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Objectives: Non-invasive prenatal testing (NIPT) allows the detection of placental chromosome aberrations. To verify whether the fetus also has the chromosome aberration, diagnostic follow-up testing is required. The aim of this retrospective study was to assess the added value of analyzing amniotic fluid (AF) cell cultures in addition to uncultured AF cells for the detection of fetal mosaicism. Method: NIPT was performed as part of the Dutch TRIDENT study. Cytogenetic studies in uncultured AF were performed using single nucleotide polymorphism (SNP)-array. Cultured AF cell colonies (in situ method) were investigated with fluorescent in situ hybridization and/or karyotyping. Clinical outcome data were collected in cases with discordant results. Results: Between April 2014 and December 2021, 368 amniocenteses were performed after a chromosomal aberration was detected with NIPT. Excluding 134 cases of common aneuploidies (confirmed by quantitative fluorescence polymerase chain reaction), 29 cases with investigation of uncultured cells only and 1 case without informed consent, 204 cases were eligible for this study. In 196 (96%) cases, the results in uncultured and cultured cells were concordant normal, abnormal or mosaic. Five cases (2%) showed mosaicism in cultured AF cells, whereas uncultured AF cells were normal. Two (1%) of these, one mosaic trisomy 13 and one mosaic trisomy 16, were considered true fetal mosaics. Conclusion: The added value of investigating AF cell cultures in addition to uncultured cells is limited to two of 204 (1%) cases in which true fetal mosaicsm would otherwise be missed. The clinical relevance of one (trisomy 13) remained unknown and the other case also showed ultrasound anomalies, which determined pregnancy management. This seems to justify limiting prenatal cytogenetic confirmatory testing to SNP arrays on uncultured AF cells, considerably shortening the reporting time.

Objective: To evaluate which cytogenetic characteristics of confined placental mosaicism (CPM) detected in the first trimester chorionic villi and/or placentas in terms of chromosome aberration, cell lineage involved and trisomy origin will lead to fetal growth restriction and low birthweight. Methods: Cohort study using routinely collected perinatal data and cytogenetic data of non-invasive prenatal testing, the first trimester chorionic villi sampling and postnatal placentas. Results: 215 CPM cases were found. Fetal growth restriction (FGR) and low birthweight below the 10 th percentile (BW < p10) were seen in 34.0% and 23.1%, respectively. Excluding cases of trisomy 16, 29.1% showed FGR and 17.9% had a BW < p10. The highest rate of FGR and BW < p10 was found in CPM type 3, but differences with type 1 and 2 were not significant. FGR and BW < p10 were significantly more often observed in cases with meiotic trisomies. Conclusion: There is an association between CPM and FGR and BW < p10. This association is not restricted to trisomy 16, neither to CPM type 3, nor to CPM involving a meiotic trisomy. Pregnancies with all CPM types and origins should be considered to be at increased risk of FGR and low BW < p10. A close prenatal fetal monitoring is indicated in all cases of CPM.

Item does not contain fulltext, BACKGROUND: About 40 disease genes have been described to date for isolated CAKUT, the most common cause of childhood CKD. However, these genes account for only 20% of cases. ARHGEF6, a guanine nucleotide exchange factor that is implicated in biologic processes such as cell migration and focal adhesion, acts downstream of integrin-linked kinase (ILK) and parvin proteins. A genetic variant of ILK that causes murine renal agenesis abrogates the interaction of ILK with a murine focal adhesion protein encoded by Parva , leading to CAKUT in mice with this variant. METHODS: To identify novel genes that, when mutated, result in CAKUT, we performed exome sequencing in an international cohort of 1265 families with CAKUT. We also assessed the effects in vitro of wild-type and mutant ARHGEF6 proteins, and the effects of Arhgef6 deficiency in mouse and frog models. RESULTS: We detected six different hemizygous variants in the gene ARHGEF6 (which is located on the X chromosome in humans) in eight individuals from six families with CAKUT. In kidney cells, overexpression of wild-type ARHGEF6 -but not proband-derived mutant ARHGEF6 -increased active levels of CDC42/RAC1, induced lamellipodia formation, and stimulated PARVA-dependent cell spreading. ARHGEF6-mutant proteins showed loss of interaction with PARVA. Three-dimensional Madin-Darby canine kidney cell cultures expressing ARHGEF6-mutant proteins exhibited reduced lumen formation and polarity defects. Arhgef6 deficiency in mouse and frog models recapitulated features of human CAKUT. CONCLUSIONS: Deleterious variants in ARHGEF6 may cause dysregulation of integrin-parvin-RAC1/CDC42 signaling, thereby leading to X-linked CAKUT.

Karin EM Diderich, Jasmijn E Klapwijk, Vyne van der Schoot, Hennie T Brüggenwirth, Marieke Joosten, Malgorzata I Srebniak Department of Clinical Genetics, Erasmus MC, Rotterdam, the NetherlandsCorrespondence: Karin EM Diderich, Department of Clinical Genetics, Erasmus MC, Wytemaweg 50, Rotterdam, the Netherlands, Tel +31 10 70 43214, Email k.diderich@erasmusmc.nlAbstract: The yield of genetic prenatal diagnosis has been notably improved by introducing whole genome chromosomal microarray (CMA) and prenatal exome sequencing (pES). However, together with increased numbers of diagnoses made, the need to manage challenging findings such as variants of unknown significance (VUS) and incidental findings (IF) also increased. We have summarized the current guidelines and recommendations and we have shown current solutions used in our tertiary center in the Netherlands. We discuss four of the most common clinical situations: fetus with normal pES results, fetus with a pathogenic finding explaining the fetal phenotype, fetus with a variant of uncertain clinical significance fitting the phenotype and fetus with a variant leading to an incidental diagnosis. Additionally, we reflect on solutions in order to facilitate genetic counseling in an NGS-era.Keywords: exome sequencing, genetic counseling, prenatal diagnosis, incidental findings, variants of unknown significance, VUS, IF

Background: Balanced chromosome aberrations are reported in about 1:30 couples with recurrent pregnancy loss (RPL). Karyotyping of both parents is necessary to identify these aberrations. Genome-wide non-invasive prenatal testing (NIPT) in case of recurrent pregnancy loss could be a more efficient way to identify couples at increased risk for carrying a balanced chromosome rearrangement. The aim of this study was to evaluate whether the potential fetal imbalances caused by parental balanced aberrations detected in our center are large enough to be detectable by genome-wide non-invasive prenatal testing (NIPT). Material and methods: From January 1970 until May 2020 our laboratory received 30,863 unique requests for karyotyping due to RPL. We have identified 16,045 couples and evaluated all abnormal cytogenetic results to assess the minimal size of the involved chromosomal segments in potential unbalanced products of the rearrangements. Results:In the presented cohort we detected 277 aberrant balanced translocations/inversions in females and 185 in males amongst 16,045 couples with RPL, which can be translated to a risk of 1:35 (2.9%, 95% CI 2.6–3.2%). Our study showed that the vast majority (98.7%, 95% CI 97.1–99.5%) of these balanced aberrations will potentially cause a fetal imbalance > 10 Mb, which is detectable with genome-wide NIPT if it was performed during one of the miscarriages. Conclusions: Our study suggests that genome-wide NIPT is able to reveal most unbalanced products of balanced chromosomal rearrangements carried by couples with RPL and therefore can potentially identify balanced chromosomal aberration carriers. Moreover, our data suggest that these couples can be offered NIPT in case they decline invasive testing in future pregnancies.

The yield of genetic prenatal diagnosis has been notably improved by introducing whole genome chromosomal microarray (CMA) and prenatal exome sequencing (pES). However, together with increased numbers of diagnoses made, the need to manage challenging findings such as variants of unknown significance (VUS) and incidental findings (IF) also increased. We have summarized the current guidelines and recommendations and we have shown current solutions used in our tertiary center in the Netherlands. We discuss four of the most common clinical situations: fetus with normal pES results, fetus with a pathogenic finding explaining the fetal phenotype, fetus with a variant of uncertain clinical significance fitting the phenotype and fetus with a variant leading to an incidental diagnosis. Additionally, we reflect on solutions in order to facilitate genetic counseling in an NGS-era.

Item does not contain fulltext, 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.

Item does not contain fulltext, PURPOSE: Genome-wide sequencing is increasingly being performed during pregnancy to identify the genetic cause of congenital anomalies. The interpretation of prenatally identified variants can be challenging and is hampered by our often limited knowledge of prenatal phenotypes. To better delineate the prenatal phenotype of Coffin-Siris syndrome (CSS), we collected clinical data from patients with a prenatal phenotype and a pathogenic variant in one of the CSS-associated genes. METHODS: Clinical data was collected through an extensive web-based survey. RESULTS: We included 44 patients with a variant in a CSS-associated gene and a prenatal phenotype; 9 of these patients have been reported before. Prenatal anomalies that were frequently observed in our cohort include hydrocephalus, agenesis of the corpus callosum, hypoplastic left heart syndrome, persistent left vena cava, diaphragmatic hernia, renal agenesis, and intrauterine growth restriction. Anal anomalies were frequently identified after birth in patients with ARID1A variants (6/14, 43%). Interestingly, pathogenic ARID1A variants were much more frequently identified in the current prenatal cohort (16/44, 36%) than in postnatal CSS cohorts (5%-9%). CONCLUSION: Our data shed new light on the prenatal phenotype of patients with pathogenic variants in CSS genes.

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.

Introduction The aim of this retrospective cohort study was to determine the potential diagnostic yield of prenatal whole exome sequencing in fetuses with structural anomalies on expert ultrasound scans and normal chromosomal microarray results. Material and methods In the period 2013-2016, 391 pregnant women with fetal ultrasound anomalies who received normal chromosomal microarray results, were referred for additional genetic counseling and opted for additional molecular testing pre- and/or postnatally. Most of the couples received only a targeted molecular test and in 159 cases (40.7%) whole exome sequencing (broad gene panels or open exome) was performed. The results of these molecular tests were evaluated retrospectively, regardless of the time of the genetic diagnosis (prenatal or postnatal). Results In 76 of 391 fetuses (19.4%, 95% CI 15.8%-23.6%) molecular testing provided a genetic diagnosis with identification of (likely) pathogenic variants. In the majority of cases (91.1%, 73/76) the (likely) pathogenic variant would be detected by prenatal whole exome sequencing analysis. Conclusions Our retrospective cohort study shows that prenatal whole exome sequencing, if offered by a clinical geneticist, in addition to chromosomal microarray, would notably increase the diagnostic yield in fetuses with ultrasound anomalies and would allow early diagnosis of a genetic disorder irrespective of the (incomplete) fetal phenotype.

Introduction: The presence of an unbalanced familial translocation can be reliably assessed in the cytotrophoblast of chorionic villi. However, carriers of a balanced translocation often decline invasive testing. This study aimed to investigate whether an unbalanced translocation can also be diagnosed in cell free DNA by whole-genome non-invasive prenatal screening (NIPS). Material and methods: Pregnant women carrying a fetus with an unbalanced familial translocation, for whom NIPS as well as microarray data were available, were included in this retrospective assessment. NIPS was performed in the course of the TRIDENT study. Results: In 12 cases, both NIPS and microarray data were available. In 10 of 12 cases the unbalanced translocation was correctly identified by NIPS without prior knowledge on parental translocation. One was missed because the fetal fraction was too low. One was missed because of technical restrictions in calling 16p gains. Conclusions: This study supports the hypothesis that routine NIPS may be used for prenatal diagnosis of unbalanced inheritance of familial translocations, especially with prior knowledge of the translocation allowing focused examination of the involved chromosomal regions. Our study showed that routine shallow sequencing designed for aneuploidy detection in cell free DNA may be sufficient for higher resolution NIPS, if specialized copy number software is used and if sufficient fetal fraction is present.

Many major structural fetal anomalies can be diagnosed by first trimester fetal anomaly scan. NIPT can accurately detect aneuploidies and large chromosomal aberrations in cfDNA in maternal blood plasma. This study shows how a patient-friendly first trimester screening for both chromosomal and structural fetal anomalies in only two outpatient visits can be provided. Genotype-first approach assures not only the earliest diagnosis of trisomy 21 (the most prevalent chromosome aberration), but also completion of the screening at 12–14 weeks. To ensure proper management and avoid unnecessary anxiety abnormal NIPT different from trisomy 21, 18 and 13 should be referred for genetic counseling.

Proteins anchored to the cell surface via glycosylphosphatidylinositol (GPI) play various key roles in the human body, particularly in development and neurogenesis. As such, many developmental disorders are caused by mutations in genes involved in the GPI biosynthesis and remodeling pathway. We describe ten unrelated families with bi-allelic mutations in PIGB, a gene that encodes phosphatidy-linositol glycan class B, which transfers the third mannose to the GPI. Ten different PIGB variants were found in these individuals. Flow cytometric analysis of blood cells and fibroblasts from the affected individuals showed decreased cell surface presence of GPI-anchored proteins. Most of the affected individuals have global developmental and/or intellectual delay, all had seizures, two had polymicrogyria, and four had a peripheral neuropathy. Eight children passed away before four years old. Two of them had a clinical diagnosis of DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures), a condition that includes sensorineural deafness, shortened terminal phalanges with small finger and toenails, intellectual disability, and seizures; this condition overlaps with the severe phenotypes associated with inherited GPI deficiency. Most individuals tested showed elevated alkaline phosphatase, which is a characteristic of the inherited GPI deficiency but not DOORS syndrome. It is notable that two severely affected individuals showed 2-oxoglutaric aciduria, which can be seen in DOORS syndrome, suggesting that severe cases of inherited GPI deficiency and DOORS syndrome might share some molecular pathway disruptions.