Evelyne Gautier

New Strategy for Prenatal Diagnosis of X-Linked Disorders

To the Editor: An invasive approach is still the gold standard for prenatal diagnosis of genetic disorders. Chorionic-villus sampling, the current procedure of choice, allows an early diagnosis, bu...

Fetal RHD genotyping in maternal serum during the first trimester of pregnancy

Summary.  Fetal RHD genotype determination is useful in the management of sensitized RhD‐negative pregnant women. It can be ascertained early during pregnancy by chorionic villus sampling (CVS) or amniocentesis. However, these procedures are invasive, resulting both in an increased risk of fetal loss and in an increased severity of immunization due to fetomaternal haemorrhage. A reliable determination of RHD genotype by fetal DNA analysis in maternal serum during the first trimester of pregnancy is reported in this study. One hundred and six sera from RhD‐negative pregnant women were obtained during the first trimester of pregnancy. These sera were tested for the presence of RHD gene using a new real‐time polymerase chain reaction assay and the results compared with those obtained later in pregnancy on amniotic fluid cells and by RHD serology of the new‐born. All sera from women carrying a RhD‐positive fetus (n = 62) gave positive results for RHD gene detection and sera from women carrying a RhD‐negative fetus (n = 40) were negative. The high level of accuracy of fetal RHD genotyping obtained in this study could enable this technique to be offered on a routine basis for the management of RhD‐negative patients during the first trimester of pregnancy.

Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes.

The diagnosis of congenital toxoplasmosis frequently relies on PCR tests of amniotic fluid (AF). A duplex real‐time quantitative PCR test based on fluorescence resonance energy transfer was developed to quantify the parasite load and to decrease the risk of contamination. An internal control based on the detection of 10 pg mouse DNA added to the AF was included to check for PCR efficiency. The relationship between the parasite load and the occurrence of ultrasonographic abnormalities in 87 samples of AF was analyzed. Seven AF (8%) had a parasitic load >103; 14 (16%) had >102–≤103; 26 (30%) had >10–≤102; and 40 (46%) had ≤10 parasites/ml. Four of the six AF with cerebral ventriculomegaly had >103 parasites/ml. The other two had 130 and 24 parasites/ml, respectively. No parasitic loads of >103 parasites/ml and no ultrasonographic abnormalities were observed in the 11 AF with maternal toxoplasmosis in the third trimester. Therefore, there is a trend to associate high parasite count with ultrasonographic abnormality, but the main concern remains early maternal infection. The importance of quantification should be better evaluated with postnatal studies. The duplex LightCycler PCR test currently provides rapid and safe results. Copyright

Hyperechogenic fetal bowel: an ultrasonographic marker for adverse fetal and neonatal outcome.

OBJECTIVE Fetal hyperchogenic bowel is associated with a variety of conditions, the incidence of which has yet to be studied. STUDY DESIGN The outcomes of 182 cases of fetal hyperechogenic bowel were reviewed. Screening for maternal toxoplasmosis, fetal karyotyping, and amniotic fluid digestive enzyme assays were performed in all cases. Eight mutations associated with cystic fibrosis were analyzed in 116 cases. RESULTS Of 135 newborns, 121 were normal, but nine underwent surgery for gastrointestinal obstruction, three had cytomegalovirus or parvovirus infection, one had a triple X chromosome, and one died from sudden infant death syndrome. In utero fetal death was observed in 24 cases. Elective termination of pregnancy was performed in 23 cases for associated anomalies. CONCLUSIONS Hyperechogenic fetal bowel was associated with increased risk for adverse outcome. Prenatal management should include ultrasonographic surveillance, fetal karyotyping, amniotic digestive enzyme assays, and screening for cystic fibrosis and infectious disease.

Cell-free DNA analysis in maternal plasma in cases of fetal abnormalities detected on ultrasound examination.

OBJECTIVE: To evaluate the utility of noninvasive prenatal testing using cell-free circulating fetal DNA for detection of the three main autosomal fetal trisomies in the setting of ultrasonographically identified fetal anomalies. METHODS: Nine hundred patients at risk for fetal aneuploidy with or without ultrasonography anomalies and who underwent invasive procedures were included in the study. Cell-free DNA analysis was performed by massive parallel sequencing during a multicenter, noninterventional, prospective study and the results were compared with a fetal karyotype. RESULTS: Among all 900 pregnancies, cell-free DNA identified 76 of 76 (100%) fetal Down syndrome, 22 of 25 (88%) trisomy 18, and 12 of 12 (100%) trisomy 13. In those with a normal ultrasonogram and normal cell-free DNA analysis, karyotype identified 2 of 483 (0.4%) additional aneuploidies other than trisomies 13, 18, and 21. In those with an abnormal ultrasonogram and a normal cell-free DNA analysis, there were 23 of 290 (7.9%) additional pathogenic karyotypes. These additional aneuploidies included sex chromosome abnormalities and triploidy. The rates of additional aneuploidies not identifiable by standard cell-free DNA screening in the two groups is significantly different at P<.01. CONCLUSION: In women with fetal abnormalities by ultrasonography, the rate of pathogenic chromosome abnormalities missed by cell-free DNA was 8%. Noninvasive prenatal testing should not be offered to women with fetal abnormalities because a negative result is falsely reassuring. LEVEL OF EVIDENCE: III

Fetal Expressed Gene Analysis in Maternal Blood: A New Tool for Noninvasive Study of the Fetus

Noninvasive approaches to prenatal diagnosis can avoid the risk of fetal loss associated with invasive procedures such as chorionic villus sampling, amniocentesis, and cordocentesis. Isolation of fetal cells from maternal blood requires further improvements before it can be applied in a clinical setting (1), but the reliability of cell-free fetal DNA analysis in maternal plasma or serum is now well established (2)(3)(4). As a result, it is currently used in specialized centers for the determination of fetal sex and fetal RhD status for the management of pregnant women at risk for X-linked disorders (5) or RhD alloimmunization (6). Because fetal DNA in maternal serum is circulating in an excess background of maternal DNA, clinical applications are restricted mainly to the detection of fetal sequences distinct from the mother’s DNA sequences. Fetal RNA in maternal blood may be an alternative source of fetal nucleic acids. Al-Mufti et al. (7) detected specific RhD mRNA in mononuclear fetal cells isolated from blood of RhD-negative pregnant women, and Lo’s group demonstrated the presence of Y-chromosome-specific ( ZFY ) mRNA in maternal plasma of women carrying a male fetus (8). These two applications, however, again require fetal sequences that differ from the mother’s. We have investigated the presence in maternal blood of fetal transcripts that may have the same sequence as that of the mother. Human chorionic gonadotropin (hCG) mRNA is a good candidate because it is a pregnancy-specific polypeptide hormone produced by the placenta and is specifically expressed in the fetal syncytiotrophoblast. We studied 43 pregnant women and 20 nonpregnant women who had previously given birth to at least one neonate. After receiving informed consent, we collected blood (2.5 mL) into PAXgeneTM tubes to reduce RNA degradation (9). All pregnancy samples were obtained before …