[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.

[Detection of hematopoietic chimera by real-time fluorescent quantitative PCR with erythrocyte Kidd blood group gene].

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.

[Discrimination of alleles in HLA-C*07:01:01G and HLA-C*07:02:01G groups through detection sequences in exons 1 to 7 of HLA-C locus by using polymerase chain reaction sequence-based typing].

This study was aimed to discriminate the alleles in the HLA-C*07:01:01G and HLA-C*07:02:01G groups and analyze their associations with HLA-B locus. Samples previously typed as HLA-C*07:01:01G and HLA-C*07:02:01G were collected. The nucleotide sequences in exons 1 to 7 of the HLA-C locus were sequenced by polymerase chain reaction sequence-based typing (PCR-SBT) and HLA-B genotyping was also preformed by PCR-SBT in these samples. The results showed that 4 samples (30.8%) were confirmed as HLA-C*07:01:01 and 9 samples (69.2%) were HLA-C*07:06 among 13 samples previously typed as HLA-C*07:01:01G. Linkage disequilibrium (LD) analysis showed that HLA-C*07:06 allele was strongly related with HLA-B*44:03. All samples were typed as C*07:02:01 among 102 individuals previously typed as C*07:02:01G. LD analysis showed that C*07:02:01 was strongly related with HLA-B*51:01, B*46:01, B*39:01, B*40:01, B*38:02, B*15:02 alleles. It is concluded that HLA-C*07:01:01 and HLA-C*07:06 alleles are confirmed in the HLA-C*07:01:01G group and HLA-C*07:02:01 is a preferred allele in the HLA-C*07:02:01G.

[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.

[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 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.

[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.

[C742T mutation of α1, 3 N-acetyl-D-galactosaminyltransferase gene is responsible for A2 subgroup].

The objective of this study was to analyze the molecular genetic basis for 2 individuals with A2B phenotype of ABO subtype. The ABO group antigens on red blood cells were identified by monoclonal antibodies and the ABO antibodies in serum were detected by the standard A, B, O cells. The exon 5 to exon 7 coding region of ABO gene was amplified by polymerase chain reaction (PCR) and the PCR product was sequenced directly after the enzymes digested. The amplified product was also cloned by TOPO TA cloning sequencing kit to split the 2 alleles apart and chosen colonies were sequencing bidirectionally for exon 6 to 7 of ABO gene. The results showed that both A and B antigen were identified on red blood cells of the individuals and there was anti-A1 antibody in their serum. There was no 261G deletion and showed 297A/G, 467C/T, 526C/G, 657C/T, 703G/A, 742C/T, 796C/A, 803G/C, 930G/A heterozygotes by direct DNA sequencing. After cloning and sequencing, it was obtained the B101 and one novel A allele. The novel allele has one nucleotide change at 742 position C to T compared with A102, which results in an amino acid Arg to Cys at 248 position and was nominated as A213. It is concluded that C742T mutation of the α1, 3 N-acetyl-D-galactosaminyl-transferase gene can lead to A2 phenotype and with anti-A1 antibody in serum.

[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.

[Para-Bombay phenotype caused by combined heterozygote of two bases deletion on fut1 alleles].

This study was purposed to investigate the molecular basis of a para-Bombay phenotype for screening and identification of rare blood group. ABO and H phenotypes of the proband were identified by serological techniques. The exon 6 to exon 7 of ABO gene and full coding region of α-1,2-fucosyltransferase (fut1) gene of the proband were analyzed by polymerase chain reaction and direct sequencing of the amplified fragments. The haplotype of compound heterozygote of fut1 was also identified by cloning sequencing. The results indicated that a rare para-Bombay phenotype was confirmed by serological techniques. Two deletion or insertion variant sites near nucleotide 547 and 880 were detected in fut1 gene. The results of cloning sequence showed that one haplotype of fut1 gene was two bases deletion at 547-552 (AGAGAG→AGAG), and another one was two bases deletion at position 880-882 (TTT→T). Both two variants caused a reading frame shift and a premature stop codon. It is concluded that a rare para-Bombay phenotype is found and confirmed in blood donor population. The molecular basis of this individual is compound heterozygote of two bases deletion on fut1 gene which weaken the activity of α-1, 2-fucosyltransferase.

[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.

[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.

[Analysis of mRNA expression profiles of megakaryocytes from human cord blood CD34+ cells ex vivo expanded using Solexa sequencing].

OBJECTIVE: To investigate the mRNA expression profiles of megakaryocytes (MKs) from human cord blood CD34+ cells ex vivo expanded using Solexa technique. METHODS: CD34+ Cells were isolated using density gradient centrifugation and magnetic activated cell sorting. Cultures were stimulated with recombinant human thrombopoietin (100 ng/ml). After 12 days, the MKs fraction was separated from the non-MKs fraction using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The mRNA expression of MKs and non-MKs was detected by Solexa sequencing. RESULTS: We obtained 3 773 147 and 3 533 805 Tags from MKs and non-MKs, respectively. The amounts of unambiguous tags were 3 291 132 and 2 967 947 and those of distinct tags were 197 769 and 245 318. The expression of 1161 genes was up-regulated and that of 902 genes down-regulated. The expression of 2717 tags was up-regulated and that of 1519 tags down-regulated. CONCLUSIONS: MKs and non-MKs have remarkably different mRNA expression profiles. The differential gene-encoded products may be involved in cellular development, adhesion, apoptosis metabolism, intra- and intercellular signal transduction, and immune response. Further studies on this topic may clarify the expression mechanism, signal transduction, and regulation mechanisms.

[Quantitative chimerism analysis of regulatory T cell subsets based on immunomagnetic sorting].

The aim of study was to explore the feasibility of quantitative chimerism analysis of regulatory T (Treg) cells using immune sorting coupling short tandem repeat (STR) method. 14 sets of artificial chimera samples were prepared by mixed lymphocytes according to different proportion. The CD4(+)CD25(+) Treg cells were harvested by negative and positive selection of immunomagnetic beads, then the STR polymorphisms of 16 loci in sorted Treg cells was analyzed. The results showed that the DNA amount extracted from sorted Treg cells was fit for STR detection. All STR alleles specific for recipient or donor could be detected and the quantitative results were consistent with theoretic values in over 10% recipient chimeras. But only partial recipient alleles could be detected and the quantitative results were different from theoretic values in less then 1% recipient chimeras. It is concluded that a quantitative chimerism analysis of Treg cell based on immune sorting is established. The sensitivity and accuracy for chimera detection are 1% to 10%, and this method can be used to monitoring hematopoietic chimerism following allogeneic hematopoietic stem cell transplantation.

[In vitro expression activity of α-(1,2) fucosyltransferase with 35C > T and 682A > G mutations].

In order to explore the effects of 35C > T and 682A > G mutations on the activity of alpha-(1,2) fucosyltransferase, the coding region of fut1 gene was amplified by polymerase chain reaction (PCR) from genomic DNA. PCR product was ligated into expression vector using TOPO TA cloning kit to obtain the recombinant plasmids. The recombinant plasmids were transfected into COS-7 cells by liposome method. After screening by using G418, H antigen expression on the COS-7 was tested by flow cytometry and fut1 mRNA was detected by real-time PCR. The results indicated that three kinds of recombinant plasmids pcDNA3.1/V5-His-wild (35C + 682A), pcDNA3.1/V5-His-35T and pcDNA3.1/V5-His-35T-682G were successfully constructed. After transfection, the H antigen expressed on membrane of COS-7 cells at the second day, with the maximum level of expression at the fourth day. When compared with pcDNA3.1/V5-His-wild transfected cells, the H antigen expression level of the 35T and 682G + 35T recombinant plasmids in the transfected cells was 52.7% and 13.3% on the fourth day, respectively. Although the level of fut1 mRNA decreased with prolonging of time, the mRNA expressed on the pcDNA3.1/V5-His-35T-682G transfected cells reached to 14% of the wild plasmids on the first day. It is concluded that 682A > G mutation obviously reduces the activity of alpha-(1,2) fucosyltransferase, while 35C > T mutation leads to partial reduction of H antigen in vitro expression.

[Probability of high resolution full match for human leukocyte antigen loci in unrelated donors and recipients with low resolution match].

This study was aimed to analyze the possibility of high resolution matching for human leukocyte antigen (HLA) loci in unrelated donor-recipient pair with low resolution match in HLA-A, -B, -DRB1 loci. Samples were genotyped for HLA-A, -B, -C, -DRB1 and -DQB1 by polymerase chain reaction sequence based typing (PCR-SBT). The results showed that the total number of patients and the donors were 166 and 274. 97 (58.43%) patients were matched for 1 donor and 47 (28.31%) patients were matched for 2 donors at low resolution level; among 274 donor-recipient pairs, HLA-A, -B, -C, -DRB1 and -DQB1 loci matching for 6/10, 7/10, 8/10, 9/10 and 10/10 were 32 (11.68%), 54 (19.71%), 62 (22.63%), 49 (17.88%) and 48 (17.52%) respectively; there were mismatch in HLA-A, -B, -C, -DRB1 and -DQB1 loci, and the most mismatch was in HLA-C locus. The number of alleles of HLA-A, -B, -C, -DRB1 and -DQB1 loci were 23, 46, 21, 30 and 17 respectively in the donors. The alleles number HLA-A, -B, -C, -DRB1 and -DQB1 loci were 20, 40, 22, 29 and 16 respectively in the patients; the haplotype number of HLA loci were 311 in the donors and 224 in the patients. The high frequency of haplotype was A*02:07-B*46:01-C*01:02-DRB1*09:01:02-DQB1*03:03 (5.63% and 6.88%). It is concluded that the probability of high resolution mismatch of HLA loci is high in unrelated donor-recipient pairs with low resolution match in HLA-A, -B, -DRB1 loci.

[Nucleotide sequence analysis of A novel HLA-B*15:124 allele confirmed].

This study was purposed to investigate the nucleotide sequences of a novel HLA-B*15:124 allele and its molecular mechanism. The genomic DNA from whole blood was extracted by using commercial DNA extraction kit. The sequences of exon 2, 3 and 4 of HLA-B locus in the proband were amplified by PCR with group-specific primers, the PCR products were purified by enzymes digestion, then exon 2 to 4 of HLA-B locus for both orientations was sequenced. The results showed that 2 HLA-B alleles of proband were gained after amplification and sequencing of group-specific primers, among them one was a B*40:03, another was a novel allele. After BLAST analysis, the novel allele showed nucleotides different from HLA-B*15:52 in exon 3 at nucleotide position 427 A > T and 440 G > T which resulted in amino acid change from Thr to Ser at codon 143 and Trp to Leu at conon 147. It is concluded that a novel HLA-B allele has two different nucleotides. This HLA-B allele is identified and has been officially named B*15:124 by the WHO Nomenclature Committee.

[Detection of fetal short tandem repeat loci in maternal plasma as gender-independent fetal DNA marker].

The aim of this study was to investigate the feasibility of using fetal short tandem repeat (STR) loci in maternal plasma as gender-independent fetal DNA marker. DNA from maternal plasma sample was extracted using QIAamp DNA Kit. AmpF1 STR profiler box was used to amplify 9 different polymorphic short tandem repeat (STR) loci (D3S1358, VWA, FGA, D5S818, D13S317, D7S820, D8S1179, D21S11, D18S51), the multiplex fluorescent PCR was used to amplify the STR alleles of fetal DNA in 36 pregnant plasma samples of pregnant women at different pregnancy. Their husbands' DNA isolated from whole blood samples were amplified at the same time. The PCR products were electrophoresis by ABI Prism 377 sequencer, the results of electrophoresis were analysed by Genscan. The presence of fetal DNA in maternal plasma by Paternally inherited fetal alleles were detected. The results showed that paternally inherited fetal alleles were detected in 4 cases in early pregnancy (4/6), 19 cases in middle pregnancy (19/20) and 9 cases in late pregnancy (9/10) respectively, the paternally inherited fetal alleles in 4 of 36 cases could not be detected. It is concluded that fluorescent multiplex PCR can be used for amplification of male and female fetal STRs in maternal plasma to obtain genetic information, which may have implication for non-invasive prenatal diagnosis of certain hereditary diseases independent of the fetal sex.

[Establishment of allele specific primer polymerase chain reaction sequence-based typing and investigation of the polymorphism in exon 3 of HLA-DRB1].

OBJECTIVE: To establish the allele specific primer polymerase chain reaction sequence-based typing (PCR-SBT) and investigate the polymorphism of exon 3 of human leukocyte antigen( HLA)-DRB1. METHODS: The fragment containing exons 2 and 3 of HLA-DRB1 gene was amplified by group specific primers. The amplified products were digested by restriction enzymes and directly sequenced in both directions. The genotype was assigned by using Assign 3.5 SBT software. RESULTS: The exon 3 sequences of HLA-DRB1*08:09 and HLA-DRB1*12:02:01 were identified for the first time. There were 27 polymorphic sites in exon 3 among the twenty-five HLA-DRB1 alleles, which was 9.56% of all nucleotides of exon 3. The method could discriminate the HLA-DRB1*14:01:01/14:54 ambiguous samples, and the HLA-DRB1*14:01:01 was identified in the Chinese population. CONCLUSION: The PCR-SBT method for exon 3 of HLA-DRB1 from the present study was reliable and there were polymorphisms in exon 3 of HLA-DRB1.