An inactivating mutation in the histone deacetylase SIRT6 causes human perinatal lethality.

It has been well established that histone and DNA modifications are critical to maintaining the equilibrium between pluripotency and differentiation during early embryogenesis. Mutations in key regulators of DNA methylation have shown that the balance between gene regulation and function is critical during neural development in early years of life. However, there have been no identified cases linking epigenetic regulators to aberrant human development and fetal demise. Here, we demonstrate that a homozygous inactivating mutation in the histone deacetylase SIRT6 results in severe congenital anomalies and perinatal lethality in four affected fetuses. In vitro, the amino acid change at Asp63 to a histidine results in virtually complete loss of H3K9 deacetylase and demyristoylase functions. Functionally, SIRT6 D63H mouse embryonic stem cells (mESCs) fail to repress pluripotent gene expression, direct targets of SIRT6, and exhibit an even more severe phenotype than Sirt6-deficient ESCs when differentiated into embryoid bodies (EBs). When terminally differentiated toward cardiomyocyte lineage, D63H mutant mESCs maintain expression of pluripotent genes and fail to form functional cardiomyocyte foci. Last, human induced pluripotent stem cells (iPSCs) derived from D63H homozygous fetuses fail to differentiate into EBs, functional cardiomyocytes, and neural progenitor cells due to a failure to repress pluripotent genes. Altogether, our study described a germline mutation in SIRT6 as a cause for fetal demise, defining SIRT6 as a key factor in human development and identifying the first mutation in a chromatin factor behind a human syndrome of perinatal lethality.

Clinical impact of additional findings detected by genome-wide non-invasive prenatal testing: Follow-up results of the TRIDENT-2 study.

In the TRIDENT-2 study, all pregnant women in the Netherlands are offered genome-wide non-invasive prenatal testing (GW-NIPT) with a choice of receiving either full screening or screening solely for common trisomies. Previous data showed that GW-NIPT can reliably detect common trisomies in the general obstetric population and that this test can also detect other chromosomal abnormalities (additional findings). However, evidence regarding the clinical impact of screening for additional findings is lacking. Therefore, we present follow-up results of the TRIDENT-2 study to determine this clinical impact based on the laboratory and perinatal outcomes of cases with additional findings. Between April 2017 and April 2019, additional findings were detected in 402/110,739 pregnancies (0.36%). For 358 cases, the origin was proven to be either fetal (n = 79; 22.1%), (assumed) confined placental mosaicism (CPM) (n = 189; 52.8%), or maternal (n = 90; 25.1%). For the remaining 44 (10.9%), the origin of the aberration could not be determined. Most fetal chromosomal aberrations were pathogenic and associated with severe clinical phenotypes (61/79; 77.2%). For CPM cases, occurrence of pre-eclampsia (8.5% [16/189] vs 0.5% [754/159,924]; RR 18.5), and birth weight <2.3rd percentile (13.6% [24/177] vs 2.5% [3,892/155,491]; RR 5.5) were significantly increased compared to the general obstetric population. Of the 90 maternal findings, 12 (13.3%) were malignancies and 32 (35.6%) (mosaic) pathogenic copy number variants, mostly associated with mild or no clinical phenotypes. Data from this large cohort study provide crucial information for deciding if and how to implement GW-NIPT in screening programs. Additionally, these data can inform the challenging interpretation, counseling, and follow-up of additional findings.

TRIDENT-2: National Implementation of Genome-wide Non-invasive Prenatal Testing as a First-Tier Screening Test in the Netherlands.

The Netherlands launched a nationwide implementation study on non-invasive prenatal testing (NIPT) as a first-tier test offered to all pregnant women. This started on April 1, 2017 as the TRIDENT-2 study, licensed by the Dutch Ministry of Health. In the first year, NIPT was performed in 73,239 pregnancies (42% of all pregnancies), 7,239 (4%) chose first-trimester combined testing, and 54% did not participate. The number of trisomies 21 (239, 0.33%), 18 (49, 0.07%), and 13 (55, 0.08%) found in this study is comparable to earlier studies, but the Positive Predictive Values (PPV)-96% for trisomy 21, 98% for trisomy 18, and 53% for trisomy 13-were higher than expected. Findings other than trisomy 21, 18, or 13 were reported on request of the pregnant women; 78% of women chose to have these reported. The number of additional findings was 207 (0.36%); these included other trisomies (101, 0.18%, PPV 6%, many of the remaining 94% of cases are likely confined placental mosaics and possibly clinically significant), structural chromosomal aberrations (95, 0.16%, PPV 32%,) and complex abnormal profiles indicative of maternal malignancies (11, 0.02%, PPV 64%). The implementation of genome-wide NIPT is under debate because the benefits of detecting other fetal chromosomal aberrations must be balanced against the risks of discordant positives, parental anxiety, and a potential increase in (invasive) diagnostic procedures. Our first-year data, including clinical data and laboratory follow-up data, will fuel this debate. Furthermore, we describe how NIPT can successfully be embedded into a national screening program with a single chain for prenatal care including counseling, testing, and follow-up.

Increased nuchal translucency before 11 weeks of gestation: Reason for referral?

OBJECTIVES: In this era of non-invasive-prenatal testing (NIPT), when dating scans are usually performed around 10 weeks of gestation, an increased NT before the official established timeframe (CRL between 45 and 84 mm) may be encountered. Information on management of these pregnancies is limited. Therefore, we evaluated the relationship between an early increased NT and adverse pregnancy outcome. Secondary, we evaluated the rate of chromosomal anomalies that might have been missed in first trimester should solely NIPT be performed as first-tier test, and the rate of adverse pregnancy outcome if NT normalizes before 14 weeks. METHODS: We performed a retrospective cohort study that included all pregnancies between January 1, 2007 and June 1, 2020 in Amsterdam UMC locations AMC and VUmc. We included fetuses with a crown-rump length (CRL) < 45 mm (∼11 weeks) and a nuchal translucency (NT) measurement ≥2.5 mm. Fetuses referred with an early increased NT and a major fetal anomaly at the dating scan were excluded, as were cases of parents with a family history of monogenetic disease(s) or recognized carriers of a balanced translocation. RESULTS: We included 120 fetuses of which 66.7% (80/120) had an adverse pregnancy outcome. Congenital anomalies were present in 56.7% (68/120), 45.8% (55/120) had a chromosomal anomaly. The prevalence of congenital anomalies was 30.3% in fetuses with NT 2.5-3.4 mm compared to 66.7% with NT ≥ 3.5 mm (p < 0.001). 16.7% (20/120) had a chromosomal anomaly that might have been missed by conventional NIPT in first trimester. We found an adverse pregnancy outcome of 24% in the group with a normalized NT compared to 78.1% in the group with a persistently increased NT (p < 0.001). CONCLUSION: An early increased NT should make the sonographer alert. In this selected cohort, an early increased NT was associated with a high probability of having an adverse pregnancy outcome. Regardless of CRL, we deem that an early increased NT ≥ 3.5 mm warrants referral to a Fetal Medicine Unit for an extensive work-up. NT normalization seems favorable, but a prospective study should define the appropriate work-up for NT in the lower range (2.5-3.4 mm).

Rapid whole exome sequencing in pregnancies to identify the underlying genetic cause in fetuses with congenital anomalies detected by ultrasound imaging.

OBJECTIVE: The purpose of this study was to explore the diagnostic yield and clinical utility of trio-based rapid whole exome sequencing (rWES) in pregnancies of fetuses with a wide range of congenital anomalies detected by ultrasound imaging. METHODS: In this observational study, we analyzed the first 54 cases referred to our laboratory for prenatal rWES to support clinical decision making, after the sonographic detection of fetal congenital anomalies. The most common identified congenital anomalies were skeletal dysplasia (n = 20), multiple major fetal congenital anomalies (n = 17) and intracerebral structural anomalies (n = 7). RESULTS: A conclusive diagnosis was identified in 18 of the 54 cases (33%). Pathogenic variants were detected most often in fetuses with skeletal dysplasia (n = 11) followed by fetuses with multiple major fetal congenital anomalies (n = 4) and intracerebral structural anomalies (n = 3). A survey, completed by the physicians for 37 of 54 cases, indicated that the rWES results impacted clinical decision making in 68% of cases. CONCLUSIONS: These results suggest that rWES improves prenatal diagnosis of fetuses with congenital anomalies, and has an important impact on prenatal and peripartum parental and clinical decision making.

Mosaic maternal 10qter deletions are associated with FRA10B expansions and may cause false-positive noninvasive prenatal screening results.

PURPOSE: Using genome-wide noninvasive prenatal screening (NIPS), we detected a 20-megabase specific deletion starting at 10q25 in eight pregnancies. The deletion could not be confirmed by invasive testing. Since all 10(q25→qter) deletions started close to the FRA10B fragile site in 10q25, we investigated whether the pregnant women were indeed carriers of FRA10B. METHODS: We performed NIPS analysis for all autosomes using single-read sequencing. Analysis was done with the WISECONDOR algorithm. Culture of blood lymphocytes with bromodeoxyuridine was used to detect FRA10B expansions. Fluorescence in situ hybridization and array analysis were used to find maternal and/or fetal deletions. RESULTS: We confirmed the presence of a FRA10B expansion in all four tested mothers. Fluorescence in situ hybridization and array analysis confirmed the presence of a maternal mosaic deletion of 10(q25→qter). CONCLUSION: The recurring 10(q25→qter) deletion detected with NIPS is a false-positive result caused by a maternal low-level mosaic deletion associated with FRA10B expansions. This has important consequences for clinical follow-up, as invasive procedures are unnecessary. Expanded maternal FRA10B repeats should be added to the growing group of variants in the maternal genome that may cause false-positive NIPS results.

Origin and clinical relevance of chromosomal aberrations other than the common trisomies detected by genome-wide NIPS: results of the TRIDENT study.

PurposeNoninvasive prenatal screening (NIPS) using cell-free DNA in maternal blood is highly sensitive for detecting fetal trisomies 21, 18, and 13. Using a genome-wide approach, other chromosome anomalies can also be detected. We report on the origin, frequency, and clinical significance of these other chromosome aberrations found in pregnancies at risk for trisomy 21, 18, or 13.MethodsWhole-genome shallow massively parallel sequencing was used and all autosomes were analyzed.ResultsIn 78 of 2,527 cases (3.1%) NIPS was indicative of trisomy 21, 18, or 13, and in 41 (1.6%) of other chromosome aberrations. The latter were of fetal (n = 10), placental (n = 22), maternal (n = 1) or unknown (n = 7). One case lacked cytogenetic follow-up. Nine of the 10 fetal cases were associated with an abnormal phenotype. Thirteen of the 22 (59%) placental aberrations were associated with fetal congenital anomalies and/or poor fetal growth (

Factors for successful implementation of population-based expanded carrier screening: learning from existing initiatives.

Background: Carrier screening for autosomal recessive disorders aims to facilitate reproductive decision-making by identifying couples with a 1-in-4 risk in every pregnancy of having an affected child. Except for a few countries or regions, carrier screening is not widely offered and is mostly ancestry-based. Technological advances enable carrier screening for multiple diseases simultaneously allowing universal screening regardless of ancestry (population-based expanded carrier screening). It is important to study how this can be successfully implemented. This study therefore aims to identify critical factors involved in successful implementation, from a user perspective, by learning from already implemented initiatives. Methods: Factors associated with successful implementation were identified by: (i) a literature review and (ii) two case studies; studying experiences with carrier screening in two high-risk communities (a Dutch founder population and the Ashkenazi Jewish population), including a survey among community members. Results: Factors identified were familiarity with (specific) genetic diseases and its availability, high perceived benefits of screening (e.g. screening avoids much suffering), acceptance of reproductive options, perceived risk of being a carrier and low perceived social barriers (e.g. stigmatization). In contrast to the Jewish community, the initial demand for screening in the Dutch founder population did not entirely come from the community itself. However, the large social cohesion of the community facilitated the implementation process. Conclusion: To ensure successful implementation of population-based expanded carrier screening, efforts should be made to increase knowledge about genetic diseases, create awareness and address personal benefits of screening in a non-directive way.

Women's Experience with Non-Invasive Prenatal Testing and Emotional Well-being and Satisfaction after Test-Results.

Increasingly, high-risk pregnant women opt for non-invasive prenatal testing (NIPT) instead of invasive diagnostic testing. Since NIPT is less accurate than invasive testing, a normal NIPT result might leave women less reassured. A questionnaire study was performed among pregnant women with elevated risk for fetal aneuploidy based on first-trimester combined test (risk ≥1:200) or medical history, who were offered NIPT in the nationwide Dutch TRIDENT study. Pre- and post-test questionnaires (n = 682) included measures on: experiences with NIPT procedure, feelings of reassurance, anxiety (State-Trait Anxiety Inventory, STAI), child-related anxiety (PRAQ-R), and satisfaction. The majority (96.1%) were glad to have been offered NIPT. Most (68.5%) perceived the waiting time for NIPT results (mean: 15 days, range 5-32) as (much) too long. Most women with a normal NIPT result felt reassured (80.9%) or somewhat reassured (15.7%). Levels of anxiety and child-related anxiety were significantly lower after receiving a normal NIPT result as compared to the moment of intake (p < 0.001). Women with inadequate health literacy or a medical history (e.g. previous child with trisomy) experienced significantly higher post-test-result anxiety (Mean (M) STAI = 31.6 and 30.0, respectively) compared to those with adequate health literacy (M = 28.6) and no medical history (M = 28.6), indicating these women might benefit from extra information and/or guidance when communicating NIPT test-results. Introducing NIPT as an alternative to invasive testing, led to an offer that satisfied and largely reassured high-risk pregnant women.