Systematic evaluation of genome sequencing for the diagnostic assessment of autism spectrum disorder and fetal structural anomalies.

Short-read genome sequencing (GS) holds the promise of becoming the primary diagnostic approach for the assessment of autism spectrum disorder (ASD) and fetal structural anomalies (FSAs). However, few studies have comprehensively evaluated its performance against current standard-of-care diagnostic tests: karyotype, chromosomal microarray (CMA), and exome sequencing (ES). To assess the clinical utility of GS, we compared its diagnostic yield against these three tests in 1,612 quartet families including an individual with ASD and in 295 prenatal families. Our GS analytic framework identified a diagnostic variant in 7.8% of ASD probands, almost 2-fold more than CMA (4.3%) and 3-fold more than ES (2.7%). However, when we systematically captured copy-number variants (CNVs) from the exome data, the diagnostic yield of ES (7.4%) was brought much closer to, but did not surpass, GS. Similarly, we estimated that GS could achieve an overall diagnostic yield of 46.1% in unselected FSAs, representing a 17.2% increased yield over karyotype, 14.1% over CMA, and 4.1% over ES with CNV calling or 36.1% increase without CNV discovery. Overall, GS provided an added diagnostic yield of 0.4% and 0.8% beyond the combination of all three standard-of-care tests in ASD and FSAs, respectively. This corresponded to nine GS unique diagnostic variants, including sequence variants in exons not captured by ES, structural variants (SVs) inaccessible to existing standard-of-care tests, and SVs where the resolution of GS changed variant classification. Overall, this large-scale evaluation demonstrated that GS significantly outperforms each individual standard-of-care test while also outperforming the combination of all three tests, thus warranting consideration as the first-tier diagnostic approach for the assessment of ASD and FSAs.

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.

Prenatal vs postnatal diagnosis of 22q11.2 deletion syndrome: cardiac and noncardiac outcomes through 1 year of age.

BACKGROUND: The 22q11.2 deletion syndrome is the most common microdeletion syndrome and is frequently associated with congenital heart disease. Prenatal diagnosis of 22q11.2 deletion syndrome is increasingly offered. It is unknown whether there is a clinical benefit to prenatal detection as compared with postnatal diagnosis. OBJECTIVE: This study aimed to determine differences in perinatal and infant outcomes between patients with prenatal and postnatal diagnosis of 22q11.2 deletion syndrome. STUDY DESIGN: This was a retrospective cohort study across multiple international centers (30 sites, 4 continents) from 2006 to 2019. Participants were fetuses, neonates, or infants with a genetic diagnosis of 22q11.2 deletion syndrome by 1 year of age with or without congenital heart disease; those with prenatal diagnosis or suspicion (suggestive ultrasound findings and/or high-risk cell-free fetal DNA screen for 22q11.2 deletion syndrome with postnatal confirmation) were compared with those with postnatal diagnosis. Perinatal management, cardiac and noncardiac morbidity, and mortality by 1 year were assessed. Outcomes were adjusted for presence of critical congenital heart disease, gestational age at birth, and site. RESULTS: A total of 625 fetuses, neonates, or infants with 22q11.2 deletion syndrome (53.4% male) were included: 259 fetuses were prenatally diagnosed (156 [60.2%] were live-born) and 122 neonates were prenatally suspected with postnatal confirmation, whereas 244 infants were postnatally diagnosed. In the live-born cohort (n=522), 1-year mortality was 5.9%, which did not differ between groups but differed by the presence of critical congenital heart disease (hazard ratio, 4.18; 95% confidence interval, 1.56-11.18; P<.001) and gestational age at birth (hazard ratio, 0.78 per week; 95% confidence interval, 0.69-0.89; P<.001). Adjusting for critical congenital heart disease and gestational age at birth, the prenatal cohort was less likely to deliver at a local community hospital (5.1% vs 38.2%; odds ratio, 0.11; 95% confidence interval, 0.06-0.23; P<.001), experience neonatal cardiac decompensation (1.3% vs 5.0%; odds ratio, 0.11; 95% confidence interval, 0.03-0.49; P=.004), or have failure to thrive by 1 year (43.4% vs 50.3%; odds ratio, 0.58; 95% confidence interval, 0.36-0.91; P=.019). CONCLUSION: Prenatal detection of 22q11.2 deletion syndrome was associated with improved delivery management and less cardiac and noncardiac morbidity, but not mortality, compared with postnatal detection.

Current controversy in prenatal diagnosis: The use of cfDNA to screen for monogenic conditions in low risk populations is ready for clinical use.

Noninvasive cfDNA testing for monogenic disorders (sgNIPT) has become integrated into the care of pregnant women at increased risk based on carrier status, known family history, or ultrasound anomalies. The availability of commercial tests for common autosomal recessive and de novo autosomal dominant conditions has led to the use of these tests in low-risk pregnancies. However, is the technology ready for use in this low-risk population? This report is a summary of the debate on this topic at the 27th International Conference on Prenatal Diagnosis and Therapy. Both expert debaters provided strong arguments in favor and against the use of sgNIPT in low-risk pregnancies. The argument in favor of sgNIPT for autosomal recessive conditions is that it allows the identification of affected pregnancies without the need for involving the partner in testing. Arguments for sgNIPT for autosomal dominant conditions include identification of affected fetuses that would have either presented later in pregnancy with fetal anomalies or not been detected prenatally given normal ultrasounds, respect for patient autonomy and patient desire for information. Strong arguments were made against offering sgNIPT screening. Given that traditional carrier screening for recessive conditions can be carried out in many jurisdictions, the added value of sgNIPT has not been clearly demonstrated. Arguments against sgNIPT for autosomal dominant conditions included the total lack of clinical validation studies and the risk of false reassurance in cases of negative results and unnecessary invasive procedures in cases of false positive results. Although there is a desire to take advantage of new technologies to improve the detection of monogenic disorders in low-risk populations, based on the discussion and the audience vote, it appears premature to offer sgNIPT to all low risk pregnant women. Further clinical validation studies are needed prior to broad implementation.

The expanded spectrum of human disease associated with GREB1L likely includes complex congenital heart disease.

OBJECTIVE: GREB1L has been linked prenatally to Potter's sequence, as well as less severe anomalies of the kidney, uterus, inner ear, and heart. The full phenotypic spectrum is unknown. The purpose of this study was to characterize known and novel pre- and postnatal phenotypes associated with GREB1L. METHODS: We solicited cases from the Fetal Sequencing Consortium, screened a population-based genomic database, and conducted a comprehensive literature search to identify disease cases associated with GREB1L. We present a detailed phenotypic spectrum and molecular changes. RESULTS: One hundred twenty-seven individuals with 51 unique pathogenic or likely pathogenic GREB1L variants were identified. 24 (47%) variants were associated with isolated kidney anomalies, 19 (37%) with anomalies of multiple systems, including one case of hypoplastic left heart syndrome, five (10%) with isolated sensorineural hearing loss, two (4%) with isolated uterine agenesis; and one (2%) with isolated tetralogy of Fallot. CONCLUSION: GREB1L may cause complex congenital heart disease (CHD) in humans. Clinicians should consider GREB1L testing in the setting of CHD, and cardiac screening in the setting of GREB1L variants.

Monogenic conditions and central nervous system anomalies: A prospective study, systematic review and meta-analysis.

OBJECTIVES: Determine the incremental diagnostic yield of prenatal exome sequencing (pES) over chromosome microarray (CMA) or G-banding karyotype in fetuses with central nervous system (CNS) abnormalities. METHODS: Data were collected via electronic searches from January 2010 to April 2022 in MEDLINE, Cochrane, Web of Science and EMBASE. The NHS England prenatal exome cohort was also included. Incremental yield was calculated as a pooled value using a random-effects model. RESULTS: Thirty studies were included (n = 1583 cases). The incremental yield with pES for any CNS anomaly was 32% [95%CI 27%-36%; I2 = 72%]. Subgroup analysis revealed apparent incremental yields in; (a) isolated CNS anomalies; 27% [95%CI 19%-34%; I2 = 74%]; (b) single CNS anomaly; 16% [95% CI 10%-23%; I2 = 41%]; (c) more than one CNS anomaly; 31% [95% Cl 21%-40%; I2 = 56%]; and (d) the anatomical subtype with the most optimal yield was Type 1 malformation of cortical development, related to abnormal cell proliferation or apoptosis, incorporating microcephalies, megalencephalies and dysplasia; 40% (22%-57%; I2 = 68%). The commonest syndromes in isolated cases were Lissencephaly 3 and X-linked hydrocephalus. CONCLUSIONS: Prenatal exome sequencing provides a high incremental diagnostic yield in fetuses with CNS abnormalities with optimal yields in cases with multiple CNS anomalies, particularly those affecting the midline, posterior fossa and cortex.

Maternal carrier screening with single-gene NIPS provides accurate fetal risk assessments for recessive conditions.

PURPOSE: The purpose of this study was to evaluate the clinical performance of carrier screening for cystic fibrosis, hemoglobinopathies, and spinal muscular atrophy with reflex single-gene noninvasive prenatal screening (sgNIPS), which does not require paternal carrier screening. METHODS: An unselected sample of 9151 pregnant individuals from the general US pregnant population was screened for carrier status, of which 1669 (18.2%) were identified as heterozygous for one or more pathogenic variants and reflexed to sgNIPS. sgNIPS results were compared with newborn outcomes obtained from parent survey responses or provider reports for a cohort of 201 pregnancies. RESULTS: Overall, 98.7% of pregnant individuals received an informative result (no-call rate = 1.3%), either a negative carrier report or, if identified as heterozygous for a pathogenic variant, a reflex sgNIPS report. In the outcomes cohort, the negative predictive value of sgNIPS was 99.4% (95% CI = 96.0%-99.9%) and average positive predictive value (PPV) of sgNIPS was 48.3% (95% CI = 36.1%-60.1%). Importantly, personalized PPVs accurately reflected the percentage of affected pregnancies in each PPV range, and all pregnancies with a sgNIPS fetal risk of >9 in 10 (90% PPV) were affected. CONCLUSION: Although traditional carrier screening is most effective when used to assess reproductive risk before pregnancy, more than 95% of the time it is pursued during a pregnancy and is complicated by incomplete uptake of paternal carrier screening (<50%) and misattributed paternity (∼10%). Even in an idealized setting, when both partners have carrier screening, the maximum risk for having an affected pregnancy is 1 in 4 (equivalent of a 25% PPV). Carrier screening with sgNIPS during pregnancy is an alternative that does not require a paternal sample and provides accurate fetal risk in a timely manner that can be used for prenatal counseling and pregnancy management.

Association of deep phenotyping with diagnostic yield of prenatal exome sequencing for fetal brain abnormalities.

PURPOSE: To evaluate whether deep prenatal phenotyping of fetal brain abnormalities (FBAs) increases diagnostic yield of trio-exome sequencing (ES) compared with standard phenotyping. METHODS: Retrospective exploratory analysis of a multicenter prenatal ES study. Participants were eligible if an FBA was diagnosed and subsequently found to have a normal microarray. Deep phenotyping was defined as phenotype based on targeted ultrasound plus prenatal/postnatal magnetic resonance imaging, autopsy, and/or known phenotypes of other affected family members. Standard phenotyping was based on targeted ultrasound alone. FBAs were categorized by major brain findings on prenatal ultrasound. Cases with positive ES results were compared with those that have negative results by available phenotyping, as well as diagnosed FBAs. RESULTS: A total of 76 trios with FBAs were identified, of which 25 (33%) cases had positive ES results and 51 (67%) had negative results. Individual modalities of deep phenotyping were not associated with diagnostic ES results. The most common FBAs identified were posterior fossa anomalies and midline defects. Neural tube defects were significantly associated with receipt of a negative ES result (0% vs 22%, P = .01). CONCLUSION: Deep phenotyping was not associated with increased diagnostic yield of ES for FBA in this small cohort. Neural tube defects were associated with negative ES results.

Diagnosis of TBC1D32-associated conditions: Expanding the phenotypic spectrum of a complex ciliopathy.

Exome sequencing is a powerful tool in prenatal and postnatal genetics and can help identify novel candidate genes critical to human development. We describe seven unpublished probands with rare likely pathogenic variants or variants of uncertain significance that segregate with recessive disease in TBC1D32, including four fetal probands in three unrelated pedigrees and three pediatric probands in unrelated pedigrees. We also report clinical comparisons with seven previously published patients. Index probands were identified through an ongoing prenatal exome sequencing study and through an online data sharing platform (Gene Matcher™). A literature review was also completed. TBC1D32 is involved in the development and function of cilia and is expressed in the developing hypothalamus and pituitary gland. We provide additional data to expand the phenotype correlated with TBC1D32 variants, including a severe prenatal phenotype associated with life-limiting congenital anomalies.

Prenatal exome and genome sequencing for fetal structural abnormalities.

As prenatal exome sequencing becomes integrated into clinical care, it is critical that providers caring for women with fetal anomalies recognize not only the benefits, but also the challenges and considerations related to this technology. This overview of prenatal sequencing includes information about indications for sequencing, methods, diagnostic yield, clinical utility, variant interpretation, ethical considerations and dilemmas, practical considerations (ie, turnaround time and cost), pre- and posttest counseling points, and psychological impact of testing on families.

Two unrelated fetuses with ITPR1 missense variants and fetal hydrops.

We describe two fetuses from unrelated families with likely pathogenic variants in ITPR1 that presented with nonimmune fetal hydrops. Trio exome sequencing revealed a de novo heterozygous likely pathogenic missense variant c.7636G > A (p.Val2531Met) in ITPR1 (NM_001378452.1) in proband 1 and a de novo heterozygous likely pathogenic missense variant c.34G > A [p.Gly12Arg] in proband 2. Variants in ITPR1 have been associated with several genetic conditions, including spinocerebellar ataxia 15, spinocerebellar ataxia 29, and Gillespie syndrome. Our report on two patients details a previously undescribed severe fetal presentation of nonimmune hydrops fetalis associated with missense variants in the ITPR1 gene.

Understanding the experiences and perspectives of prenatal screening among a diverse cohort.

INTRODUCTION: Rapid advances in prenatal genetic screening technology make it difficult for providers to deliver adequate prenatal counseling. The aim of this study was to understand how prenatal screening educational approaches can meet the needs of patients. METHODS: Qualitative content analysis was conducted on a diverse population who were interviewed to explore their perceived experiences and preferences for prenatal screening educational delivery. RESULTS: Twenty-two women from three US sites were interviewed. Participants were racially/ethnically diverse with 22.7% identifying as Black or African American (n = 5), 40.9% as Hispanic (n = 9), and 4.5% as Pacific Islander (n = 1). Four themes were identified: prenatal screening education, prenatal screening decision-making, return of results, and suggestions for creating a decision aid. Most results were consistent with previous research not targeting a diverse population. DISCUSSION/CONCLUSION: Our results indicate that learning style preferences vary between patients and that current methods are not consistently satisfying patient's desire for understanding, particularly with 'high-risk' results, suggesting that a standardized tool could improve knowledge and decrease decisional conflict. This diverse cohort suggested a list and description of each of the testing options offered, information about each condition being screened for, a timeline for the testing and return of results, costs associated, and non-technical language.

Diagnostic yield and psychological outcomes among women pursuing trio-exome sequencing: Do women with recurrent anomalous fetal phenotypes experience more negative psychological outcomes?

PURPOSE: To describe psychological outcomes among people with recurrent anomalous pregnancies pursuing trio-exome sequencing (exome sequencing (ES)) compared to those with one affected. METHODS: We analyzed data from a prospective ES cohort, enrolling patients with major fetal anomaly and normal microarray. Participants completed validated scales before and after ES. We (1) compared responses of those with multiple anomalous pregnancies to those with one affected and (2) conducted linear regression to examine associations between multiple affected pregnancies and post-ES constructs. RESULTS: Of 166 trios, 61 (37%) received results from ES. Forty (24%) had more than one affected pregnancy and 45% of those received a result explaining the fetal phenotype. All participants had clinically significant presequencing generalized psychological distress. For the 93 who completed the post-ES surveys, those with multiple affected pregnancies had higher psychological adaptation scores but worse test related distress scores (9.3 (6.2) versus 7.1(5.6), p = 0.12) and (14.3 (1.5) versus 15.4 (1.4), p = 0.01). In linear regression models, there were no significant differences in post-ES constructs after adjusting for clinically relevant covariates. CONCLUSIONS: All individuals experienced significant generalized psychological distress in the pre-ES period, extending our knowledge of how pregnancy history contributes to parental sequencing outcomes.

Prenatal phenotype of 47, XXY (Klinefelter syndrome).

OBJECTIVE: There is a paucity of knowledge regarding the prenatal presentation of Klinefelter syndrome, or 47, XXY. Accurate prenatal counseling is critical and in utero diagnosis is currently limited by a poor understanding of the prenatal phenotype of this condition. METHODS: This is a case series of fetuses with cytogenetically confirmed 47, XXY in the prenatal period or up to age 5 years, with prenatal records available for review from four academic institutions between 2006 and 2019. Ultrasound reports were reviewed in detail to assess for increased nuchal translucency and structural abnormalities. Additionally, we reviewed results of cell-free DNA and serum analyte testing when performed to inform our understanding of the detection of fetal 47, XXY through standard genetic screening tests. RESULTS: Forty-one cases with confirmed cytogenetic diagnosis of 47, XXY and prenatal records available for review were identified: 37 had a prenatal diagnosis and 4 had a postnatal diagnosis. Nuchal translucency was increased ≥3.0 mm in 23.1% (6/26) of cases with a documented measurement. In 29.2% (7/24) of cases with a second trimester anatomical ultrasound available for review, a fetal abnormality was identified (3 brain anomalies, 1 cardiac abnormality, 1 echogenic bowel, and 2 limb abnormalities). Among those who had cell-free DNA and serum analytes performed, 92.6% (25/27) and 36.3% (4/11) had an abnormal result respectively. CONCLUSION: This case series expands our knowledge of the prenatal presentation of 47, XXY by identifying first and second trimester fetal sonographic abnormalities. Prenatal identification of this condition enables accurate counseling, focused prenatal management, and early postnatal interventions to ameliorate some of the known complications.

Sex-Dependent Differences in Mouse Placental Gene Expression following a Maternal High-Fat Diet.

OBJECTIVE: In utero fetal exposures may have sex-specific placental gene responses. Our objective was to measure sex-based differences in placental gene expression from dams fed high-fat diet (HFD) versus control diet (CD). STUDY DESIGN: We fed timed pregnant Friend virus B-strain dams either a CD (n = 5) or an HFD (n = 5). We euthanized dams on embryonic day 17.5 to collect placentas. We extracted placental RNA and hybridized it to a customized 96-gene Nanostring panel focusing on angiogenic, inflammatory, and growth genes. We compared normalized gene expression between CD and HFD, stratified by fetal sex, using analysis of variance. Pathway analysis was used to further interpret the genomic data. RESULTS: Pups from HFD-fed dams were heavier than those from CD-fed dams (0.97 ± 0.06 vs 0.84 ± 0.08 g, p < 0.001). Male pups were heavier than females in the HFD (0.99 ± 0.05 vs 0.94 ± 0.06 g, p = 0.004) but not CD (0.87 ± 0.08 vs 0.83 ± 0.07 g, p = 0.10) group. No sex-based differences in placental gene expression in CD-fed dams were observed. Among HFD-fed dams, placentas from female pups exhibited upregulation of 15 genes (q = 0.01). Network analyses identified a cluster of genes involved in carbohydrate metabolism, cellular function and maintenance, and endocrine system development and function (p = 1 × 10-23). The observed female-specific increased gene expression following in utero HFD exposure was predicted to be regulated by insulin (p = 5.79 × 10-13). CONCLUSION: In female compared with male pups, in utero exposure to HFD upregulated placental gene expression in 15 genes predicted to be regulated by insulin. Sex-specific differences in placental expression of these genes should be further investigated. KEY POINTS: · Male pups were heavier than female pups at the time of sacrifice when dams were fed an HFD.. · HFD was associated with upregulated gene expression in female placentas.. · Female-specific increased gene was predicted to be regulated by insulin..

Insulin Elevates ID2 Expression in Trophoblasts and Aggravates Preeclampsia in Obese ASB4-Null Mice.

Obesity is a risk factor for preeclampsia. We investigated how obesity influences preeclampsia in mice lacking ankyrin-repeat-and-SOCS-box-containing-protein 4 (ASB4), which promotes trophoblast differentiation via degrading the inhibitor of DNA-binding protein 2 (ID2). Asb4-/- mice on normal chow (NC) develop mild preeclampsia-like phenotypes during pregnancy, including hypertension, proteinuria, and reduced litter size. Wild-type (WT) and Asb4-/- females were placed on a high-fat diet (HFD) starting at weaning. At the age of 8-9 weeks, they were mated with WT or Asb4-/- males, and preeclamptic phenotypes were assessed. HFD-WT dams had no obvious adverse outcomes of pregnancy. In contrast, HFD-Asb4-/- dams had significantly more severe preeclampsia-like phenotypes compared to NC-Asb4-/- dams. The HFD increased white fat weights and plasma leptin and insulin levels in Asb4-/- females. In the HFD-Asb4-/- placenta, ID2 amounts doubled without changing the transcript levels, indicating that insulin likely increases ID2 at a level of post-transcription. In human first-trimester trophoblast HTR8/SVneo cells, exposure to insulin, but not to leptin, led to a significant increase in ID2. HFD-induced obesity markedly worsens the preeclampsia-like phenotypes in the absence of ASB4. Our data indicate that hyperinsulinemia perturbs the timely removal of ID2 and interferes with proper trophoblast differentiation, contributing to enhanced preeclampsia.

Discovering a new part of the phenotypic spectrum of Coffin-Siris syndrome in a fetal cohort.

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.

RNA-Seq of amniotic fluid cell-free RNA: a discovery phase study of the pathophysiology of congenital cytomegalovirus infection.

BACKGROUND: Congenital cytomegalovirus infection is the most common perinatal infection and a significant cause of sensorineural hearing loss, cerebral palsy, and neurodevelopmental disability. There is a paucity of human gene expression studies examining the pathophysiology of cytomegalovirus infection. OBJECTIVE: This study aimed to perform a whole transcriptomic assessment of amniotic fluid from pregnancies with live fetuses to identify differentially expressed genes and enriched Gene Ontology categories associated with congenital cytomegalovirus infection. STUDY DESIGN: Amniotic fluid supernatant was prospectively collected from pregnant women undergoing amniocentesis for suspected congenital cytomegalovirus infection because of first-trimester maternal primary infection or ultrasound features suggestive of fetal infection. Women who had received therapy to prevent fetal infection were excluded. Congenital cytomegalovirus infection was diagnosed via viral polymerase chain reaction of amniotic fluid; cytomegalovirus-infected fetuses were paired with noninfected controls, matched for gestational age and fetal sex. Paired-end RNA sequencing was performed on amniotic fluid cell-free RNA with the Novaseq 6000 at a depth of 30 million reads per sample. Following quality control and filtering, reads were mapped to the human genome and counts summarized across genes. Differentially expressed genes were identified using 2 approaches: voomWithQualityWeights in conjunction with limma and RUVSeq with edgeR. Genes with a false discovery rate <0.05 were considered statistically significant. Differential exon use was analyzed using DEXSeq. Functional analysis was performed using gene set enrichment analysis and Ingenuity Pathway Analysis. Manual curation of differentially regulated genes was also performed. RESULTS: Amniotic fluid samples were collected from 50 women; 16 (32%) had congenital cytomegalovirus infection confirmed by polymerase chain reaction. After excluding 3 samples without matched controls, 13 cytomegalovirus-infected samples collected at 18 to 23 weeks and 13 cytomegalovirus-negative gestation-matched controls were submitted for RNA sequencing and analysis (N=26). Ten of the 13 pregnancies with cytomegalovirus-infected fetuses had amniocentesis because of serologic evidence of maternal primary infection with normal fetal ultrasound, and 3 had amniocentesis because of ultrasound abnormality suggestive of cytomegalovirus infection. Four cytomegalovirus-infected pregnancies ended in termination (n=3) or fetal death (n=1), and 9 resulted in live births. Pregnancy outcomes were available for 11 of the 13 cytomegalovirus-negative controls; all resulted in live births of clinically-well infants. Differential gene expression analysis revealed 309 up-regulated and 32 down-regulated genes in the cytomegalovirus-infected group compared with the cytomegalovirus-negative group. Gene set enrichment analysis showed significant enrichment of multiple Gene Ontology categories involving the innate immune response to viral infection and interferon signaling. Of the 32 significantly down-regulated genes, 8 were known to be involved in neurodevelopment and preferentially expressed by the brain. Six specific cellular restriction factors involved in host defense to cytomegalovirus infection were up-regulated in the cytomegalovirus-infected group. Ingenuity Pathway Analysis predicted the activation of pathways involved in progressive neurologic disease and inflammatory neurologic disease. CONCLUSION: In this next-generation sequencing study, we revealed new insights into the pathophysiology of congenital cytomegalovirus infection. These data on the up-regulation of the intraamniotic innate immune response to cytomegalovirus infection and the dysregulation of neurodevelopmental genes may inform future approaches to developing prognostic markers and assessing fetal responses to in utero therapy.

Parental motivations for and adaptation to trio-exome sequencing in a prospective prenatal testing cohort: Beyond the diagnosis.

PURPOSE: To understand motivations for and parental interpretation of results from trio-exome sequencing (ES) for fetal anomalies with a negative standard genetic diagnosis. METHODS: Analysis of an ongoing, prospective prenatal trio-ES study of pregnancies with ultrasound-identified congenital anomalies and lack of a standard genetic diagnosis. After determination of pregnancy disposition, participants completed questionnaires and a semi-structured interview pre- and post-sequencing. Interviews were analyzed using a constructivist grounded theory methodology to identify themes. Associations between themes and ES result were also examined. RESULTS: One hundred twenty-six trios have been sequenced. Of those, 45 (36%) resulted in fetal diagnosis. One hundred twenty-five women completed pre-sequencing surveys, and 91 women completed post-sequencing surveys. The main themes identified include (1) variable reasons to pursue ES, (2) limited expectations but high hopes from ES, (3) parental adaptation to uncertain results, (4) impact on personal health and reproduction, and (5) gratitude for the process. CONCLUSION: Participants pursued ES for various reasons, most often to identify a diagnosis and guide reproduction. Post-sequencing, most participants described the process, their interpretation of results, and the impact of receiving the results. Less frequently, but of most concern, participants expressed anxiety about testing and implications for themselves, relationships, and other family members, thus identifying an area of high need for additional support among patients undergoing prenatal ES.

Noninvasive prenatal exome sequencing diagnostic utility limited by sequencing depth and fetal fraction.

OBJECTIVE: Sequencing cell-free DNA now allows detection of large chromosomal abnormalities and dominant Mendelian disorders in the prenatal period. Improving upon these methods would allow newborn screening programs to begin with prenatal genetics, ultimately improving the management of rare genetic disorders. METHODS: As a pilot study, we performed exome sequencing on the cell-free DNA from three mothers with singleton pregnancies to assess the viability of broad sequencing modalities in a noninvasive prenatal setting. RESULTS: We found poor resolution of maternal and fetal genotypes due to both sampling and technical issues. CONCLUSION: We find broad sequencing modalities inefficient for noninvasive prenatal applications. Alternatively, we suggest a more targeted path forward for noninvasive prenatal genotyping.