Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21

The trials performed worldwide toward noninvasive prenatal diagnosis (NIPD) of Down's syndrome (or trisomy 21) have shown the commercial and medical potential of NIPD compared to the currently used invasive prenatal diagnostic procedures. Extensive investigation of methylation differences between the mother and the fetus has led to the identification of differentially methylated regions (DMRs). In this study, we present a strategy using the methylated DNA immunoprecipitation (MeDiP) methodology in combination with real-time quantitative PCR (qPCR) to achieve fetal chromosome dosage assessment, which can be performed noninvasively through the analysis of fetal-specific DMRs. We achieved noninvasive prenatal detection of trisomy 21 by determining the methylation ratio of normal and trisomy 21 cases for each tested fetal-specific DMR present in maternal peripheral blood, followed by further statistical analysis. The application of this fetal-specific methylation ratio approach provided correct diagnosis of 14 trisomy 21 and 26 normal cases.     

Ring chromosome 21 in a normal female

In the present paper we report a phenotypically normal woman with a ring chromosome 21 in her karyotype, who sought genetic counseling because of her previous reproductive failure and whose third, prenatally diagnosed, pregnancy resulted in the delivery of a healthy and karyotypically normal boy.     

Correlations between pregnancy pathology, study of the placenta, antenatal echography and examination of the product of conception in a series of 175 congenital malformations

In order to verify the hypothesis that during pregnancy in a woman without peculiar history, signs could be discovered when the fetus is malformed we have reviewed the files of 175 women who had a malformed child and of 300 controls. All of these women had at least one clinical examination and one ultrasonographic examination during pregnancy. Two clinical symptoms were more often discovered in the mother of the malformed fetus (p less than 0.001): decrease of fetal movements and small for date fetus. The placenta is never abnormal in the mother with normal fetus. Placenta is abnormal in 31% of the mother with malformed fetus but the abnormalities are not specific. Ultrasonographic examinations allowed more often the discovery of a malformation when hydramnios (p less than 0.001) or fetal hypotrophy (p less than 0.01) or an anomaly of the morphology of the fetus is discovered. Accuracy of prenatal diagnostic is considered for the different categories of congenital malformations.     

Second prenatal diagnosis in a familial form of male pseudohermaphroditism caused by 17 keto-reductase deficiency: prediction confirmed by a normal third male infant

In the first child of this family, the diagnosis of male pseudo-hermaphroditism due to 17 keto-reductase deficiency was established at two months of age after HCG test. During the second pregnancy, amniocentesis was performed for fetal karyotype and steroid determination in the amniotic fluid: an affected male fetus was suspected and this prediction was confirmed at birth. For the third pregnancy, a prenatal diagnosis was requested again and made, according to the same procedure: a normal male fetus was predicted and this diagnosis was confirmed at birth; this study demonstrates the feasibility and reliability of a prenatal diagnosis for 17 keto-reductase deficiency.     

Prenatal diagnosis of genetic disorders of the skin by means of electron microscopy

Summary This paper summarizes the ultrastructural aspects of prenatal diagnosis of inherited skin disorders by means of electron microscopy on fetal skin biopsies obtained under fetoscopy. The investigation comprises skin samples of a series of 26 fetoscopies performed at the Departments of Gynecology at the Universities of Giessen and Lund. Among them, seven fetoscopies were performed for prenatal diagnosis in pregnancies at risk of epidermolysis bullosa or ichthyosis during the 19th and 22nd week of gestational age. Positive prenatal diagnosis was made in one fetus at risk of epidermolysis bullosa of unknown genetic type; this was based on dermolytic blister formation and collagenolysis diagnostic of EB dystrophica Hallopeau-Siemens. In a pregnancy with a fetal risk of bullous congenital ichthyosiform erythroderma, positive prenatal diagnosis was based on cytolysis and tonofilament clumping in the fetal epidermis and verified clinically and ultrastructurally after interruption; in another pregnancy bullous ECI was excluded ultrastructurally by the normal ultrastructural constitution of the fetal epithelium. In three fetuses at risk of the Herlitz syndrome and in one at risk of epidermolysis bullosa atrophicans inversa with pronounced sites of predilection, prenatal exclusion of the disorder was possible; this was based on the ultrastructural demonstration of the regular presence of normal hemidesmosomes with well-developed sub-basal dense plates at the dermo-epidermal junction in all four cases. One of these children has since been born and has normal skin; no scarring was found after birth on the sites of fetal skin biopsies.Possible application of this kind of prenatal diagnosis to other groups of genetic disorders is discussed in which biochemical defects are still unknown. Even more important is the possibility of excluding genetic disorders by demonstration of normal ultrastructural features in fetal skin biopsies and so avoiding abortion of healthy children.Awarded the Hans-Nachtsheim-Preis 1981In cooperation with R. Rauskolb and V. Jovanovic, Zentrum für Frauenheilkunde der Universität Giessen, 6300 Giessen, Federal Republic of Germany, and B. Gustavii, E. Cordesius, and L. Löfberg, Department of Gynecology, University of Lund, Sweden     

Evaluation of 100 autopsies performed in the maternity service of the H?tel-Dieu in Lyon during an 18 month period

100 Necropsies have been performed from January 1983 to June 1984, on 53 abortus and stillborn and 47 therapeutic terminations of pregnancy. All fetuses came from the same obstetric unit. Half spontaneous fetal deaths remained of unknown aetiology; in 18 cases (34%) placental, maternal or pregnancy pathology existed; fetal abnormalities were discovered in 10 (18%). As for therapeutic interruptions of pregnancy (the indications of which are detailed) the importance of ultrasonography emphasized since this technique allowed 25 of the 47 prenatal diagnosis. The importance of necropsy to help precise diagnosis and subsequent counselling is also recalled.     

Prenatal diagnosis of chromosomic defects in Costa Rica

This is an historical overview of prenatal cytogenetic diagnosis in Costa Rica. It started in 1984 at the Institute for Health Research of the University of Costa Rica. This is the only fetal cytogenetic diagnosis facility in the country and serves social security as well as private patients. Perinatologists send amniotic fluid and fetal blood samples from high risk pregnancies, mainly due to abnormal ultrasound assessment, sonographic markers of aneuploidy and advanced maternal age. Second and third trimester diagnosis allows the development of coping strategies for the families of affected fetuses, since pregnancy interruption is not permitted. Normal fetal cytogenetic results provide reassurance to the parents.     

Rapid detection of trisomies 21 and 18 and sexing by quantitative fluorescent multiplex PCR

Aneuploidies involving chromosomes 21, 18, 13, X and Y account for over 95% of all chromosomal abnormalities in live-born infants. Prenatal diagnosis of these disorders is usually accomplished by cytogenetic analysis of amniotic or chorionic cells but this is a lengthy procedure requiring great technical expertise.In this paper, we assess the diagnostic value of using a quantitative fluorescent polymerase chain reaction (PCR) suitable for the simultaneous and rapid diagnosis of trisomies 21 and 18 together with the detection of DNA sequences derived from the X and Y chromosomes. Samples of DNA, extracted from amniotic fluid, fetal blood or tissues, and peripheral blood from normal adults were investigated by quantitative fluorescent PCR amplification of polymorphic small tandem repeats (STRs) specific for two loci on each of chromosomes 21 and 18. Quantitative analysis of the amplification products allowed the diagnosis of trisomies 21 and 18, while sexing was performed simultaneously using PCR amplification of DNA sequences derived from the chromosomes X and Y. These results indicate the advantages of using two sets of STR markers for the detection of chromosome 21 trisomies and confirmed the usefulness of quantitative fluorescent multiplex PCR for the rapid prenatal diagnosis of selected chromosomal abnormalities. Received: 23 January 1996 / Revised: 21 February 1996     

Prenatal diagnosis of spinal muscular atrophy in Macedonian families

Spinal muscular atrophy (SMA) is the second most common lethal autosomal recessive disorder of childhood, affecting approximately 1 in 6,000-10,000 births, with a carrier frequency of 1 in 40-60. There is no effective cure or treatment for this disease. Thus, the availability of prenatal testing is important. The aim of this study was to establish an efficient and rapid method for prenatal diagnosis of SMA and genetic counseling in families with risk for having a child with SMA. In this paper we present the results from prenatal diagnosis in Macedonian SMA families using direct analysis of fetal DNA. The probands of these families were previously found to be homozygous for a deletion of exons 7 and 8 of SMN1 gene. DNA obtained from chorionic villas samples and amniocytes was analyzed for deletions in SMN gene. SMN exon 7 and 8 deletion analysis was performed by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). Of the 12 prenatal diagnoses, DNA analysis showed normal results in eight fetuses. Four of the fetuses were homozygote for a deletion of exons 7 and 8 of SMN1. After genetic counseling, the parents of the eight normal fetuses decided to continue the pregnancy, while in the four families with affected fetuses, the pregnancy was terminated. The results were confirmed after birth.     

Prenatal diagnosis of de novo unbalanced translocation 8p;21q using subtelomeric probes

We report an unbalanced translocation involving chromosomes 8 and 21 in a fetus showing ultrasonographic abnormalities in the second trimester of pregnancy. A 41-year-old pregnant woman, gravida 1 para 0, was referred to the Genetics Clinic at the 16th week of gestation because of advanced maternal age and fetal pelvicaliectasis on ultrasonographic examination. Pregnancy had occurred following ICSI treatment. After genetic counseling amniocentesis was performed. Fetal karyotype analysis revealed a 46,XY,8p+ karyotype. Ultrasonographic examination was repeated at the 20th week of gestation and showed intrauterine growth retardation, ventriculomegaly, cerebellar structural abnormality and pelvicaliectasis. Chromosomes of both parents were normal. Molecular cytogenetic studies (FISH) using chromosome-specific subtelomere probes showed a terminal deletion of 8p and trisomy of the 21q subtelomeric region. Further analysis with Down Syndrome specific region probes revealed two signals. The couple decided to terminate the pregnancy. This is the first prenatally diagnosed case of unbalanced t(8p;21q) of de novo origin.     

Down syndrome: interaction between culture, demography, and biology in determining the prevalence of a genetic trait

The incidence of Down syndrome (DS) at conception is highly dependent upon the maternal age distribution and age-specific pregnancy rates. Live-birth prevalence of DS reflects these factors and fetal deaths. Since the introduction of prenatal diagnosis in the early 1970s, the role of fetal deaths in the equation has increased. Between 1920 and the early 1980s, DS live-birth prevalence decreased in many populations due to declining fertility rates, particularly among older women. In the late-1970s the trend reversed, as the median age of populations and birth rates among older women steadily increased. This paper illustrates these interactions using data we have analyzed for New York State (NYS) and comparative data obtained from the literature. Between 1983 and 1997 DS live-birth prevalence in NYS remained stable at about 9.9 per 10,000 live births. The number of prenatal tests performed increased by 158%, and the number of DS fetuses detected prenatally more than quadrupled. Fertility rates of women aged 35-49 continued to increase. The proportion of DS cases born to these older mothers increased from 23% in 1985 to 43% in 1997. We estimated that without prenatal diagnosis, DS live-birth prevalence would have been 17.0 per 10,000 live births by 1995. Cultural factors influence demographic trends, birthing technologies, physician practices, and women's decision-making regarding prenatal screening and diagnosis for DS.     

Corticoid formation by the monkey fetal adrenal: Evaluation of 17-, 21- and 11-hydroxylase activities

There is indirect evidence that cortisol synthesis in the fetal rhesus monkey adrenal gland is limited at Day 135 of gestation but increases thereafter. This study was conducted to ascertain whether a reduced synthetic capacity is caused by a deficiency in 17-, 21- or 11-hydroxylase activity. For the sake of comparison 11- and 21-hydroxylases were also estimated in adult adrenals. 11-, 21-Hydroxylases were measured in the entire adrenal by the oxidation of NADPH by mitochondria and microsomes, respectively. 17-Hydroxylase was evaluated in outer and inner regions of the fetal gland by the formation of [³H]17-hydroxyprogesterone, -11-deoxycortisol, -cortisol and -androstenedione from [³H]progesterone. The maximum velocity of both the 11- and 21-hydroxylase was similar in fetal and adult glands indicating that corticoid formation in the fetus is not constrained by levels of these enzymes.[³H]Progesterone was extensively metabolized to -17-hydroxyprogesterone, -androstenedione, -11-deoxycortisol and -cortisol by homogenates from both regions of the fetal adrenal. The ratio of [³H]-cortisol to [³H]11-deoxycortisol was consistently higher in incubations of the inner glandular area. Together, these findings indicate that 17-hydroxylase is also active at Day 135 and that the 11-hydroxylase may be more concentrated in the fetal cortex. These data suggest in addition that the restriction in cortisol formation occurs at a step prior to the metabolism of progesterone to cortisol.     

Prenatal diagnosis of Sanfilippo disease type B

Summary The prenatal diagnosis of a fetus affected with Sanfilippo disease type B is described. The deficiency of -N-acetylglucosaminidase in the cultured amniotic fluid cells was shown by a microassay enabling early prenatal diagnosis. In addition an increased level of heparan sulphate was demonstrated in the amniotic fluid by two-dimensional electrophoresis of glycosaminoglycans. The latter result confirmed the value of this test as an adjunctive method in the prenatal diagnosis. The pregnancy was terminated and the prenatal diagnosis was confirmed by enzyme analysis of cultured fetal fibroblasts and fetal liver.     

Probing the fetal genome: progress in non-invasive prenatal diagnosis

Progress in our understanding of the molecular basis of heritable diseases, through identification of specific mutations, has provided a foundation for the development of DNA-based prenatal diagnosis. Genetic analysis of fetal DNA is now routinely performed from chorionic villus samples obtained as early as the tenth week of gestation or by amniocentesis from week 15 onwards. However, both of these approaches involve invasive procedures with increased risk of fetal loss. To avoid such complications, attempts have been made to develop non-invasive tests through the identification, characterization and isolation of fetal cells or free fetal DNA from the maternal circulation. Recently, progress has been made towards the development of novel strategies that are expected to provide non-invasive means for early prenatal diagnosis in pregnancy.     

Vulnerability of placental antibody transfer and fetal complement synthesis to disturbance of the pregnant monkey

Maternal and fetal/infant antibody levels were assessed across pregnancy and at birth to evaluate the prenatal transmission of IgG in the rhesus monkey. Although some antibody was evident in the fetus by midpregnancy, the marked increase in IgG occurred primarily during the last two weeks of pregnancy. This delay until the end of pregnancy would result in low antibody titers in premature infants. In contrast, when gestation length was normal, the placental transfer of IgG was resistant to both dexamethasone treatment and a prolonged period of stress during pregnancy. This resiliency occurred despite an effect of prenatal stress on other aspects of infant development, including physical growth and the fetal synthesis of complement proteins.     

Expression of ras oncogene p21 during human fetal development as determined by monoclonal antibodies RAP-5, Y13-259, and DWP

In this report we describe the expression of the ras proto-oncogene p21 protein in various tissues during normal fetal development. Conventional, formalin fixed and paraffin-embedded sections of normal organs were examined from fetuses ranging 9 to 42 weeks of gestation. Immunohistochemical localization of ras p21 was accomplished using the broadly reactive, mouse monoclonal antibodies RAP-5 and Y13-259. The monoclonal antibody DWP, which is specific for a mutated form of ras p21 having a valine/cysteine at amino acid position 12, was also used. Detectable expression of the p21 protein was seen at different time periods during fetal development depending on the tissue. The expression of ras p21 (as detected by RAP-5 and Y13-259) was noted in a wide range of cell types and tissues; intense immunostaining was noted in epithelial cells of the gastrointestinal tract, exocrine and endocrine pancreas, renal tubules and transitional urotheliem, as well as in other tissues. This immunostaining generally, but not invariably, corresponded with patterns previously reported in benign and/or malignant neoplasms of adult tissues. In most instances ras p21 expression, when present, occurred during periods of rapid growth in given organ systems. However, some actively proliferating fetal tissues such as thymus and spleen, failed to express detectable ras p21 suggesting that factors other than cell cycle may influence its expression. No reactivity with DWP was noted in any of the tissues, suggesting that the mutated forms detected by this monoclonal antibody are not expressed during normal human embryogenesis. These data show that there is regulated expression, and broad distribution of this gene product in normal developing human fetal tissue.     

Contribution of ultrasonographic examination to the prenatal detection of trisomy 21: experience from 19 European registers

The objective of this study was to evaluate the contribution of ultrasound scanning to the prenatal detection of trisomy 21 in a large unselected European population. Data from 19 congenital malformation registers in 11 European countries were included. The prenatal ultrasound screening programs in the countries ranged from no routine screening to three ultrasound investigations per patient. Routine serum screening was offered in four of the 11 countries and routine screening on the basis of maternal age amniocentesis in all. The results show that overall 53% of cases of trisomy 21 were detected prenatally with a range from 3% in Lithuania to 88% in Paris. Ninety-eight percent of women whose babies were diagnosed before 24 weeks gestation chose to terminate the pregnancy. Centres/countries that offer serum screening do not have a significantly higher detection rate of trisomy 21 when compared to those that offer maternal age amniocentesis and anomaly scanning only. Fifty percent of trisomy 21 cases were born to women aged 35 years or more. In conclusions, second trimester ultrasound plays an important role in the prenatal diagnosis of trisomy 21. Of those cases prenatally diagnosed, 64% of cases in women <35 years and 36% of those in women >or=35 years were detected because of an ultrasound finding. Ultrasound soft markers accounted for 84% of the scan diagnoses. There is evidence of increasing maternal age across Europe with 50% of cases of trisomy 21 born to women aged 35 years or more.     

False Negative NIPT Results: Risk Figures for Chromosomes 13, 18 and 21 Based on Chorionic Villi Results in 5967 Cases and Literature Review

Non-invasive prenatal testing (NIPT) demonstrated a small chance for a false negative result. Since the “fetal” DNA in maternal blood originates from the cytotrophoblast of chorionic villi (CV), some false negative results will have a biological origin. Based on our experience with cytogenetic studies of CV, we tried to estimate this risk. 5967 CV samples of pregnancies at high risk for common aneuplodies were cytogenetically investigated in our centre between January 2000 and December 2011. All cases of fetal trisomy 13, 18 and 21 were retrospectively studied for the presence of a normal karyotype or mosaicism < 30% in short-term cultured (STC-) villi. 404 cases of trisomies 13, 18 and 21 were found amongst 5967 samples (6,8%). Of these 404 cases, 14 (3,7%) had a normal or low mosaic karyotype in STC-villi and therefore would potentially be missed with NIPT. It involved 2% (5/242) of all trisomy 21 cases and 7.3% (9/123) of all trisomy 18 cases. In 1:426 (14/5967) NIPT samples of patients at high risk for common aneuploidies, a trisomy 18 or 21 will potentially be missed due to the biological phenomenon of absence of the chromosome aberration in the cytotrophoblast.     

Prenatal diagnosis of trisomy 21: registration results from a single genetic center

This is a retrospective review of all collected amniotic fluid samples, chorionic villus samples and other fluid-aspirations (hygroma colli fluid/urine from megacystis) over an 11-year period (1996-2006) in a single Genetic Center (University Hospital Gasthuisberg, Leuven), looking at the prenatal diagnosis of trisomy 21. In this study a total of 404 diagnoses of trisomy 21 were made on 29696 samples (1.4%). The prenatal diagnosis of trisomy 21 increased over the years with 0.88% (21/2363) in 1996 and 1.99% (50/2512)in 2006. Also the type of invasive testing changed over the years with an increase of the proportion of trisomy 21- diagnoses by chorionic villussampling from 2001. Looking at the registry for perinatal activities in Flanders for the year 2006 the live birth incidence for trisomy 21 was 1/1782 and this is lower than the often reported incidence oftrisomy 21 at birth of 1/800: it is likely that the use of more sensitive screening methods for the prenatal detection of trisomy 21 and the election of termination for most affected pregnancies affects the birth incidence oftrisomy 21.