Expanded targeted preconception screening panel in Israel: findings and insights.

BACKGROUND: We aimed to analyse the efficacy and added value of a targeted Israeli expanded carrier screening panel (IL-ECSP), beyond the first-tier test covered by the Israeli Ministry of Health (IMOH) and the second-tier covered by the Health Maintenance Organisations (HMOs). METHODS: A curated variant-based IL-ECSP, tailored to the uniquely diverse Israeli population, was offered at two tertiary hospitals and a major genetics laboratory. The panel includes 1487 variants in 357 autosomal recessive and X-linked genes. RESULTS: We analysed 10 115 Israeli samples during an 18-month period. Of these, 6036 (59.7%) were tested as couples and 4079 (40.3%) were singles. Carriers were most frequently identified with mutations in the following genes: GJB2/GJB6 (1:22 allele frequency), CFTR (1:28), GBA (1:34), TYR (1:39), PAH (1:50), SMN1 (1:52) and HEXA (1:56). Of 3018 couples tested, 753 (25%) had no findings, in 1464 (48.5%) only one partner was a carrier, and in 733 (24.3%) both were carriers of different diseases. We identified 79 (2.6%) at-risk couples, where both partners are carriers of the same autosomal recessive condition, or the female carries an X-linked disease. Importantly, 48.1% of these would not have been detected by ethnically-based screening tests currently provided by the IMOH and HMOs, for example, variants in GBA, TYR, PAH and GJB2/GJB6. CONCLUSION: This is the largest cohort of targeted ECSP testing, tailored to the diverse Israeli population. The IL-ECSP expands the identification of couples at risk and empowers their reproductive choices. We recommend endorsing an expanded targeted panel to the National Genetic Carrier Screening programme.

Genetic diagnosis and detection rates using C9orf72 repeat expansion and a multi-gene panel in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder. It is mostly sporadic, with the C9orf72 repeat expansion being the most common genetic cause. While the prevalence of C9orf72-ALS in patients from different populations has been studied, data regarding the yield of C9orf72 compared to an ALS gene panel testing is limited.We aimed to explore the application of C9orf72 versus a gene panel in the general Israeli population. A total of 140 ALS patients attended our Neurogenetics Clinic throughout 2018-2023. Disease onset was between ages 60 and 69 years for most patients (34%); however, a quarter had an early-onset disease (< 50 years). Overall, 119 patients (85%) were genetically evaluated: 116 (97%) were tested for the C9orf72 repeat expansion and 64 (54%) underwent gene panel testing. The C9orf72 repeat expansion had a prevalence of 21% among Ashkenazi Jewish patients compared to 5.7% in non-Ashkenazi patients, while the gene panel had a higher yield in non-Ashkenazi patients with 14% disease-causing variants compared to 5.7% in Ashkenazi Jews. Among early-onset ALS patients, panel testing was positive in 12% compared to 2.9% for C9orf72.We suggest a testing strategy for the Israeli ALS patients: C9orf72 should be the first-tier test in Ashkenazi Jewish patients, while a gene panel should be considered as the first step in non-Ashkenazi and early-onset patients. Tiered testing has important implications for patient management, including prognosis, ongoing clinical trials, and prevention in future generations. Similar studies should be implemented worldwide to uncover the diverse ALS genetic architecture and facilitate tailored care.

Exploring the factors affecting classification and reporting of uncertain prenatal microarray findings, using a "virtual fetus" model-a pilot study.

OBJECTIVE: Significant discrepancy exists between laboratories in classification and reporting of copy number variants (CNVs). Studies exploring factors affecting prenatal CNV management are rare. Our "virtual fetus" pilot study examines these factors. METHOD: Ten prenatally diagnosed CNVs of uncertain significance (VUS) > 1Mb, encompassing OMIM-morbid genes, inherited from healthy parents, were classified by 15 MD geneticists from laboratory, prenatal, and preimplantation genetic testing (PGT) units. Geneticists addressed factors affecting classification, obligation to report, and recommendation for invasive testing or PGT. RESULTS: CNVs were classified likely benign (10.7%), VUS (74.7%), likely pathogenic (8.7%), or pathogenic (6.0%). Classification discrepancy was higher for losses versus gains. Classifying pathogenic/likely pathogenic was more common for losses (adjusted odds ratio [aOR] 10.9, 95% CI 1.55-76.9), and geneticists specializing in gynecology (aOR 4.9, 95% CI 1.03-23.3). 84.0% of respondents would report CNVs, depending on classification and family phenotype. Invasive testing in pregnancies was recommended for 29.3% of CNVs, depending on the classification and geneticist's specialization. PGT was recommended for 32.4%, depending on classification, experience years, and family's phenotype (38.0% for patients undergoing in vitro fertilization irrespectively, 26.7% otherwise). CONCLUSION: Factors affecting CNV classification/reporting are mainly dosage, family phenotype, geneticist specialization and experience. Understanding factors from our pilot study may facilitate developing an algorithm for clinical consensus and optimal management.

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.

SMARCC1 is a susceptibility gene for congenital hydrocephalus with an autosomal dominant inheritance mode and incomplete penetrance.

A Jewish couple of mixed origin was referred for genetic counseling following termination of pregnancy at 18 weeks of gestation due to severe ventriculomegaly with aqueduct stenosis. Trio exome sequencing revealed a loss-of-function heterozygous variant in the SMARCC1 gene inherited from an unaffected mother. The SMARCC1 gene is associated with embryonic neurodevelopmental processes. Recent studies have linked perturbations of the gene with autosomal dominant congenital hydrocephalus, albeit with reduced penetrance. However, these studies were not referenced in the SMARCC1 OMIM record (*601732) and the gene was not considered, at the time, an OMIM morbid gene. Following our case and appeal, SMARCC1 is now considered a susceptibility gene for hydrocephalus. This allowed us to reclassify the variant as likely pathogenic and empowered the couple to make informed reproductive choices.

Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease.

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

Diagnostic yield with exome sequencing in prenatal severe bilateral ventriculomegaly: a systematic review and meta-analysis.

OBJECTIVE: This study aimed to determine the incremental diagnostic yield of prenatal exome sequencing after negative chromosomal microarray analysis results in prenatally diagnosed bilateral severe ventriculomegaly or hydrocephalus; another objective was to categorize the associated genes and variants. DATA SOURCES: A systematic search was performed to identify relevant studies published until June 2022 using 4 databases (Cochrane Library, Web of Science, Scopus, and MEDLINE). STUDY ELIGIBILITY CRITERIA: Studies in English reporting on the diagnostic yield of exome sequencing following negative chromosomal microarray analysis results in cases of prenatally diagnosed bilateral severe ventriculomegaly were included. METHODS: Authors of cohort studies were contacted for individual participant data, and 2 studies provided their extended cohort data. The incremental diagnostic yield of exome sequencing was assessed for pathogenic/likely pathogenic findings in cases of: (1) all severe ventriculomegaly; (2) isolated severe ventriculomegaly (as the only cranial anomaly); (3) severe ventriculomegaly with other cranial anomalies; and (4) nonisolated severe ventriculomegaly (with extracranial anomalies). To be able to identify all reported genetic associations, the systematic review portion was not limited to any minimal severe ventriculomegaly case numbers; however, for the synthetic meta-analysis, we included studies with ≥3 severe ventriculomegaly cases. Meta-analysis of proportions was done using a random-effects model. Quality assessment of the included studies was performed using the modified STARD (Standards for Reporting of Diagnostic Accuracy Studies) criteria. RESULTS: A total of 28 studies had 1988 prenatal exome sequencing analyses performed following negative chromosomal microarray analysis results for various prenatal phenotypes; this included 138 cases with prenatal bilateral severe ventriculomegaly. We categorized 59 genetic variants in 47 genes associated with prenatal severe ventriculomegaly along with their full phenotypic description. There were 13 studies reporting on ≥3 severe ventriculomegaly cases, encompassing 117 severe ventriculomegaly cases that were included in the synthetic analysis. Of all the included cases, 45% (95% confidence interval, 30-60) had positive pathogenic/likely pathogenic exome sequencing results. The highest yield was for nonisolated cases (presence of extracranial anomalies; 54%; 95% confidence interval, 38-69), followed by severe ventriculomegaly with other cranial anomalies (38%; 95% confidence interval, 22-57) and isolated severe ventriculomegaly (35%; 95% confidence interval, 18-58). CONCLUSION: There is an apparent incremental diagnostic yield of prenatal exome sequencing following negative chromosomal microarray analysis results in bilateral severe ventriculomegaly. Although the greatest yield was found in cases of nonisolated severe ventriculomegaly, consideration should also be given to performing exome sequencing in cases of isolated severe ventriculomegaly as the only brain anomaly identified on prenatal imaging.

Community data-driven approach to identify pathogenic founder variants for pan-ethnic carrier screening panels.

BACKGROUND: The American College of Medical Genetics and Genomics (ACMG) recently published new tier-based carrier screening recommendations. While many pan-ethnic genetic disorders are well established, some genes carry pathogenic founder variants (PFVs) that are unique to specific ethnic groups. We aimed to demonstrate a community data-driven approach to creating a pan-ethnic carrier screening panel that meets the ACMG recommendations. METHODS: Exome sequencing data from 3061 Israeli individuals were analyzed. Machine learning determined ancestries. Frequencies of candidate pathogenic/likely pathogenic (P/LP) variants based on ClinVar and Franklin were calculated for each subpopulation based on the Franklin community platform and compared with existing screening panels. Candidate PFVs were manually curated through community members and the literature. RESULTS: The samples were automatically assigned to 13 ancestries. The largest number of samples was classified as Ashkenazi Jewish (n = 1011), followed by Muslim Arabs (n = 613). We detected one tier-2 and seven tier-3 variants that were not included in existing carrier screening panels for Ashkenazi Jewish or Muslim Arab ancestries. Five of these P/LP variants were supported by evidence from the Franklin community. Twenty additional variants were detected that are potentially pathogenic tier-2 or tier-3. CONCLUSIONS: The community data-driven and sharing approaches facilitate generating inclusive and equitable ethnically based carrier screening panels. This approach identified new PFVs missing from currently available panels and highlighted variants that may require reclassification.

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.

Upgrading an intronic TMEM67 variant of unknown significance to likely pathogenic through RNA studies and community data sharing.

FETAL PHENOTYPE: A couple of Ashkenazi Jewish descent was referred for an early anatomy scan at 14 + 2 weeks of gestation following a previous pregnancy termination due to posterior encephalocele and enlarged kidneys. The index pregnancy was also positive for several fetal abnormalities, including enlarged kidneys with cystic dysplasia and abnormal cerebellar morphology highly suggestive of Joubert syndrome. GENETIC DIAGNOSTIC TEST PERFORMED, RESULT, AND INTERPRETATION: Trio exome sequencing revealed compound heterozygosity for variants in the TMEM67 gene: a known pathogenic maternally inherited variant found in trans with a paternal intronic variant of unknown significance. RNA analysis revealed that the intronic variant creates a cryptic acceptor splice site in intron 12, leading to the insertion of 22 bp and causing a frameshift with a premature stop codon. This analysis enabled the reclassification of the intronic variant to likely pathogenic. IMPLICATIONS AND NOVELTY: This information empowered the couple to make informed reproductive choices and opt for preimplantation genetic testing (PGT) for future pregnancies.

Whole Genome Sequencing Applied in Familial Hamartomatous Polyposis Identifies Novel Structural Variations.

Hamartomatous polyposis syndromes (HPS) are rare cancer-predisposing disorders including Juvenile polyposis (JPS), Peutz-Jeghers (PJS) and PTEN hamartomatous syndromes (PHS). Penetrant mutations in corresponding genes (SMAD4, BMPR1A, STK11, PTEN and AKT1), are usually diagnosed via a next-generation-sequencing gene panel (NGS-GP) for tailored surveillance and preimplantation testing for monogenic disorders (PGT-M). Five probands with HPS phenotype, with no genetic diagnosis per genetic workup, underwent whole-genome sequencing (WGS) that identified structural genetic alterations: two novel inversions in BMPRA1 and STK11, two BMPR1A-deletions, known as founders among Bukharan Jews, and BMPR1A microdeletion. BMPR1A inversion was validated by "junction fragment" amplification and direct testing. PGT-M was performed via multiplex-PCR and enabled successful birth of a non-carrier baby. WGS may be considered for HPS patients with no NGS-GP findings to exclude structural alterations.

Economic impact of using maternal plasma cell-free DNA testing to guide further workup in recurrent pregnancy loss.

OBJECTIVE: We have previously demonstrated that maternal-plasma cell-free DNA (cfDNA)-testing can detect chromosomal anomalies in recurrent pregnancy loss (RPL) with 81.8% sensitivity and 90.3% specificity. Here we assess whether this is cost effective in guiding further workup in RPLs. METHOD: A decision-analytic model was developed to compare the cost of various RPL management pathways: (1) current American Society for Reproductive Medicine (ASRM) RPL workup; (2) microarray or karyotyping analysis of products of conception (POCs) and RPL workup only for euploid cases; and (3) cfDNA testing and RPL workup only for euploid cases. Sample accessibility, failure rates, and sensitivity were specified for each test. Costs of sample collection, genetic tests, and RPL workup were considered. Analysis outcomes included detection rate of chromosomal anomaly and cost per patient tested. RESULTS: In comparison to existing cytogenetic testing on POCs, cfDNA testing pathway allowed for better sample accessibility with a lower cost per patient. In addition, using cfDNA to guide further workup significantly increases the number of causative fetal chromosome anomalies detected, reducing the number of patients undergoing unnecessary workup resulting in an overall cost savings. CONCLUSION: Our study showed that inclusion of cfDNA testing is a cost-effective approach to guide RPL workup.

Familial Beckwith-Wiedemann syndrome: Prenatal manifestation and a possible expansion of the phenotype.

We describe a case of Beckwith-Wiedemann syndrome (BWS) demonstrating pre- and post-natal intra-familial variability. Our first encounter with the family occurred in the 1990s following the birth of 3 affected offspring. The first two pregnancies presented with exomphalos and elevated second trimester maternal serum alpha-fetoprotein (msAFP, 3.43 and 4.01 MOM, respectively) as well as elevated maternal human chorionic gonadotrophin (mhCG, 4.33 and 8.8 MOM, respectively). The diagnosis of BWS was confirmed postnatally in both cases. The third ongoing pregnancy presented only with elevated mhCG (7.09 MOM) and no malformation. Nonetheless BWS was suspected. The diagnosis was confirmed postnatally with clinical manifestations including macroglossia and cleft palate. Two affected female siblings were also diagnosed with Mullerian agenesis in adulthood. Suspecting a common genetic etiology, sequencing of the CDKN1C gene revealed a maternally inherited, likely pathogenic variant (NM_000076.2: c.367_385del; p.(Ala123Serfs*143)) causative of BWS. Chromosomal microarray and whole exome sequencing did not reveal any other pathogenic variant that would explain the Mullerian agenesis. One of the affected females underwent successful preimplantation genetic testing (PGT) with a surrogate and gave birth to a healthy female. To the best of our knowledge, this is the first report of Mullerian agenesis as a possible rare expansion of the BWS phenotype. In addition, this case highlights the potential role of abnormal second trimester biochemical markers (msAFP, mHCG) as possible indicators of BWS, especially in familial cases.

Ethnicity has a multiplex impact upon the risk of a full mutation expansion among female heterozygotes for FMR1 premutation.

PURPOSE: To evaluate whether ethnicity affects the risk of full mutation expansion among females heterozygous for FMR1 premutation. METHODS: Women who carry the FMR1 premutation alelle of Jewish origin who underwent fragile X prenatal diagnosis between 2011 and 2018 in two medical centers in Israel were included. The heterozygote women and fetuses were analyzed for the number of CGG repeats and AGG interruptions. RESULTS: Seven hundred sixty-six subjects were included. Parental ethnicity was fully concordant in 592 cases (Jewish, Ashkenazi, and non-Ashkenazi). Ashkenazi compared with non-Ashkenazi heterozygotes have a significantly higher mean number of CGG repeats (68 ± 8.7, 64 ± 6.4 respectively, P = 0.03) and a lower mean number of AGG interruptions (0.89 ± 0.83, 1.60 ± 1.18 respectively, p = 0.0001). Overall, 56/198 (28.2%) fetuses of Ashkenazi heterozygotes had an expansion to a full mutation compared with 6/98 among the non-Ashkenazi (6.1%) (p = 0.001). Multivariate analysis demonstrated that, in addition to CGG repeats and AGG interruptions (which contributed 68.3% of variance), ethnicity is an independent risk factor for a full mutation expansion (odds ratio [OR] = 2.04, p < 0.001) and accounted for 9% of the variation of a full mutation expansion. CONCLUSION: Apart from significant differences regarding the number of CGG repeats and AGG interruptions between Ashkenazi and non-Ashkenazi heterozygotes, ethnicity independently affects the risk of a full mutation.

Improving renal phenotype and evolving extra-renal features of 17q12 deletion encompassing the HNF1B gene.

BACKGROUND: HNF1B deletion/intragenic mutations are the most commonly identified genetic cause of congenital anomalies of the kidney and urinary tract (CAKUT) suggested by fetal ultrasound findings such as: parenchymal hyperechogenicity, overt cystic changes or gross morphological urinary system (UT) abnormalities. The postnatal evolution of these 17q12 deletions encompassing the HNF1B gene-associated findings has not been assessed in depth. METHODS: In this observational study, we present postnatal follow-up findings in 5 of 6 cases (one pregnancy was terminated on parental request) of fetal-onset cystic/hyperechogenic kidneys eventually diagnosed with 17q12 microdeletion encompassing the HNF1B gene between 2009 and 2017. RESULTS: Complete normalization of kidney parenchymal abnormalities and of depressed neonatal renal function was observed in 4/5 and 5/5 patients within 2-4.9 years and 1.5-8 months, respectively. All 5 patients had preserved normal renal function at 3-11 years of follow-up. The evolving later-onset renal features included: hypomagnesemia, hyperuricemia, urinary tract infection (UTI), and bilateral grade 3-4 vesicoureteral reflux and bladder diverticula in 3, 3, 2, and 1 patient, respectively. HNF1B gene deletion-associated extra-renal manifestations with delayed presentation were global developmental delay/autistic spectrum disorder (ASD), rolandic-type seizures, overweight, and borderline fasting hyperglycemia observed in 1-2 patients each. Family history was positive for small-size or asymptomatic cystic kidneys with normal function, diabetes mellitus, seizures, and mental/psychiatric problems in 3/6 cases. CONCLUSIONS: Fetal-onset HNF1B deletion-associated kidneys' parenchymal abnormalities confirmed postnatally with initially depressed renal function might undergo complete resolution within several years and few months, respectively. However, later-onset urinary tract, metabolic, and neurodevelopmental features of this mutation might appear over years. Therefore, genetic molecular evaluation/diagnosis and continuous follow-up for evolving features are mandatory in affected children.