Application of quantitative fluorescent polymerase chain reaction analysis for the rapid confirmation of trisomy 13 of maternal origin in a pregnancy with fetal holoprosencephaly,cyclopia, polydactyly,omphalocele and cell culture failure

ObjectiveWe present the application of quantitative fluorescent polymerase chain reaction (QF-PCR) for the rapid confirmation of trisomy 13 of maternal origin in a pregnancy with fetal holoprosencephaly (HPE), cyclopia, polydactyly, omphalocele and cell culture failure.Case reportA 21-year-old, gravida 2, para 0, woman was referred for termination of the pregnancy at 17 weeks of gestation because of the abnormal ultrasound finding of alobar HPE. The pregnancy was subsequently terminated, and a 118-g malformed male fetus was delivered with cyclopia, bilateral postaxial polydactyly of the hands and ruptured omphalocele. Postmortem cell culture of the placental tissue and umbilical cord was not successful. The parental karyotypes were normal. QF-PCR analysis using the polymorphic DNA markers of D13S1810, D13S790 and D13S251 on the DNA extracted from placenta, umbilical cord and parental bloods showed trisomy 13 of maternal origin.ConclusionPerinatal diagnosis of concomitant HPE, polydactyly and omphalocele should raise a suspicion of fetal trisomy 13. QF-PCR analysis is useful for rapid confirmation of trisomy 13 and the parental origin especially under the circumstance of cell culture failure, and the information acquired is very useful for genetic counseling of the parents.     

Rapid confirmation of trisomy 13 of maternal origin by QF-PCR following postmortem tissue cell culture failure in a pregnancy with trisomy 13 at amniocentesis and fetal postaxial polydactyly and facial cleft

ObjectiveWe present rapid confirmation of trisomy 13 of maternal origin by quantitative fluorescent polymerase chain reaction (QF-PCR) following postmortem tissue cell culture failure in a pregnancy with trisomy 13 at amniocentesis and fetal postaxial polydactyly and facial cleft.Case ReportA 34-year-old woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Cytogenetic analysis of cultured amniocytes revealed a karyotype of 47,XX,+13. Prenatal ultrasound revealed postaxial polydactyly. The pregnancy was subsequently terminated, and a malformed fetus was delivered with facial cleft and postaxial polydactyly of the hand and foot. Postmortem cytogenetic analysis of the fetal tissue revealed no growth of the cells due to culture failure, but QF-PCR analysis on the DNA extracted from placenta, umbilical cord and parental bloods confirmed trisomy 13 and maternal origin of the extra chromosome 13.ConclusionQF-PCR analysis is useful for rapid perinatal confirmation of trisomy 13 and the parental origin of the extra chromosome 13, especially under the circumstance of tissue cell culture failure, and the acquired information is useful for genetic counseling.     

Rapid diagnosis of trisomy 18 of maternal origin by quantitative fluorescent polymerase chain reaction analysis following tissue culture failure for conventional cytogenetic analysis in a fetus with holoprosencephaly,ventricular septal defect,arthrogryposis of bilateral wrists and aplasia of the thumbs

ObjectiveWe present rapid diagnosis of trisomy 18 of maternal origin by quantitative fluorescent polymerase chain reaction (QF-PCR) analysis following tissue culture failure for conventional cytogenetic analysis in a fetus with holoprosencephaly (HPE), ventricular septal defect (VSD), arthrogryposis of bilateral wrists and aplasia of the thumbs.Case reportA 22-year-old, primigravid woman was referred for first-trimester ultrasound screening at 13 weeks of gestation, and the fetus was found to have HPE and VSD. The pregnancy was subsequently terminated at 14 weeks of gestation, and a malformed fetus was delivered with cebocephaly, arthrogryposis of bilateral wrists and aplasia of the thumbs. The umbilical cord and placental tissues were collected for genetic analysis. However, tissue culture failure for conventional cytogenetic analysis occurred because of contamination. QF-PCR analysis using the polymorphic DNA markers of D18S1369 (18q12.2) and D18S1361 (18q22.3) confirmed trisomy 18 of maternal origin.ConclusionQF-PCR analysis is useful for rapid confirmation of trisomy 18 and the parental origin when tissue culture failure for conventional cytogenetic analysis occurs in pregnancy suspicious of fetal trisomy 18.     

Detection of paternal origin of fetal trisomy 21 in a pregnancy with isolated ventriculomegaly but without advanced parental age

ObjectiveWe present detection of paternal origin of fetal trisomy 21 in a pregnancy with isolated ventriculomegaly but without advanced parental age.Case reportA 29-year-old pregnant woman was admitted to the hospital at 18 weeks of gestation for tocolytic treatment because of irregular uterine contractions. Her husband was 30 years old. The couple had a healthy daughter. Prenatal ultrasound incidentally found isolated ventriculomegaly, and subsequent amniocentesis revealed a karyotype of 47,XX,+21 in 20/20 colonies of cultured amniocytes. The pregnancy was terminated, and the fetus manifested characteristic craniofacial appearance of Down syndrome and hyposplastic middle phalanx of the fifth finger. Postnatal polymorphic DNA marker analysis on the DNAs extracted from the cord blood and parental bloods using quantitative fluorescent polymerase chain reaction (QF-PCR) showed a paternal origin of fetal trisomy 21. The father had a karyotype of 46, XY in 40/40 blood lymphocytes.ConclusionQF-PCR is useful for rapid confirmation of prenatally detected fetal trisomy 21 and determination of paternal origin of fetal trisomy 21 especially in pregnancies with fetal structural abnormalities but without advanced parental age.     

Rapid detection of trisomy 21 by gene dosage analysis using quantitative real-time polymerase chain reaction

AIM: Rapid detection of fetal aneuploidy helps inform a mother's choice about the course of her pregnancy. Obtaining results by fluorescent in situ hybridization (FISH) requires more than 24 h, and thus a more rapid method is needed. METHODS: Conventional G-banding and FISH for chromosome 21 were performed for cultured amniocytes. Genomic DNA was extracted from uncultured amniocytes obtained from 23 patients. TaqMan polymerase chain reaction (PCR) primers were designed to amplify the potassium voltage gated channel gene on chromosome 21q22.12 and the ribosomal phosphoprotein gene on 18q21.1. Quantitative real-time PCR was performed for these two gene fragments and the differences of the threshold cycle (Ct) of the two genes (Ct 18-Ct 21) were calculated for each sample. RESULTS: G-banding revealed that 19 patients had a normal karyotype and four had trisomy 21. FISH resulted in one case of a false positive. The Delta Ct values (Ct 18-Ct 21) of trisomy 21 patients were significantly higher than the values of individuals with normal karyotypes (P < 0.001) and there was no overlapping. CONCLUSIONS: Fetal trisomy 21 is rapidly detectable by gene dosage analysis from amniocytes using quantitative real-time PCR.     

Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of fetal infection with Toxoplasma gondii

OBJECTIVE: Our purpose was to evaluate Toxoplasma gondii concentration in amniotic fluid (AF) samples as a prognostic marker of congenital toxoplasmosis. STUDY DESIGN: A retrospective study was carried out in 88 consecutive AF samples from 86 pregnant women, which were found positive by prospective polymerase chain reaction (PCR) testing. Parasite AF concentrations were estimated by real-time quantitative PCR and analyzed in relation to the clinical outcome of infected fetuses during pregnancy and at birth, taking into account the gestational age at maternal infection. RESULTS: A significant negative linear regression was observed between gestational age at maternal infection and T gondii DNA loads in AF. After adjusting for time at maternal seroconversion by multivariate analysis, higher parasite concentrations were significantly associated with a severe outcome of congenital infection (odds ratio [OR]=15.38/log (parasites/mL AF) [95% CI=2.45-97.7]). CONCLUSION: PCR quantification of T gondii in AF can be highly contributive for early prognosis of congenital toxoplasmosis. Maternal infections acquired before 20 weeks with a parasite load greater than 100/mL of AF have the highest risk of severe fetal outcome.