Further description of two individuals with de novo p.(Glu127Lys) missense variant in the ASCL1 gene.

Achaete-Scute Family basic-helix-loop-helix (bHLH) Transcription Factor 1 (ASCL1) is a proneural transcription factor involved in neuron development in the central and peripheral nervous system. While initially suspected to contribute to congenital central hypoventilation syndrome-1 (CCHS) with or without Hirschsprung disease (HSCR) in three individuals, its implication was ruled out by the presence, in one of the individuals, of a Paired-like homeobox 2B (PHOX2B) heterozygous polyalanine expansion variant, known to cause CCHS. We report two additional unrelated individuals sharing the same sporadic ASCL1 p.(Glu127Lys) missense variant in the bHLH domain and a common phenotype with short-segment HSCR, signs of dysautonomia, and developmental delay. One has also mild CCHS without polyalanine expansion in PHOX2B, compatible with the diagnosis of Haddad syndrome. Furthermore, missense variants with homologous position in the same bHLH domain in other genes are known to cause human diseases. The description of additional individuals carrying the same variant and similar phenotype, as well as targeted functional studies, would be interesting to further evaluate the role of ASCL1 in neurocristopathies.

Early prenatal diagnosis of causative homozygous variants in ASCC1 in a fetus with cystic hygroma and additional homozygous variants of unknown significance associated with a neurological phenotype not visible in early gestation: Dual diagnosis or not?

A consanguineous couple was referred at 10 weeks of gestation (WG) for prenatal genetic investigations due to isolated cystic hygroma. Prenatal trio exome sequencing identified causative homozygous truncating variants in ASCC1 previously implicated in spinal muscular atrophy with congenital bone fractures. Prenatal manifestations in ASCC1 can usually include hydramnios, fetal hypo-/akinesia, arthrogryposis, contractures and limb deformities, hydrops fetalis and cystic hygroma. An additional truncating variant was identified in CSPP1 associated with Joubert syndrome. Presentations in CSPP1 include cerebellar and brainstem malformations with vermis hypoplasia and molar tooth sign, difficult to visualize in early gestation. A second pregnancy was marked by the recurrence of isolated increased nuchal translucency at 10 + 2 WG. Sanger prenatal diagnosis targeted on ASCC1 and CSPP1 variants showed the presence of the homozygous familial ASCC1 variant. In this case, prenatal exome sequencing analysis is subject to a partial ASCC1 phenotype and an undetectable CSPP1 phenotype at 10 weeks of gestation. As CSPP1 contribution is unclear or speculative to a potentially later in pregnancy or postnatal phenotype, it is mentioned as a variant of uncertain significance. The detection of pathogenic or likely pathogenic variants involved in severe disorders but without phenotype-genotype correlation because the pregnancy is in the early stages or due to prenatally undetectable phenotypes, will encourage the clinical community to define future practices in molecular prenatal reporting.

Multiple molecular diagnoses in the field of intellectual disability and congenital anomalies: 3.5% of all positive cases.

PURPOSE: Wide access to clinical exome/genome sequencing (ES/GS) enables the identification of multiple molecular diagnoses (MMDs), being a long-standing but underestimated concept, defined by two or more causal loci implicated in the phenotype of an individual with a rare disease. Only few series report MMDs rates (1.8% to 7.1%). This study highlights the increasing role of MMDs in a large cohort of individuals addressed for congenital anomalies/intellectual disability (CA/ID). METHODS: From 2014 to 2021, our diagnostic laboratory rendered 880/2658 positive ES diagnoses for CA/ID aetiology. Exhaustive search on MMDs from ES data was performed prospectively (January 2019 to December 2021) and retrospectively (March 2014 to December 2018). RESULTS: MMDs were identified in 31/880 individuals (3.5%), responsible for distinct (9/31) or overlapping (22/31) phenotypes, and potential MMDs in 39/880 additional individuals (4.4%). CONCLUSION: MMDs are frequent in CA/ID and remain a strong challenge. Reanalysis of positive ES data appears essential when phenotypes are partially explained by the initial diagnosis or atypically enriched overtime. Up-to-date clinical data, clinical expertise from the referring physician, strong interactions between clinicians and biologists, and increasing gene discoveries and improved ES bioinformatics tools appear all the more fundamental to enhance chances of identifying MMDs. It is essential to provide appropriate patient care and genetic counselling.

FOXG1 variants can be associated with milder phenotypes than congenital Rett syndrome with unassisted walking and language development.

Since 2008, FOXG1 haploinsufficiency has been linked to a severe neurodevelopmental phenotype resembling Rett syndrome but with earlier onset. Most patients are unable to sit, walk, or speak. For years, FOXG1 sequencing was only prescribed in such severe cases, limiting insight into the full clinical spectrum associated with this gene. Next-generation sequencing (NGS) now enables unbiased diagnostics. Through the European Reference Network for Rare Malformation Syndromes, Intellectual and Other Neurodevelopmental Disorders, we gathered data from patients with heterozygous FOXG1 variants presenting a mild phenotype, defined as able to speak and walk independently. We also reviewed data from three previously reported patients meeting our criteria. We identified five new patients with pathogenic FOXG1 missense variants, primarily in the forkhead domain, showing varying nonspecific intellectual disability and developmental delay. These features are not typical of congenital Rett syndrome and were rarely associated with microcephaly and epilepsy. Our findings are consistent with a previous genotype-phenotype analysis by Mitter et al. suggesting the delineation of five different FOXG1 genotype groups. Milder phenotypes were associated with missense variants in the forkhead domain. This information may facilitate prognostic assessments in children carrying a FOXG1 variant and improve the interpretation of new variants identified with genomic sequencing.

De Novo Variants in CNOT1, a Central Component of the CCR4-NOT Complex Involved in Gene Expression and RNA and Protein Stability, Cause Neurodevelopmental Delay.

CNOT1 is a member of the CCR4-NOT complex, which is a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. We report on 39 individuals with heterozygous de novo CNOT1 variants, including missense, splice site, and nonsense variants, who present with a clinical spectrum of intellectual disability, motor delay, speech delay, seizures, hypotonia, and behavioral problems. To link CNOT1 dysfunction to the neurodevelopmental phenotype observed, we generated variant-specific Drosophila models, which showed learning and memory defects upon CNOT1 knockdown. Introduction of human wild-type CNOT1 was able to rescue this phenotype, whereas mutants could not or only partially, supporting our hypothesis that CNOT1 impairment results in neurodevelopmental delay. Furthermore, the genetic interaction with autism-spectrum genes, such as ASH1L, DYRK1A, MED13, and SHANK3, was impaired in our Drosophila models. Molecular characterization of CNOT1 variants revealed normal CNOT1 expression levels, with both mutant and wild-type alleles expressed at similar levels. Analysis of protein-protein interactions with other members indicated that the CCR4-NOT complex remained intact. An integrated omics approach of patient-derived genomics and transcriptomics data suggested only minimal effects on endonucleolytic nonsense-mediated mRNA decay components, suggesting that de novo CNOT1 variants are likely haploinsufficient hypomorph or neomorph, rather than dominant negative. In summary, we provide strong evidence that de novo CNOT1 variants cause neurodevelopmental delay with a wide range of additional co-morbidities. Whereas the underlying pathophysiological mechanism warrants further analysis, our data demonstrate an essential and central role of the CCR4-NOT complex in human brain development.

High efficiency and clinical relevance of exome sequencing in the daily practice of neurogenetics.

OBJECTIVE: To assess the efficiency and relevance of clinical exome sequencing (cES) as a first-tier or second-tier test for the diagnosis of progressive neurological disorders in the daily practice of Neurology and Genetic Departments. METHODS: Sixty-seven probands with various progressive neurological disorders (cerebellar ataxias, neuromuscular disorders, spastic paraplegias, movement disorders and individuals with complex phenotypes labelled 'other') were recruited over a 4-year period regardless of their age, gender, familial history and clinical framework. Individuals could have had prior genetic tests as long as it was not cES. cES was performed in a proband-only (60/67) or trio (7/67) strategy depending on available samples and was analysed with an in-house pipeline including software for CNV and mitochondrial-DNA variant detection. RESULTS: In 29/67 individuals, cES identified clearly pathogenic variants leading to a 43% positive yield. When performed as a first-tier test, cES identified pathogenic variants for 53% of individuals (10/19). Difficult cases were solved including double diagnoses within a kindred or identification of a neurodegeneration with brain iron accumulation in a patient with encephalopathy of suspected mitochondrial origin. CONCLUSION: This study shows that cES is a powerful tool for the daily practice of neurogenetics offering an efficient (43%) and appropriate approach for clinically and genetically complex and heterogeneous disorders.

Copy number variants calling from WES data through eXome hidden Markov model (XHMM) identifies additional 2.5% pathogenic genomic imbalances smaller than 30 kb undetected by array-CGH.

It has been estimated that Copy Number Variants (CNVs) account for 10%-20% of patients affected by Developmental Disorder (DD)/Intellectual Disability (ID). Although array comparative genomic hybridization (array-CGH) represents the gold-standard for the detection of genomic imbalances, common Agilent array-CGH 4 × 180 kb arrays fail to detect CNVs smaller than 30 kb. Whole Exome sequencing (WES) is becoming the reference application for the detection of gene variants and makes it possible also to infer genomic imbalances at single exon resolution. However, the contribution of small CNVs in DD/ID is still underinvestigated. We made use of the eXome Hidden Markov Model (XHMM) software, a tool utilized by the ExAC consortium, to detect CNVs from whole exome sequencing data, in a cohort of 200 unsolved DD/DI patients after array-CGH and WES-based single nucleotide/indel variant analyses. In five out of 200 patients (2.5%), we identified pathogenic CNV(s) smaller than 30 kb, ranging from one to six exons. They included two heterozygous deletions in TCF4 and STXBP1 and three homozygous deletions in PPT1, CLCN2, and PIGN. After reverse phenotyping, all variants were reported as causative. This study shows the interest in applying sequencing-based CNV detection, from available WES data, to reduce the diagnostic odyssey of additional patients unsolved DD/DI patients and compare the CNV-detection yield of Agilent array-CGH 4 × 180kb versus whole exome sequencing.

Haploinsufficiency of ARFGEF1 is associated with developmental delay, intellectual disability, and epilepsy with variable expressivity.

PURPOSE: ADP ribosylation factor guanine nucleotide exchange factors (ARFGEFs) are a family of proteins implicated in cellular trafficking between the Golgi apparatus and the plasma membrane through vesicle formation. Among them is ARFGEF1/BIG1, a protein involved in axon elongation, neurite development, and polarization processes. ARFGEF1 has been previously suggested as a candidate gene for different types of epilepsies, although its implication in human disease has not been well characterized. METHODS: International data sharing, in silico predictions, and in vitro assays with minigene study, western blot analyses, and RNA sequencing. RESULTS: We identified 13 individuals with heterozygous likely pathogenic variants in ARFGEF1. These individuals displayed congruent clinical features of developmental delay, behavioral problems, abnormal findings on brain magnetic resonance image (MRI), and epilepsy for almost half of them. While nearly half of the cohort carried de novo variants, at least 40% of variants were inherited from mildly affected parents who were clinically re-evaluated by reverse phenotyping. Our in silico predictions and in vitro assays support the contention that ARFGEF1-related conditions are caused by haploinsufficiency, and are transmitted in an autosomal dominant fashion with variable expressivity. CONCLUSION: We provide evidence that loss-of-function variants in ARFGEF1 are implicated in sporadic and familial cases of developmental delay with or without epilepsy.

Second-tier trio exome sequencing after negative solo clinical exome sequencing: an efficient strategy to increase diagnostic yield and decipher molecular bases in undiagnosed developmental disorders.

Developmental disorders (DD), characterized by malformations/dysmorphism and/or intellectual disability, affecting around 3% of worldwide population, are mostly linked to genetic anomalies. Despite clinical exome sequencing (cES) centered on genes involved in human genetic disorders, the majority of patients affected by DD remain undiagnosed after solo-cES. Trio-based strategy is expected to facilitate variant selection thanks to rapid parental segregation. We performed a second step trio-ES (not only focusing on genes involved in human disorders) analysis in 70 patients with negative results after solo-cES. All candidate variants were shared with a MatchMaking exchange system to identify additional patients carrying variants in the same genes and with similar phenotype. In 18/70 patients (26%), we confirmed causal implication of nine OMIM-morbid genes and identified nine new strong candidate genes (eight de novo and one compound heterozygous variants). These nine new candidate genes were validated through the identification of patients with similar phenotype and genotype thanks to data sharing. Moreover, 11 genes harbored variants of unknown significance in 10/70 patients (14%). In DD, a second step trio-based ES analysis appears an efficient strategy in diagnostic and translational research to identify highly candidate genes and improve diagnostic yield.

Next-generation sequencing approaches and challenges in the diagnosis of developmental anomalies and intellectual disability.

Recent advances in next-generation sequencing (NGS) technologies have revolutionized the field of human genetics. Alongside a broad panel of bioinformatics tools and databases, NGS technologies have unprecedentedly improved the molecular diagnosis rate and the identification of new genes associated with rare disorders. However, about 50% of patients remain without a final diagnosis. Here, we highlight the utility of NGS applications in developmental anomalies and intellectual disability, illustrating their main advantages and pitfalls. Through specific examples, we suggest novel strategies and tools for identifying the molecular bases in the remaining patients, and we outline future challenges.

Novel KIAA1033/WASHC4 mutations in three patients with syndromic intellectual disability and a review of the literature.

In 2011, KIAA1033/WASHC4 was associated with autosomal recessive intellectual disability (ARID) in a large consanguineous family comprising seven affected individuals with moderate ID and short stature. Since then, no other cases of KIAA1033 variants have been reported. Here we describe three additional patients (from two unrelated families) with syndromic ID due to compound heterozygous KIAA1033 variants ascertained by exome sequencing (ES). Two sisters, aged 4 and 5.5 years, had a stop-gain and a missense variants, each inherited from one parent (p.(Gln442*) and p.(Asp1048Gly)). Both had learning disabilities, macrocephaly, dysmorphic features, skeletal anomalies, and subependymal heterotopic nodules. In addition, the younger sibling had a congenital absence of the right internal carotid and bilateral sensorineural hearing loss. The third patient was aged 34 years and had two missense variants, one inherited from each parent (p.(Lys1079Arg) and p.(His503Arg)). This patient presented with mild ID, short stature, and microcephaly. KIAA1033 encodes a large protein (WASHC4), which is part of the WASH complex. The WASH complex is involved in the regulation of the fission of tubules that serve as transport intermediates during endosome sorting. Another member of the WASH complex, KIAA0196/WASHC5, has already been implicated in ARID with brain and cardiac malformations, under the designation of 3C or Ritscher-Schinzel syndrome (MIM#20210). ES has proved efficient for finding replications of genes with insufficient data in the literature to be defined as new OMIM genes. We conclude that KIAA1033 is responsible for a heterogeneous ARID phenotype, and additional description will be needed to refine the clinical phenotype.

2.5 years' experience of GeneMatcher data-sharing: a powerful tool for identifying new genes responsible for rare diseases.

PURPOSE: Exome sequencing (ES) powerfully identifies the molecular bases of heterogeneous conditions such as intellectual disability and/or multiple congenital anomalies (ID/MCA). Current ES analysis, combining diagnosis analysis restricted to disease-causing genes reported in OMIM database and subsequent research investigation extended to other genes, indicated causal and candidate genes around 40% and 10%. Nonconclusive results are frequent in such ultrarare conditions that recurrence and genotype-phenotype correlations are limited. International data-sharing permits the gathering of additional patients carrying variants in the same gene to draw definitive conclusions on their implication as disease causing. Several web-based tools have been developed and grouped in Matchmaker Exchange. In this study, we report our current experience as a regional center that has implemented ES as a first-line diagnostic test since 2013, working with a research laboratory devoted to disease gene identification. METHODS: We used GeneMatcher over 2.5 years to share 71 novel candidate genes identified by ES. RESULTS: Matches occurred in 60/71 candidate genes allowing to confirm the implication of 39% of matched genes as causal and to rule out 6% of them. CONCLUSION: The introduction of user-friendly gene-matching tools, such as GeneMatcher, appeared to be an essential step for the rapid identification of novel disease genes responsible for ID/MCA.

Hearing impairment as an early sign of alpha-mannosidosis in children with a mild phenotype: Report of seven new cases.

Alpha-mannosidosis (AM) is a very rare (prevalence: 1/500000 births) autosomal recessive lysosomal storage disorder. It is characterized by multi-systemic involvement associated with progressive intellectual disability, hearing loss, skeletal anomalies, and coarse facial features. The spectrum is wide, from very severe and lethal to a milder phenotype that usually progresses slowly. AM is caused by a deficiency of lysosomal alpha-mannosidase. A diagnosis can be established by measuring the activity of lysosomal alpha-mannosidase in leucocytes and screening for abnormal urinary excretion of mannose-rich oligosaccharides. Genetic confirmation is obtained with the identification of MAN2B1 mutations. Enzyme replacement therapy (LAMZEDER ) was approved for use in Europe in August 2018. Here, we describe seven individuals from four families, diagnosed at 3-23 years of age, and who were referred to a clinical geneticist for etiologic exploration of syndromic hearing loss, associated with moderate learning disabilities. Exome sequencing had been used to establish the molecular diagnosis in five cases, including a two-sibling pair. In the remaining two patients, the diagnosis was obtained with screening of urinary oligosaccharides excretion and the association of deafness and hypotonia. These observations emphasize that the clinical diagnosis of AM can be challenging, and that it is likely an underdiagnosed rare cause of syndromic hearing loss. Exome sequencing can contribute significantly to the early diagnosis of these nonspecific mild phenotypes, with advantages for treatment and management.

Episignatures in practice: independent evaluation of published episignatures for the molecular diagnostics of ten neurodevelopmental disorders.

Variants of uncertain significance (VUS) are a significant issue for the molecular diagnosis of rare diseases. The publication of episignatures as effective biomarkers of certain Mendelian neurodevelopmental disorders has raised hopes to help classify VUS. However, prediction abilities of most published episignatures have not been independently investigated yet, which is a prerequisite for an informed and rigorous use in a diagnostic setting. We generated DNA methylation data from 101 carriers of (likely) pathogenic variants in ten different genes, 57 VUS carriers, and 25 healthy controls. Combining published episignature information and new validation data with a k-nearest-neighbour classifier within a leave-one-out scheme, we provide unbiased specificity and sensitivity estimates for each of the signatures. Our procedure reached 100% specificity, but the sensitivities unexpectedly spanned a very large spectrum. While ATRX, DNMT3A, KMT2D, and NSD1 signatures displayed a 100% sensitivity, CREBBP-RSTS and one of the CHD8 signatures reached <40% sensitivity on our dataset. Remaining Cornelia de Lange syndrome, KMT2A, KDM5C and CHD7 signatures reached 70-100% sensitivity at best with unstable performances, suffering from heterogeneous methylation profiles among cases and rare discordant samples. Our results call for cautiousness and demonstrate that episignatures do not perform equally well. Some signatures are ready for confident use in a diagnostic setting. Yet, it is imperative to characterise the actual validity perimeter and interpretation of each episignature with the help of larger validation sample sizes and in a broader set of episignatures.

Further clinical and molecular characterization of an XLID syndrome associated with BRWD3 variants, a gene implicated in the leukemia-related JAK-STAT pathway.

BACKGROUND: Since the first description of a BRWD3-associated nonsydromic intellectual disability (ID) disorder in 2007, 21 additional families have been reported in the literature. METHODS: Using exome sequencing (ES) and international data sharing, we identified 14 additional unrelated individuals with pathogenic BRWD3 variants (12 males and 2 females, including one with skewed X-inactivation). We reviewed the 31 previously published cases in the literature with clinical data available, and describe the collective phenotypes of 43 males and 2 females, with 33 different BRWD3 variants. RESULTS: The most common features in males (excluding one patient with a mosaic variant) included ID (39/39 males), speech delay (24/25 males), postnatal macrocephaly (28/35 males) with prominent forehead (18/25 males) and large ears (14/26 males), and obesity (12/27 males). Both females presented with macrocephaly, speech delay, and epilepsy, while epilepsy was only observed in 4/41 males. Among the 28 variants with available segregation reported, 19 were inherited from unaffected mothers and 9 were de novo. CONCLUSION: This study demonstrates that the BRWD3-related phenotypes are largely non-specific, leading to difficulty in clinical recognition of this disorder. A genotype-first approach, however, allows for the more efficient diagnosis of the BRWD3-related nonsyndromic ID. The refined clinical features presented here may provide additional diagnostic assistance for reverse phenotyping efforts.

Patient satisfaction, experience and preferences in the implementation of genetics teleconsultations in the North-eastern region of France.

INTRODUCTION: In France, few centres per region offer genetics consultations. Consequently, each centre covers a large area, often requiring patients to take a day off to travel long distances. In certain situations, genetic counselling in particular, a physical exam is not required. In these cases, teleconsultations between medical professional and patients, at the patient's location of choice, are an interesting offer. The COVID-19 pandemic has accelerated the implementation and the use of this type of consultation. With the aim of developing teleconsultation for certain types of referrals, a study of patient satisfaction, experience and preferences has been set up in our region. METHODS: 2307 patients who had a teleconsultation by phone or videoconferencing with professionals from one of five genetic centres in North-eastern France between March and December 2020 were asked by e-mail or by post to answer an online survey. RESULTS: 20% of the patients (n = 465) responded to the survey (80% women, 55% over 40 years old). In 64% of the cases (n = 299), the teleconsultation replaced a physical consultation due to the pandemic. In 56% of cases (n = 217), the consultations were conducted by videoconference. The teleconsultation involved the disclosure of results in 56% of cases (n = 260), a first consultation in 30% of cases (n = 138), and a follow-up consultation in 14% of cases (n = 67). The satisfaction rate was 96% (n = 447), with a rating of "excellent" in 72% of responses (n = 290) and "good" in 24% of responses (n = 157). Only 22% of the patients (n = 103), particularly patients who lived near the hospital or who were older than 70 years, would have preferred a physical consultation. Half of respondents (n = 232) declared that they avoided more than 1.5 h of transport, and 69% (n = 321) avoided taking a work day off. Patients were less often accompanied by a relative than if the consultation had taken place face-to-face (43%; n = 201 vs. 61%; n = 285). There was no change in responses during or after lock-down. CONCLUSION: This collection of feedback and analysis of patients' preferences has validated the long-term implementation of medical genetics teleconsultations in certain circumstances and indications, for patients who prefer this approach.

Combining globally search for a regular expression and print matching lines with bibliographic monitoring of genomic database improves diagnosis.

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.

Stepwise use of genomics and transcriptomics technologies increases diagnostic yield in Mendelian disorders.

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.

The neurodevelopmental and facial phenotype in individuals with a TRIP12 variant.

Haploinsufficiency of TRIP12 causes a neurodevelopmental disorder characterized by intellectual disability associated with epilepsy, autism spectrum disorder and dysmorphic features, also named Clark-Baraitser syndrome. Only a limited number of cases have been reported to date. We aimed to further delineate the TRIP12-associated phenotype and objectify characteristic facial traits through GestaltMatcher image analysis based on deep-learning algorithms in order to establish a TRIP12 gestalt. 38 individuals between 3 and 66 years (F = 20, M = 18) - 1 previously published and 37 novel individuals - were recruited through an ERN ITHACA call for collaboration. 35 TRIP12 variants were identified, including frameshift (n = 15) and nonsense (n = 6) variants, as well as missense (n = 5) and splice (n = 3) variants, intragenic deletions (n = 4) and two multigene deletions disrupting TRIP12. Though variable in severity, global developmental delay was noted in all individuals, with language deficit most pronounced. About half showed autistic features and susceptibility to obesity seemed inherent to this disorder. A more severe expression was noted in individuals with a missense variant. Facial analysis showed a clear gestalt including deep-set eyes with narrow palpebral fissures and fullness of the upper eyelids, downturned corners of the mouth and large, often low-set ears with prominent earlobes. We report the largest cohort to date of individuals with TRIP12 variants, further delineating the associated phenotype and introducing a facial gestalt. These findings will improve future counseling and patient guidance.

Prenatal diagnosis by trio exome sequencing in fetuses with ultrasound anomalies: A powerful diagnostic tool.

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.