RESUMO
The Wnt gene family is involved in a wide range of developmental processes. Despite its significance, the evolution and function of Wnt genes remain largely unclear. Here, an exhaustive survey of Wnt genes was conducted in Tenebrio molitor and 17 other beetle genomes. A total of 146 Wnt genes were identified, creating a comprehensive coleopteran Wnt gene catalog. Comparative genomics indicates that dynamic evolutionary patterns of Wnt gene loss and duplication occurred in Coleoptera, leading to the diverse Wnt gene repertoire in various beetles. A striking loss of particular Wnt gene subfamilies occurs in Coleoptera. Remarkably, Wnt gene duplication was discovered for the first time in insects. Further analysis of Wnt gene expression in T. molitor indicates that each Wnt gene, including the duplicated ones, has a unique spatial or temporal expression pattern. The current study provides valuable insight into the evolution and functional validation of Wnt genes in Coleoptera.
Assuntos
Besouros , Tenebrio , Animais , Besouros/genética , Genoma , Tenebrio/genética , Tenebrio/metabolismoRESUMO
PURPOSE: Recent genetic association studies showed conflicting results on the relationship of miRNA single-nucleotide polymorphisms (SNPs) and congenital heart disease (CHD) risk. The purpose of the present systematic review was to collect the current available evidences to evaluate the association between miRNA polymorphisms and CHD risk. METHODS: Four electronic databases including PubMed, EMBASE, ISI Web of Science, and CENTRAL were extensively searched for relevant studies published before February, 2019. Observational studies determining the association between miRNA polymorphisms and risk of CHD were included. Risk of bias was evaluated using the Newcastle-Ottawa Scale by 2 independent researchers. Major characteristics of each study and estimation of effect size of individual locus polymorphism were summarized. In addition, meta-analysis was performed to quantify the associations between miRNA polymorphisms and CHD risk. RESULTS: Nine studies containing 6502 CHD patients and 6969 healthy controls were included in this systematic review. Ten loci in 9 miRNAs were reported. Only rs11614913 in miR-196a2 was determined to have significant associations with CHD susceptibility, which was supported by meta-analysis (CC vs CT+TT: odds ratio 1.54, 95% confidence interval 1.30, 1.82; Pâ<â.00001). A strong evidence indicated lack of association between rs2910164 in miR-146a and CHD. Limited or conflicting evidences were found for the associations of the other variants (rs11134527, rs139365823, rs76987351, rs3746444, rs4938723, rs2292832, rs41291957, rs895819) and risk of CHD. CONCLUSIONS: Locus polymorphisms in miRNAs are not generally associated with CHD. Only rs11614913 was found to have significant associations with CHD. Further studies will be needed, using larger populations of different ethnicities, to obtain a better understanding of these associations.
Assuntos
Cardiopatias Congênitas/genética , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Razão de ChancesRESUMO
OBJECTIVE: A previous meta-analysis concluded that TNF-α 238A/G and TNF-α 308A/G polymorphisms were not associated with the risk of juvenile idiopathic arthritis (JIA) in the overall population or Caucasian subjects. With the publication of a fair number of studies on the association between TNF-α polymorphisms and JIA in recent years, we conducted this updated meta-analysis to make a more accurate evaluation of such relationship. METHODS: We adopted PubMed, EMBASE, ISI Web of Science and CNKI to identify observational studies that addressed the association between TNF-α polymorphisms and risk for JIA. The allelic effect of variant A for the risk of JIA was expressed as odds ratio (OR) along with the associated 95% confidence interval (95% CI). Meta-analyses were performed by pooling ORs and 95%CI from included studies using RevMan 5.3 software. The stratified-analysis based on ethnicity was performed to confirm the ethnicity-dependent effect on the relationship. RESULTS: A total of 15 case-control studies including 2845 patients in JIA groups and 4771 patients in control groups were included in our study. The findings indicated a statistically significant association between the A allele of the TNF-alpha 238A/G polymorphism and the decreased JIA risk in Caucasians (Pâ=â.0002). The study in Iranian showed similar results (Pâ=â.0002) whereas the studies in other ethnicities failed to replicate this finding: Han (Pâ=â.29), Mexican (Pâ=â.64) and Turkish population (Pâ=â.32). TNF-α 308A/G was not statistically associated with JIA in overall subjects or Caucasians. CONCLUSION: Our study confirmed the protective role of the A allele in TNF-α 238A/G but not TNF-α 308A/G against the occurrence of JIA in the Caucasian population. To exactly validate the correlation between TNF-α polymorphisms and JIA in other ethnic backgrounds, additional studies are required.
Assuntos
Artrite Juvenil/diagnóstico , Artrite Juvenil/genética , Fator de Necrose Tumoral alfa/genética , Alelos , Artrite Juvenil/etnologia , Estudos de Casos e Controles , Indicadores Básicos de Saúde , Humanos , Irã (Geográfico)/epidemiologia , Estudos Observacionais como Assunto , Polimorfismo de Nucleotídeo Único/genética , População Branca/genéticaRESUMO
The aim of the present study was to determine whether High mobility group box 1 (HMGB1) polymorphism was associated with cancer susceptibility. PubMed, Embase, and ISI Web of Science were extensively searched without language restriction. Data were extracted using a standardized data collection sheet after two reviewers scanned studies independently. The association between HMGB1 polymorphism and cancer risks was indicated as odds ratio (OR) along with its related 95% confidence interval (95%CI). Meta-analysis was conducted via RevMan 5.3 software. A total of ten studies comprising 4530 cases and 5167 controls were included in our study. Meta-analysis revealed no statistical association between rs1045411, rs1360485, rs1412125, or rs2249825 polymorphisms in HMGB1 gene and risk of cancer, either did subgroup analysis of rs1045411 stratified by cancer types and ethnic groups. Our results revealed no statistical association between current four polymorphism loci and cancer risks, suggesting that the attempt of applying HMGB1 variants as a therapeutic target or a prognosis predictor might still require a second thought. However, HMGB1 is deemed to play pleiotropic roles in cancers, we strongly call for large-scale studies with high evidence level to uncover the exact relationship between HMGB1 gene variants and cancer progression.
Assuntos
Proteína HMGB1/genética , Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Predisposição Genética para Doença , HumanosRESUMO
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Assuntos
Humanos , Alelos , Éxons , Frequência do Gene , Genótipo , Cadeias beta de HLA-DQ , Genética , Cadeias HLA-DRB1 , Genética , Cadeias HLA-DRB3 , GenéticaRESUMO
<p><b>OBJECTIVE</b>To explore the molecular basis of an individual featuring an ABx variant of ABO blood group system.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The 808T>A mutation of the glycosyltransferase B gene may decrease the enzymatic activity and result in the Bx variant.</p>
Assuntos
Adulto , Feminino , Humanos , Sistema ABO de Grupos Sanguíneos , Genética , Éxons , Glicosiltransferases , Genética , Haplótipos , MutaçãoRESUMO
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.
Assuntos
Humanos , Quimera , Eritrócitos , Sistema do Grupo Sanguíneo Kidd , Genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
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.
Assuntos
Humanos , Alelos , Sequência de Bases , Éxons , Antígenos HLA-B , Genética , Antígenos HLA-C , Genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Métodos , Análise de Sequência de DNARESUMO
<p><b>OBJECTIVE</b>To analyze the molecular basis for an individual with ABx09 phenotype of ABO subtype.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>G to A at nt889 of alpha-1,3 galactosyltransferasegene can result in Bx09 phenotype, with the presence of anti-B antibody in serum.</p>
Assuntos
Adolescente , Feminino , Humanos , Sistema ABO de Grupos Sanguíneos , Genética , Metabolismo , Alelos , Sequência de Bases , Tipagem e Reações Cruzadas Sanguíneas , Frequência do Gene , Genótipo , Dados de Sequência Molecular , Linhagem , FenótipoRESUMO
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Assuntos
Animais , Células COS , Chlorocebus aethiops , Fucosiltransferases , Genética , Metabolismo , Regulação da Expressão Gênica , Vetores Genéticos , Genética , Proteínas Mutantes , Genética , Metabolismo , RNA Mensageiro , GenéticaRESUMO
<p><b>OBJECTIVE</b>To investigate the recombination events between human leukocyte antigen (HLA) loci within two families.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Assuntos
Feminino , Humanos , Masculino , Povo Asiático , Etnologia , Genética , China , Etnologia , Etnicidade , Genética , Loci Gênicos , Genética , Antígenos HLA-A , Genética , Antígenos HLA-C , Genética , Haplótipos , Genética , Linhagem , Recombinação Genética , GenéticaRESUMO
<p><b>OBJECTIVE</b>To elucidate the serological characteristics and molecular mechanism of a blood donor with weaken B antigen.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The methylation in the CpG island of ABO gene promoter region may cause weak expression of the B antigen.</p>
Assuntos
Humanos , Sistema ABO de Grupos Sanguíneos , Genética , Alergia e Imunologia , Alelos , Anticorpos Monoclonais , Alergia e Imunologia , Doadores de Sangue , Ilhas de CpG , Genética , Eritrócitos , Alergia e Imunologia , Regulação da Expressão Gênica , Alergia e Imunologia , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
<p><b>OBJECTIVE</b>To analyze the sequence of the exons 2-4 of human leukocyte antigen (HLA) novel allele HLA-B*15:129.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>A novel HLA-B allele was identified and has been officially named B15:129 by the WHO Nomenclature Committee.</p>
Assuntos
Humanos , Masculino , Alelos , Sequência de Bases , Primers do DNA , Éxons , Antígenos HLA-B , Genética , Dados de Sequência Molecular , Tipagem Molecular , Reação em Cadeia da PolimeraseRESUMO
<p><b>OBJECTIVE</b>To investigate the mRNA expression profiles of megakaryocytes (MKs) from human cord blood CD34+ cells ex vivo expanded using Solexa technique.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>
Assuntos
Humanos , Antígenos CD34 , Células Cultivadas , Sangue Fetal , Biologia Celular , Megacariócitos , Biologia Celular , Metabolismo , RNA Mensageiro , Genética , TranscriptomaRESUMO
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.
Assuntos
Humanos , Sistema ABO de Grupos Sanguíneos , Genética , Alergia e Imunologia , Alelos , Anticorpos Anti-Idiotípicos , Alergia e Imunologia , Doadores de Sangue , Éxons , Genótipo , Heterozigoto , Dados de Sequência Molecular , Mutação , N-Acetilgalactosaminiltransferases , Genética , Fenótipo , Análise de Sequência de DNARESUMO
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>There were polymorphisms in the 5'-UTR of ABO gene and the nucleotide sequences near the proximal promoter were conservative.</p>
Assuntos
Humanos , Regiões 5' não Traduzidas , Genética , Sistema ABO de Grupos Sanguíneos , Genética , Clonagem Molecular , Genótipo , Haplótipos , Polimorfismo Genético , Análise de Sequência de DNARESUMO
<p><b>OBJECTIVE</b>To investigate the polymorphisms of platelet membrane glycoprotein genes related to human platelet alloantigen (HPA)-1 to 17w.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Assuntos
Humanos , Antígenos de Plaquetas Humanas , Genética , Isoantígenos , Genética , Glicoproteínas da Membrana de Plaquetas , Genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
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.
Assuntos
Feminino , Humanos , Sistema ABO de Grupos Sanguíneos , Genética , Alelos , Pareamento de Bases , Fucosiltransferases , Genética , Genótipo , Heterozigoto , Mutação , Fenótipo , Deleção de SequênciaRESUMO
This study was purposed to construct the recombinant retroviral vector containing human homeobox gene hoxA10 and to establish the packaging cell lines which stably produce viruses. The whole coding region of hoxA10 gene was amplified by PCR and inserted into the retroviral vector MSCVneo. The recombinant vector was identified by DNA sequencing. The recombinant and control retroviral vectors were transfected into the packaging cell line PT67 by liposome Lipofectamine(TM) 2000. These cell lines stably producing retrovirus were isolated following G418 selection. The viral suspension was harvested and the viral titer was determined by NIH3T3 cells. The results showed that the recombinant retroviral vector was proved to encode hoxA10 genes by sequencing. The cell lines efficiently producing virus were screened by G418 and designated as PT67/MSCVneo and PT67/MSCVneo-hoxA10. The titers of them were 5 x 10(5) CFU/ml and 4 x 10(4) CFU/ml respectively. It is concluded that the recombinant retroviral vector containing homeobox gene hoxA10 and the stably packaging cell lines which efficiently and correctly produce viruses are successfully constructed, which provides a basis for further exploration of the hoxA10 gene function in the regulation of hematopoiesis.
Assuntos
Humanos , Linhagem Celular , Genes Homeobox , Vetores Genéticos , Proteínas de Homeodomínio , Genética , Retroviridae , Genética , TransfecçãoRESUMO
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.