Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8–14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligo-nucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F₅₀₈ deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F₅₀₈ primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.     

Congenital myotonic dystrophy: An often unsuspected cause of severe polyhydramnios

Two cases of severe polyhydramnios are presented in which the unsuspected diagnosis of myotonic dystrophy was made in the patient and the fetus.     

Prenatal diagnosis of inborn errors in peroxisomal β-oxidation

In recent years, an increasing number of inherited diseases in man have been recognized in which there is an impairment in the peroxisomal β-oxidation of very-long-chain fatty acids. In general, these disorders are associated with severe neurological and physical abnormalities and death within the first years of life. In this paper we describe our experience with regard to the prenatal diagnosis of a number of different inborn errors of peroxisomal β-oxidation. Eleven pregnancies at risk were monitored by measuring very-long-chain fatty acid levels as well as very-long-chain fatty acid β-oxidation in cultured chorionic villous fibroblasts and/or amniotic fluid cells. Five affected fetuses were identified. It is concluded that prenatal diagnosis in this group of diseases can be done reliably using cultured chorionic villous fibroblasts or amniotic fluid cells.     

Early amniocentesis at 11–14 weeks' gestation for the diagnosis of fetal chromosomal abnormality—a clinical evaluation

Early arnniocentesis between 11 and 14 weeks' gestation was offered to 110 women at risk of a chromosomally abnormal fetus due to maternal age. Four were found to be unsuitable for the procedure, and 106 early amniocenteses were performed. In 102 cases, clear amniotic fluid was obtained with a single tap. There were two dry taps and two bloodstained tapis; sampling was repeated in three of these cases before 15 weeks. In the fourth case, placental biopsy was performed at 16 weeks. Thus, we were able to obtain a satisfactory sample in all but three cases(2.8 percent). Karyotyping of cells harvested from the early amniotic fluid samples was successful in all the 105 cases. Cell culture from the initial samples revealed a normal karyotype in 99 cases, two balanced translocations, two tetraploid karyotypes, and two cases of pseudomosaicism. Of the 105 pregnancies successfully sampled, there have been two losses to date (1–8 per cent). Two further patients presented with premature rupture of membranes, both pregnancies having successful outcomes. Sixty-two babies have delivered to date, with four congenital anomalies. There were no respiratory problems. Twenty-nine pregnancies are continuing without known complications, and details are not yet available on the remaining 12. The results indicate that early arnniocentesis may replace the traditional test at 15–17 weeks.     

Prenatal diagnosis and outcomes of five cases of mosaicism for an isochromosome of 20q

Five cases of mosaicism for an isochromosome of 20q have been detected from a total of 50 000 cases analysed for prenatal diagnosis by amniocentesis. Karyotypes were designated mos 46,X_/46,X_,i(20q). In all cases, the abnormal cell line was detected in more than one primary culture, thus fulfilling the criterion for true (level III) mosaicism. Indications for prenatal diagnosis were parental anxiety (two cases), low maternal serum alpha-fetoprotein (AFP) (two cases), and high maternal serum AFP (one case). Level II ultrasounds on all five fetuses were normal, and the abnormal cell line was never detected in fetal blood and/or cord blood. All five pregnancies were continued and had normal outcomes, with birth weights ranging from 2.4 to 3.8 kg. The development of all five children has been normal, with the oldest child in the study now 4 years of age. We suggest that the abnormal cell line in each case was of extrafetal origin, and that this may be one of the more common examples of this phenomenon, occurring in approximately 1/10000 prenatal diagnoses. Mosaicism i(20q) may have been missed in the past because of the higher resolution necessary to detect this subtle change.     

Chorionic villus sampling: Analysis of fetal losses to delivery,placental pathology,and cervical microbiology

A population of 1639 patients were seen for chorionic villus sampling (CVS). Embryonic death was identified at ultrasound in 5.3 per cent of patients. The number of patients undergoing CVS was 1551, with 1416 transcervical procedures and 135 transabdominal procedures. The most common indication for CVS was advanced maternal age. Spontaneous pregnancy losses identified by increased risk of pregnancy loss with increasing aspiration attempts. The total fetal loss for this population was 5.4 per cent with the pregnancy loss estimated due to procedure being 1.2 per cent. Analysis of placentae from patients having CVS and amniocen-tesis showed no differences. Microbiological assessment prior to CVS was similar to previous publications.     

Uncommon chromosomal mosaicism in chorionic villi

Three cases of unusual chromosomal mosaicism are reported for which the cytogenetic data show inconsistent findings between CVS and AC or fetal tissue, and which cannot be explained simply by non-disjunction. For case 1, in CVS the karyotype was 46,XY, whereas lymphocytes and fibroblasts revealed 69,XXY. DNA fingerprinting indicated one paternal and two maternal chromosome sets, the latter most probably due to omission of maternal meiosis II. For case 2, in CVS mos 46,XX/47,XX,+mar de novo was observed. Amniotic fluid cells had the karyotype 46,XX. The origin of the marker chromosome might be explained by at least two events of unknown order (a somatic chromosome/chromatid deletion and non-disjunction of the homologous chromosome). In case 3 (CVS: mos 46,XY/46,XY,19q+ de novo; amniotic fluid cells, lymphocytes, and fibroblasts: 46,XY), the surplus of chromosome material in 19q+ might be explained on the basis of a somatic translocation. The idea of a chimera is less convincing, as the mosaic finding is restricted to one tissue. Furthermore, there was no hint of a vanishing twin. Hitherto, no case of structural chromosome mosaicism in CVS has been reconfirmed in fetal tissues.     

Mosaicism and accuracy of prenatal cytogenetic diagnoses after chorionic villus sampling and placental biopsies

Discrepant chromosome findings in placenta and fetus (false negative and false positive) after chorionic villus sampling (CVS) are mainly due to confined mosaicism. Non-mosaic normal or abnormal chromosome counts after direct preparation and culture nearly always correctly reflect the fetal chromosome constitution. False-negative results have almost exclusively been restricted to cytotrophoblast cells not representing a fetal chromosome abnormality. Diagnosis of placental mosaicism definitely requires an adequate follow-up by amniocentesis, fetal blood sampling, or sonography before a pregnancy is terminated. When direct preparations and cultured cells are used for cytogenetic diagnoses and placental mosaicism is not taken as proof for a chromosomal abnormality in the fetus, CVS is an accurate diagnostic tool.     

Vitamins,folic acid and neural tube defects: Comments on investigations in the United States

No clear answer concerning whether multivitamin/folate supplementation prevents neural tube defects (NTDs) is provided by three studies in the United States. All these studies are occurrence in nature, no recurrence studies having been conducted. The Atlanta Birth Defects Study is subject to pronounced memory and recall biases, the length between event and interview being as long as 16 years. In a second study (Boston University), objections can be raised to certain aspects of the experimental design, and the claim that 22 per cent of women started vitamins sufficiently early after pregnancy diagnosis to influence NTD formation is suspicious. Our NICHD case control study of 541 women in California and Illinois revealed no evidence for multivitamins or folic acid preventing NTDs. U.S. public policy-makers face difficulties in applying results of recurrence or occurrence studies in high-risk areas to low-risk areas in the U.S.