Localisation of the classical DNA satellites on human chromosomes as determined by primed in situ labelling (PRINS).

The chromosomal localisation and relative amounts in humans of the classical DNA satellites I, II and III have been determined by using the primed in situ labelling reaction with a variety of oligonucleotide primers. The centromeres of seven of the human chromosomes, viz. nos 6, 8, 11, 12, 18, 19 and X, are not identifiably marked by any of the primers. A possible phylogenetic explanation of this is suggested and the possible relationship of the classical satellites to the function of the centromere is discussed.

Extreme variant of the short arm of chromosome 15.

Using fluorescence in situ hybridization, primed in situ labelling, and conventional cytogenetic staining we have characterized an excessively enlarged short arm of chromosome 15. The likely mechanism explaining this variant chromosome involves amplification of rDNA sequences followed by inverted insertional translocation between the enlarged sister chromatids of the short arm of chromosome 15.

Selective miscarriage of Down's syndrome fetuses in women aged 35 years and older.

OBJECTIVE: To estimate the fetal loss of Down's syndrome fetuses between the time of chorionic villus sampling (10 weeks gestation) and the time of amniocentesis (16 weeks gestation) and term in women aged 35 years and older. DESIGN: The age specific prevalence rates in the first trimester of Down's syndrome were estimated using the Danish cytogenetic register in combination with results from four published studies. These were compared with the reported prevalence at the time of amniocentesis and at birth. SUBJECTS: 5927 singleton pregnancies undergoing chorionic villus sampling (71 cases of Down's syndrome and 5856 unaffected cases). This was combined with published data on a further 231 cases of Down's syndrome and 16,620 unaffected cases. MAIN OUTCOME MEASURES: Age specific prevalences at the time of chorionic villus sampling. Proportion of pregnancies lost between the time of chorionic villus sampling and the time of amniocentesis and term. RESULTS: Thirty-two percent of Down's syndrome pregnancies are lost between the time of chorionic villus sampling (10 weeks) and the time of amniocentesis (16 weeks) and 54% are lost by term. CONCLUSIONS: The high fetal loss rates of Down's syndrome between the time of chorionic villus sampling and term introduce problems when evaluating first trimester screening tests with respect to their effective detection rates at term. A recommendation for quoting term risks is made.

Partial deletion 11q: report of a case with a large terminal deletion 11q21-qter without loss of telomeric sequences, and review of the literature.

We describe the cytogenetic findings and the dysmorphic features in a stillborn girl with a large de novo terminal deletion of the long arm of chromosome 11. The karyotype was 46,XX,del(11)(q21qter). By reviewing previous reports of deletion 11q, we found that cleft lip and palate are most frequently seen in proximal 11q deletions involving 11q21. Telomeric staining using the PRINS technique demonstrated normal telomeric sequences in the deleted chromosome 11.

Staining of human telomeres with primed in situ labeling (PRINS).

As described, the PRINS method is a very rapid and reliable way of staining human telomeres. To obtain the maximum frequency of stained telomeres, the primer (CCCTAA)7 should be used, although the average frequency never quite reaches 100%. The frequency is strongly dependent on the age of the individual, being significantly higher in children and newborns than in adults. A difference between the (CCCTAA)7 primer and the complementary primer is demonstrated and a possible explanation is proposed, namely, that gaps in the C-rich strand cause chain elongation termination after the addition of only one dTTP molecule.

Ring chromosome 20 with loss of telomeric sequences detected by multicolour PRINS.

A ring chromosome 20 in a male infant with epileptic seizures, mental and somatic growth retardation, and behavioural disturbances is described. Conventional cytogenetics revealed the karyotype to be 46,XY,r(20)(pter-->qter) and no signs of mosaicism were found. Fluorescence in situ hybridisation using the clone p20Z1 identified the ring to be derived from chromosome 20. By counting 111 metaphases, only 7% were found to be missing the ring. The absence of telomeric sequences in the ring chromosome was demonstrated by multicolour PRINS: a three-step PRimed IN Situ labelling technique, using unlabelled primers. A terminal deletion of both arms thus seems to be the cause of the ring formation in the proband. Bivariate flow-analysis of chromosomes verified a deletion of the ring chromosome. The clinical and cytogenetic findings are compared with previous cases. A specific ring 20 syndrome seems justified.

A routine method for storing lymphocytes for repeated isolation of DNA.

A protocol for easy storage and later expansion of lymphocyte populations is given. Compared with methods using transformed cell lines, the method has a number of advantages for repeated production of cells for the isolation of DNA in amounts sufficient for use in diagnostic DNA technology.

Prenatal cytogenetic diagnosis after transabdominal chorionic villus sampling in the first trimester.

First trimester prenatal cytogenetic diagnosis was attempted in 350 pregnancies after transabdominal chorionic villus sampling. The cytogenetic investigation was performed using both a short-term method (24 h incubation) and cell culture. Adequate samples were obtained in 99.1 per cent and in all these cases the fetal karyotype was established. A chromosome abnormality was found in 2.0 per cent of cases. A discrepancy between the karyotype obtained after 24 h incubation and the karyotype in cell culture was observed in 2.3 per cent. Maternal cell contamination in the cultures was confirmed in 13 of 181 cases where the 24 h incubation revealed a male karyotype. Studies of culture morphology showed that colonies of convoluted cells may serve as a marker for contamination with maternal cells in culture. For the present, we recommend using a short-term method as well as cell culture for cytogenetic investigation until the problems with karyotype discrepancy and maternal cell contamination have been further clarified.

Convoluted cells as a marker for maternal cell contamination in CVS cultures.

In order to identify cells of maternal origin in CVS cultures, tissue from 1st trimester abortions were cultivated and the cultures stained in situ for X-chromatin. Convoluted cells and maternal fibroblasts were found to be positive. By chromosome analysis of cultures from 105 diagnostic placenta biopsies, obtained by the transabdominal route, metaphases of maternal origin were found in nine cases. In eight of these cases colonies of convoluted cells were observed. We conclude that convoluted cells are of maternal origin and are a reliable marker for maternal cell contamination in CVS cultures.

Spontaneous abortion following mid-trimester amniocentesis. Clinical significance of placental perforation and blood-stained amniotic fluid.

The clinical significance of placental perforation and blood-stained amniotic fluid was studied in a group of 7238 Danish women undergoing mid-trimester amniocentesis for prenatal diagnosis under ultrasound guidance. The risk of spontaneous abortion was significantly increased both in pregnancies where the placenta was perforated and in those with blood-stained amniotic fluid. The risk estimate nearly doubled after placental perforation and more than doubled with a bloody tap. It is concluded that for women at relatively low risk of a fetal genetic abnormality, the indication of the amniocentesis should be reconsidered if a placental perforation is unavoidable.

Admixture of maternal metaphases in first trimester direct chromosome preparations?

The autoradiographic labelling of different cell types in chorionic villi and decidual tissue was investigated after [3H]-thymidine incorporation in vitro. Although the extent of labelling was found to be lower in decidual than in villus tissue the possibility that direct chromosome preparations may contain maternal metaphases should be considered. The need for careful selection of villi for direct cytogenetic analysis was stressed.

Transabdominal chorionic villi sampling for first trimester fetal diagnosis. First 26 pregnancies followed to term.

A consecutive series of 26 women followed to term after first trimester transabdominal chorionic villi sampling is presented. The clinical application of transabdominal chorionic villi sampling (TA-CVS) seems to have certain advantages, especially from the patients' point of view, but also in regard to successful sampling and to the complication ratio. The results in this clinical trial revealed no cases of abortions, no signs of placental damage and no cases of vaginal bleeding or infections.

Results of in vitro fertilization and culture of human ova.

Using a technique with different media for fertilization and subsequent culture, cleavage of at least one oocyte was achieved in 35 (83%) out of 42 consecutive patients. A total of 138 oocytes (74 pre-ovulatory, 20 non-ovulatory and 44 atretic) were inseminated in vitro. Fifty-two (70%) of the pre-ovulatory oocytes clove and of these 37 (72%) developed apparently normally to the morula or blastocyst stage, while cleavage of the remaining 15 (28%) was characterized by an abnormal growth curve or abnormal morphology of the embryo. Only one (5%) of the 20 non-ovulatory oocytes clove, and this embryo displayed an abnormal growth curve. None of the atretic oocytes clove. It is concluded that evaluation of morphology and growth curve seems to be of value in distinguishing between apparently normally and abnormally developing embryos in vitro.

Selective inhibition of fibroblasts by spermine in primary cultures of normal human skin epithelial cells.

Overgrowth with fibroblasts has been a major problem in the cultivation of normal human skin epithelium. In the present study it is shown that the addition of spermine to the culture medium in micromolar concentrations has a differential cytotoxic effect on fibroblasts allowing the cultivation of human skin epithelial cells in primary culture without fibroblastic overgrowth. Putrescine, another polyamine, is shown to be equally cytotoxic to fibroblasts and epithelial cells when added in millimolar concentrations; below this concentration range no cytotoxic effect could be demonstrated. This difference in cytotoxicity between spermine and putrescine is suggested to depend on the conversion of spermine, but not putrescine, and to highly cytotoxic products by an amine oxidase present in fetal bovine serum.

An attempt to define 1qh+, 9qh+, and 16qh+.

The lengths of the secondary constrictions of chromosomes 1, 9, and 16 vary with the degree of contraction of the chromosomes but these constrictions contract to a lesser degree than the euchromatic portions of the chromosomes. The regression coefficient for the regression of the length of the secondary constriction on the length of the euchromatic part of the chromosomes is shown to be larger for large constrictions. It is furthermore shown that there is a linear correlation between the regression coefficient and the size of the secondary constriction in question. This linear correlation makes it possible to correct the lengths of the secondary constrictions to the lengths expected when contraction is average. The correction method is used in a sample of 30 couples, and on the basis of this sample, the normal limits for the lengths of the secondary constrictions in chromosomes 1, 9, and 16 are defined.