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Introduction: Exome sequencing has a diagnostic yield ranging from 25% to 70% in rare diseases and regularly implicates genes in novel disorders. Retrospective data reanalysis has demonstrated strong efficacy in improving diagnosis, but poses organizational difficulties for clinical laboratories.Patients and methods: We applied a reanalysis strategy based on intensive prospective bibliographic monitoring along with direct application of the GREP command-line tool (to “globally search for a regular expression and print matching lines”) in a large ES database. For 18 months, we submitted the same five keywords of interest [(intellectual disability, (neuro)developmental delay, and (neuro)developmental disorder)] to PubMed on a daily basis to identify recently published novel disease–gene associations or new phenotypes in genes already implicated in human pathology. We used the Linux GREP tool and an in-house script to collect all variants of these genes from our 5,459 exome database.Results: After GREP queries and variant filtration, we identified 128 genes of interest and collected 56 candidate variants from 53 individuals. We confirmed causal diagnosis for 19/128 genes (15%) in 21 individuals and identified variants of unknown significance for 19/128 genes (15%) in 23 individuals. Altogether, GREP queries for only 128 genes over a period of 18 months permitted a causal diagnosis to be established in 21/2875 undiagnosed affected probands (0.7%).Conclusion: The GREP query strategy is efficient and less tedious than complete periodic reanalysis. It is an interesting reanalysis strategy to improve diagnosis.

Introduction: Prenatal ultrasound (US) anomalies are detected in around 5%–10% of pregnancies. In prenatal diagnosis, exome sequencing (ES) diagnostic yield ranges from 6% to 80% depending on the inclusion criteria. We describe the first French national multicenter pilot study aiming to implement ES in prenatal diagnosis following the detection of anomalies on US.Patients and methods: We prospectively performed prenatal trio-ES in 150 fetuses with at least two US anomalies or one US anomaly known to be frequently linked to a genetic disorder. Trio-ES was only performed if the results could influence pregnancy management. Chromosomal microarray (CMA) was performed before or in parallel.Results: A causal diagnosis was identified in 52/150 fetuses (34%) with a median time to diagnosis of 28 days, which rose to 56/150 fetuses (37%) after additional investigation. Sporadic occurrences were identified in 34/56 (60%) fetuses and unfavorable vital and/or neurodevelopmental prognosis was made in 13/56 (24%) fetuses. The overall diagnostic yield was 41% (37/89) with first-line trio-ES versus 31% (19/61) after normal CMA. Trio-ES and CMA were systematically concordant for identification of pathogenic CNV.Conclusion: Trio-ES provided a substantial prenatal diagnostic yield, similar to postnatal diagnosis with a median turnaround of approximately 1 month, supporting its routine implementation during the detection of prenatal US anomalies.

Purpose: Multi-omics offer worthwhile and increasingly accessible technologies to diagnostic laboratories seeking potential second-tier strategies to help patients with unresolved rare diseases, especially patients clinically diagnosed with a rare OMIM (Online Mendelian Inheritance in Man) disease. However, no consensus exists regarding the optimal diagnostic care pathway to adopt after negative results with standard approaches.Methods: In 15 unsolved individuals clinically diagnosed with recognizable OMIM diseases but with negative or inconclusive first-line genetic results, we explored the utility of a multi-step approach using several novel omics technologies to establish a molecular diagnosis. Inclusion criteria included a clinical autosomal recessive disease diagnosis and single heterozygous pathogenic variant in the gene of interest identified by first-line analysis (60%–9/15) or a clinical diagnosis of an X-linked recessive or autosomal dominant disease with no causative variant identified (40%–6/15). We performed a multi-step analysis involving short-read genome sequencing (srGS) and complementary approaches such as mRNA sequencing (mRNA-seq), long-read genome sequencing (lrG), or optical genome mapping (oGM) selected according to the outcome of the GS analysis.Results: SrGS alone or in combination with additional genomic and/or transcriptomic technologies allowed us to resolve 87% of individuals by identifying single nucleotide variants/indels missed by first-line targeted tests, identifying variants affecting transcription, or structural variants sometimes requiring lrGS or oGM for their characterization.Conclusion: Hypothesis-driven implementation of combined omics technologies is particularly effective in identifying molecular etiologies. In this study, we detail our experience of the implementation of genomics and transcriptomics technologies in a pilot cohort of previously investigated patients with a typical clinical diagnosis without molecular etiology.

Purpose: Patients with rare or ultra-rare genetic diseases, which affect 350 million people worldwide, may experience a diagnostic odyssey. High-throughput sequencing leads to an etiological diagnosis in up to 50% of individuals with heterogeneous neurodevelopmental or malformation disorders. There is a growing interest in additional omics technologies in translational research settings to examine the remaining unsolved cases.Methods: We gathered 30 individuals with malformation syndromes and/or severe neurodevelopmental disorders with negative trio exome sequencing and array comparative genomic hybridization results through a multicenter project. We applied short-read genome sequencing, total RNA sequencing, and DNA methylation analysis, in that order, as complementary translational research tools for a molecular diagnosis.Results: The cohort was mainly composed of pediatric individuals with a median age of 13.7 years (4 years and 6 months to 35 years and 1 month). Genome sequencing alone identified at least one variant with a high level of evidence of pathogenicity in 8/30 individuals (26.7%) and at least a candidate disease-causing variant in 7/30 other individuals (23.3%). RNA-seq data in 23 individuals allowed two additional individuals (8.7%) to be diagnosed, confirming the implication of two pathogenic variants (8.7%), and excluding one candidate variant (4.3%). Finally, DNA methylation analysis confirmed one diagnosis identified by genome sequencing (Kabuki syndrome) and identified an episignature compatible with a BAFopathy in a patient with a clinical diagnosis of Coffin-Siris with negative genome and RNA-seq results in blood.Conclusion: Overall, our integrated genome, transcriptome, and DNA methylation analysis solved 10/30 (33.3%) cases and identified a strong candidate gene in 4/30 (13.3%) of the patients with rare neurodevelopmental disorders and negative exome sequencing results.