OBJECTIVE: To investigate the role of miRNAs in amniotic fluid exosomes in growth and development of fetuses with Down syndrome (DS). METHODS: Amniotic fluid were collected from 20 fetuses with DS and 20 normal fetuses (control) to extract amniotic exosome miRNA. MicroRNA sequencing technique was used to identify the differentially expressed miRNAs between the two groups, for which gene ontology (GO) and pathway analysis was performed. Three differentially expressed miRNAs with the strongest correlation with DS phenotype were selected for qPCR verification. Dual luciferase reporter assay was used to verify the activity of let-7d-5p for targeted regulation of BACH1. RESULTS: We identified 15 differentially expressed miRNAs in DS as compared with the control group, among which 7 miRNAs were up-regulated and 8 were down-regulated. Target gene prediction results showed that the differentially expressed miRNAs targeted 17 DS-related genes. GO analysis revealed that the main functions of the target genes involved protein binding, protein transport, ATP binding, transferase activity and synapses. Pathway analysis revealed that the functional pathways were closely related with the development of the nervous system. qPCR results showed that the expression levels of miR-140-3p and let-7d-5p were significantly lower in DS group than in the control group (P < 0.05), as was consistent with miRNA sequencing results; the expression level of miR-4512 was significantly higher in DS group than in control group (P < 0.05), which was contrary to miRNA sequencing results. The results of double luciferase reporter gene assay confirmed that let-7d-5p was capable of targeted regulation of BACH1 expression. CONCLUSION Let-7d-5p in amniotic fluid exosomes may promote oxidative stress events in the brain of fetuses with DS by regulating BACH1 expression.
Amniotic Fluid/metabolism* ; Down Syndrome/genetics* ; Exosomes ; Female ; Humans ; MicroRNAs/metabolism* ; Pregnancy

Down syndrome is caused by partial or complete triplication of genes located on chromosome 21. Its incidence increases dramatically with the age of women. Hypotheses proposed for this have included abnormal homologous recombination, defective spindle assembly, biological aging, reduction of cohesion complexes, endocrine disorders, oocyte selection model, and single nucleotide polymorphisms of genes that maintain chromosome stability, etc. A literature review is provided here.
Aging ; genetics ; Chromosomes, Human, Pair 21 ; genetics ; Down Syndrome ; genetics ; Female ; Humans ; Maternal Age ; Oocytes ; metabolism ; Polymorphism, Single Nucleotide ; genetics

BACKGROUNDBrain hypoplasia and mental retardation in Down syndrome (DS) can be attributed to a severe and selective disruption of neurogenesis. Secondary disruption of the transcriptome, as well as primary gene dosage imbalance, is responsible for the phenotype. MicroRNA (miRNA) expression is relatively abundant in brain tissue. Perturbed miRNA expression might contribute to the cellular events underlying the pathology in DS.

METHODSMiRNA expression profiles in the cerebrum of Ts65Dn mice, a DS model, were examined with a real-time RT-PCR array. MiRNA target gene expression was detected by real-time quantitative PCR and Western blotting. Based on the prediction of their cerebrum-specific targets, the functions of the misregulated miRNAs were annotated by Gene Ontology (GO) enrichment analysis.

RESULTSA total of 342 miRNAs were examined. Among them, 20 miRNAs showed decreased expression in the brains of Ts65Dn mice, and some of these belonged to the same family. Two known targets of the miR-200 family, Lfng and Zeb2, were specifically selected to compare their expression in the cerebrum of Ts65Dn mice with those of euploids. However, no significant difference was found in terms of mRNA and protein expression levels of these genes. By enrichment analysis of the cerebrum-specific targets of each miRNA, we found that 15 of the differential miRNAs could significantly affect target genes that were enriched in the GO biological processes related to nervous system development.

CONCLUSIONPerturbed expression of multiple functionally cooperative miRNAs contributes to the cellular events underlying the pathogenesis of DS.

Animals ; Brain ; metabolism ; Disease Models, Animal ; Down Syndrome ; etiology ; genetics ; Gene Expression Regulation ; Mice ; MicroRNAs ; physiology

BACKGROUNDAmniotic fluid (AF) supernatant contains cell-free fetal DNA (cffDNA) fragments. This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis, which could be combined with simple quantitative fluorescent polymerase chain reaction (QF-PCR) to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome.

METHODSAF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes. Peripheral blood samples of the parents were collected at the same time. Short tandem repeat (STR) fragments on chromosome 21 were amplified by QF-PCR. Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks.

RESULTSThe sensitivity of the assay for the aneuploid was 93% (26/28; confidence interval, CI: 77%-98%) and the specificity was 100% (26/26; CI: 88%-100%). The determination rate of the origin of the extra chromosome was 69%. The sensitivity and the specificity of the assay in the euploid were 100% (27/27).

CONCLUSIONSTrisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant. This karyotype analysis method greatly reduces the requirement for the specimen size. It will be a benefit for early amniocentesis and could avoid pregnancy complications. The method may become an ancillary method for prenatal diagnosis of trisomy 21.

Amniotic Fluid ; metabolism ; Chromosomes, Human, Pair 21 ; genetics ; Down Syndrome ; diagnosis ; genetics ; Female ; Humans ; Microsatellite Repeats ; genetics ; Polymerase Chain Reaction ; methods ; Pregnancy ; Prenatal Diagnosis ; methods

OBJECTIVETo screen and diagnose Down's syndrome during mid-term pregnancy to reduce the number of babies with Down's syndrome.

METHODSWith the multi-level of stratified cluster sampling, twenty thousand and eight hundred and three women at 15-20 weeks gestation were screened by maternal serum AFP and beta-hCG using the time resolved fluoroimmunoassay (TRFIA). Then the screened high-risk women were diagnosed by amniocentesis, cell culture and chromosome analyses. The born children were diagnosed by follow-up and peripheral blood chromosome analyses.

RESULTSSix fetuses were diagnosed by serum screening and amniotic fluid chromosome analyses, and 3 born children were diagnosed by follow-up and peripheral blood chromosome analyses. Nine cases of Down's syndrome were detected in total, with the positive prenatal screen rate being 67% (6/9).

CONCLUSIONThe prenatal screening and diagnosis can reduce the birth of Down's syndrome patients and improve the population quality. However, the diagnosis accuracy still needs to be improved to further reduce the false negative rate and prevent misdiagnosis.

Adult ; Chorionic Gonadotropin, beta Subunit, Human ; blood ; Chromosome Aberrations ; Down Syndrome ; blood ; diagnosis ; genetics ; metabolism ; Female ; Fluoroimmunoassay ; Humans ; Pregnancy ; Prenatal Diagnosis ; methods ; Young Adult ; alpha-Fetoproteins ; metabolism

OBJECTIVETo explore the relationship between genetic polymorphisms in methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR), the central enzymes in folate metabolism that affects DNA methylation and synthesis, and the risk of Down syndrome in China.

METHODSGenomic DNA was isolated from the peripheral lymphocytes of 64 mothers of children with Down syndrome and 70 age matched control subjects. Polymerase chain reaction and restriction fragment length polymorphism were used to examine the polymorphisms of MTHFR 677C-->T, MTRR 66A-->G and the relationship between these genotypes and the risk of Down syndrome was analyzed.

RESULTSThe results show that the MTHFR 677C-->T polymorphism is more prevalent among mothers of children with Down syndrome than among control mothers, with an odds ratio of 3.78 (95% confidence interval (CI), 1.78 approximately 8.47). In addition, the homozygous MTRR 66A-->G polymorphism was independently associated with a 5.2-fold increase in estimated risk (95% CI, 1.90 approximately 14.22). The combined presence of both polymorphisms was associated with a greater risk of Down syndrome than the presence of either alone, with an odds ratio of 6.0 (95% CI, 2.058 approximately 17.496). The two polymorphisms appear to act without a multiplicative interaction.

CONCLUSIONMTHFR and MTRR gene mutation alleles are related to Down syndrome, and CT, TT and GG gene mutation types increase the risk of Down syndrome.

Alleles ; Case-Control Studies ; China ; Down Syndrome ; diagnosis ; ethnology ; genetics ; Female ; Ferredoxin-NADP Reductase ; genetics ; Folic Acid ; metabolism ; Genetic Predisposition to Disease ; Genotype ; Homozygote ; Humans ; Lymphocytes ; metabolism ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Polymorphism, Genetic ; Risk Factors

OBJECTIVETo compare the results of fluorescence in situ hybridization (FISH) assay and conventional karyotyping analysis for the detection of chromosomal aneuploidies.

METHODSIn total 2607 amniotic fluid samples were subjected to an improved FISH technique. Meanwhile, karyotype analysis was also ordered for each sample.

RESULTSOf the 2607 samples, 62 abnormalities were identified by FISH, which included 62 cases of trisomy 21, 5 cases of 45,X, 12 cases of trisomy 18, 3 cases of trisomy 13, and 1 case of 47, XYY. Conventional karyotyping analysis has identified 63 cases of trisomy 21, 5 cases of 45,X, 12 cases of trisomy 18, 3 cases of trisomy 13, 1 case of 47, XYY, and 57 cases of balanced translocations. The success rate of FISH detection was 98.4% for trisomy 21, and 100% for 45,X, trisomy 18 and trisomy 13.

CONCLUSIONFor the detection of chromosomal aneuploidies, FISH assay is quick, simple, accurate and can reduce workload when aminocyte culture has failed. As an auxiliary method for amniocytic analysis, it can provide reference for the consultation of those with advanced age and high pregnancy risk.

Adult ; Amniocentesis ; methods ; Amniotic Fluid ; cytology ; metabolism ; Chromosomes, Human, Pair 18 ; genetics ; Chromosomes, Human, Pair 3 ; genetics ; Chromosomes, Human, Y ; genetics ; Down Syndrome ; genetics ; Female ; Fetal Diseases ; diagnosis ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Karyotype ; Karyotyping ; methods ; Middle Aged ; Pregnancy ; Reproducibility of Results ; Sensitivity and Specificity ; Sex Chromosome Aberrations ; Trisomy ; genetics ; Trisomy 18 Syndrome ; Turner Syndrome ; genetics ; Young Adult

OBJECTIVETo investigate whether the polymorphism in methylenetetrahydrofolate reductase (MTHFR) gene involved in folate metabolism is associated with Down syndrome (DS).

METHODSOne hundred Chinese mothers who gave birth to babies with DS and 100 control mothers were chosen. Genotype of MTHFR 677 was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and concentration of plasma homocysteine (HCY) was measured by chemiluminescence.

RESULTSThe MTHFR 677T allele frequency was significantly different among case mothers, compared with control mothers (P=0.002); the odds ratio for the heterozygous CT genotype was 2.12 (95%CI: 1.14-3.94), whereas for the homozygous TT genotype, the odds ratio was 3.43 (95%CI:1.41-8.36). The mean plasma HCY concentration [(9.04 +/- 3.85) mu mol/L] of cases was significantly different from that of controls [(6.53 +/- 2.06) mu mol/L](P <0.01). The presence of the 677C>T substitution in one or both alleles was associated with increased plasma HCY both in case mothers and control mothers (P < 0.01). Interestingly, although both being MTHFR 677CC, the plasma HCY concentrations were higher in case mothers than in control mothers, the increase was not dependent on MTHFR genotype (P < 0.01).

CONCLUSIONOur results provide evidences that plasma HCY and genetic polymorphism in gene of folate pathway are risk factors for mothers to have a DS child in China.

Adult ; Alleles ; Base Sequence ; Case-Control Studies ; Down Syndrome ; blood ; enzymology ; genetics ; metabolism ; Female ; Folic Acid ; metabolism ; Genetic Predisposition to Disease ; Genotype ; Homocysteine ; blood ; Humans ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Mutation ; Polymorphism, Genetic ; Pregnancy

OBJECTIVETo evaluate the use of real-time fluorescence quantitative PCR (FQ-PCR) accompanied with comparison of Delta CT as a method for diagnosis of Down's syndrome from a single cell.

METHODSSingle lymphocyte was isolated from the peripheral or umbilical cord blood samples of 22 clinically diagnosed Down's syndrome patients or fetus and 40 normal controls by micromanipulation techniques. Primer extension preamplification (PEP) and real-time FQ-PCR were used to amplify the S100B and DCSR1 located on chromosome 21, and GAPDH located on chromosome 12. Two pairs of DeltaCT values were compared between the two groups. The ratios of S100B/GAPDH and DSCR1/GAPDH products were calculated in trisomy 21 group.

RESULTSThe DeltaCT values of Down's syndrome patients were significantly lower than those of normal controls. The difference between the two groups was statistically significant (P<0.01). The ratios of S100B/GAPDH and DSCR1/GAPDH products for trisomy 21 were 1.891(1.563-2.287) and 1.840 (1.562-2.168), respectively.

CONCLUSIONReal-time FQ-PCR is a reliabe method that may provide a new way for non-invasive prenatal diagnosis and preimplantation genetic diagnosis for Down's syndrome.

Child, Preschool ; Down Syndrome ; blood ; diagnosis ; genetics ; Female ; Fetal Blood ; cytology ; metabolism ; Humans ; Infant ; Lymphocytes ; cytology ; metabolism ; Male ; Microsatellite Repeats ; genetics ; Polymerase Chain Reaction ; methods ; Pregnancy ; Prenatal Diagnosis ; methods ; Reproducibility of Results ; Sensitivity and Specificity

The microRNA-21 (miR-21) is known to play a major role in cancer progression; however, its function in the cardiovascular system appears to be even more complex and conflicting. To characterize miR-21 expression in the plasma of individuals with or without metabolic syndrome (MetS), 58 MetS cases and 96 non-MetS controls were investigated. Expression levels of miR-21 were significantly decreased in the circulation of MetS subjects (OR=0.52, 95% CI: 0.29-0.92) compared with that of non-MetS subjects. Body mass index (BMI) and the number of MetS components had a negative correlation with the level of miR-21, whereas age was inversely related to the level of miR-21. No significant difference was detected in miR-21 levels between the sexes (P=0.056). MiR-21 might be a negative regulating factor in MetS.
Adult ; Aged ; Down-Regulation ; Female ; Humans ; Male ; Metabolic Syndrome ; blood ; etiology ; genetics ; MicroRNAs ; blood ; genetics ; metabolism ; Middle Aged ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Risk Factors