Familial transmission of a translocation Y/14

Summary A translocation of heterochromatic material, brightly fluorescent after actinomycin D-DAPI staining, to the short arm of chromosome 14 was prenatally detected during cytogenetic examination of cells obtained by amniocentesis on the indication of advanced maternal age. Besides this abnormal chromosome, 43 autosomes and two X chromosomes were present. Silver staining made clear that an active nucleolus-organizing region was included in the translocation product. Both the intense fluorescence and the size of the translocated extra heterochromatic block were indicative of a Yq origin. Upon cytogenetic investigation of the parents, the mother appeared to carry the same t(Y;14) chromosome. Therefore, we expected a normal girl to be born. This was confirmed after birth.     

Prenatal prevention of drepanocytosis: analysis of cellular DNA in 2 cases

Direct analysis of fetal DNA using restriction endonucleases constitutes a major area of progress in prenatal diagnosis. This recent technology may permit the precise identification of a mutant allele for some diseases, whereas in others it allows the familial segregation of a pathogenic allele to be followed by its linkage to a DNA sequence polymorphism. This type of analysis, available in a few centers, is currently used, among others, for the prenatal diagnosis of hemoglobinopathies such as sickle cell anemia. After fetal cells have been obtained by choriocentesis or amniocentesis, the extracted DNA is exposed to selected restriction enzymes. In the diagnosis of sickle cell anemia the mutant codon responsible for the substitution of glutamic acid by valine in the beta hemoglobin chain is no longer cut by the enzyme Mst II, due to its variance with the normal codon; this difference in fragment length is detected by DNA electrophoresis, and the particular fragments are identified by molecular hybridization with appropriate radioactive probes. Utilizing these methods the genotype of a homozygous normal fetus can be distinguished from that of a homozygote affected or a heterozygote for the sickle mutation of the beta hemoglobin chain. We have recently applied this prenatal methodology to the pregnancies of two couples from Zaire, in which each member was a proven sickle cell carrier. Fetal material was obtained in both cases by amniocentesis at the 16th week of gestation and followed by cell culture. In the first case, a 46, XX fetus, DNA (10 mcg) revealed a heterozygous sickle cell carrier genotype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromosome Analysis before Birth and its Value in Genetic Counselling

Chromosome analysis of amniotic cell cultures was achieved in 29 out of 30 consecutive patients who were referred for genetic counselling during pregnancy. Amniocentesis was performed without any apparent untoward maternal or fetal complication. The only pregnancy terminated was that of a carrier of X-linked granulomatous disease, in whom the amniotic cells showed that the fetus was male and also had Down''s syndrome (trisomy G). Chromosome analysis in the remaining 28 patients showed normal karyotypes. The interval between amniocentesis and a definitive karyotype varied from 7 to 31 (average 18·4) days.The reliability of chromosome analysis from amniotic cell culture and of fetal sex determination by means of the sex chromatin and Y-fluorescence techniques was studied further in amniotic fluid from cases of therapeutic abortion and of rhesus incompatibility. The fetal sex was correctly determined in all cases. It is concluded that antenatal diagnosis of genetic disease by amniocentesis now permits a more practical approach to genetic counselling.     

Amniotic Fluid Cells; Prenatal Sex Prediction and Culture

Sex chromatin studies were carried out on small amounts of amniotic fluid obtained by amniocentesis or from intact amniotic sacs removed at hysterotomy. Provided that satisfactory preparations were obtained the accuracy of fetal sexing was 87%. Nevertheless, in the management of a pregnancy in which there is a risk of a serious X-linked recessive disorder, repeat amniocentesis may be necessary to ensure satisfactory specimens.Of 90 samples of fluid cultured, satisfactory growth was obtained in 49; the success rate was not increased by the addition of stimulants to the culture medium. It is suggested that between the 13th and the 16th week of pregnancy is the optimum time for amniocentesis to obtain cells for culture, since sufficient cells are then present in a small volume of fluid and therapeutic abortion would still be possible once the results were available.     

The influence of androgen status on metabolic activity in the rat epididymis

Forty-two bovine fetal fluid samples (29 by transsacrosciatic amniocentesis, 2 by transischiorectal amniocentesis, 11 from slaughter-house material) were set up in tissue culture. Thirteen cultures failed due to either incubator failure, bacterial or fungal contamination, or the paucity of culturable cells. Sufficient growth for sex chromosome analysis was obtained in 29 smaples. Twenty-four of these were correctly sexed, three could not be verified and two were incorrectly sexed.Contamination of fetal fluid samples with maternal cells was found to be a major problem which could lead to the misinterpretation of results. To minimize this possibility: the first 1–2 ml of aspirated fetal fluids must be discarded; multiple cultures should be initiated from each sample; and when male cells are not observed, numerous metaphase spreads from each culture and from several passages must be analyzed.     

Maternal cell contamination in amniotic fluid samples as a consequence of the sampling technique

Maternal cell contamination in amniotic fluid samples is easily detected by in situ hybridization if the karyotype of the fetus differs from the karyotype of the mother. One out of two amniotic fluid samples appears to contain more than 20% maternal cells. Bloody samples often contain even more than 50% maternal cells. Maternal cells can also be identified on the basis of their nuclear morphology. Maternal cell contamination is regularly observed in pregnancies with an anterior placenta, whereas it is rare in posterior placenta pregnancies. The maternal cells are therefore thought to be artificially introduced into the amniotic fluid sample, as a result of placental bleeding during amniocentesis. The implications of maternal cell contamination for prenatal diagnosis using uncultured amniotic fluid samples are discussed.     

Familial inversion of chromosome No. 8: an affected child and a carrier fetus.

An infant with multiple congenital anomalies was found to have a duplication-deficiency disorder involving chromosome No. 8. The abnormality was identified as an unbalanced recombinant inherited from the mother who was a carrier of a pericentric inversion of chromosome No. 8. The inversion was observed in several members of this family, including a fetus who was diagnosed by an amniocentesis. The inverted chromosome was demonstrated only with the use of a differential staining technique, in this case, by trypsin-Giemsa banding.     

The Antepartum Assessment of Fetal Well-being

Modern obstetrics is concerned with the health and well-being of both the mother and the unborn child. During the first two thirds of this century there was a pronounced decrease in maternal mortality and morbidity related to pregnancy. More recently, using the techniques of amniocentesis, antepartum fetal heart rate testing and ultrasound, obstetricians have been able to more accurately assess the health of a fetus. This knowledge has resulted in decreased perinatal mortality and morbidity and, when rigorously applied, a decrease in fetal loss.     

Periodic health examination, 1996 update: 1. Prenatal screening for and diagnosis of Down syndrome. Canadian Task Force on the Periodic Health Examination.

OBJECTIVE: To make recommendations to physicians providing prenatal care on (1) whether prenatal screening for and diagnosis of Down syndrome (DS) is advisable and (2) alternative screening and diagnosis manoeuvres. OPTIONS: "Triple-marker" screening of maternal serum levels of alpha-fetoprotein, human chorionic gonadotropin and unconjugated estriol; fetal ultrasonographic examination; amniocentesis; and chorionic villus sampling (CVS). OUTCOMES: Accuracy of detection of DS in fetuses, and risks to the mother, including psychologic distress, and to the fetus from the screening and diagnostic interventions. EVIDENCE: A MEDLINE search for relevant articles published from Jan. 1, 1966, to Mar. 31, 1994, with the use of MeSH terms "Down syndrome," "prenatal diagnosis," "screening," "prevention," "amniocentesis," "chorionic villus sampling," "ultrasonography," "anxiety," "depression" and "psychological stress" and a manual search of bibliographies, recent issues of key journals and Current Contents. VALUES: The evidence-based methods and values of the Canadian Task Force on the Periodic Health Examination were used. A high value was placed on providing pregnant women with the opportunity to determine whether they are carrying a fetus with DS and to make choices concerning the termination of the pregnancy. The economic issues involved are complex and were not considered. BENEFITS, HARMS AND COSTS: Triple-marker screening identifies an estimated 58% of fetuses with DS, but it has an estimated rate of true-positive results of 0.1% and of false-positive results of 3.7% (given a risk cut-off of one chance in 190 of DS). These rates vary with maternal age and the risk cut-off chosen. Women with a known risk of having a fetus with DS (e.g., those who have had a previous child with DS) may benefit from a reduction in anxiety after confirmation that their fetus does not have DS. Screening allows women at low risk of having a child with DS to detect fetuses with the syndrome, but may cause psychologic distress if there is a false-positive screening test result. Up to 20% of women with positive results of screening tests may decline to undergo a subsequent amniocentesis. Amniocentesis and CVS are very accurate in diagnosing DS in fetuses and have a very low rate of serious complications for the mother. Amniocentesis is associated with a 1.7% rate of fetal loss when it is performed after 16 weeks'' gestation, whereas the rate among controls is 0.7% (for a difference of 1%, 95% confidence interval 0.3% to 1.5%). CVS entails a greater risk of fetal loss than amniocentesis (odds ratio 1.32, 95% confidence interval 1.11 to 1.57). There is little evidence from controlled trials of significant associations between amniocentesis or CVS and neonatal morbidity or malformations; however, samples have been too small to show differences in rare outcomes. Results from some case-control studies suggest that CVS increases the risk of transverse limb deficiency. Costs were not considered because they are beyond the scope of this review. RECOMMENDATIONS: There is fair evidence to offer triple-marker screening through a comprehensive program to pregnant women under 35 years of age (grade B recommendation). Women given detailed information about serum-marker screening show more satisfaction with the screening than those not given this information. There is fair evidence to offer amniocentesis or CVS to pregnant women 35 years of age and older and to women with a history of a fetus with DS or of a chromosome 21 anomaly (grade B recommendation). Information on the limitations and advantages of each procedure should be offered. Triple-marker screening may be offered as an alternative to CVS or amniocentesis to pregnant women over 35. VALIDATION: Recommendations concerning prenatal diagnosis are similar to those of the US Preventive Services Task Force, the Society of Obstetricians and Gynaecologists of Canada, the Canadian College of Medical Geneticists and the Cochrane Pregnancy and Childbirth Group. No previous specific recommendations concerning triple-maker screening exist. SPONSORS: These guidelines were developed and endorsed by the Canadian Task Force on the Periodic Health Examination, which is funded by Health Canada and the National Health Research and Development Program.     

The effect of different iron compounds on transferrin receptor expression in term human cytotrophoblast cells

During pregnancy, the mother is faced with an increased food demand. A good example of this increased demand is iron (Fe). Fe is needed in all growing cells. During pregnancy, the Fe transport to the fetus increases enormously. This amount can easily induce an Fe deficiency in the mother. Fe suppletion is very important for her, but not for the Fe status of the fetus, which is protected against Fe toxicity as well as deficiency. The placenta seems to be autonomous in Fe uptake. Likely there is a regulation mechanism. The human placenta is hemomonochorial. The cell layer of the fetus in contact with the maternal blood is formed by syncytiotrophoblasts. Fe is transported to the placenta by transferrin. Transferrin binds to a transferrin receptor on the trophoblast membrane and is internalized via an endocytic pathway. During this cycle, Fe is released from transferrin and the transferrin-transferrin receptor complex is recycled to the membrane. Isolated trophoblast cells from term placentas form a syncytium in vitro, and transferrin receptors are expressed. Expression depends on the number of cells in culture, culture time, the amount of Fe available, and the Fe compound. By regulation of the number of transferrin receptors, trophoblasts are able to control their Fe uptake.     

Contribution of amniocentesis to prenatal screening of neural tube closing defects: evaluation of 10 years of a center's experience

The authors report retrospective data on analysis of amniotic fluid DFTN markers (AFP and AChE) from 306 cases. Data were obtained from 261 amniocentesis done because there was a recurrence risk of DFTN and from 45 amniocentesis done because an anomaly as DFTN was diagnosed with ultrasonography. Results first show that the risk of recurrence is 3.03% in Rh?ne-Alpes area. In utero exposure to valproate appears as new indication for amniocentesis, in view of the possible association between Spina-Bifida and prenatal-valproate exposure (1/8 in the study). In contrast to anencephaly, Spina Bifida can be difficult to diagnose with ultrasonography before 20 weeks and require amniocentesis with AFP and AChE study.     

Mating between two balanced translocation carriers in two unrelated families

Partial trisomy 9p and a 13/14 translocation occurred in the daughter of a t(5;9)(p15;p12) mother and a t(13;14)(p11;q11) father. Two additional offspring displayed a normal karyotype and a translocation trisomy 13 respectively. Two first cousins, selected for chromosome analysis because of a spontaneous abortion, were found to have an identical translocation t(14;21)(p11;q11). Their second pregnancy was monitored by midtrimester amniocentesis and disclosed a balanced fetus. The different zygotic chromosome constitutions and the counselling problems in the marriages between two balanced translocation carriers are discussed.     

Use of genetic markers to certify fetal origin of cultured amniotic fluid cells

Summary Phenotypes of five polymorphic enzymes: red cell acid phosphatase, phosphoglucomutase, esterase D, adenosine deaminase, and 6-phosphogluconate dehydrogenase were determined in extracts of 24 amniotic fluid cell cultures and in the corresponding maternal red cells. Twenty-one of the 24 fetus/mother pairs can be distinguished by at least one of the markers. Thus, polymorphic enzyme markers may be useful in affirmation of fetal origin of cultured cells and to avoid possible diagnostic errors.Work supported by USPHS grants GM 15253 and AI 12617     

MATURATION OF THE RAT FETAL THYROID

Maturation of the rat fetal thyroid was studied with the aid of I¹³¹ and of fluorescence and electron microscopy. The I¹³¹ concentration of the fetal gland increased exponentially from day 17 to day 20 of gestation and was related to the weight of the fetus (and presumably the weight of the thyroid) and also to the quantity of I¹³¹ accumulated by the fetus. In the 17-day gland, thyroglobulin or immunologically similar material was sparsely present in the incipient lumens of some cell clusters. With maturation, this material increased and was also observed within follicular cells on days 18 to 19 of gestation. On day 20, the specifically reacting material was present in the follicular lumens and was absent from the cytoplasm of follicular epithelium. Ultrastructurally, the earliest thyroid cells examined were replete with all the organelles found in the more mature epithelium. No direct correlation could be made between the cytoplasmic structures and the presence of thyroglobulin, although the granular endoplasmic reticulum was most likely the organelle responsible for synthesis of thyroglobulin. Thyroglobulin or a precursor was found in fetal thyroid cells before measurable quantities of I¹³¹ were concentrated and before cytoplasmic droplets appeared.     

Prenatal diagnosis of cystic fibrosis using linked DNA markers and microvillar intestinal enzyme analysis

Summary Prenatal dignosis was performed for 47 pregnancies with 1 in 4 risk of cystic fibrosis, including 7 cases analyzed with linked DNA markers, 16 cases analyzed by microvillar intestinal enzyme testing, and 24 cases where both methods of testing were attempted. DNA was obtained by chorionic villus sampling in 10 cases and by amniocentesis in 21 cases, and diagnosis was based on analysis with the tightly linked DNA markers D7S8 and met. DNA analysis using these probes was fully informative in 74.4% of 90 couples with 1 in 4 risk. In 18 cases where both DNA results and microvillar intestinal enzyme data were diagnostic, there was agreement regarding the predicted status of the fetus. No adequate diagnosis was achieved in two cases where both diagnostic tests were attempted. Ourcome is known for 24 pregnancies including 10 where DNA analysis was diagnostic, and no errors in diagnosis were detected. Prenatal diagnosis of cystic fibrosis using DNA markers is highly informative and accurate, but microvillar intestinal enzyme analysis remains a valuable part of a complete diagnostic program.     

Microarray analysis of cell-free fetal DNA in amniotic fluid: a prenatal molecular karyotype

Metaphase karyotype analysis of fetal cells obtained by amniocentesis or chorionic villus sampling is the current standard for prenatal cytogenetic diagnosis, particularly for the detection of trisomy 21. We previously demonstrated that large quantities of cell-free fetal DNA (cffDNA) are easily extracted from amniotic fluid (AF). In this study, we explored potential clinical applications of AF cffDNA by testing its ability to hybridize to DNA microarrays for comparative genomic hybridization (CGH) analysis. cffDNA isolated from 11 male fetuses showed significantly increased hybridization signals on SRY and decreased signals on X-chromosome markers, compared with female reference DNA. cffDNA isolated from six female fetuses showed the reverse when compared with male reference DNA. cffDNA from three fetuses with trisomy 21 had increased hybridization signals on the majority of the chromosome 21 markers, and cffDNA from a fetus with monosomy X (Turner syndrome) had decreased hybridization signals on most X-chromosome markers, compared with euploid female reference DNA. These results indicate that cffDNA extracted from AF can be analyzed using CGH microarrays to correctly identify fetal sex and aneuploidy. This technology facilitates rapid screening of samples for whole-chromosome changes and may augment standard karyotyping techniques by providing additional molecular information.     

Familial Down's syndrome.

A family is reported in which the same mother conceived two children with trisomy 21. The pregnancy with the second affected child was interrupted after diagnostic amniocentesis. Maternal chromosome analysis was normal. This family and those previously reported suggest that there is an increased recurrence risk of trisomy 21 after the birth of an affected individual, possibly caused by a genetic tendency for non-disjunction. After the birth of a child with Down's syndrome, amniocentesis and chromosome analysis of cultured amniotic fluid cells is indicated in each further pregnancy, irrespective of maternal age.     

Estimation of Fermentation Biomass Concentration by Measuring Culture Fluorescence

The fluorescence of a fermentation culture was studied for its application as an estimator of biomass concentration. The measurement was obtained by irradiating the culture with ultraviolet light (366 nm) through a glass window and detecting fluorescent light at the window surface at 460 nm. It was estimated that over one-half of the fluorescent material was intercellular reduced nicotinamide adenine dinucleotide, with the remainder being reduced nicotinamide adenine dinucleotide phosphate and other unidentified intercellular and extracellular fluorophores. The culture fluorescence was found to be a function of biomass concentration, together with environmental factors, which presumably act at the cellular metabolic level to modify intercellular reduced nicotinamide adenine dinucleotide pools (e.g., dissolved oxygen tension, energy substrate concentration, and inhibitors). When these environmental conditions were controlled, a linear relationship was obtained between the log of the biomass concentration and the log of the fluorescence. Under these conditions, this relationship has considerable potential as a method to provide real-time biomass concentration estimates for process control and optimization since the fluorescence data is obtained on line. When environmental conditions are variable, the fluorescence data may be a sensitive index of overall culture activity because of its dependence on intercellular reduced nicotinamide adenine dinucleotide reserves and metabolic rates. This index may provide information about the period of maximum specific productivity for a specific microbial product.     

Carrier detection and prenatal molecular diagnosis in a Duchenne muscular dystrophy family without any affected relative available.

In this paper we report a family where the affected DMD patients were not available for study and a molecular strategy was used for female carriers detection and for prenatal diagnosis. Linkage analysis was performed with two markers within the DMD gene, in all family members screened. DMD markers used (pERT87.8/Taq1 and pERT87.15/Xmn1) seemed not to be informative because the propositas mother (II-2) was homozygous for the minor allele at each marker (T2 and X2), however, the proposita and one sister carried only the major allele, which was inherited from the father. These results suggested that a deletion involving both markers could be present, and was inherited from the mother to both daughters. Quantitative multiplex PCR confirmed the deletion in female carriers, involving at least exons 12 to 17. DNA studies of cultured amniotic fluid cells at 14 weeks gestation, by amplification of specific Y-chromosome sequences, followed by multiplex PCR, lead to the diagnosis of a male fetus affected by DMD.     

Distribution of chitin in the yeast cell wall. An ultrastructural and chemical study

The distribution of chitin in Saccharomyces cervisiae primary septa and cell walls was studied with three methods: electron microscopy of colloidal gold particles coated either with wheat germ agglutinin or with one of two different chitinases, fluorescence microscopy with fluorescein isothiocyanate derivatives of the same markers, and enzymatic treatments of [14C]glucosamine-labeled cells. The septa were uniformly and heavily labeled with the gold-attached markers, an indication that chitin was evenly distributed throughout. To study the localization of chitin in lateral walls, alkali-extracted cell ghosts were used. Observations by electron and fluorescence microscopy suggest that lectin-binding material is uniformly distributed over the whole cell ghost wall. This material also appears to be chitin, on the basis of the analysis of the products obtained after treatment of 14C-labeled cell ghosts with lytic enzymes. The chitin of lateral walls can be specifically removed by treatment with beta-(1 leads to 6)-glucanase containing a slight amount of chitinase. During this incubation approximately 7% of the total radioactivity is solubilized, about the same amount liberated when lateral walls of cell ghosts are completely digested with snail glucanase yield primary septa. It is concluded that the remaining chitin, i.e., greater than 90% of the total, is in the septa. The facilitation of chitin removal from the cell wall by beta-(1 leads to 6)-glucanase indicates a strong association between chitin and beta-(1 leads to 6)-glucan. Covalent linkages between the two polysaccharides were not detected but cannot be excluded.