Genomic futures of prenatal screening: ethical reflection.

The practice of prenatal screening is undergoing important changes as a result of the introduction of genomic testing technologies at different stages of the screening trajectory. It is expected that eventually it will become possible to routinely obtain a comprehensive 'genome scan' of all fetuses. Although this will still take several years, there are clear continuities between present developments and this future scenario. As this review shows, behind the still limited scope of screening for common aneuploidies, a rapid widening of the range of conditions tested for is already taking shape at the invasive testing stage. But the continuities are not just technical; they are also ethical. If screening for Down's syndrome is a matter of providing autonomous reproductive choice, then why would providing the choice to have a full fetal genome scan be something entirely different? There is a clear need for a sustainable normative framework that will have to answer three challenges: the indeterminateness of the autonomy paradigm, the need to acknowledge the future child as an interested stakeholder, and the prospect of broad-scope genomic prenatal screening with a double purpose: autonomy and prevention.

Unexplained False Negative Results in Noninvasive Prenatal Testing: Two Cases Involving Trisomies 13 and 18.

Noninvasive prenatal testing (NIPT) validation studies show high sensitivity and specificity for detection of trisomies 13, 18, and 21. False negative cases have rarely been reported. We describe a false negative case of trisomy 13 and another of trisomy 18 in which NIPT was commercially marketed directly to the clinician. Both cases came to our attention because a fetal anatomy scan at 20 weeks of gestation revealed multiple anomalies. Karyotyping of cultured amniocytes showed nonmosaic trisomies 13 and 18, respectively. Cytogenetic investigation of cytotrophoblast cells from multiple placental biopsies showed a low proportion of nontrisomic cells in each case, but this was considered too small for explaining the false negative NIPT result. The discordant results also could not be explained by early gestational age, elevated maternal weight, a vanishing twin, or suboptimal storage or transport of samples. The root cause of the discrepancies could, therefore, not be identified. The couples involved experienced difficulties in accepting the unexpected and late-adverse outcome of their pregnancy. We recommend that all parties involved in caring for couples who choose NIPT should collaborate to clarify false negative results in order to unravel possible biological causes and to improve the process of patient care from initial counseling to communication of the result.

Analysis of false-positive results of fetal RHD typing in a national screening program reveals vanishing twins as potential cause for discrepancy.

OBJECTIVES: We aim to elucidate causes of false-positive fetal RHD screening results obtained with cell-free DNA. METHODS: Fetal RHD screening was performed in 32,222 samples from RhD-negative women by multiplex real-time PCR in triplicate for RHD exons 5 and 7 using cell-free DNA isolated from maternal plasma obtained in the 27th gestational week. PCR results were compared with cord blood serology in 25,789 pregnancies (80.04%). False-positive cases were analyzed. Known biological causes (RHD variant genes), technical causes of discordance, and errors around blood sampling were investigated with leukocyte DNA from maternal and cord blood, and cell-free DNA from stored maternal plasma. RESULTS: Not only RHD but also Y-chromosome (DYS14) sequences were present in four plasma samples from RHD-negative women bearing an RHD-negative girl. Sample mix-up and other sampling errors could be excluded in three samples. CONCLUSIONS: These results indicate that false-positive fetal RHD screening results can be caused by cell-free DNA fragments in maternal plasma derived from a third cell line that is not representative for either the maternal genome or the genome of the vital fetus. We propose that remaining (cyto)trophoblasts of a vanishing twin are the underlying mechanism, and we estimate a frequency of this phenomenon of 0.6%.

Detection of fetal chromosomal anomalies: does nuchal translucency measurement have added value in the era of non-invasive prenatal testing?

OBJECTIVES: The objective of this study is to determine what percentage of fetal chromosomal anomalies remains undetected when first trimester combined testing is replaced by non-invasive prenatal testing for trisomies 13, 18, and 21. We focused on the added clinical value of nuchal translucency (NT) measurement. METHODS: Data on fetal karyotype, ultrasound findings, and pregnancy outcome of all pregnancies with an NT measurement ≥3.5 mm were retrospectively collected from a cohort of 25,057 singleton pregnancies in which first trimester combined testing was performed. RESULTS: Two hundred twenty-five fetuses (0.9 %) had an NT ≥3.5 mm. In 24 of these pregnancies, a chromosomal anomaly other than trisomy 13, 18, or 21 was detected. Eleven resulted in fetal demise, and ten showed fetal ultrasound anomalies. In three fetuses with normal ultrasound findings, a chromosomal anomaly was detected, of which one was a triple X. CONCLUSIONS: In three out of 25,057 pregnancies (0.01%), non-invasive prenatal testing and fetal ultrasound would have missed a chromosomal anomaly that would have been identified by NT measurement. © 2015 John Wiley & Sons, Ltd.

Noninvasive fetal genotyping of human platelet antigen-1a.

We describe a reliable noninvasive fetal human platelet antigen (HPA)-1a genotyping assay on a real-time polymerase chain reaction (PCR) platform using cell-free fetal DNA isolated from maternal blood. Nonspecific amplification of maternal cell-free DNA is overcome by pre-PCR digestion of the cell-free DNA with the Msp1 restriction enzyme. Noninvasive fetal HPA-1a genotyping offers a safe method for alloimmunised pregnant women to determine whether their fetus is at risk of fetal or neonatal alloimmune thrombocytopenia (FNAIT) and whether interventions to prevent intracranial haemorrhage are required. The availability of this test is relevant to the ongoing debate on screening pregnancies for HPA-1a-mediated FNAIT.

Noninvasive fetal blood group genotyping of rhesus D, c, E and of K in alloimmunised pregnant women: evaluation of a 7-year clinical experience.

OBJECTIVE: To evaluate the diagnostic performance of noninvasive fetal blood group genotyping. DESIGN: Descriptive analysis. SETTING: Dutch national reference laboratory for pregnancies complicated by alloimmunisation. POPULATION: All consecutive alloimmunised pregnant women for whom fetal blood group genotyping of rhesus D, c, E or of K in maternal plasma was performed from 2003 up to 2010. METHODS: The test results of each individual assay were collected. Real-time polymerase chain reaction was performed for RHD exon 5 and RHD exon 7, or the specific allele of the RHCE or KEL gene. A stringent diagnostic algorithm was applied. In the case of a negative result, the presence of fetal DNA was ascertained by the analysis of the Y chromosome-specific SRY gene or other paternal genetic markers. Results were compared with available serology after birth or genotyping results of amniotic fluid cells. MAIN OUTCOME MEASURES: Percentage of conclusive test results and diagnostic accuracy. RESULTS: A total of 362 tests was performed (D: n = 168; c: n = 49; E: n = 85; K: n = 60). The median gestational age was 17 weeks (range 7-38 weeks). In 351 women (97%), a test result was issued: in seven samples, the presence of fetal DNA could not be confirmed; in two samples, non-specific amplification in the K assay led to an inconclusive result; in two samples, a maternal silent RHD gene prevented fetal RHD genotyping. No false-positive or false-negative results were found among those women for whom cord blood serology or genotyping results of amniotic fluid cells were available (n = 212). CONCLUSIONS: Noninvasive fetal blood group genotyping is accurate and applicable in a clinical diagnostic setting.

[Genetic cancer syndromes and reproductive choice: dialogue between parents and politicians on preimplantation genetic diagnosis]. / Erfelijke kankersyndromen en kinderwens: ouders en politiek in dialoog over pre-implantatiegenetische diagnostiek.

Genetic cancer syndromes have identical clinical severity, limited therapeutic options, reduced life expectancy, and risks of genetic transmission, as do other genetic or congenital diseases for which prenatal genetic diagnosis or preimplantation genetic diagnosis (PGD) is allowed in the Netherlands. That was implied in the certification of one Dutch PGD centre at Maastricht University Hospital by the Dutch Ministry of Health, Welfare and Sport in 2003. A report by the Health Council of the Netherlands in 2006 confirmed this view with scientific and ethical evaluation. However, in 2006 the State Secretary for Health strongly objected to PGD for cancer, and disease risks of 50-100% for gene carriers, i.e. for highly, but not always fully penetrant genes. In 2006, the Maastricht centre discontinued PGD for cancer and couples were referred to other countries; prenatal genetic diagnosis remained available, however. On 26 May 2008, the present State Secretary proposed to parliament that the Health Council of the Netherlands report from 2006 be followed. This once again clashed with the fears of some Christian parties for a slippery slope and embryo selection for 'only a risk and not certainty of disease'. Yet no firm evidence for the existence of such a slope has been found. The Dutch framework for handling the ethical and medical evaluation of new reproductive and genetic technologies by the Health Council of the Netherlands Advisory Committees, professional and patient organisations, and the Ministry, has functioned for over 30 years without leading to any wrongdoing. There is no actual need for a new government body to license genetic tests on a case-by-case or per disease basis.

[Need for blood transfusion in premature infants in 2 Dutch perinatology centres particularly determined by blood sampling for diagnosis]. / Bloedtransfusiebehoefte bij prematuren in 2 Nederlandse perinatologische centra sterk bepaald door bloedafname voor diagnostiek.

OBJECTIVE: Determination of factors related to the need for transfusion in premature infants. DESIGN: Descriptive. METHOD: The need for transfusion in premature infants was determined in 2 academic centres: University Medical Center Utrecht and Leiden University Medical Center, The Netherlands. The data had been acquired in another study. The factors under study were: hospital, pregnancy duration, birth weight, gender, time of clamping of the umbilical cord, total volume of blood sampled for diagnostic purposes, number of days of mechanical ventilation, total duration of admission and duration of the admission to the Neonatal Intensive care unit. Both hospitals followed the national interdisciplinary practice guideline 'Blood transfusion'. RESULTS: The total volume ofsampled blood for diagnosis, the duration of the mechanical ventilation and the admission period were related to a greater need for transfusion. On the other hand, the chance of transfusions diminished with longer pregnancy duration or increased birth weight. The difference in need for blood transfusion between both centres was significant. The total volume of transfused erythrocytes showed a strong correlation with the volume sampled for diagnostic procedures. CONCLUSION: Anaemia in neonates is strongly related to the amount of blood taken for diagnostic procedures. Alternatives for blood transfusions in premature infants, and consequently for the reduction of the number of donors per child, are to be sought in delayed clamping of the umbilical cord, use of erythropoietin and use ofautologous umbilical cord blood.

A prospective study on parental coping 4 months after termination of pregnancy for fetal anomalies.

OBJECTIVE: To identify short-term factors influencing psychological outcome of termination of pregnancy for fetal anomaly, in order to define those patients most vulnerable to psychopathology. STUDY DESIGN: A prospective cohort of 217 women and 169 men completed standardized questionnaires 4 months after termination. Psychological adjustment was measured by the Inventory of Complicated Grief (ICG), the Impact of Event Scale (IES), the Edinburgh Postnatal Depression Scale (EPDS), and the Symptom Checklist-90 (SCL-90). RESULTS: Women and men showed high levels of posttraumatic stress (PTS) symptoms (44 and 22%, respectively) and symptoms of depression (28 and 16%, respectively). Determinants of adverse psychological outcome were the following: high level of doubt in the decision period, inadequate partner support, low self-efficacy, lower parental age, being religious, and advanced gestational age. Whether the condition was Down syndrome or another disability was irrelevant to the outcome. Termination did not have an important effect on future reproductive intentions. Only 2% of women and less than 1% of men regretted the decision to terminate. CONCLUSION: Termination of pregnancy (TOP) for fetal anomaly affects parents deeply. Four months after termination a considerable part still suffers from posttraumatic stress symptoms and depressive feelings. Patients who are at high risk could benefit from intensified support.

Rapid detection of chromosomal aneuploidies in uncultured amniocytes by multiplex ligation-dependent probe amplification (MLPA).

OBJECTIVE: To test whether multiplex ligation-dependent probe amplification (MLPA) can be used for the detection of aneuploidy of chromosomes 13, 18, 21, X, and Y in uncultured amniocytes. METHODS: We performed a prospective study based on 527 amniotic fluid samples. Chromosome copy numbers were determined by analysing the relative amount of PCR product of chromosome-specific MLPA probes. Results were available within 48 h and were compared with those of karyotyping. RESULTS: There were 517 conclusive MLPA tests. In 514 tests, results were concordant with those of karyotyping. There were two cases of 69,XXX triploidy that could not be detected by MLPA and there was one false-positive result. Here, MLPA indicated a 47,XXY fetus, whereas the karyotype was 46,XY. We correctly identified all 23 cases of autosomal trisomy and the single case of monosomy X in samples collected from 16 up to 36 weeks of gestation. In 10 cases (2%), the result was inconclusive owing to an insufficient amount of DNA. CONCLUSION: Sensitivity, specificity, and failure rate of MLPA were comparable to those of FISH and QF-PCR. Aneuploidy screening in uncultured amniocytes by MLPA is feasible in a clinical diagnostic setting, yielding an informative and rapid result in 98% of cases.

[The advisory report 'Neonatal screening' from the Health Council of The Netherlands]. / Het advies 'Neonatale screening' van de Gezondheidsraad.

The Health Council of the Netherlands has published an advisory report on neonatal screening in view of developments in diagnostics, therapy and the prevalence of neonatal diseases. Currently it involves screening for phenylketonuria, congenital hypothyroidism and congenital adrenal hyperplasia. Because screening may lead to considerably better outcomes in affected newborns, the council recommends expanding current screening to include medium-chain acyl-CoA dehydrogenase deficiency, sickle-cell disease and 12 other rare disorders: biotinidase deficiency, galactosaemia, glutaricaciduria type I, HMG-CoA lyase deficiency, holocarboxylase-synthetase deficiency, homocystinuria, isovaleric-acidaemia, long-chain hydroxyacyl-CoA dehydrogenase deficiency, maple syrup urine disease, 3-methylcrotonyl-CoA carboxylase deficiency, tyrosinaemia I and very-long-chain acyl-CoA dehydrogenase deficiency. A better detection method for cystic fibrosis must be developed before it is included in screening to restrict the number of sweat-test referrals of unaffected newborns. The council recommends providing information on neonatal screening during pregnancy and gives special attention to the possibility of detecting carriership in the parents.

Improved prenatal detection of a fetal point mutation for achondroplasia by the use of size-fractionated circulatory DNA in maternal plasma--case report.

INTRODUCTION: The efficacious analysis of fetal loci involving point mutations from circulatory fetal DNA in maternal plasma is hindered by the preponderance of maternal DNA. It has recently been shown that the size difference between fetal and maternal DNA species can be used for the selective enrichment of circulatory fetal DNA in maternal plasma. We have now tested this approach for the detection of a fetal point mutation in the fibroblast growth factor receptor 3 (FGFR3) gene that causes achondroplasia. METHODS: Circulatory DNA was extracted from maternal plasma and size-fractionated by agarose gel electrophoresis. The fraction with a size less than 300 bp was examined by a touchdown PCR assay specific for the FGFR3 gene, and the mutation was identified by SfcI restriction analysis. RESULT: Our analysis indicated that although the fetal mutation was discernible in the analysis of total plasma DNA, the result using size-fractionated DNA was much more evident. CONCLUSION: The enrichment of circulatory fetal DNA in maternal plasma by size-fractionation facilitates the detection of subtle feto-maternal genetic differences, such as those involving point mutations. This approach can easily be extended for the non-invasive prenatal determination of other fetal loci.