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1.
Biomed Res Int ; 2017: 3043476, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28758112

RESUMO

BACKGROUND: Congenital heart defect (CHD) is one of the most common birth defects in the world. The methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes are two of the most important candidate genes for fetal CHD. However, the correlations between the two genes and fetal CHD were inconsistent in various reports. Therefore, this study is aimed to evaluate the parental effects of the two genes on fetal CHD via three genetic polymorphisms, MTHFR 677C>T (rs1801133), MTHFR 1298 A>C (rs1801131), and MTRR 66A>G (rs1801394). METHODS: Parents with pregnancy history of fetal CHD were divided into two subgroups: ventricular septal defect (VSD) (21) and non-VSD groups (78). VSD, non-VSD, and 114 control parents (controls) were analyzed in this study. Genotyping of these genetic polymorphisms was done by sequencing. RESULTS: The MTHFR 677C>T polymorphism of either mothers or fathers was independently associated with fetal non-VSD (P < 0.05) but not VSD, while the MTRR 66A>G polymorphism was independently associated with fetal VSD (P < 0.05) but not non-VSD. No significance was found for MTHFR 1298A>C polymorphism. CONCLUSION: In either maternal or paternal group, the MTHFR 677C>T polymorphism was independently related to fetal non-VSD, while the MTRR 66A>G polymorphism was independently related to fetal VSD.


Assuntos
Ferredoxina-NADP Redutase/genética , Feto , Defeitos dos Septos Cardíacos/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo Genético , Adulto , Feminino , Humanos , Masculino
2.
Mol Med Rep ; 15(6): 3989-3998, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28440505

RESUMO

The discovery of cell-free DNA fetal (cff DNA) in maternal plasma during pregnancy provides a novel perspective for the development of non­invasive prenatal diagnosis (NIPD). Against the background of maternal DNA, the use of the relatively low concentration of cff DNA is limited in NIPD. Therefore, in order to overcome the complication of the background of maternal DNA and expand the scope of cff DNA application in clinical practice, it is necessary to identify novel universal fetal­specific DNA markers. The GeneChip Human Promoter 1.0R Array set was used in the present study to analyze the methylation status of 12 placental tissue and maternal peripheral blood whole­genome DNA samples. In total, 5 fetus differential hypermethylation regions and 6 fetus differential hypomethylation regions were identified. In order to verify the 11 selected methylation regions and detect the differential CpG sites in these regions, a bisulfate direct sequencing strategy was used. In total, 87 fetal differential methylation CpG sites were identified from 123 CpG sites. The detection of fetal differential methylation DNA regions and CpG sites may be instrumental in the development of efficient NIPD and in the expansion of its application in other disorders.


Assuntos
Metilação de DNA , Epigênese Genética , Epigenômica , Estudo de Associação Genômica Ampla , Diagnóstico Pré-Natal , Adulto , Biomarcadores , Biologia Computacional/métodos , Ilhas de CpG , Epigenômica/métodos , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla/métodos , Idade Gestacional , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Diagnóstico Pré-Natal/métodos , Regiões Promotoras Genéticas , Análise de Sequência de DNA
3.
Medicine (Baltimore) ; 96(50): e8814, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29390271

RESUMO

BACKGROUND: Patients with Duchenne muscular dystrophy (DMD) usually have severe and fatal symptoms. At present, there is no effective treatment for DMD, thus it is very important to avoid the birth of children with DMD by effective prenatal diagnosis. We identified a de novo DMD gene mutation in a Chinese family, and make a prenatal diagnosis. METHODS: First, multiplex ligation-dependent probe amplification (MLPA) was applied to analyze DMD gene exon deletion/duplication in all family members. The coding sequences of 79 exons in DMD gene were analyzed by Sanger sequencing in the patient; and then according to DMD gene exon mutation in the patient, DMD gene sequencing was performed in the family members. On the basis of results above, the pathogenic mutation in DMD gene was identified. RESULTS: MLPA showed no DMD gene exon deletion/duplication in all family members. Sanger sequencing revealed c.2767_2767delT [p.Ser923LeufsX26] mutation in DMD gene of the patient. Heterozygous deletion mutation (T/-) at this locus was observed in the pregnant woman and her mother and younger sister. The analyses of amniotic fluid samples indicated negative Y chromosome sex-determining gene, no DMD gene exon deletion/duplication, no mutations at c.2767 locus, and the inherited maternal X chromosome different from that of the patient. CONCLUSION: The pathogenic mutation in DMD gene, c.2767_2767delT [p.Ser923LeufsX26], identified in this family is a de novo mutation. On the basis of specific conditions, it is necessary to select suitable methods to make prenatal diagnosis more effective, accurate, and economic.


Assuntos
Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Diagnóstico Pré-Natal/métodos , Adolescente , Adulto , Pré-Escolar , China , Éxons , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Linhagem , Gravidez , Deleção de Sequência
4.
J Biomed Sci ; 23(1): 48, 2016 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-27266699

RESUMO

BACKGROUNDS: Down syndrome (DS), caused by triplication of human chromosome 21, is the most common aneuploidies. The main characteristic of DS patients is intellectual disability. MicroRNAs (miRNAs) play important regulatory roles in various biological processes, such as embryonic development, cell differentiation, proliferation and apoptosis. Several miRNAs have shown association with DS. However, the role of miRNAs in DS patients has not been well elaborated. METHODS: In this research, total RNA extracted from fetal hippocampal tissues was used to analyze miRNA and mRNA expression via Affymetrix miRNA 4.0 and PrimeView Human Gene Expression Array, respectively. Then miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their predicted target mRNAs. Microarray data were further validated by real-time PCR. Regulation of zeste homolog 2 (EZH2) expression by hsa-miR-138 was determined by luciferase reporter assay. RESULTS: We analyzed microRNA expression profile in hippocampal tissues from DS fetal using miRNA microarray. Further with the use of mRNA microarray data, we integrate miRNA expression and mRNA expression in hippocampus of trisomy 21 fetus to elucidate the mechanism that underlying DS abnormalities. We characterized the repertoire of specific miRNAs involved in hippocampus in trisomy 21 patients, highlighting hsa-miR-138 and hsa-miR-409, in particular the importance of hsa-miR-138, especially the -5p strand. Furthermore, the expression level of predicted target genes of hsa-miR-138-5p in trisomy 21 fetus, including zeste homolog 2 (EZH2) were further confirmed. In addition, luciferase assay indicated that EZH2 was a direct target of hsa-miR-138 in HEK293T cells. CONCLUSION: The function of hsa-miR-138-5p and its target EZH2 was involved in hippocampus in DS patients. Our findings provide a clue to study the underlying molecular mechanisms in DS patients, and its potential contribution in improving understanding of intellectual disability development in DS patients.


Assuntos
Síndrome de Down/embriologia , Proteína Potenciadora do Homólogo 2 de Zeste/biossíntese , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/embriologia , MicroRNAs/biossíntese , RNA Mensageiro/biossíntese , Síndrome de Down/genética , Síndrome de Down/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Feminino , Feto/patologia , Perfilação da Expressão Gênica , Hipocampo/patologia , Humanos , Masculino , MicroRNAs/genética , RNA Mensageiro/genética
5.
Taiwan J Obstet Gynecol ; 55(6): 867-870, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28040136

RESUMO

OBJECTIVE: The aim of this report is to describe the phenotype-genotype correlation of chromosome 9p deletion syndrome cases, particularly the prenatal cases. MATERIALS AND METHODS: A 30-year-old woman was referred to a hospital at 19+1 weeks of gestation because of omphalocele detected in the fetus. The conventional karyotyping analysis and array comparative genomic hybridization (aCGH) were utilized for the prenatal diagnosis and genetic counseling in the fetus. The prenatal abnormality and cytogenetic findings in the fetus were compared with other patients with 9p deletion. RESULTS: Karyotype analysis of the fetus cell showed a karyotype of 46,XX,del(9)(p22). aCGH analysis detected a deletion as arr[hg19] 9p24.2p22.2(226,7812-1,7466,907)×1. Individuals with 9p deletions tend to have features with widely variable expressivity. The common clinical manifestations of the 9p deletion include development delay, learning difficulties, hypotonia and trigonocephaly. CONCLUSION: Phenotypes of 9p deletion cases are broadly in line. The prenatal diagnosis of the omphalocele provides evidence for a correlation with distal 9q deletion.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 9 , Doenças Fetais/diagnóstico , Doenças Fetais/genética , Anormalidades Múltiplas/genética , Adulto , Criança , Pré-Escolar , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Feminino , Idade Gestacional , Hérnia Umbilical/diagnóstico por imagem , Hérnia Umbilical/genética , Humanos , Deficiência Intelectual/genética , Cariotipagem , Masculino , Gravidez , Síndrome , Ultrassonografia Pré-Natal
6.
J Matern Fetal Neonatal Med ; 29(3): 493-503, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-25731651

RESUMO

OBJECTIVE: To provide a basis for evaluating the prognosis of small left heart system development in fetuses, we analyzed its related factors. METHODS: The fetal echocardiogram was performed in 3859 pregnant women, and then small left heart system development was identified in 69 fetuses. The data of prenatal and postnatal echocardiograms, postnatal cardiac surgical treatment, chromosome and autopsy after induced labor were analyzed in the 69 fetuses. RESULTS: Except 1320 cases losing follow-up, 2539 cases had complete data. Among the 2539 cases, small left heart system development was identified in 69 fetuses. Of the 69 fetuses, 12 had hypoplastic left heart syndrome, 20 premature closure of foramen ovale, 13 total anomalous pulmonary venous drainage, 2 common pulmonary vein lumen atresia, 21 aortic coarctation or interruption and 1 right pulmonary hypoplasia. Among the 69 fetuses, chromosome abnormality was found in 7. CONCLUSION: There are many etiological factors causing small left heart system development. The prognosis is poor in the fetuses with hypoplastic left heart syndrome, common pulmonary vein lumen atresia, pulmonary hypoplasia, other malformations or/and chromosome abnormality. Fetal echocardiography combined with chromosome examination can provide important bases for making diagnosis and evaluating the prognosis regarding small left heart system development.


Assuntos
Doenças Fetais/etiologia , Cardiopatias Congênitas/etiologia , Adolescente , Adulto , Ecocardiografia , Feminino , Doenças Fetais/diagnóstico por imagem , Cardiopatias Congênitas/diagnóstico por imagem , Humanos , Cariótipo , Pessoa de Meia-Idade , Gravidez , Prognóstico , Estudos Prospectivos , Reprodutibilidade dos Testes , Ultrassonografia Pré-Natal , Adulto Jovem
7.
BMC Med Genomics ; 7: 18, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24731722

RESUMO

BACKGROUND: DNA methylation is a crucial epigenetic modification of the genome which is involved in embryonic development, transcription, chromatin structure, X chromosome inactivation, genomic imprinting and chromosome stability. Consistent with these important roles, DNA methylation has been demonstrated to be required for vertebrate early embryogenesis and essential for regulating temporal and spatial expression of genes controlling cell fate and differentiation. Further studies have shown that abnormal DNA methylation is associated with human diseases including the embryonic development diseases. We attempt to study the DNA methylation status of CpG islands in fetus related to fetus growth and development. METHODS: GeneChip® Human Tiling 2.0R Array set is used for analysis of methylated DNA in a whole-genome wide in 8 pairs amniotic fluid and maternal blood DNA samples. RESULTS: We found 1 fetus hypermethylation DNA markers and 4 fetus hypomethylation DNA markers though a Genome-wide analysis. These DNA markers all found to be associated with the critical genes for fetus growth and development (SH2D3C gene, EML3 gene, TRIM71 gene, HOXA3 gene and HOXA5 gene). CONCLUSIONS: These genes can be used as a biomarker for association studying of embryonic development, pathological pregnancy and so on. The present study has provided new and fundamental insights into the roles that DNA methylation has in embryonic development and in the pathological pregnancy.


Assuntos
Metilação de DNA/genética , Feto/metabolismo , Genoma Humano/genética , Adulto , Cromossomos Humanos Par 21/genética , Feminino , Marcadores Genéticos , Humanos , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Software
8.
Arch Virol ; 159(6): 1393-401, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24385156

RESUMO

The etiological role of human papillomavirus (HPV) in cervical cancer has been well established. However, it is inconclusive whether HPV plays the same role in esophageal carcinogenesis. In this study, we detected HPV infection in 145 frozen esophageal tissues, including 30 normal epithelium (ENOR), 37 dysplasia (DYS) and 78 invasive squamous cell carcinoma (ESCC), and in 143 frozen cervical tissues composed of 30 normal epithelium (CNOR), 38 intraepithelial neoplasia (CIN) and 75 invasive squamous cell carcinoma (CSCC). The patients and symptom-free subjects enrolled in this study were from a high-incidence area for both ESCC and CSCC, Linzhou City, Northern China, from 2007 to 2009. The HPV infection analysis was conducted by using an HPV GenoArray Test Kit. We found that the high-risk HPV types accounted for more than 90 % of the HPV-positive lesions of esophagus and cervix tissues. The prevalence of high-risk HPV types increased significantly during the progression of both esophageal and cervical carcinogenesis (positive rate in esophageal tissues: 33 % ENOR, 70 % in DYS and 69 % in ESCC; positive rate in cervical tissues: 27 % in CNOR, 82 % in CIN and 88 % in CSCC; P < 0.001, respectively). Infection with the high-risk HPV types increased the risk for both DYS and ESCC by 4-fold (DYS vs. ENOR: OR = 4.73, 95 %CI = 1.68-13.32; ESCC vs. ENOR: OR = 4.50, 95 %CI = 1.83-11.05) and increased the risk for both CIN and CSCC by 12-fold and 20-fold (CIN vs. CNOR: OR = 12.18, 95 %CI = 3.85-38.55; CSCC vs. CNOR: OR = 20.17, 95 %CI = 6.93-58.65), respectively. The prevalence of high-risk types in ESCC patients was lower than that in CSCC patients (P = 0.005) and was significantly associated with the degree of ESCC tumor infiltration (P = 0.001). HPV 16 was the most prevalent subtype in both esophageal and cervical tissues. Single HPV infection increased significantly along with the progression of ESCC and maintained a high level in cervical tissues, regardless of whether they were CNOR or CSCC tissues. Our results showed that infection with HPV, especially the high-risk types, was positively associated with both esophageal and cervical cancers, suggesting that HPV also plays a role in the etiology of ESCC in the high-incidence area.


Assuntos
Neoplasias Esofágicas/epidemiologia , Neoplasias Esofágicas/virologia , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/virologia , China/epidemiologia , Neoplasias Esofágicas/etiologia , Feminino , Humanos , Papillomaviridae/classificação , Papillomaviridae/genética , Infecções por Papillomavirus/complicações , Prevalência , Medição de Risco , Neoplasias do Colo do Útero/etiologia
9.
Mol Biol Rep ; 41(4): 2371-5, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24469714

RESUMO

We obtained the allelic frequencies and forensic efficiency data for eight mini short tandem repeat loci including Penta E, D12S391, D6S1043, D2S1338, D19S433, CSF1PO, Penta D and D19S253 loci from a sample of 128 unrelated Uyghur individuals from China. The amplification products of the eight STR loci are <240 bp in size. A total of 94 alleles were observed and the corresponding allelic frequencies ranged from 0.0039 to 0.3438 in the present study. Observed genotype distributions for each locus do not show deviations from Hardy-Weinberg equilibrium expectations. The combined power of discrimination, combined power of exclusion and combined matching probability of the eight STR loci equaled to 0.999999999963373, 0.9997770 and 3.6627 × 10(-11), respectively. Because of the small fragment length of PCR products and the high degree of polymorphisms, the eight STR loci are highly beneficial for the forensic analysis of degraded DNA samples which are commonly observed in forensic cases. The STR data of the Uyghur group were compared with the previously published population STR data of other groups from different ethnic or areas, and significant differences were observed among these groups at some loci.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Grupos Étnicos/genética , Genética Forense , Repetições de Microssatélites , Polimorfismo Genético , Alelos , China , Frequência do Gene , Humanos , Desequilíbrio de Ligação
10.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 30(4): 439-42, 2013 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-23926012

RESUMO

OBJECTIVE: To analyze CYP17A1 gene mutations in a child patient with 17 alpha-hydroxylase/17, 20-lyase deficiency (17OHD), and to review characteristics of CYP17A1 gene mutations in Chinese patients with 17OHD. METHODS: Clinical data were collected. PCR and DNA sequencing were performed to detect mutations in the patient. RESULTS: The patient has presented classical features of 17OHD including hypertension, hypokalemia, decreased sex hormones and plasma cortisol, and elevated blood adrenocorticotrophic hormone. A compound heterozygous mutation c.987C>A and c.985del was detected in the CYP17A1 gene, which resulted in two premature stop codons at positions 328 and 417. CONCLUSION: A compound mutation, c.987C>A and c.985del, has been identified in a patient with 17OHD. Among CYP17A1 gene mutations identified in Chinese patients, missence mutations have been most common, and exons 5 and 8 have been the mutation hotspots.


Assuntos
Hiperplasia Suprarrenal Congênita/enzimologia , Liases/genética , Mutação , Esteroide 17-alfa-Hidroxilase/genética , Adolescente , Hiperplasia Suprarrenal Congênita/genética , Sequência de Bases , Feminino , Humanos , Liases/deficiência , Dados de Sequência Molecular
11.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 30(2): 199-202, 2013 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-23568735

RESUMO

OBJECTIVE: To identify potential mutations of retinoschisis 1 (RS1) gene responsible for X-linked retinoschisis (XLRS) in two Chinese families. METHODS: The 6 exons and flanking intronic regions were analyzed with PCR and direct sequencing. RESULTS: Two RS1 mutations were identified in the two families, which included 1 frameshift mutation (c.573delG, p.Pro192fs) and 1 missense mutation (c.626G>A, p.Arg209His). CONCLUSION: Two RS1 mutations have been identified, among which Pro192fs mutation is discovered for the first time in Chinese population. Above results may enrich our understanding of the clinical manifestations of XLRS and facilitated early diagnosis and genetic counseling for the disease.


Assuntos
Proteínas do Olho/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação , Diagnóstico Pré-Natal , Retinosquise/genética , Adolescente , Adulto , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Retinosquise/diagnóstico
12.
Zhonghua Fu Chan Ke Za Zhi ; 48(2): 86-91, 2013 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-23544487

RESUMO

OBJECTIVE: To assess the frequency and significance of maternal cell contamination (MCC) in the invasive prenatal diagnosis, and to analysis the MCC effect on prenatal diagnosis results. METHODS: Totally 519 amniotic fluid samples from second trimester pregnancy, 57 chorionic villus samples from first trimester pregnancy, and 576 blood samples from corresponded pregnant women were collected and genotyped by Promega PowerPlex 16 system. MCC was determined according to the genotyping results. Karyotypic and molecular diagnosis results were contrasted between MCC and non-MCC specimen of the same fetal. RESULTS: MCC presented in 3.1% (16/519) uncultured amniotic fluid, 1.3% (7/519) cultured amniotic fluid and 5% (3/57) villi specimens. In the study of fetal karyotype, MCC had no significant effect on normal female fetus; but for male fetus and abnormal female fetus, there were risk of erroneous results of mosaics. As to molecular diagnosis, MCC resulted in more complex effects for the different diagnostic methods. And 10%MCC had led to misdiagnosis. CONCLUSIONS: For the prenatal cytogenetic tests, MCC should be excluded when there were mosaicism karyotype results or suspicious MCC of chorionic villi samples. The effects of MCC had more seriously impact on prenatal molecular testing, which suggesting the recommend regular identity test for MCC should be carried out.


Assuntos
Líquido Amniótico/citologia , Artefatos , Erros de Diagnóstico/prevenção & controle , Diagnóstico Pré-Natal/métodos , Manejo de Espécimes/métodos , Adulto , Amniocentese/métodos , Amniocentese/normas , Líquido Amniótico/química , Células Cultivadas , Amostra da Vilosidade Coriônica/métodos , Amostra da Vilosidade Coriônica/normas , DNA/análise , Contaminação por DNA , Feminino , Humanos , Cariotipagem , Masculino , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase/métodos , Gravidez , Diagnóstico Pré-Natal/normas , Manejo de Espécimes/normas , Sequências de Repetição em Tandem
13.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 30(1): 40-4, 2013 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-23450477

RESUMO

OBJECTIVE: To investigate the efficiency of multiplex ligation-dependent probe amplification (MLPA) combined with short tandem repeat (STR) linkage analysis for the prenatal diagnosis for Duchenne muscular dystrophy (DMD). METHODS: Gender of the fetus was first determined by the presence of Y chromosome sex-determining gene (SRY). Subsequently, combined MLPA and STR linkage analysis were applied for the probands, pregnant women and fetuses in 45 affected families. RESULTS: Among the 45 families, 31 SRY-positive fetuses were identified, among whom six were diagnosed with DMD. For 14 SRY-negative fetuses, four were diagnosed as carriers. The remainders were normal. CONCLUSION: MLPA can detect mutations in the exons of dystrophin gene, whilst STR linkage analysis can determine whether the fetus has inherited the maternal X chromosome bearing the mutant gene. As the result, the method can detect affected fetuses in which no exonic mutations are detected with MLPA. By combining the two methods, the diagnostic rate for DMD can be greatly improved.


Assuntos
Repetições de Microssatélites , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofina/genética , Éxons , Feminino , Ligação Genética , Heterozigoto , Humanos , Masculino , Reação em Cadeia da Polimerase Multiplex , Mutação , Gravidez , Diagnóstico Pré-Natal
15.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 29(4): 435-8, 2012 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-22875501

RESUMO

OBJECTIVE: To determine the feasibility and accuracy of detecting numerical chromosomal abnormalities by high-flux sequencing analysis of free fetal DNA from maternal plasma. METHODS: High-flux sequencing was applied to analyze fetal chromosome sequence copy numbers in 153 pregnant women. Fetal karyotyping was also carried out on amniocentesis samples. RESULTS: Six cases were detected with fetal chromosomal abnormalities by high-flux sequencing analysis, among which five were confirmed by karyotyping to be chromosomal aneuploidies (47,XYY; 45,X; 47,XY,+18; 47,XY,+21 and 47,XY,+13), 1 case was confirmed to be structural rearrangement, i.e., 46,XY,der(13;21)(q10;q10),+21. Furthermore, 3 chromosomal polymorphisms (one 46,XY,21p+ and two 46,XY,Yqh-) were identified. The two methods yielded similar results on fetal chromosome copy number detection. CONCLUSION: High-flux sequencing analysis of free DNA derived from maternal plasma is efficient for detecting fetal chromosomal aneuploidies, and is non-invasive, highly sensitive and specific. It therefore has a broad application in antenatal diagnosis.


Assuntos
Aneuploidia , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , DNA/química , DNA/genética , Adulto , Amniocentese/métodos , Feminino , Feto , Humanos , Gravidez , Diagnóstico Pré-Natal/métodos , Adulto Jovem
16.
Zhonghua Fu Chan Ke Za Zhi ; 47(11): 813-7, 2012 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-23302120

RESUMO

OBJECTIVE: To investigate the clinical value of non-invasive prenatal diagnosis using cell free fetal DNA (cff-DNA) in maternal blood. METHODS: From Sep. 2010 to Mar. 2012, 103 pregnant women who came to Henan Province People's Hospital in the first trimester for prenatal diagnosis of sex-linked inherited diseases were included in the first trimester group. From Oct. 2010 to Jan. 2012, 205 pregnant women undergoing amniotic fluid sampling for fetal karyotype analysis in the same hospital were included in the second trimester group. Real time quantitative PCR and fluorescent PCR were used to detect sex determining region of Y chromosome gene (SRY) and amelogenin gene (AML) on cff-DNA of the first trimester group. Moreover, 12 Y chromosome STR loci analysis were performed for 33 male fetuses and their fathers. Massively Parallel Signature Sequencing (MPSS) was used for aneuploidy analysis in cff-DNA of the second trimester group. RESULTS: (1) In the first trimester group, there were 53 SRY positive and 50 SRY negative. Compared with the results of cff-DNA of chorionic villus samples, there was one SRY false positive and one false negative results, with a sensitivity of 98% and specificity of 98%. For the AML gene test, there were two PCR products of male fetuses:102 bp fragment originating from X chromosome (AML X) and 108 bp fragment from Y chromosome (AML Y); but only AML X was found in products from female fetuses. In the first trimester group, 102 bp and 108 bp fragments were detected in 52 cases, and only 102 bp fragment was found in the other cases. Compared to AML results from chorionic villus samples, there were 2 false negative results, with a sensitivity of 96% and specificity of 100%. (2) For cff-DNA with plasma SRY over 30 copy/ml, Y STR loci were analyzed on cff-DNA of 33 fetuses and their fathers. The Y STR loci less then 200 bp were successfully detected, while Y STR loci with PCR products between 200-300 bp showed low signal or could not be amplicated; and no PCR products more than 300 bp were detected from cff-DNA. Comparing the detected Y STR loci of cff-DNA to the fathers, 32 fetuses were concordant with their fathers'. Exogenous contamination was found in the rest one sample. (3) In the second trimester group, 6 fetuses with abnormal karyotype (two trisomy 21, three trisomy 18 and one 45, XO) were detected by cff-DNA and were proved by karyotype analysis. Moreover, the MPSS results of cff-DNA revealed one 45, Y and one trisomy 16 whose karyotype analysis showed normal results. And in one case, MPSS suggested less chrX or chrY, that was proved to be 47, XYY by karyotype analysis. CONCLUSIONS: (1) Cff-DNA in maternal blood can be used to determine fetal gender in early prenancy with considerable sensitivity and specificity. But the trace cff-DNA and the high maternal DNA background might have impact on the result. (2) Analysis of cff-DNA in maternal blood of the second trimester women showed that MPSS could be used for prenatal screening of trisomy 21 and trisomy 18. However, further research should be done for other chromosomes aneuploidy detection.


Assuntos
Aneuploidia , Transtornos Cromossômicos/diagnóstico , DNA/sangue , Cariotipagem , Diagnóstico Pré-Natal/métodos , Adulto , Transtornos Cromossômicos/sangue , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Y/genética , Feminino , Humanos , Masculino , Testes para Triagem do Soro Materno , Gravidez , Primeiro Trimestre da Gravidez , Segundo Trimestre da Gravidez , Sensibilidade e Especificidade , Trissomia/diagnóstico
17.
Zhonghua Fu Chan Ke Za Zhi ; 47(10): 742-6, 2012 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-23302731

RESUMO

OBJECTIVE: To explore the relationship between the polymorphism of methionine synthase reductase (MTRR) A66G and the susceptibility to unexplained repeated spontaneous abortion (URSA). METHODS: Total of 200 Henan Han couples with URSA (URSA group) and 76 Henan Han healthy couples without URSA (control group) were enrolled in this study. Their MTRR A66G genotypes were determined by PCR restriction fragment length polymorphism (PCR-RFLP). RESULTS: (1) The allele frequencies of MTRR A66G: the frequencies of allele A and allele G in URSA group were 76.5% (153/200) in husband and 72.8% (146/200) in wife, 23.5% (47/200) in husband and 27.2% (54/200) in wife, respectively. The frequencies of allele A and allele G in control group were 78.9% (60/76) in husband and 78.3% (59/76) in wife, 21.1% (16/76) in husband and 21.7% (16/76) in wife, respectively. The frequencies of allele A and allele G were not significantly different between female and male subjects within the same experimental group (P > 0.05), and also there were not significantly different between the same gender subjects at URAS and control groups (P > 0.05). (2) The genotype frequencies of MTRR A66G: the frequencies of genotype AA, AG and GG in URSA group were 57.0% (114/200) in husband and 52.0% (104/200) in wife, 39.0% (78/200) in husband and 41.5% (83/200) in wife, 4.0% (8/200) in husband and 6.5% (13/200) in wife, prepectively. The frequencies of genotype AA, AG and GG in control group were 59.2% (45/76) in husband and 59.2% (50/76) in wife, 39.5% (30/76) in husband and 38.2% (29/76) in wife; 1.3% (1/76) in husband and 2.6% (2/76) in wife, prepectively. The frequencies of genotype AA, AG and GG were not significantly different between female and male subjects within the same group (P > 0.05), and also there were not significantly different between the same gender subjects at URSA and control groups (P > 0.05).(3)Combined genotype of couples: the combined genotype frequencies of GG + GG, GG + AG, GG + AA, AG + AG, AG + AA and AA + AA in URSA group were 1.0% (2/200), 2.5% (5/200), 6.0% (12/200), 20.0% (40/200), 38.0% (76/200), and 32.5% (65/200), prepectively; the combined genotype frequencies in control group were 0, 1.3% (1/76), 2.6% (2/76), 17.1% (13/76), 42.1% (32/76), 36.8% (28/76), prepectively. The combined genotype analysis between the two groups were also not significantly different (P > 0.05). CONCLUSION: The polymorphism of MTRR A66G gene was not associated with the susceptibility to URSA (P > 0.05), and so it was not the inherited genetic risk factor of URSA.


Assuntos
Aborto Habitual/genética , Ferredoxina-NADP Redutase/genética , Polimorfismo Genético , Aborto Habitual/patologia , Adulto , Grupo com Ancestrais do Continente Asiático , Estudos de Casos e Controles , China , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Reação em Cadeia da Polimerase , Gravidez , Fatores de Risco , Adulto Jovem
18.
BMC Med Genet ; 12: 68, 2011 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-21575262

RESUMO

BACKGROUND: Chromosome abnormalities, especially trisomy of chromosome 21, 13, or 18 as well as sex chromosome aneuploidy, are a well-established cause of pregnancy loss. Cultured cell karyotype analysis and FISH have been considered reliable detectors of fetal abnormality. However, results are usually not available for 3-4 days or more. Multiplex ligation-dependent probe amplification (MLPA) has emerged as an alternative rapid technique for detection of chromosome aneuploidies. However, conventional MLPA does not allow for relative quantification of more than 50 different target sequences in one reaction and does not detect mosaic trisomy. A multiplexed MLPA with more sensitive detection would be useful for fetal genetic screening. METHODS: We developed a method of array-based MLPA to rapidly screen for common aneuploidies. We designed 116 universal tag-probes covering chromosomes 13, 18, 21, X, and Y, and 8 control autosomal genes. We performed MLPA and hybridized the products on a 4-well flow-through microarray system. We determined chromosome copy numbers by analyzing the relative signals of the chromosome-specific probes. RESULTS: In a blind study of 161 peripheral blood and 12 amniotic fluid samples previously karyotyped, 169 of 173 (97.7%) including all the amniotic fluid samples were correctly identified by array-MLPA. Furthermore, we detected two chromosome X monosomy mosaic cases in which the mosaism rates estimated by array-MLPA were basically consistent with the results from karyotyping. Additionally, we identified five Y chromosome abnormalities in which G-banding could not distinguish their origins for four of the five cases. CONCLUSIONS: Our study demonstrates the successful application and strong potential of array-MLPA in clinical diagnosis and prenatal testing for rapid and sensitive chromosomal aneuploidy screening. Furthermore, we have developed a simple and rapid procedure for screening copy numbers on chromosomes 13, 18, 21, X, and Y using array-MLPA.


Assuntos
Aneuploidia , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 21 , Cromossomos Humanos X , Cromossomos Humanos Y , Sondas de DNA , Feminino , Humanos , Cariotipagem , Masculino , Mosaicismo , Gravidez
19.
Beijing Da Xue Xue Bao Yi Xue Ban ; 37(1): 39-41, 2005 Feb 18.
Artigo em Chinês | MEDLINE | ID: mdl-15719038

RESUMO

OBJECTIVE: To Investigate the performance of prenatal screening for chromosomal abnormalities in first trimester. METHODS: Maternal serum were collected from 2 739 pregnant women between 11 and 14 weeks gestation. Free beta human chorionic gonadotrophin(beta-hCG), pregnancy-associated plasma protein(PAPP-A) from materal serum were measured using time resolved fluorescence immunoassay(TRFIA) and fetal nuchal translucency(NT) thickness were measured using transabdominal or transvaginal ultrasound. 22 chromosomal defects were diagnosed in 22 cases using karyotyping. The levels of three markers were analyzed among 22 cases and 870 controls. RESULTS: The level of three markers were significant difference between affected and unaffected pregnancies. In affected cases, the value or level of NT and free beta-hCG were higher, while the level of PAPP-A was lower. We found that screening for chromosomal defects using a combination of NT and serum biochemistry was associated with a detection rate of 91.67% for all types of chromosomal defects, with a false-positive rate of 11.16%. CONCLUSION: A combination of nuchal translucency measurement with materal serum biochemistry markers provides an effective method of screening for chromosomal defects.


Assuntos
Gonadotropina Coriônica Humana Subunidade beta/sangue , Aberrações Cromossômicas , Medição da Translucência Nucal , Proteína Plasmática A Associada à Gravidez/metabolismo , Diagnóstico Pré-Natal/métodos , Adulto , Biomarcadores/sangue , Cromossomos Humanos Par 22/genética , Feminino , Testes Genéticos , Humanos , Gravidez , Primeiro Trimestre da Gravidez/sangue
20.
Yi Chuan Xue Bao ; 29(4): 303-6, 2002 Apr.
Artigo em Japonês | MEDLINE | ID: mdl-11985262

RESUMO

To increase the efficiency of in vitro transformation of human lymphocytes by Epstein-Barr virus (EBV) and establish permanent lymphoblastoid cell lines from patients with abnormal chromosome karyotype, B lymphoid cells were prepared from cryopreserved heparinized blood samples. The lymphoid cell pellet was resuspended with 0.5 ml medium of RPMI with 20% fetal calf serum(FCS), and added 2 ml virus-containing superatant of the EB virus-producing cell lines by filtrated, and mixed. Four 25 cm2 cell culture bottles were put upright. A total of 2.5 ml of RPMI with 20% FCS was put in each of them. The blood-virus mixture was distributed among the four cell culture bottles as follows: Bottle I, Bottle II, Bottle III and Bottle IV were added with 0.3 ml, 0.6 ml, 1.2 ml and the rest respectively. The cells culture bottles were put into the cell culture incubator in an upright position. After 3 days the cells were puting new medium with 20% FCS as follows: Bottle I 3 ml, Bottle II 4 ml, Bottle III 5 ml and Bottle IV 6 ml. After one week, the medium was changed again as described above. The medium change was conducted until the cells grew very fast. The right ratio between blood cells and virus titer can not be exactly determined for every blood sample, and therefore a dilution series with four different blood/virus ratios was set up. Due to the dilution series, addition of immune inhibitors like cyclosporine, was not necessary. Forty-seven permanent lymphoblastoid cell lines of patients with abnormal chromosome karyotype. Transformed cells were found in only one or two of the four cell culture bottles. The total successive rate increased up to 97.87%. Of the four cell culture bottles, Bottle I, Bottle II, Bottle III and Bottle IV, the successive rates were 6.39%, 61.70%, 31.91% and 8.51% respectively. This method can be used for preserving large number of lymphoblastoid cell lines, and also provide enough research materials for further studies.


Assuntos
Linhagem Celular Transformada , Transformação Celular Viral , Aberrações Cromossômicas , Herpesvirus Humano 4/fisiologia , Linfócitos/citologia , Humanos , Cariotipagem
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