Application of fluorescent in situ hybridization for ‘de novo’ anomalies in prenatal diagnosis

Fluorescent in situ Hybridization (FISH) was carried out for three cases of abnormal karyotypes in prenatal studies. Two concerned de novo structural anomalies and the third a marker chromosome. The origin of the extra material could be defined in all three cases, which gives a better insight into the relationship between genotype and phenotype and makes more adequate genetic counselling possible.     

Routine cytogenetic prenatal diagnosis using dynamic banding (RBG-GBG): A highly reproducible method for amniocytes,fetal cord blood,and chorionic villus investigations

Dynamic banding (RBG-GBG) using pulse 5-bromodeoxyuridine (5-BrdU) incorporation during part of the last S-phase before harvesting has been used in prenatal investigations. This method has already been routinely applied in 1344 cytogenetic investigations. GBG and RBG bandings produced almost identical patterns to classical G- and R-banding methods except for heterochromatic portions and some euchromatic segments. Nevertheless, these discordances may be somewhat helpful for cytogenetic diagnosis (i.e., X numerical abnormalities). The results showed particularly good contrast and staining; 5-BrdU incorporation did not prevent additional staining. Likewise, previous RBG or GBG disclosure allowed further chromosomal identification with C-banding or nucleolar organizer staining. Simplicity and reproducibility were very helpful in cases with a low mitotic index. 5-BrdU had no significant effect on in-vitro damage because only 0.31 per cent of cells were affected; so, we believe that dynamic banding should be used more extensively in cytogenetic investigations. Moreover, the staining and contrast qualities were very suitable for automatic methods of analysis now in use: i.e., metaphase finding and computer-assisted karyogram creation.     

An improved method of direct chromosome preparation from chorionic villus and high resolution banding technique

The trophoblast was dissociated from the underlying mesenchymal layer either with acetic acid after short-term prefixation or with mechanical power after fixation twice. The colcemid treatment time was shorted to 16 min and trypsin solution of low pH (6.2) was used for banding. By these steps, the quality of chromosome banding was greatly improved and complete standard chromosome diagnoses were made in 24 of 24 cases. With the modified technique, high resolution banding chromosomes were consistently obtained after short-term incubation.     

Prenatal identification of a 45,X/46,Xder(Y) mosaicism and confirmation by high resolution cytogenetics and fluorescence in situ hybridization

A 45,X/46,Xder(Y) mosaicism detected prenatally was shown to have a rare Y inversion- duplication or Y/Y translocation which can only be identified by a combination of high resolution cytogenetics and fluorescence in situ hybridization. The present data indicate the usefulness and importance of chromosome-specific probes in the identification and characterization of chromosome rearrangements.     

A culture vessel for amniotic fluid cells allowing faster preparation of chromosome slides

A new culture vessel for amniotic fluid culture is presented (flaskette). It consists of a microscope slide, on top of which a culture chamber is mounted. Amniotic fluid cell cultures using in situ technique in the flaskette were compared to subcultured samples in ordinary (Falcon) tissue culture bottles. Working time was reduced by using this new culture vessel because of a very simple harvest procedure allowing simultaneous harvest of 15 samples. The interval between amniocentesis and harvest was shorter for the in situ technique than for the subcultivation technique. The frequency of aneuploidy in individual metaphases was higher with the subcultivation technique. while there was no difference in the frequency of structural anomalies.     

Rapid karyotyping in prenatal diagnosis: A comparative study of the ‘pipette method’ and the ‘in situ’ technique for chromosome harvesting

Rapid karyotyping in the second and third trimesters has important implications for the management of pregnancies at risk. From September 1985 to March 1992, 735 amniotic fluid samples sent to our laboratory for rapid karyotyping from 64 different diagnostic centres of the Federal Republic of Germany were included in a comparative study on harvesting for chromosome analysis using the ‘pipette method’ or the ‘in situ’ technique. The average time between preparation of the amniotic fluid and verbal notification of the analysed karyotype was 5·41 days. The ‘pipette method’ needed on average 4·65 days, and the ‘in situ’ technique 5·97 days. In comparison with other more invasive techniques available for rapid karyotyping such as cordocentesis and placental biopsy, amniocentesis and subsequent chromosome harvesting using the ‘pipette method’ and/or the ‘in situ’ technique proved very useful and efficient. The overall incidence of chromosome aberrations was 15·3 per cent. The high rate of structural chromosome aberrations and uncommon aneuploidies found in our investigation (12 per cent) indicates that for rapid karyotyping in the second and third trimesters, conventional cytogenetic techniques cannot be replaced by faster techniques based on fluorescent in situ hybridization on interphase cells in the near future.     

Evaluation of X,Y, 18, and 13/21 alpha satellite DNA probes for interphase cytogenetic analysis of uncultured amniocytes by fluorescence in situ hybridization

The major aneuploidies diagnosed prenatally involve the autosomes 13, 18, and 21, and sex chromosomes. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. This prospective study evaluated the use of four commercially available centromeric DNA probes (DXZ1, DYZ1, D18Z1, and D13Z1/D21Z1) for direct analysis of uncultured amniocytes. One hundred and sixteen amniotic fluid samples were analysed by FISH and standard cytogenetics. This evaluation demonstrated that FISH with, X, Y, and 18 alpha satellite DNA probes could accurately and rapidly detect aneuploidies involving these chromosomes and could be used in any prenatal clinical laboratory. In contrast, the 13/21 alpha satellite DNA probe hybridizing both chromosomes 13 and 21 was unreliable for prenatal diagnosis in uncultured amniocytes.     

Measurement of cholinesterases in amniotic fluid using a naphthyl acetate as substrate: A possible initial test for prenatal diagnosis of open neural tube defects

A quantitative method for cholinesterases in amniotic fluid using the non-specific substrate α naphthyl acetate and the cholinesterase-specific inhibitor, eserine, is described. This assay was used to test 671 samples of amniotic fluid. The diagnoses for fetal ONTDs, based on the levels of AChE + ChE, were compared with those made for the same samples by the AFP method. Correct diagnoses were made by both methods with amniotic fluid from 35 women carrying fetuses with ONTDs and 631 carrying normal fetuses. There were five false-positive test results for normal fetuses by both methods when the cut-off points were 5 standard deviations above the mean for AFP and above the upper limit of the normal range (7. 5 milliunits) for cholinesterase (AChE + ChE). None of the false-positive samples from either method had the acetylcholinesterase band of activity characteristic of ONTDs after gel electrophoresis. In addition to the above 671 samples, 37 pregnancies with serious fetal abnormalities other than ONTDs were tested. Two were identified by both the AFP and AChE + ChE methods, two more by AFP assay and one other by the AChE + ChE assay.     

A further case with a small supernumerary marker chromosome (sSMC) derived from chromosome 1—evidence for high variability in mosaicism in different tissues of sSMC carriers

A prenatally ascertained case with a de novo small supernumerary marker chromosome (sSMC) derived from chromosome 1 is reported. Due to a fetal heart defect the parents decided in favour of an induced abortion. Postmortem, a molecular cytogenetic study on eleven formalin fixed, paraffin-embedded tissues of the fetus was performed, to further characterize the levels of mosaicism of the sSMC(1). sSMC presence varied between 13 and 62% within different tissues of sSMC carriers. This finding is something common in sSMC carriers and could explain why up to the present no clinical correlations for sSMC mosaicism and clinical outcome in the corresponding carriers could be established. Copyright © 2007 John Wiley & Sons, Ltd.     

Maternal age specific rates for chromosome aberrations and factors influencing them: Report of a collaborative european study on 52 965 amniocenteses

Maternal age specific rates for all major chromosome aberrations have been determined in 52 965 pregnancies in mothers 35 years of age and over at the time of amniocentesis. Rates increase exponentially with advancing maternal age for trisomies 21, 18 and 13, and for the XXX and XXY syndromes, but in the autosomal trisomies this rise appears to be followed by a levelling off at the upper end of the age range. A significant inverse relationship with maternal age is found for 45,X cases. It is postulated that these various patterns are the result of the interaction of three principal factors: a maternal age effect acting particularly on first meiotic nondisjunction: a higher spontaneous abortion rate with advancing maternal age for aneuploid as compared to euploid conceptions; and an increased probability of spontaneous abortion before the time of amniocentesis for conceptions with more extensive chromosome imbalance. A stepwise logistic regression analysis of 13 299 pregnancies in which both parental ages are known shows that the father's age does not influence these maternal age specific rates, with the possible exception of the 47,XXY syndrome.     

A simple,rapid, and highly informative PCR-based procedure for prenatal diagnosis and carrier screening of phenylketonuria

The polymorphic information content (PIC) and the degree of heterozygosity of several polymorphic systems within the phenylalanine hydroxylase (PAH) gene were determined in 85 European Caucasian and 19 Chinese phenylketonuria (PKU) kindreds. The first system examined, a short tandem repeat (STR), had a PIC of 80 and 73 per cent in these Caucasian and Chinese samples, respectively. The degree of heterozygosity actually observed for this system was 81 and 64 per cent in the Caucasian and Chinese PKU families, respectively. Through the addition of a second polymorphism based on a variable number of tandem repeats (VNTR), the PIC was increased to 90 per cent in Caucasians, but only to 75 per cent in Chinese. The degree of heterozygosity observed for this combination was 94 per cent in European PKU families and 67 per cent in Chinese PKU families. The further addition of an Xmnl RFLP increased both the PIC and the level of heterozygosity in Caucasians to 95 per cent, but did not change either of these measures in Chinese. The combined use of these three polymorphisms significantly increases the informativity of prenatal diagnostic and carrier screening procedures in both Caucasian and Chinese PKU kindreds. Furthermore, since each of these polymorphisms can be studied by PCR-based methods, these new tests can be performed more quickly and easily than previous Southern-based procedures.     

Estimating the risk of a fetal autosomal trisomy at mid-trimester using maternal serum alpha fetoprotein and age: A retrospective study of 142 pregnancies

Risks appropriate for mid-trimester prenatal screening for autosomal trisomies have been estimated from a combination of maternal age and maternal serum (MS) alpha-fetoprotein (AFP) levels at 16–20 weeks gestation. Published data on the frequency of Down's syndrome births relative to maternal age were modified to include the additional age-related frequency of trisomy 18 and trisomy 13 cases to provide an overall risk for an autosomal trisomy at midtrimester. MSAFP results from a retrospective study of 142 affected (114 trisomy 21, 19 trisomy 18, and 9 trisomy 13)and 113 000 unaffected pregnancies were converted to multiples of the appropriate gestational median (MOM). The AFP levels in the autosomal trisomy pregnancies were found to be significantly reduced at 0.72 MOM of the unaffected pregnancies. Risks (likelihood ratios) were derived from the overlapping log Gaussian distributions for affected and unaffected pregnancies and combined with maternal age risks to give the overall odds of an affected pregnancy. A mid-trimester cut-off risk of 1:280 gave an estimated 37 per cent detection rate for autosomal trisomies in the west of Scotland population for a follow-up (false-positive) rate of 6.6 per cent. These figures compare with a 30 per cent detection and 6.7 per cent false-positive rate if age 35 years and over is used as the sole criterion for selection of at-risk pregnancies.     

Is selective abortion for a genetic disease an issue for the medical profession? A comparative study of Quebec and France

This article discusses the results of a study of the stand and attitudes of physicians from the Picardie, Nord-Pas-de-Calais region in France and the province of Quebec (Canada) regarding abortion following the diagnosis of a fetal anomaly by ultrasound, amniocentesis, or chorionic villus sampling. The study examined the degree of acceptability of abortion for several specific conditions as well as the physicians' perceptions of their role in the women's decision to abort. The study shows a consensus (over 75 per cent of the physicians surveyed) for aborting a fetus with trisomy 21. There is a similar consensus, except among Francophones in Quebec, for muscular dystrophy, cystic fibrosis, and Huntington disease. Conversely, there is no consensus (below 60 per cent) for several anomalies. In these cases, Quebec Anglophone physicians find abortion more acceptable than Quebec Francophone or French physicians. Concerning the role of the practitioners in the decision to abort, physicians in France tend to be much more directive than their overseas colleagues. Several hypotheses are suggested to explain the difference between the three groups surveyed.     

A simple and cheap method for preparation of coupled ZrO2/ZnO with high photocatalytic activities

The objective of this study was to prepare a new photocatalyst with high activities for degradation of organic pollutants. Coupled ZrO₂/ZnO photocatalyst was prepared with a simple precipitation method with cheap raw materials zinc acetate and zirconium oxychloride, and was characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM). Reactive brilliant red X-3B was used as a model compound to investigate the photocatalytic activity of synthesized catalysts in water under 254 nm UV irradiation. Results show that the optimal calcination temperature and coupling molar ratio of Zr were 350°C And 2.5%, respectively. At the calcination temperature of 350°C, ZrO₂ was dispersed on the surface of hexagonal ZnO in the form of amorphous clusters. The particle size of ZrO₂/ZnO decreased with the decrease of calcination temperature and the increase of Zr coupling amount. ZrO₂/ZnO has better photocatalytic activity for degradation of reactive brilliant red (RBR) X-3B than pure ZnO and P25-TiO₂.     

A 10-year survey, 1980–1990, of prenatally diagnosed small supernumerary marker chromosomes,identified by fish analysis. Outcome and follow-up of 14 cases diagnosed in a series of 12 699 prenatal samples

A cytogenetic survey and follow-up studies were made of 14 cases with supernumerary marker chromosomes, identified among 12 699 prenatal samples, investigated at our institution over a 10-year period from 1980 to 1990. FISH (fluorescence in situ hybridization) techniques were employed to identify the chromosomal origin of the marker chromosomes. Five cases were familial, all derived from acrocentric chromosomes, and all without apparent phenotypic effects in the children. Nine cases represented de novo aberrations. In two cases (one with a marker from chromosome 14 or 22, the other with a ring-like marker derived from chromosome 17), the pregnancies continued and apparently normal babies were delivered at term, but the child with a marker derived from chromosome 17 showed slight psychomotor retardation at 2 years of age. All other pregnancies with de novo markers were terminated. In three cases, significant abnormalities were found at autopsy. One of these had an isochromosome 12p and the phenotype was consistent with Pallister-Killian syndrome. In conclusion, marker chromosome identification, as well as clinical follow-up, is essential for the purpose of improving genetic counselling.     

A novel, simple and sensitive resonance scattering spectral method for the determination of chlorite in water by means of rhodamine B

A new resonance scattering method was proposed for the determination of chlorite, basing on the resonance scattering effect of rhodamine dye. In HCl-sodium acetate buffer solution, chlorite oxidizes 1- into 12 and the reaction of 12 and excess 1- results in If. It is respectively combined with rhodaminc dyes, including rhodamine B （RhB）, butyl rhodamine B （b-RhB）, rhodamine G （RhG） and rhodamine S （RhS）, to form association complex particles, which exhibit stronger resonance scattering （RS） effect at 400 nm. The chlorite concentration of ClO2 in the range of 0.00726-0.218 μg/ml, 0.0102-0.292 μg/ml, 0.00726-0.145 μg/ml and 0.0290- 0.174 μg/ml is respectively linear to the RS intensity of association complex particle systems at 400 nm for the RhB, b-RhB, RhG and RhS. The detection limits of the four systems were respectively 0.00436, 0.00652, 0.00580 and 0.01450μg/ml ClO2^-. In the four systems, the RhB system possesses good stability and high sensitivity. It has been applied to the analysis of chlorite in wastewater with satisfactory results.     

A simple and sensitive method for the determination of 4-n-octylphenol based on multi-walled carbon nanotubes modified glassy carbon electrode

A simple and sensitive electroanalytical method was presented for the determination of 4-n-octylphenol (OP) based on multi-walled carbon nanotubes (MWCNTs) modified glassy carbon electrode (GCE). OP was directly oxidized on the MWCNTs/GCE, and the electrochemical oxidation mechanism was demonstrated by a one-electron and one-proton process in the reaction. The oxidation peak current of OP was significantly enhanced by the use of MWCNTs/GCE compared with those of bare glassy carbon electrode, suggesting that the modified electrode can remarkably improve the performance for OP determination. Factors influencing the detection processes were optimized. Under these optimal conditions, a linear relationship between concentration of OP and current response was obtained in the range of 5×10^-8 to 1×10^-5 mol/L with a detection limit of 1.5×10^-8 mol/L and correlation coefficient 0.9986. The modified electrode showed good selectivity, sensitivity, reproducibility and high stability.