ABSTRACT
We have prospectively examined the diagnostic accuracy of a conventional multiplex polymerase chain reaction (PCR) analysis of maternal plasma for fetal gender determination in daily practice. Plasma DNAs were obtained from 168 pregnant women between five and 32 weeks of gestation. The 198-bp-specific sequence on Y-chromosome and the 261 bp ATL1-gene-specific sequence on X-chromosome were coamplified in a multiplex nested PCR manner. The results of fetal sex prediction were compared with routine analysis of fetal tissues and birth outcomes. The ATL1-specific sequences were detected in all cases but Y-chromosome-specific signals were observed in 95 of 97 plasma samples of women carrying male fetuses, leading to the sensitivity of 97.9, 100 and 100% for the first, second and third trimesters, respectively, and the specificity of 100% at all gestational stages. Therefore, fetal gender detection in maternal plasma with a conventional multiplex PCR assay is highly accurate in daily practice which should prove useful for non-invasive prenatal screening of sex-linked genetic disorders.
Subject(s)
Chromosomes, Human, X/genetics , Chromosomes, Human, Y/genetics , DNA/genetics , Polymerase Chain Reaction/methods , Sex Determination Analysis/methods , Adult , Chromosomes, Human, X/chemistry , Chromosomes, Human, Y/chemistry , DNA/blood , Female , Gene Amplification , Gestational Age , Humans , Male , Predictive Value of Tests , Pregnancy/blood , Pregnancy Trimesters/blood , Prenatal Diagnosis , Prospective Studies , Sensitivity and SpecificityABSTRACT
To establish simple noninvasive prenatal diagnosis of common beta-thalassemia in Southeast Asia, we have evaluated the possibility of identifying the 3 most common beta-thalassemia genes [beta(E), beta(17A-T), and beta(41/42(-CTCC))] by analysis of fetal DNA in maternal plasma using combined conventional polymerase chain reaction (PCR) and real-time quantitative PCR. Maternal plasma was obtained from peripheral blood of Thai pregnant women collected during the first and second trimesters of gestation. DNA was prepared from 200 microL plasma using a QIAmp Blood Mini Kit. Identifications of the beta(E), beta(41/42(-CTTT)), and beta(17A-T) in plasma DNA were carried out using semi-nested (for beta(E)) and nested (for beta(41/42) and beta(17)) real-time allele-specific PCR methodologies, and the results were compared with those obtained on fetal tissue analysis with routine invasive procedure. Twenty-six fetal beta(E) genes were correctly identified by maternal plasma DNA analysis of 39 pregnant women investigated. The fetal beta(41/42) and beta(17) mutations were detectable in 6 of 12 and 4 of 9 maternal plasma specimens, respectively, which were in concordance with the results obtained by routine invasive procedure. The noninvasive prenatal diagnostic methods developed should potentially prove useful for detection of paternally inherited mutation and for providing the exclusion of pregnancies at risk for this common genetic disorder in the region.
Subject(s)
DNA/blood , Hemoglobin E/genetics , Prenatal Diagnosis , beta-Thalassemia/diagnosis , Adult , Chromosome Mapping , DNA Mutational Analysis , Female , Hemoglobin E/metabolism , Humans , Maternal-Fetal Exchange , Pregnancy , Pregnancy Trimester, First , Pregnancy Trimester, Second , Reverse Transcriptase Polymerase Chain Reaction , Thailand , beta-Thalassemia/blood , beta-Thalassemia/geneticsABSTRACT
In order to provide a noninvasive prenatal diagnosis of alpha(0)-Thalassemia (Southeast Asian [SEA] deletion), we have developed a real-time quantitative semi-nested polymerase chain reaction (PCR) method for identifying the fetal alpha(0)-Thalassemia in maternal plasma. Analysis was performed using DNA extracted from 200 muL plasma from 13 pregnant women during 8-20 weeks of gestation who carried fetuses with normal (2), alpha(0)-Thalassemia carrier (8), Hb H disease (1), and homozygous alpha(0)-Thalassemia (Hb Bart's hydrops fetalis (2). The alpha(0)-Thalassemia was detected using a two-step PCR. Plasma DNA was amplified conventionally using alpha(0)-Thalassemia-specific primers and a portion of the first PCR product was subjected to a semi-nested real-time q-PCR using the SYBR green I chemistry for fluorescence detection. Calibration curve for alpha(0)-Thalassemia quantification was prepared by assaying serial dilution of genomic DNA of an alpha(0)-Thalassemia carrier. Differences in the C(T) (threshold cycle) values and calculated concentrations of amplified DNA among normal fetus, alpha(0)-Thalassemia carrier, Hb H disease, and homozygous alpha(0)-Thalassemia were clearly observed, which could help in prenatal prediction of the fetal genotype. This noninvasive prenatal detection of alpha(0)-Thalassemia in maternal plasma should enhance prenatal diagnostic options for this common genetic disorder in routine DNA diagnostic setting.
Subject(s)
DNA/blood , Polymerase Chain Reaction/methods , Prenatal Diagnosis , alpha-Thalassemia/diagnosis , alpha-Thalassemia/genetics , Female , Humans , Maternal-Fetal Exchange , Pregnancy , ThailandABSTRACT
BACKGROUND: In order to provide a reliable non-invasive method for fetal sex determination in a routine setting, we evaluated the possibility of identifying the fetal Y chromosome-specific sequence in maternal plasma using conventional PCR analysis. METHODS: Fetal gender was determined in 31 pregnant women including one with a dizygotic twin pregnancy between 7 and 32 weeks of gestation using DNA extracted from 200 microl of each maternal plasma. The 198 bp SRY gene-specific sequence on Y chromosome and the 261 bp ATL1 gene-specific sequence on X chromosome were co-amplified in a multiplex nested PCR manner. The result was confirmed by routine analysis of fetal tissue obtained by invasive procedure or examination of newborns after delivery. RESULTS: The 198 bp SRY-specific sequence was detected in 15 plasma samples obtained from pregnant women carrying male fetuses. In the remaining cases bearing female fetuses, only the 261 bp ATL1 gene sequence was detected, producing 100% sensitivity and specificity of fetal sex prediction. The result was completely concordant with the conventional fetal tissue analysis and examination of the newborns after delivery. CONCLUSIONS: A conventional nested PCR analysis of maternal plasma could be used for accurate fetal gender detection and enable a reliable prospective non-invasive fetal sex determination which should enhance prenatal diagnostic options especially for sex-linked disorders.
Subject(s)
DNA/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Sex Determination Analysis/methods , Adult , Chromosomes, Human, X/chemistry , Chromosomes, Human, X/genetics , Chromosomes, Human, Y/chemistry , Chromosomes, Human, Y/genetics , DNA/blood , DNA Primers , Electrophoresis, Polyacrylamide Gel , Female , Humans , Pregnancy , Prenatal DiagnosisABSTRACT
In order to provide a noninvasive prenatal diagnosis of the hemoglobin E (Hb E) related disorder, we have evaluated the possibility of identifying the fetal beta(E)-globin gene in maternal plasma. The analysis was performed during 8 to 18 weeks of gestation using DNA extracted from 200 micro L of plasma from pregnant women whose husbands carried Hb E. The beta(E)-globin mutation in maternal plasma was detected by a nested PCR amplification followed by the Mnl I restriction analysis. The result was compared with that of routine analysis of the CVS specimens. Among the five pregnant women examined, the fetal beta(E)-globin gene was identified in maternal plasma in three of them and the result was completely concordant with the conventional CVS analysis. This simple noninvasive prenatal detection of the fetal beta(E)-globin gene should prove useful in a prevention and control program of Hb E/beta-thalassemia in countries where the beta(E)-globin gene is prevalent.
