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.     

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.     

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.     

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.     

Prenatal diagnosis in advanced maternal age. Amniocentesis or CVS,a patient's choice or lack of information?

Ninety-six women of advanced maternal age were interviewed about the way they obtained information on prenatal diagnosis and about how the decision was made as to what procedure was to be performed (transabdominal chorionic villus sampling (TA-CVS) or amnio-centesis). In the CVS group, women visited their physician or midwife earlier in pregnancy (mean 7.1 weeks) than those in the amniocentesis group (mean 10.7 weeks). The availability of prenatal diagnosis was not mentioned during the first antenatal visit in 55 per cent of women from the amniocentesis group as opposed to 25 per cent from the TA-CVS group. Approximately 40 per cent of women eligible for prenatal diagnosis did not receive any information from the referring body prior to counselling at our centre. Only 29 per cent of women who underwent amniocentesis had actually chosen this procedure; 71 per cent were too late to undergo TA-CVS at 12 weeks. It is concluded that information to the patient must be improved in order to ensure early referral for prenatal diagnosis.     

Comparative audit of booking and mid-trimester ultrasound scans in the prenatal diagnosis of congenital anomalies

During 1988 and 1989, 3565 women booked under consultants who performed an ultrasound scan at booking, whilst 4984 booked under consultants who performed a formal mid-trimester scan between 16 and 18 weeks. All significant anomalies diagnosed prenatally and in the neonatal period were recorded, the incidence in each group being 12.9/1000 and 9.83/1000, respectively (NS). The sensitivity of diagnosis before 20 weeks was 45 per cent in the ‘mid-trimester’ group (kappa 0.62) compared with 30 per cent in the ‘booking’ group (kappa 0.46), overall sensitivity of prenatal diagnosis, however, being similar in both groups (63 vs. 65 per cent, kappa 0.77 vs. 0.79). Cardiac anomalies were the single largest group which were not detected equally prenatally in both groups. This study shows that formal mid-trimester scanning leads to anomalies being detected significantly earlier in the antenatal period. Although not statistically significant, three lethal anomalies were missed prenatally in the ‘booking’ group which we would have expected to diagnose on a mid-trimester scan. These figures are discussed in the light of previous reports.     

A Bayesian assessment of the effect of highway bypasses in Iowa on crashes and crash rate

Introduction

A common contention is that the construction of highway bypasses negatively impacts the economy of local communities by reducing pass-by traffic for businesses. However, as access to specific business' account records is limited, this impact is difficult to quantify. Another common contention is that bypasses contribute to a reduction in overall crashes in the community and in the surrounding areas. Even though a large number of bypasses have been constructed in the State of Iowa over the past several years, their actual impact in terms of traffic safety has not been quantified.

Objectives

This study seeks answers to the following questions: (a) Are bypasses in Iowa associated with a reduction in crash frequencies and crash rates on the bypassed highway? (b) Do bypasses in Iowa introduce a reduction of overall crash frequencies and rates or do they merely shift crashes from the highways through the communities to the bypasses with no significant overall reduction?

Method

We obtained crash information from the Iowa DOT at 19 sites on which a bypass was constructed sometime during the past 23 years. We also obtained the same information at six sites used as comparison sites on which no bypasses were constructed at least until 2005. We them employed a Bayesian approach to estimating the association between the construction of the bypass and crash rates, while also accounting for other factors.

Results

The construction of bypasses in Iowa is associated with a significant increase in traffic safety both on the main road through town and on the combined main road and bypass roadway.