Anne-Sophie Denommé-Pichon

Pathogenic variants in RUBCN , encoding the Run domain Beclin-1 interacting and cysteine-rich domain-containing protein (Rubicon) have been implicated in autosomal recessive spinocerebellar ataxia 15 (SCAR15). However, the molecular mechanisms underlying disease pathogenesis remain poorly understood. Here, we report 18 individuals from 15 unrelated families harbouring biallelic RUBCN variants, who present with an aggressive neurodevelopmental disorder variably characterized by seizures, developmental delay, intellectual disability and movement abnormalities that cause regression, progressive brain atrophy and neurodegenerative features. Through functional characterization, we demonstrate that a subset of disease-associated putative truncating variants disrupt autophagy regulation. In Caenorhabditis elegans models, loss-of-function RUBCN variants result in an increased autophagic flux and impaired neuronal function, recapitulating key features in humans. Correspondingly, cellular assays reveal that nonsense and frameshift RUBCN variants lead to defective autophagy inhibition, underscoring a crucial role for RUBCN as a key negative autophagy regulator. Molecular dynamics simulations rank the eleven missense variants by structural effect, with p.Arg813Trp alone altering the target protein at both the local and the regional level and lying within the RAB7A-binding module that the truncating alleles remove altogether. Our findings establish and expand the RUBCN -related disorders as a clinically and molecularly distinct subset of autophagy-related diseases. By delineating both the genetic landscape and cellular consequences of Rubicon dysfunction, this study enhances our understanding of autophagy-related neurodevelopmental disorders and provides a foundation for future therapeutic investigations.

Les analyses pangénomiques d’exome et de génome ont permis d’augmenter considérablement le rendement diagnostique des maladies rares d’origine génétique au cours des dernières années, mais plus de 50 % des patients restent en impasse diagnostique (Wright, FitzPatrick, et Firth 2018). Pour les patients qui bénéficient d’un diagnostic, le délai d’obtention reste long, dépassant 5 ans pour 25 % des patients (Ministère des Solidarités et de la Santé 2019). L’absence de diagnostic ou le délai avant celui-ci sont une réelle perte de chance pour le patient et les familles, en termes de prise en charge de soin, mais également en termes de prise en charge sociale.L’objectif de la thèse est d’optimiser l’analyse de données génomiques dans les maladies rares, par l’implémentation de nouvelles approches, qu’elles soient organisationnelles, biologiques ou bioinformatiques, en vue d’obtenir un diagnostic plus rapidement dans un contexte d’urgence, et d’augmenter le rendement diagnostique. La thèse est divisée en trois axes d’optimisation.Le premier axe, l’optimisation organisationnelle, consiste en l’étude Fastgen, qui évalue la faisabilité de l’analyse accélérée de génomes short read en trio chez des enfants en réanimation ou soins intensifs, nécessitant un diagnostic génétique précoce pour orienter leur prise en charge. L’objectif était d’obtenir un résultat en moins de 45 jours. Un séquençage de génomes en trio a été réalisé chez 37 familles, conduisant à un diagnostic moléculaire chez 18/37 (49 %) enfants, avec une durée médiane de rendu de résultat de 42 jours.Le deuxième axe, l’optimisation biologique, consiste en l’étude de la cohorte unsolved cases du projet Solve-RD, qui vise à réduire l’impasse diagnostique par la réanalyse de données d’exomes de patients sans diagnostic et par la mise à disposition des données génomiques aux équipes du projet. Les données de 3576 exomes (1522 cas index et 2054 parents) ont été réanalysées selon une stratégie low-hanging fruit qui vise à rechercher les variations déjà rapportées dans ClinVar comme étant pathogènes ou probablement pathogènes. La réanalyse a permis d’identifier les variations causales chez 59 patients (3,9 %), dont 9 pour lesquels nous présentons les raisons de l’absence de diagnostic au moment de la première analyse et les leçons à en tirer.Le partage des données au sein de Solve-RD a facilité la construction d’une cohorte qui a permis d’établir la responsabilité du gène YWHAE dans un trouble du neurodéveloppement avec ou sans anomalies cérébrales, ce qui a mené à un diagnostic chez 7 sujets avec des variations perte de fonction de ce gène.Le troisième axe, l’optimisation bioinformatique, consiste en l’implémentation d’un pipeline utilisant les meilleurs outils de génotypage d’expansions de microsatellites à partir de données de génome short read, dans le but d’établir de nouveaux diagnostics. Nous avons évalué les 10 principaux outils de la littérature, en avons conservé 4 et les avons testés sur 23 locus impliqués en pathologie humaine sur notre cohorte de 323 génomes de sujets atteints de troubles du neurodéveloppement. Le pipeline développé a mené à l’identification d’une expansion de microsatellites pathologique pouvant prédisposer à une maladie neurodégénérative, considérée comme une donnée incidente car sans lien avec le phénotype du patient.Notre stratégie d’optimisation de l’analyse des données génomiques dans les maladies rares s’avère efficace : elle a permis de réduire l’errance diagnostique chez des enfants dans un contexte d’urgence et d’augmenter le rendement diagnostique du séquençage de l’exome et du génome short read par le développement de nouvelles stratégies d’analyse, de réanalyse et par le partage de données à large échelle.

Small nuclear RNAs (snRNAs) are essential components of the spliceosome. De novo variants in snRNA genes RNU4-2 (ReNU syndrome), RNU5B-1 and RNU2-2 have been linked to dominant neurodevelopmental disorders (NDDs), revealing a large unexpected contribution of noncoding RNA genes to genetic diseases. Here, through international collaborations, we analyze systematically 200 potentially functional snRNA genes in a French cohort of 34,329 people with rare disorders. We report RNU2-2 variants in 141 individuals, including 35 with recurrent dominant pathogenic variants and 91 affected members from 73 families with biallelic variants. Recessive RNU2-2 NDD is at least twice as frequent as the dominant form and often involves a de novo variant in trans with an inherited allele, consistent with the high mutability of snRNA genes. Dominant and recessive RNU2-2 NDDs share overlapping clinical features, with frequent epilepsy. Blood transcriptomics and DNA methylation analyses revealed subtle, variant-specific effects on splicing and episignatures. Our results support a gradient-of-impact model bridging dominant and recessive inheritance, and establish RNU2-2 variants as a principal contributor to NDDs, nearly as prevalent as ReNU syndrome.

Genetic disorders affecting the epigenetic machinery constitute a major group of neurodevelopmental conditions. Pathogenic variants in several ARID transcription factors—particularly ARID1A, ARID1B, and ARID2—cause Coffin–Siris syndromes, all characterized by intellectual disability (ID). These genes encode core subunits of the BRG1/BRM-associated factor (BAF) chromatin remodeling complex. In contrast, ARID family members that function in other regulatory complexes have remained largely unexplored in neurodevelopmental disease. Here, we identify 29 individuals carrying heterozygous ARID5B variants, of which 24 (83%) introduce premature termination codons in the exceptionally long final exon, one affects the exon 9 splice donor site, and four are missense variants in conserved domains within the N-terminal half of the protein. Using a CRISPR–Cas9 knock-in mouse model harboring the p.Q522Ter variant, together with in vitro assays, we investigated the functional consequences of C-terminal ARID5B truncations. All affected individuals presented with global developmental delay or ID—most commonly mild—and frequent speech and language impairment. Recurrent features included kidney malformations, behavioral difficulties, and recurrent infections of the respiratory and urinary tracts. Two individuals experienced central nervous system inflammation, and two infants presented with persistent pulmonary hypertension. Remarkably, 19 of 29 variants (66%) cluster within the first quarter of exon 10, are de novo, and escape nonsense-mediated mRNA decay (NMD), which we confirmed for two variants affecting seven individuals. Variants outside this region were inherited. Heterozygous mice exhibited developmental and behavioral abnormalities, while homozygous mutations was perinatally lethal. Truncations and a small deletion within a predicted nuclear localization signal (NLS) caused cytosolic mislocalization of ARID5B, whereas the isolated C-terminal half retained nuclear localization, suggesting an independent distal NLS. Collectively, these findings define ARID5B-related neurodevelopmental disorder as a distinct clinical entity and reveal how disruption of specific ARID5B domains impacts protein localization, mammalian development, immune and neurobehavioral function.

Cohesin is a fundamental genome-organizing complex that orchestrates three-dimensional chromosome folding and gene expression via DNA loop extrusion. Alterations to genes encoding cohesin subunits and cohesin loaders cause Mendelian disorders, including Cornelia de Lange syndrome (CdLS). By contrast, disruption of factors that remove cohesin from DNA, including WAPL and its binding partners PDS5A and PDS5B , have not yet been associated with human disease. Here, we explored the relevance of these cohesin release factors in Mendelian disease by establishing a rare disease cohort of deeply phenotyped individuals with heterozygous, predicted damaging variants in WAPL (n=27), PDS5A (n=8), and PDS5B (n=8), by modeling WAPL deficiency in human cell lines and mice, and by aggregating rare disease association statistics from consortia studies. We identified a WAPL -related disorder characterized by developmental delay, intellectual disability, and risk of other developmental anomalies including clubfoot. Similarities between individuals with damaging WAPL variants and those with large, recurrent 10q22.3q23.2 (10q) deletions (which encompass WAPL ) nominate WAPL as a driver gene within this genomic disorder region. While carriers of PDS5A or PDS5B variants exhibited features of developmental disorders, neither cohort-based statistics nor case phenotyping associated these genes with specific phenotypes. We used CRISPR engineering to generate truncating variants in WAPL , as well the 7.8 Mb 10q deletion or duplication in human iPSCs and induced neurons. Transcriptomic analyses identified differentially expressed genes in both models, with highly significant overlap between WAPL haploinsufficiency and 10q deletion signatures. Mice with 50% residual Wapl expression exhibited mild deficits of growth and learning/memory, whereas those with 25% residual Wapl expression displayed birth defects and postnatal lethality, revealing a dosage liability threshold below the level of heterozygosity. In summary, we delineated a novel genetic condition caused by cohesin release factor deficiency, nominated WAPL as a driver gene within a genomic disorder region, and further illuminated dosage sensitivity of human cohesin.

SF3B1 is an essential and ubiquitous splicing factor that plays a pivotal role in the early steps of pre-mRNA splicing. Recurrent somatic missense mutations in SF3B1 are frequent in cancers, but no constitutional variant has been reported so far. We describe here a cohort of 26 individuals with neurodevelopmental disorders, harbouring SF3B1 constitutional heterozygous variants that appeared mostly de novo. Patients present with a global developmental delay, associated with variable neurological and facial dysmorphic traits. A dichotomy may emerge between patients harbouring predicted loss of function ( n = 9) and missense variants ( n = 17), the latter being associated with a more severe and syndromic phenotype, including heart and gastrointestinal anomalies. We focused on de novo SF3B1 missense variants, which were largely distinct from those reported in cancer. Functional complementation assays show that de novo SF3B1 missense variants did not cause a loss of function of the protein. Targeted and genome-wide analysis of RNA splicing reveal that they affect canonical and alternative splicing more moderately than somatic variants, and subtly modify the splicing of many transcripts. These findings place SF3B1 among the rare U2 snRNP components implicated in both cancer and neurodevelopmental disorders, highlighting its critical and multifaceted role in human disease.

Genetic disorders affecting the epigenetic machinery constitute a major group of neurodevelopmental conditions. Pathogenic variants in several ARID transcription factors—particularly ARID1A , ARID1B , and ARID2 —cause Coffin–Siris syndromes, all characterized by intellectual disability (ID). These genes encode core subunits of the BRG1/BRM-associated factor (BAF) chromatin remodeling complex. In contrast, ARID family members that function in other regulatory complexes have remained largely unexplored in neurodevelopmental disease. Here, we identify 29 individuals carrying heterozygous ARID5B variants, of which 24 (83%) introduce premature termination codons in the exceptionally long final exon, one affects the exon 9 splice donor site, and four are missense variants in conserved domains within the N-terminal half of the protein. Using a CRISPR–Cas9 knock-in mouse model harboring the p.Q522Ter variant, together with in vitro assays, we investigated the functional consequences of C-terminal ARID5B truncations. All affected individuals presented with global developmental delay or ID—most commonly mild—and frequent speech and language impairment. Recurrent features included kidney malformations, behavioral difficulties, and recurrent infections of the respiratory and urinary tracts. Two individuals experienced central nervous system inflammation, and two infants presented with persistent pulmonary hypertension. Remarkably, 19 of 29 variants (66%) cluster within the first quarter of exon 10, are de novo, and escape nonsense-mediated mRNA decay (NMD), which we confirmed for two variants affecting seven individuals. Variants outside this region were inherited. Heterozygous mice exhibited developmental and behavioral abnormalities, while homozygous mutations was perinatally lethal. Truncations and a small deletion within a predicted nuclear localization signal (NLS) caused cytosolic mislocalization of ARID5B, whereas the isolated C-terminal half retained nuclear localization, suggesting an independent distal NLS. Collectively, these findings define ARID5B -related neurodevelopmental disorder as a distinct clinical entity and reveal how disruption of specific ARID5B domains impacts protein localization, mammalian development, immune and neurobehavioral function. Abstract Figure:

BACKGROUND: Up to 15% of strokes occur in young adults, with more cryptogenic cases, raising the possibility of rare causes, such as genetic diseases. Although available in everyday practice, genetic analyses are usually reserved for patients with evocative personal or family history. We aimed to assess the interest of systematic genetic analyses in young adults with cryptogenic stroke and estimate the true frequency of genetic disorders in such patients. METHODS: We conducted a retrospective observational cohort study with additional prospective genetic testing. We screened all patients under 50 admitted to Dijon University Hospital stroke unit between 2018 and 2021. Those already genetically tested during etiological work-up were included in a first cohort (cohort 1). Among the remaining patients, those with unexplained intracerebral hemorrhage or stroke (ie, cryptogenic stroke), or a stroke subtype known to have monogenic forms, were offered exome sequencing to form cohort 2. Monogenic diagnoses were defined by likely pathogenic/pathogenic variants according to American College of Medical Genetics and Genomics criteria. RESULTS: Among 305 patients with stroke screened, 24 had prior genetic testing (cohort 1) with exome sequencing, genome sequencing, gene panels or targeted gene analyses, leading to a molecular diagnosis in 8 (33%). Of the remaining 281 patients, 71 met eligibility criteria and 35 consented to the study (cohort 2). Exome sequencing identified pathogenic variants in 4 of them (11%). The overall diagnostic yield of genetic tests was 20.3% (12/59 patients tested across both cohorts). In total, monogenic causes explained 3.9% (12/305) of all young stroke cases. Notably, most diagnosed patients (66%) had no family history of stroke. Genetic cardiopathies and conditions conferring increased cardiovascular risk factors accounted for 50% of diagnoses. CONCLUSIONS: Genetic analyses should be considered in all unexplained strokes or in stroke subtypes with known genetic forms (eg, moyamoya syndrome, cardiopathy, small vessel disease), even without an evocative family history.