cCD79a and cMPO-negative, they also co-expressed CD13 and CD33. The mantle cell lymphoma cases were positive for CD3, CD13 and CD33. Of the 8 B-cell leukemia cases, 4 were positive for CD5, 3 for CD13 and 1 for CD13 and CD33. The 18 acute non-lymphoid leukemia cases (including 1 acute non-lymphoid leukemia case involving lymph nodes) were cMPO-positive and cCD79a and cCD3epsilon-negative. Eight of the 18 expressed T-cell markers (including 1 case of acute non-lymphoid leukemia involving lymph nodes), 8 expressed B-cell markers, 2 expressed both T and B-cell markers.

CONCLUSIONSFlow cytometry can demonstrate aberrant antigen expression in lymphoma/leukemia cells and is helpful in delineating their cell origin. The technique is thus useful in the differential diagnosis of lymphoma/leukemia.

Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; CD13 Antigens ; metabolism ; CD3 Complex ; metabolism ; CD5 Antigens ; metabolism ; CD79 Antigens ; metabolism ; Diagnosis, Differential ; Flow Cytometry ; Humans ; Leukemia, B-Cell ; diagnosis ; immunology ; Leukemia, T-Cell ; diagnosis ; immunology ; Lymphoma, Mantle-Cell ; diagnosis ; immunology ; Peroxidase ; metabolism ; Retrospective Studies ; Sialic Acid Binding Ig-like Lectin 3

This study was purposed to investigate the molecular genetics basis for para-Bombay phenotype. The para-Bombay phenotype of two probands was identified by routine serological techniques. The full coding region of alpha (1, 2) fucosyltransferase gene (FUT1 and FUT2) in the probands was amplified by polymerase chain reaction and the amplified fragments were directly sequenced, meanwhile the mutations of FUT1 were also identified by TOPO TA cloning sequence method. The results indicated that two heterozygous mutations were detected by directly sequencing in two probands: AG deletion at position 547 - 552 and C to T mutation at position 658. Two different mutations were confirmed to be true compound heterozygotes with each mutation on a separate homologous chromosome by TOPO TA cloning sequence method. AG deletion at position 547 - 552 caused a reading frame shift and a premature stop codon. C658T mutation resulted in Arg-->Cys at amino acid position 220. It is suggested that the FUT1 mutation of two probands are compound heterozygous mutation with different chromosomes, which are named h1h3 and may be the genetics basis of para-Bombay phenotype.
ABO Blood-Group System ; genetics ; Alleles ; Frameshift Mutation ; Fucosyltransferases ; genetics ; Gene Deletion ; Heterozygote ; Humans ; Male ; Mutation, Missense