Study of the molecular basis for an individual with Bel variant due to deletion of B glycosyltransferase gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the molecular basis of an individual with Bel variant of the ABO blood group.</p><p><b>METHODS</b>The ABO antigen and serum antibody of the individual were detected by serological method. All coding regions and flanking introns of the ABO gene were amplified with PCR and sequenced bidirectionally. The haplotypes of the individual were analyzed by cloning and sequencing. A three dimensional model of the mutant protein was constructed and analyzed.</p><p><b>RESULTS</b>The individual has expressed a very weak B antigen on its red blood cells by absorption and elution testing, which was identified as a Bel variant phenotype. The heterozygous sites in exon 6 (261del/G) and exon 7 (297A/G, 484del/G, 526C/G, 657C/T, 703G/A, 796C/A, 803G/C, 930G/A) of the coding region of the ABO gene were identified by direct sequencing. Haplotype analysis showed that the individual has carried an O01 allele and a novel B allele. The sequence of the novel B allele was identical to B101 except for a del G at nucleotide position 484 (484delG), which was nominated as B120 by the Blood Group Antigen Gene Mutation Database (dbRBC NCBI). The 484delG mutation of the B allele has led to a reading frame shift and created a premature terminal codon for the glycosyltransferase (GT) enzyme. Prediction of the 3D structure suggested that the GT enzyme has become an incomplete protein only with its N-terminal region.</p><p><b>CONCLUSION</b>The 484delG mutation of the glycosyltransferase B gene has probably abolished or reduced the enzymatic activity and resulted in the Bel variant phenotype.</p>

Molecular basis for an individual with rare p phenotype in P1Pk blood group system / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the molecular basis for an individual with rare p phenotype in the P1Pk blood group system.</p><p><b>METHODS</b>Erythrocyte blood group antigens and antibodies in serum were identified in the proband and five family members with a serological method. Coding regions and flanking untranslated regions of the α1,4-galactosyltransferase gene (A4GALT) encoding P1Pk antigens were amplified with polymerase chain reaction and directly sequenced. The haplotypes of A4GALT in the parents of the proband were also analyzed by cloning sequencing.</p><p><b>RESULTS</b>The proband was found with a rare p phenotype with anti-Tja antibody in his serum by serological method. The other family members all had a common P2 phenotype. The results of DNA sequencing showed that a cytosine was inserted at nucleotide position 1026 to 1029 (1026_1029insC) of both alleles of the A4GALT gene in the proband. The mutation has caused a reading frame shift and formed a mutant protein by extending 92 amino acid residues. The other family members were either heterozygous for the insertion or of the wild type at above position.</p><p><b>CONCLUSION</b>The 1026_1029insC mutation of the A4GALT gene is probably responsible for the p phenotype identified for the first time in Chinese population. The individual with the p phenotype possesses anti-Tja antibody.</p>

A rare Pk phenotype caused by a 433 C>T mutation of the β-1,3-N-acetylgalactosyltransferase gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the serological characteristics and molecular mechanism for a rare Pk phenotype of the P1Pk blood group system.</p><p><b>METHODS</b>The blood group of the proband was identified by serological techniques. The coding region and flanking intronic sequences of the β-1,3-N-acetylgalactosyltransferase gene (B3GALANT1) associated with the Pk phenotype were analyzed using polymerase chain reaction sequence-based typing.</p><p><b>RESULTS</b>The proband was identified as having a rare Pk phenotype including anti-P in her serum. The blood group of her daughter and husband showed a P2 phenotype. The nucleotide sequences of the B3GALANT1 gene of her husband and two randomly-chosen individuals were the same as the reference sequence (GenBank AB050855). Nucleotide position 433 C>T homozygous mutation in the B3GALANT1 was found in the proband, which has resulted in a stop codon at amino acid position 145, which may produce a premature protein capable of decreasing or inhibiting the activity of the β -1,3-N-acetylgalactosyltransferase. The nucleotide position 433 C/T heterozygous in the B3GALANT1 was found in her daughter.</p><p><b>CONCLUSION</b>The Pk phenotype resulted from 433 C>T mutation in the B3GALANT1 gene has been identified.</p>

Analysis of erythroid-specific blood group genes using un-mobilized peripheral stem cells cultured in vitro / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze specific expression of blood group genes using nucleated erythroid cells cultured from un-mobilized peripheral stem cells in vitro.</p><p><b>METHODS</b>Hematopoietic stem cells(HSC) bearing the CD34 antigen were isolated from peripheral blood by centrifugation and magnetic beads sorting, followed by suspension culture in vitro. Cells were collected from medium on various stages and analyzed by immunofluorescence. The RNA transcription of RH and ABO blood group genes was analyzed using culture cells on day 12.</p><p><b>RESULTS</b>A total of(3.19±0.13) ×10 (4) CD34+cells were isolated from about 50 mL peripheral blood with a recovery rate of 67.3%±2.7%. The cells amount in erythroid-lineage culture system on day 9 reached a plateau of a 237.1±15.5-fold amplification of the initial cell input. The stem cell-specific CD34 antigen was dropped off, while the erythroid-specific CD235a and CD240D antigens were increased in culture period. RHD/CE and ABO genes can be amplified using RNA extracted from culture cells on day 12, and genotypes of Rh and ABO systems by DNA sequencing were consistent with their serologic phenotypes.</p><p><b>CONCLUSION</b>A method was established to analyze the gene expression of erythroid blood group derived from un-mobilized peripheral stem cells cultured in vitro. It can be used to study the expression of various erythroid-specific genes.</p>

A rare p phenotype caused by a 26-bp deletion in α 1,4-galactosyltransferase gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To delineate serological features and genetic basis for a rare p phenotype of P1Pk blood group system found in a Chinese individual.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>A 26 bp deletion at position 972 to 997 of the A4GALT gene has been identified in a Chinese individual with p phenotype.</p>

Molecular basis of the B(A)phenotype and its pedigree analysis / 中华检验医学杂志

Objective To investigate the serological characteristics and molecular basis of the B (A)phenotype in ABO blood group and provide the data for clinical transfusion of individuals with B(A) phenotype.Methods The ABO group antigens on red cells of the proband,family members and donors were identified by monoclonal antibodies and the ABO antibodies in sera were detected by the standard A,B,O cells.The compatibility testing for the proband and donors was detected by salted test,polybrene test and antiglobulin test.The coding region of exon 6 to exon 7 in ABO gene was amplified by polymerase chain reaction(PCR) and the PCR products were sequenced.The haplotypes of proband were analyzed by cloning and sequencing.Results It was showed that both A and B antigens were detected on red cells of the proband and her two family members,and there was anti-A_1 antibody in their sera.The serological phenotype of the samples are identified as the A_2B.DNA sequencing showed 261 G/del,297A/G,526C/G,657C/T,700C/G,703G/A,796C/A,803G/C,930G/A heterozygotes in exon 6 to exon 7.It can be deduced that genotype in the proband is B(A)_(02)/O_(01).The genotypes of her mother and grandmother-in-law were B(A)_(02)/B_(101) and B(A)_(02)/O_(01),respectively.After cloning and sequencing,two alleles B(A)_(02) and O_(01) in proband was showed.B(A)_(02) has snigle nucleotide change(700 C>G),which resets replacement of proline with alanine at position 234.Two donors with phenotype A_2B were identified as genotype B(A)_(02)/O_(01) and A_(208)/B_(101),respectively.The results of crossmatch testing is in accordane between the proband and two donors and there was no clinical adverse reaction after transfusion.Conclusions 700C>G in α-1,3galactosyltransferase allele(B allde)can result in B(A)phenotype in individuals with the phenotype of A_2B.The donors in the transfusion for the individuals with B(A) phenotype should include individuals with A_2B phenotype.

Establishment and application of hnman platelet antigen genotyping with PCR sequencing-basod typing method / 中华检验医学杂志

Objective To establish a PCR sequencing-based typing (PCR-SBT) method for simultaneous genotyping of human platelet antigen HPA-1 to HPA-16w.Methods All DNA polymorphism sites of HPA-1 to HPA-16w were obtained from the immuno polymorphism database.The specific primers were designed using Primer Premier 5.0 software to amplify nucleotide acid fragments encompassing each HPA polymorphism site.The primer sequence and PCR condition were optimized to obtain specific and single amplification product.The PCR product was purified and then sequenced to determine the HPA genotypes.Two standard DNA samples were detected using the HPA PCR-SBT method to examine the accuracy d this method.Sixteen reference samples (including 6 interference samples with HPA gene mutations) provided by 14th platelet immunology workshop of international society of blood transfusion (ISBT) in 2008 were also tested by this home-brew HPA PCR-SBT method.Results Total eleven pairs of primers were designed to amplify and sequence the sixteen HPA systems.The HPA genotypes of two standard samples were 1aa/2aa/3ab/4aa/5ab/6aa/7aa/8aa/9aa/10aa/11aa/12aa/13aa/14aa/15aa/16aa and 1aa/ 2aa/3aa/4aa/5aa/6aa/7aa/8aa/9aa/10aa/11aa/12aa/13aa/14aa/15aa/16aa,respectively.The 256 HPA genotypes of 16 reference samples were clear.128 genotypes among them were completely accordance with the results provided by ISBT report.Conclusions The PCR-SBT assay combining high-throughput DNA sequencer established in the study provides a simple,rapid and accurate method for HPA-1 to HPA-16w systems genotyping.The assay is suitable for routine clinical HPA genotyping and shows a broad prospect in further applications.