Lethal phenotypes in Mendelian disorders.

PURPOSE: Existing resources that characterize the essentiality status of genes are based on either proliferation assessment in human cell lines, viability evaluation in mouse knockouts, or constraint metrics derived from human population sequencing studies. Several repositories document phenotypic annotations for rare disorders; however, there is a lack of comprehensive reporting on lethal phenotypes. METHODS: We queried Online Mendelian Inheritance in Man for terms related to lethality and classified all Mendelian genes according to the earliest age of death recorded for the associated disorders, from prenatal death to no reports of premature death. We characterized the genes across these lethality categories, examined the evidence on viability from mouse models and explored how this information could be used for novel gene discovery. RESULTS: We developed the Lethal Phenotypes Portal to showcase this curated catalog of human essential genes. Differences in the mode of inheritance, physiological systems affected, and disease class were found for genes in different lethality categories, as well as discrepancies between the lethal phenotypes observed in mouse and human. CONCLUSION: We anticipate that this resource will aid clinicians in the diagnosis of early lethal conditions and assist researchers in investigating the properties that make these genes essential for human development.

Amniocentesis in pregnancies at or beyond 24 weeks: An international multicenter study.

BACKGROUND: Amniocentesis for genetic diagnosis is most commonly done between 15 and 22 weeks of gestation, but can be performed at later gestational ages. The safety and genetic diagnostic accuracy of amniocentesis have been well-established through numerous large-scale, multicenter studies for procedures before 24 weeks, but comprehensive data on late amniocentesis remain sparse. OBJECTIVES: To evaluate the indications, diagnostic yield, safety, and maternal and fetal outcomes associated with amniocentesis performed at or beyond 24 weeks of gestation. STUDY DESIGN: We conducted an international, multicenter retrospective cohort study examining pregnant individuals who underwent amniocentesis for prenatal diagnostic testing at gestational ages between 24w0d and 36w6d. The study, spanning from 2011 to 2022, involved nine referral centers. We included singleton or twin pregnancies with documented outcomes, excluding cases where other invasive procedures were performed during pregnancy or if amniocentesis was conducted for obstetric indications. We analyzed indications for late amniocentesis, types of genetic tests performed, their results, and the diagnostic yield, along with pregnancy outcomes and post-procedure complications. RESULTS: Of the 752 pregnant individuals included in our study, late amniocentesis was primarily performed for the prenatal diagnosis of structural anomalies (91.6%), followed by suspected fetal infection (2.3%) and high-risk findings from cell-free DNA screening (1.9%). The median gestational age at the time of the procedure was 28w5d, and 98.3% of pregnant individuals received results of genetic testing before birth or pregnancy termination. The diagnostic yield was 22.9%, and a diagnosis was made 2.4 times more often for fetuses with anomalies in multiple organ systems (36.4%) compared to those with anomalies in a single organ system (15.3%). Additionally, the diagnostic yield varied depending on the specific organ system involved, with the highest yield for musculoskeletal anomalies (36.7%) and hydrops fetalis (36.4%) when a single organ system or entity was affected. The most prevalent genetic diagnoses were aneuploidies (46.8%), followed by copy number variants (26.3%) and monogenic disorders (22.2%). The median gestational age at delivery was 38w3d, with an average of 59 days between the procedure and delivery date. The overall complication rate within two weeks post-procedure was 1.2%. We found no significant difference in the rate of preterm delivery between pregnant individuals undergoing amniocentesis between 24-28 weeks and those between 28-32 weeks, reinforcing the procedure's safety across these gestational periods. CONCLUSIONS: Late amniocentesis, at or after 24 weeks gestation, especially for pregnancies complicated by multiple congenital anomalies, has a high diagnostic yield and a low complication rate, underscoring its clinical utility. It provides pregnant individuals and their providers with a comprehensive diagnostic evaluation and results before delivery, enabling informed counseling and optimized perinatal and neonatal care planning.

International Society for Prenatal Diagnosis 2022 debate 3-Fetal genome sequencing should be offered to all pregnant patients.

Prenatal trio exome sequencing (ES) has become integrated into the care for pregnant women when the fetus has structural anomalies. Details regarding optimizing indications for prenatal exome sequencing, its detection rates with different categories of fetal anomalies, and principles of interpretation of pathogenicity of sequence variants are still under investigation. However, there is now growing consensus about its benefits for finding the cause of fetal structural anomalies. What is not established, is whether exome or genome sequencing (GS) has a place in the care of all pregnant women. This report is a summary of the debate on this topic at the 26th International Conference on Prenatal Diagnosis and Therapy. Both expert debaters considered the advantages and disadvantages. Advantages include the ability to diagnose serious childhood conditions without a prenatally observable phenotype, which creates the potential of early treatments. Disadvantages include difficulties with variant classification, counseling complexities, healthcare cost, and the burden on healthcare systems and families, in particular with the discovery of adult-onset disorders or variants of uncertain significance. Although both debaters weighed the balance of these conflicting arguments differently, they agreed that more research is needed to further explore the clinical utility and ethical aspects of GS for all pregnant women.

Multicenter clinical experience with non-invasive cell-free DNA screening for monosomy X and related X-chromosome variants.

OBJECTIVE: We aimed to investigate how the presence of fetal anomalies and different X chromosome variants influences Cell-free DNA (cfDNA) screening results for monosomy X. METHODS: From a multicenter retrospective survey on 673 pregnancies with prenatally suspected or confirmed Turner syndrome, we analyzed the subgroup for which prenatal cfDNA screening and karyotype results were available. A cfDNA screening result was defined as true positive (TP) when confirmatory testing showed 45,X or an X-chromosome variant. RESULTS: We had cfDNA results, karyotype, and phenotype data for 55 pregnancies. cfDNA results were high risk for monosomy X in 48/55, of which 23 were TP and 25 were false positive (FP). 32/48 high-risk cfDNA cases did not show fetal anomalies. Of these, 7 were TP. All were X-chromosome variants. All 16 fetuses with high-risk cfDNA result and ultrasound anomalies were TP. Of fetuses with abnormalities, those with 45,X more often had fetal hydrops/cystic hygroma, whereas those with "variant" karyotypes had different anomalies. CONCLUSION: Both, 45,X or X-chromosome variants can be detected after a high-risk cfDNA result for monosomy X. When there are fetal anomalies, the result is more likely a TP. In the absence of fetal anomalies, it is most often an FP or X-chromosome variant.

Cell-based Noninvasive Prenatal Testing (cbNIPT)-A Review on the Current Developments and Future Prospects.

Considering the diagnostic limitations of cfDNA-based noninvasive prenatal testing (NIPT), scientists have long been interested in isolating and analyzing rare intact fetal and trophoblast cells from maternal blood or endocervical samples to diagnose fetal genetic conditions. These cells may be scarce and difficult to isolate, but they are a direct source of pure fetal genetic material. In this review, we summarize the history of cell-based NIPT, present an updated review on its current developments, evaluate its genetic diagnostic potential, and discuss its future prospects for clinical use.

Validation Studies for Single Circulating Trophoblast Genetic Testing as a Form of Noninvasive Prenatal Diagnosis.

It has long been appreciated that genetic analysis of fetal or trophoblast cells in maternal blood could revolutionize prenatal diagnosis. We implemented a protocol for single circulating trophoblast (SCT) testing using positive selection by magnetic-activated cell sorting and single-cell low-coverage whole-genome sequencing to detect fetal aneuploidies and copy-number variants (CNVs) at ∼1 Mb resolution. In 95 validation cases, we identified on average 0.20 putative trophoblasts/mL, of which 55% were of high quality and scorable for both aneuploidy and CNVs. We emphasize the importance of analyzing individual cells because some cells are apoptotic, in S-phase, or otherwise of poor quality. When two or more high-quality trophoblast cells were available for singleton pregnancies, there was complete concordance between all trophoblasts unless there was evidence of confined placental mosaicism. SCT results were highly concordant with available clinical data from chorionic villus sampling (CVS) or amniocentesis procedures. Although determining the exact sensitivity and specificity will require more data, this study further supports the potential for SCT testing to become a diagnostic prenatal test.

Paralog Studies Augment Gene Discovery: DDX and DHX Genes.

Members of a paralogous gene family in which variation in one gene is known to cause disease are eight times more likely to also be associated with human disease. Recent studies have elucidated DHX30 and DDX3X as genes for which pathogenic variant alleles are involved in neurodevelopmental disorders. We hypothesized that variants in paralogous genes encoding members of the DExD/H-box RNA helicase superfamily might also underlie developmental delay and/or intellectual disability (DD and/or ID) disease phenotypes. Here we describe 15 unrelated individuals who have DD and/or ID, central nervous system (CNS) dysfunction, vertebral anomalies, and dysmorphic features and were found to have probably damaging variants in DExD/H-box RNA helicase genes. In addition, these individuals exhibit a variety of other tissue and organ system involvement including ocular, outer ear, hearing, cardiac, and kidney tissues. Five individuals with homozygous (one), compound-heterozygous (two), or de novo (two) missense variants in DHX37 were identified by exome sequencing. We identified ten total individuals with missense variants in three other DDX/DHX paralogs: DHX16 (four individuals), DDX54 (three individuals), and DHX34 (three individuals). Most identified variants are rare, predicted to be damaging, and occur at conserved amino acid residues. Taken together, these 15 individuals implicate the DExD/H-box helicases in both dominantly and recessively inherited neurodevelopmental phenotypes and highlight the potential for more than one disease mechanism underlying these disorders.

Parental mosaicism for apparent de novo genetic variants: Scope, detection, and counseling challenges.

The disease burden of de novo mutations (DNMs) has been evidenced only recently when the common application of next-generation sequencing technologies enabled their reliable and affordable detection through family-based clinical exome or genome sequencing. Implementation of exome sequencing into prenatal diagnostics revealed that up to 63% of pathogenic or likely pathogenic variants associated with fetal structural anomalies are apparently de novo, primarily for autosomal dominant disorders. Apparent DNMs have been considered to primarily occur as germline or zygotic events, with consequently negligible recurrence risks. However, there is now evidence that a considerable proportion of them are in fact inherited from a parent mosaic for the variant. Here, we review the burden of DNMs in prenatal diagnostics and the influence of parental mosaicism on the interpretation of apparent DNMs and discuss the challenges with detecting and quantifying parental mosaicism and its effect on recurrence risk. We also describe new bioinformatic and technological tools developed to assess mosaicism and discuss how they improve the accuracy of reproductive risk counseling when parental mosaicism is detected.

International Society for Prenatal Diagnosis Updated Position Statement on the use of genome-wide sequencing for prenatal diagnosis.

The research and clinical use of genome-wide sequencing for prenatal diagnosis of fetuses at risk for genetic disorders have rapidly increased in recent years. Current data indicate that the diagnostic rate is comparable and for certain indications higher than that of standard testing by karyotype and chromosomal microarray. Responsible clinical implementation and diagnostic use of prenatal sequencing depends on standardized laboratory practices and detailed pre-test and post-test counseling. This Updated Position Statement on behalf of the International Society for Prenatal Diagnosis recommends best practices for the clinical use of prenatal exome and genome sequencing from an international perspective. We include several new points for consideration by researchers and clinical service and laboratory providers.

Circulating trophoblast numbers as a potential marker for pregnancy complications.

OBJECTIVE: To explore the potential of circulating trophoblasts (TBs) as a non-invasive tool to assess placental health and predict obstetric complications. METHODS: We retrospectively reviewed maternal characteristics and pregnancy outcomes of 369 women who enrolled in our original cell-based NIPT (cbNIPT) study. The number of circulating TBs recovered from the maternal blood samples was recorded and expressed as fetal cell concentration (FCC). We evaluated if FCC can be used to predict pregnancy outcomes such as hypertensive disorders of pregnancy (HDP), fetal growth restriction, placental abruption, preterm labor, and pregnancy loss. RESULTS: Receiver operating characteristic (ROC) analysis was performed to find the best cut off value to classify FCC into a low and high FCC group, and this cut-off point was calculated as 11.1 cells per 100 ml of blood. The adjusted odds ratio (aOR) for the composite morbidity was significantly increased for the high FCC group at an aOR of 1.6. CONCLUSION: Circulating TB have the potential of predicting obstetrical complications such as HDP. Future studies, with larger sample sizes, should focus on the study of these cells as a biomarker for placental health and a possible screening or diagnostic tool for fetal genetic conditions.

Lower fetal fraction in clinical cell-free DNA screening results is associated with increased risk of hypertensive disorders of pregnancy.

OBJECTIVE: To evaluate if fetal fraction (FF) reported on cell-free DNA (cfDNA) screening is a marker for adverse obstetric outcomes. METHODS: We retrospectively reviewed medical records from a cohort of women with singleton pregnancies who had cfDNA screening. We evaluated if reported FF could predict the following pregnancy complications: hypertensive disorders of pregnancy (HDP), fetal growth restriction, preterm delivery, gestational diabetes mellitus, or a composite maternal morbidity, defined as the presence of at least one of these outcomes. RESULTS: Receiver operating curve analysis was performed on FF from 534 women to define the FF that differentiated a low FF group (<10%; N = 259) and a high FF group (≥10%; N = 275). Hypertensive disorders of pregnancy were more common for women in the low FF group (32.0% vs. 11.6% and p < 0.001), who had a two-fold odds of developing HDP (p = 0.006). Composite maternal morbidity was also more common for women in the low FF group (51.4% vs. 30.2% and p < 0.001), who had a 1.7-fold odds of developing any of the adverse obstetrical outcomes (p = 0.014). CONCLUSION: We found that low FF on cfDNA screening is associated with an increased risk of HDP. Fetal fraction reported that cfDNA screening reports have potential as a predictive marker for the development of HDP and adverse outcomes.

Information is power: The experiences, attitudes and needs of individuals who chose to have prenatal genomic sequencing for fetal anomalies.

OBJECTIVE: This study sought to evaluate the experiences of individuals who chose to participate in a study and receive prenatal genomic sequencing (pGS) for fetuses with congenital structural anomalies. METHOD: Individuals who received research results of prenatal sequencing were invited to participate in semi-structured interviews about their experiences. A constructivist grounded theory approach was used to code and analyze interviews. RESULTS: Thirty-three participants from 27 pregnancies were interviewed. Participants were motivated to enroll in the study to find out more about their fetus' condition and prepare for the future. The waiting period was a time of significant anxiety for participants. Most participants felt relief and closure upon receiving results, regardless of the category of result, and had a clear understanding of the implications of the results. CONCLUSION: Participants' experiences with pGS were often intertwined with the experience of having a fetus with an abnormality. Participants were satisfied with the decision to participate in research and the support they received from the healthcare team, although waiting for results was associated with anxiety. The healthcare team plays an integral role in setting expectations and validating feelings of anxiety, fear and uncertainty.

Prenatal phenotyping of fetal tubulinopathies: A multicenter retrospective case series.

OBJECTIVE: Tubulinopathies refer to conditions caused by genetic variants in isotypes of tubulin resulting in defective neuronal migration. Historically, diagnosis was primarily via postnatal imaging. Our objective was to establish the prenatal phenotype/genotype correlations of tubulinopathies identified by fetal imaging. METHODS: A large, multicenter retrospective case series was performed across nine institutions in the Fetal Sequencing Consortium. Demographics, fetal imaging reports, genetic screening and diagnostic testing results, delivery reports, and neonatal imaging reports were extracted for pregnancies with a confirmed molecular diagnosis of a tubulinopathy. RESULTS: Nineteen pregnancies with a fetal tubulinopathy were identified. The most common prenatal imaging findings were cerebral ventriculomegaly (15/19), cerebellar hypoplasia (13/19), absence of the cavum septum pellucidum (6/19), abnormalities of the corpus callosum (6/19), and microcephaly (3/19). Fetal MRI identified additional central nervous system features that were not appreciated on neurosonogram in eight cases. Single gene variants were reported in TUBA1A (13), TUBB (1), TUBB2A (1), TUBB2B (2), and TUBB3 (2). CONCLUSION: The presence of ventriculomegaly with cerebellar abnormalities in conjunction with additional prenatal neurosonographic findings warrants additional evaluation for a tubulinopathy. Conclusive diagnosis can be achieved by molecular sequencing, which may assist in coordination, prognostication, and reproductive planning.

Lack of consensus among healthcare professionals at a large academic medical center on the use of exome sequencing for prenatal diagnosis.

Prenatal exome sequencing (ES) is increasingly used for prenatal diagnosis because emerging data indicate it has incremental diagnostic benefit in pregnancies with fetal anomalies without identified genetic abnormalities by karyotyping and chromosomal microarray analysis. The aim of this study was to evaluate the medical community's attitude toward the clinical utility and use of exome sequencing for prenatal diagnosis and to address differences in attitudes and responses by type of practitioner, level of training, and years passed since last full-time training. We analyzed the answers of 109 trainees and professionals in the fields of genetic counseling, laboratory science, and medicine to an online survey addressing these topics. Multiple-choice questions asked participants about their awareness of prenatal ES and what genetic test they would choose to order in certain scenarios. Likert-scale questions assessed participants' opinions of statements asserting when prenatal ES should be used for diagnostic testing. Attitude toward the use of prenatal ES statistically differed (p < 0.05) by type of participant and level of training. Practicing genetic counselors and physicians were more selective in their recommendations for prenatal ES than laboratory scientists. Genetic counseling students and practicing genetic counselors felt similarly about indications for the use of prenatal ES, whereas medical students were more liberal in their recommendations for prenatal ES than practicing physicians. This study shows a lack of consensus among the medical community regarding the clinical utility and indications for prenatal ES.

Causative Mutations and Mechanism of Androgenetic Hydatidiform Moles.

Androgenetic complete hydatidiform moles are human pregnancies with no embryos and affect 1 in every 1,400 pregnancies. They have mostly androgenetic monospermic genomes with all the chromosomes originating from a haploid sperm and no maternal chromosomes. Androgenetic complete hydatidiform moles were described in 1977, but how they occur has remained an open question. We identified bi-allelic deleterious mutations in MEI1, TOP6BL/C11orf80, and REC114, with roles in meiotic double-strand breaks formation in women with recurrent androgenetic complete hydatidiform moles. We investigated the occurrence of androgenesis in Mei1-deficient female mice and discovered that 8% of their oocytes lose all their chromosomes by extruding them with the spindles into the first polar body. We demonstrate that Mei1-/- oocytes are capable of fertilization and 5% produce androgenetic zygotes. Thus, we uncover a meiotic abnormality in mammals and a mechanism for the genesis of androgenetic zygotes that is the extrusion of all maternal chromosomes and their spindles into the first polar body.

Overview and recent developments in cell-based noninvasive prenatal testing.

Investigators have long been interested in the natural phenomenon of fetal and placental cell trafficking into the maternal circulation. The scarcity of these circulating cells makes their detection and isolation technically challenging. However, as a DNA source of fetal origin not mixed with maternal DNA, they have the potential of considerable benefit over circulating cell-free DNA-based noninvasive prenatal genetic testing (NIPT). Endocervical trophoblasts, which are less rare but more challenging to recover are also being investigated as an approach for cell-based NIPT. We review published studies from around the world describing both forms of cell-based NIPT and highlight the different approaches' advantages and drawbacks. We also offer guidance for developing a sound cell-based NIPT protocol.

The current and future impact of genome-wide sequencing on fetal precision medicine.

Next-generation sequencing and other genomic technologies are transforming prenatal and reproductive screening and testing for fetal genetic disorders at an unprecedented pace. Original approaches of screening and testing for fetal genetic and genomic disorders were focused on a few more prevalent conditions that were easily diagnosable with pre-genomic era diagnostic tools. First, chromosomal microarray analysis and then next-generation sequencing brought technology capable of more detailed genomic evaluation to prenatal genetic screening and diagnosis. This has facilitated parallel introduction of a variety of new tests on maternal blood samples, including expanded carrier screening and cell-free DNA-based non-invasive screening for fetal aneuploidy, selected copy number variants, and single-gene disorders. Genomic tests on fetal DNA samples, obtained primarily through amniocentesis or chorionic villus sampling, include chromosomal microarray analysis and gene panel and exome sequencing. All these form the diagnostic pillar of the emerging field of fetal precision medicine, but their implementation is associated with ethical, counseling and healthcare resource utilization challenges. We discuss where in the reproductive and prenatal care continuum these exciting new technologies are integrated, along with associated challenges. We propose areas of priority for research to gain the data in support of their responsible implementation into clinical reproductive and prenatal care.

Current controversies in prenatal diagnosis 2: The 59 genes ACMG recommends reporting as secondary findings when sequencing postnatally should be reported when detected on fetal (and parental) sequencing.

Genome sequencing is increasingly being used to aid genetic diagnosis in fetuses with structural abnormalities detected on ultrasound examination. However, with clinical exome and genome sequencing, there is potential for the recognition and reporting of incidental or secondary findings unrelated to the indication for ordering the sequencing, but of potential medical value for patient care. In the postnatal setting, the American College of Medical Genetics and Genomics (ACMG) has clear guidelines that state that when offering sequencing, secondary findings should be reported in 59 genes for which ACMG consider there is a clinical evidence that pathogenic variants may result in disease that might be prevented or treated, with the option to opt out of receiving this information. However, these guidelines specifically exclude prenatal sequencing. Here, we report the debate on whether or not pathogenic findings in these 59 genes should or should not be reported in the prenatal setting. Although more were in favour of reporting before the debate, there was no significant consensus from the audience. After the debate there was a swing toward not reporting, but a slim majority (55%) remained in favour, indicating that this is an area requiring further research and the development of evidence-based guidelines applicable to prenatal proband and trio sequencing.

Prenatal testing in pregnancies conceived by in vitro fertilization with pre-implantation genetic testing.

OBJECTIVE: Women with pregnancies resulting from in vitro fertilization (IVF) with normal pre-implantation genetic testing for aneuploidy (PGT-A) are advised to undergo prenatal screening and testing during pregnancy. It is not well known how many follow these recommendations. Our objective was to study prenatal testing decisions made by women with pregnancies conceived through IVF with PGT-A. METHODS: We performed a retrospective review of women who received genetic counseling during pregnancies conceived through IVF with normal PGT-A. We excluded those who received genetic counseling preconceptionally prior to IVF. Statistical analysis included descriptive statistics and after testing for normality by the Kolmogorov-Smirnov test, independent t test, Mann-Whitney U test, or Chi-square/Fisher's exact test. RESULTS: Data from 83 women were included. Of these, 53 (63.9%) had at least one of the following prenatal tests: first trimester combined screening (16.9%), non-invasive prenatal screening (NIPS) (45.8%), second trimester serum screening (6%), and invasive diagnostic testing (6%). 10.8% had more than one of the above tests and 36.1% declined all tests. CONCLUSION: Almost two-thirds of women who were pregnant after IVF with normal PGT-A had prenatal aneuploidy screening or testing. Future prospective studies with larger cohorts are needed to further ascertain decision making in this population.

The effect of maternal body mass index and gestational age on circulating trophoblast yield in cell-based noninvasive prenatal testing.

OBJECTIVE: To examine the effects of maternal body mass index (BMI) and gestational age (GA) on the number of single circulating trophoblasts (SCT). METHODS: Maternal blood was collected in 20 to 40 mL. All singleton pregnant women at any gestation were recruited. Trophoblasts were recovered by immunomagnetic enrichment and stained for cytokeratin and CD45. Candidate trophoblasts were identified by fluorescence microscopy. RESULTS: Blood samples were collected from 425 singleton pregnancies from April 2018 to December 2019. At least one candidate cell was identified in 88% (373/425). There was an inverse correlation between trophoblasts yield and increasing BMI (r = -0.19, P < .001). The mean ± SD number of trophoblasts/mL was 0.12 ± 0.22 in the underweight group (n = 5), 0.23 ± 0.25 in the normal weight (n = 169), 0.18 ± 0.19 in the overweight (n = 114), and 0.13 ± 0.15 in the obese (n = 109). Significantly more cells were identified in the normal weight than those in the obese (P = .001). In addition, the mean ± SD number of cells/mL was 0.21 ± 0.21 at GA of 10 to 14 weeks (n = 260), 0.14 ± 0.23 at GA ≥15 (n = 102) and 0.12 ± 0.12 at GA <10 (n = 63); P < .001. CONCLUSION: The lower number of SCT was identified from the samples of women with a high BMI. Cell recovery for SCT testing seems optimal at GA of 10 to 14 weeks, but earlier and later testing is still possible.