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    Diane Van Opstal

    ObjectivePlacental cytogenetic studies may reveal the origin of discordant noninvasive prenatal testing (NIPT). We performed placental studies to elucidate discordances between NIPT showing a structural chromosome aberration and the fetus... more
    ObjectivePlacental cytogenetic studies may reveal the origin of discordant noninvasive prenatal testing (NIPT). We performed placental studies to elucidate discordances between NIPT showing a structural chromosome aberration and the fetus having a different chromosome aberration in three cases.MethodDiagnostic testing with genomic SNP microarray was performed in three cases with NIPT showing a duplication on 4q (case 1), a terminal deletion of 13q (case 2), and a terminal deletion of 15q (case 3). Placental studies involved SNP array analysis of cytotrophoblast and mesenchymal core of chorionic villi of four placental quadrants. Clinical follow‐up was performed as well.ResultsAmniotic fluid revealed a different structural chromosome aberration than predicted by NIPT: a terminal 2q deletion (case 1), a segmental uniparental isodisomy of 13q (case 2), and a terminal duplication of 15q and of 13q (case 3). Placental studies revealed the aberration detected with NIPT in the cytotrophobl...
    Milder ovarian stimulation for in-vitro fertilization reduces aneuploidy in the human preimplantation
    Oesophageal atresia (OA) with or without tracheoesophageal fistula (TOF) are rare anatomical congenital malformations whose cause is unknown in over 90% of patients. A genetic background is suggested, and among the reported genetic... more
    Oesophageal atresia (OA) with or without tracheoesophageal fistula (TOF) are rare anatomical congenital malformations whose cause is unknown in over 90% of patients. A genetic background is suggested, and among the reported genetic defects are copy number variations (CNVs). We hypothesized that CNVs contribute to OA/TOF development. Quantifying their prevalence could aid in genetic diagnosis and clinical care strategies. Therefore, we profiled 375 patients in a combined Dutch, American and German cohort via genomic microarray and compared the CNV profiles with their unaffected parents and published control cohorts. We identified 167 rare CNVs containing genes (frequency<0.0005 in our in-house cohort). Eight rare CNVs - in six patients - were de novo, including one CNV previously associated with oesophageal disease. (hg19 chr7:g.(143820444_143839360)_(159119486_159138663)del) 1.55% of isolated OA/TOF patients and 1.62% of patients with additional congenital anomalies had de novo C...
    ABSTRACT Fluorescence in situ hybridization (FISH) on uncultured amniotic fluid (AF) cells is a widespread technique for the rapid prenatal detection of specific chromosome aberrations. During a 6-year period (1993-1998) we used FISH for... more
    ABSTRACT Fluorescence in situ hybridization (FISH) on uncultured amniotic fluid (AF) cells is a widespread technique for the rapid prenatal detection of specific chromosome aberrations. During a 6-year period (1993-1998) we used FISH for quick follow-up investigations in uncultured AF cells after finding an uncertain chromosome aberration in a first chorionic villus (CV) or AF sample in 79 cases. These FISH results were compared with conventional cytogenetic results of the AF cell cultures in all cases. We found discrepant FISH and cytogenetic results in four instances. In general, FISH on uncultured AF cells proved to be a reliable technique for the rapid differentiation between confined placental mosaicism and true fetal mosaicism, and between pseudomosaicism and true mosaicism, respectively. Uncultured cells may sometimes even better reflect chromosomal mosaicism than cultured cells, since they are not subject to culture induced selection mechanisms. However, we found evidence that exceptional cases of tissue confined mosaicism may go undetected in uncultured cells.
    In the population of children born after prenatal cytogenetic investigation in chorionic villi at our department from 1992 to 1995 (N = 3940), three are known to us with uniparental disomy. One case of maternal heterodisomy 16 was... more
    In the population of children born after prenatal cytogenetic investigation in chorionic villi at our department from 1992 to 1995 (N = 3940), three are known to us with uniparental disomy. One case of maternal heterodisomy 16 was prenatally discovered because of trisomy 16 in direct chorionic villi with subsequently normal amniotic fluid cells. The other two had normal karyotypes in chorionic villi. Maternal heterodisomy 15 was postnatally detected in one of them because of Prader-Willi syndrome. Maternal hetero/isodisomy 16 was accidentally encountered in the other case in the course of prenatal DNA analysis of the tuberous sclerosis complex 2 region at 16p13.3. A model is presented for the understanding of the various combinations of karyotypes in direct chorionic villi, cultured chorionic villi and the fetus in the case of successful and unsuccessful trisomic zygote rescue.
    Chromosomal abnormalities are an important cause of multiple congenital anomalies (MCA). However, conventional cytogenetic analysis using culture is unsuccessful in 10% to 40% of the cases. The purpose of this study was to examine if... more
    Chromosomal abnormalities are an important cause of multiple congenital anomalies (MCA). However, conventional cytogenetic analysis using culture is unsuccessful in 10% to 40% of the cases. The purpose of this study was to examine if retrospective chromosomal analysis was possible on paraffin-embedded autopsy material with new techniques, including comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH). We investigated 92 patients, including 71 patients with MCA, 17 patients with an isolated congenital anomaly, and 4 normal controls, by conventional CGH analysis and/or FISH. The karyotype was known in 52 cases, of which 26 patients were normal and 26 had chromosomal anomalies. Comparative genomic hybridization or FISH confirmed all but 2 cases, which were not interpretable. In 40 patients the karyotype was unknown but could be analyzed successfully in 36 cases (90%) by CGH. In this series, we found 1 additional chromosomal aberration, 45,X (Turner syndr...
    Objective: Investigation of the normal frequency of tetraploid metaphases in semidirect (STC) and cultured (LTC) chorionic villi. Methods: Fifty metaphases in STC- and in LTC-villi slides of 100 women of advanced maternal age were... more
    Objective: Investigation of the normal frequency of tetraploid metaphases in semidirect (STC) and cultured (LTC) chorionic villi. Methods: Fifty metaphases in STC- and in LTC-villi slides of 100 women of advanced maternal age were screened for tetraploidy. Results: Up to three tetraploid metaphases were encountered in 27% of the STC-villi preparations; the scores fitted a Poisson distribution. In all LTC-villi preparations tetraploid cells were seen; the scores fitted a log-Gaussian distribution. Conclusions: On the basis of these distributions, we propose a protocol for the management of tetraploid metaphases in chorionic villi, strongly reducing the number of prenatal follow-up investigations.
    The occurrence of nasopharyngeal teratomas (NPT) is an infrequent event and prenatal detection of such tumors is even rarer. We present a case report and review of the literature (N = 78 cases), in which we describe the cytogenetic, DNA,... more
    The occurrence of nasopharyngeal teratomas (NPT) is an infrequent event and prenatal detection of such tumors is even rarer. We present a case report and review of the literature (N = 78 cases), in which we describe the cytogenetic, DNA, and pathological findings of a fetus with a mature NPT which was detected prenatally by ultrasound investigation following complaints of severe polyhydramnios by the mother.
    We report on a prenatally detected case of ring chromosome 18 [46,XX,r(18)] in amniotic fluid cells of a fetus with an abnormal facial profile on ultrasound as the only malformation. The chromosome 18 origin of the ring chromosome, of a... more
    We report on a prenatally detected case of ring chromosome 18 [46,XX,r(18)] in amniotic fluid cells of a fetus with an abnormal facial profile on ultrasound as the only malformation. The chromosome 18 origin of the ring chromosome, of a supernumerary marker chromosome in some cells, and of micronuclei was demonstrated by fluorescent in situ hybridization with a whole chromosome 18 paint (Cambio) and 18 centromere probe L1.84. DNA investigations showed deletions of 18p as well as 18q material of r(18), which turned out to be of paternal origin. Autopsy of the fetus after termination of pregnancy at 20 weeks of gestation showed no additional malformations, in agreement with the previous ultrasound findings.
    To evaluate preferences and decision-making amongst high-risk pregnant women offered a choice between Non-Invasive Prenatal Testing (NIPT), invasive testing or no further testing. Nationwide implementation study (TRIDENT) offering NIPT as... more
    To evaluate preferences and decision-making amongst high-risk pregnant women offered a choice between Non-Invasive Prenatal Testing (NIPT), invasive testing or no further testing. Nationwide implementation study (TRIDENT) offering NIPT as contingent screening test for women at increased risk for fetal aneuploidy based on first-trimester combined testing (>1:200) or medical history. A questionnaire was completed after counseling assessing knowledge, attitudes and participation following the Multidimensional Measure of Informed Choice. 1091/1253 (87%) women completed the questionnaire. Of these, 1053 (96.5%) underwent NIPT, 37 (3.4%) invasive testing and 1 (0.1%) declined testing. 91.7% preferred NIPT because of test safety. 77.9% made an informed choice, 89.8% had sufficient knowledge and 90.5% had positive attitudes towards NIPT. Women with intermediate (Odds Ratio(OR) = 3.51[1.70-7.22],p < 0.001) or high educational level (OR = 4.36[2.22-8.54],p < 0.001) and women with ade...
    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 chori-onic villi (CV), some false negative results will have a... more
    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 chori-onic villi (CV), some false negative results will have a biological origin. Based on our experi-ence 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 sam...
    Prenatal diagnostics has been impacted by technological changes in the past decade, which have affected the diagnostic yield. The aim of this study was to evaluate the impact of SNP array and noninvasive prenatal testing (NIPT) on the... more
    Prenatal diagnostics has been impacted by technological changes in the past decade, which have affected the diagnostic yield. The aim of this study was to evaluate the impact of SNP array and noninvasive prenatal testing (NIPT) on the diagnostic yield and the number of invasive tests in our center. The frequency of pathogenic fetal unbalanced chromosome aberrations was studied in 10,005 cases referred for prenatal testing in 2009-2015. Chromosomal SNP microarray analysis replaced karyotyping in all invasively tested pregnancies and since 2014 a choice between NIPT and diagnostic testing with microarray was offered to women with an increased risk for common aneuploidy. The introduction of microarray led to an additional yield of submicroscopic pathogenic chromosome aberrations: 3.6% in fetuses with ultrasound anomalies and 1.9% in fetuses without ultrasound anomalies. The introduction of NIPT led to a decrease of invasive tests and of diagnostic yield. Moreover, a diagnostic delay in...
    To establish the frequency of pathogenic submicroscopic chromosome aberrations in fetuses that are not at increased risk for unbalanced structural chromosome aberrations, a systematic literature search was performed. The aim was to... more
    To establish the frequency of pathogenic submicroscopic chromosome aberrations in fetuses that are not at increased risk for unbalanced structural chromosome aberrations, a systematic literature search was performed. The aim was to determine whether high resolution testing for submicroscopic aberrations is beneficial in a general pregnant population. On 3rd June 2016 Embase and PubMed databases were systematically searched for all relevant articles on prevalence of pathogenic submicroscopic CNVs in fetuses tested due to advanced maternal age or parental anxiety. Relevant full text articles were analyzed and based on the extracted data the prevalence of submicroscopic CNVs was calculated. Meta-analysis was conducted in a pooled cohort of 10,614 fetuses based on the 10 largest studies (N > 300) of a total of 19 relevant studies. In 0.84%, 95%CI [0.55%, 1.30%] of fetuses a submicroscopic pathogenic aberration was detected prenatally. The onset/penetrance of the submicroscopic findin...
    Whole genome array testing not only provides an increased diagnostic yield of pathogenic causative findings, but it may also reveal so called susceptibility loci (SL) for neurodevelopmental disorders. The goal of this study was to... more
    Whole genome array testing not only provides an increased diagnostic yield of pathogenic causative findings, but it may also reveal so called susceptibility loci (SL) for neurodevelopmental disorders. The goal of this study was to evaluate the pregnancy outcomes in SL cases and to establish a protocol for pregnancy management, follow-up and additional investigations. Fifty seven cases were evaluated: 34 with and 23 without ultrasound anomalies at referral. Each pregnant couple received pretest counseling and extensive posttest genetic counseling. After diagnosis of SL, parental testing and an additional ultrasound examination were offered. The severity of the ultrasound anomalies and not the diagnosis of SL was the most important factor contributing to the decision on pregnancy continuation. In the majority of cases with milder or no ultrasound anomalies, the pregnancy was continued and a normal outcome after birth was observed. The diagnosis of a SL did not seem to be a reason for ...
    Since non-invasive prenatal testing (NIPT) in maternal blood became available, we evaluated which chromosome aberrations found in our cohort of fetuses with an enlarged NT in the first trimester of pregnancy (tested with SNP microarray)... more
    Since non-invasive prenatal testing (NIPT) in maternal blood became available, we evaluated which chromosome aberrations found in our cohort of fetuses with an enlarged NT in the first trimester of pregnancy (tested with SNP microarray) could be detected by NIPT as well. 362 fetuses were referred for cytogenetic testing due to an enlarged NT (≥3.5 mm). Chromosome aberrations were investigated using QF-PCR, karyotyping and whole genome SNP array. After invasive testing a chromosomal abnormality was detected in 137/362 (38 %) fetuses. 100/362 (28 %) cases concerned trisomy 21, 18 or 13, 25/362 (7 %) an aneuploidy of sex chromosomes and 3/362 (0.8 %) triploidy. In 6/362 (1.6 %) a pathogenic structural unbalanced chromosome aberration was seen and in 3/362 (0.8 %) a susceptibility locus for neurodevelopmental disorders was found. We estimated that in 2-10 % of fetuses with enlarged NT a chromosome aberration would be missed by current NIPT approaches. Based on our cohort of fetuses with...
    The aim of this study was to evaluate whether unexpected diagnoses (UD) made by prenatal array testing contribute to pregnancy management. In 2010-2015 in 19/4043 (0.5%) pregnancies an UD was made. The clinical usefulness of UDs was... more
    The aim of this study was to evaluate whether unexpected diagnoses (UD) made by prenatal array testing contribute to pregnancy management. In 2010-2015 in 19/4043 (0.5%) pregnancies an UD was made. The clinical usefulness of UDs was assessed based on the couple's responses during post-test counseling and their decisions. In 16/19 cases, the UD was helpful either for the couples in making a decision about the course of their pregnancy, for perinatal management or family genetic counseling. The majority of the pregnant couples found the UDs relevant for pregnancy management and genetic counseling. This adds another motive for offering whole genome array during pregnancy in patients who wish broad testing of their fetus.
    ... Malgorzata Srebniak*, Petra Noomen, Pedro dos Santos, Dicky Halley, Raoul van de Graaf, Lutgarde Govaerts, Cokkie Wouters, Robert-Jan Galjaard and ... Chorion villi sampling (CVS) was performed and both short-term cultured (STC) and... more
    ... Malgorzata Srebniak*, Petra Noomen, Pedro dos Santos, Dicky Halley, Raoul van de Graaf, Lutgarde Govaerts, Cokkie Wouters, Robert-Jan Galjaard and ... Chorion villi sampling (CVS) was performed and both short-term cultured (STC) and long-term cultured (LTC) villi were ...
    500 women with multiple pregnancies underwent amniocentesis or chorionic villus (CV) sampling at our department between January 1988 and July 1997. The aim of this retrospective study was to evaluate the laboratory aspects and the... more
    500 women with multiple pregnancies underwent amniocentesis or chorionic villus (CV) sampling at our department between January 1988 and July 1997. The aim of this retrospective study was to evaluate the laboratory aspects and the consequences of discordant results in these pregnancies in relation to the method of sampling. Uncertain results in one or both samples, requiring further investigation were more frequent in CV samples (eight times in 163 paired samples, 5 per cent) than in amniotic fluid (AF) samples (once in 298 paired samples, 0.3 per cent). Sampling one fetus twice (erroneous sampling) was seen only once among 163 pregnancies with two CV samples in our study. Cross contamination due to mixed sampling was discovered in two of seven pregnancies that underwent DNA diagnosis in CV and might be a rather regular occuring phenomenon. In none of the 500 pregnancies mixed sampling caused diagnostic dilemmas. A third sampling problem, maternal cell contamination caused a diagnostic problem once among the AF samples. Selective fetal reduction appeared safer after CV sampling than after amniocentesis. Subsequently, CV sampling instead of amniocentesis has become the method of choice for prenatal diagnosis in multiple pregnancies in our department.

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