BACKGROUND:Potocki-Lupski syndrome (PTLS) is a rare genetic disorder, with an estimated prevalence of 1:25 000. Detection of a duplication at position 17p11.2 comprising the RAI1 gene establishes the diagnosis. Deletion of this same region is responsible for Smith-Magenis syndrome (SMS). Hitherto, the non-specific clinical features included psychomotor and growth retardation and multiple congenital anomalies. Our aim was to further delineate the clinical spectrum of PLTS. METHODS:We gathered a series of 56 individuals carrying a 17p11.2 duplication, one of the largest reported to date. We collected detailed phenotypic data and established a phenotypic comparison with individuals already described in the literature. RESULTS:We corroborated the main clinical signs associated with PTLS and highlighted additional features present in a significant proportion in our series, such as intrauterine growth retardation or low birth weight, musculoskeletal and ophthalmological anomalies, and abnormalities of the skin appendages. In line with previous reports, behavioural disorders were frequently identified (23%). Yet unexpectedly, self-aggressive and hetero-aggressive behaviours, characteristic features of SMS, were found in a small number of individuals. Forty-six individuals harboured the recurrent duplication (85%), five had larger duplications (9%) and three had smaller duplications (6%). We did not identify inherited duplications when parental information was available (n=43). CONCLUSION:Our study refined the clinical features of PTLS and their relative frequencies. Our findings therefore contribute to improving management of people with PTLS. These open up new pathophysiological hypotheses involving RAI1 gene dosage of the genesis and control of behaviour, as well as new, more complex regulatory pathways.

BACKGROUND:KRAS mosaic activating variants are the main cause of non-ossifying fibromas (NOFs), the most common benign lesion of the growing skeleton. Multifocal NOFs cause bone fragility, and no specific treatment is currently available. METHODS:We report two children, carrying mosaic KRAS variants (p.G13D and p.A146T), presenting with oculoectodermal syndrome and recurrent fractures due to progressive polyostotic NOFs. To assess the impact of these mutations on KRAS function, we conducted transient KRAS overexpression in HEK293 cells and then tested the effect of the MEK-inhibitor trametinib at the cellular level. RESULTS:We show that trametinib yields, in vitro, significant reduction of RAS-pathway hyperactivation induced by the two KRAS variants and, in vivo, remarkable clinical and radiological improvement with no recurrence of fractures and reossification of NOFs under treatment; resurgence of lesions is observed one year after stopping treatment. CONCLUSIONS:Hence, trametinib constitutes a promising precision therapeutic approach for severe KRAS-related NOFs.

PURPOSE:Biallelic variants in the minor spliceosomal gene RNU4ATAC were successively identified in Taybi-Linder/Microcephalic osteodysplastic primordial dwarfism type I, Roifman, and Lowry-Wood syndromes, which are characterized by variable microcephaly, short stature, neurodevelopmental impairment, skeletal dysplasia, and immunodeficiency. Two-thirds of the reported individuals present with Taybi-Linder syndrome, the first-described and most severe form. METHODS:We collected clinical and molecular data from individuals with biallelic RNU4ATAC variants through various French and European networks and clinics to refine the phenotypic spectrum of RNU4ATAC-opathies. RESULTS:We enrolled 69 participants and identified 18 new pathogenic variants. We report a significant proportion of attenuated or atypical presentations, novel rare symptoms, and, unexpectedly, a broad spectrum of autoimmune or inflammatory manifestations, affecting nearly half of the participants. Integrating our data with the 109 published cases, we propose a novel classification based on the main manifestations, immunodeficiency, and microcephalic primordial dwarfism. Using computer-assisted facial analysis, we also demonstrated the existence of a specific dysmorphic pattern in RNU4ATAC-opathies that is distinct among some sub-syndromes. CONCLUSION:We present a large cohort of individuals with RNU4ATAC-opathies and expand the phenotypic spectrum to paucisymptomatic forms, indicating that these diseases are likely to remain underdiagnosed.

The non-homologous end joining (NHEJ) pathway is essential to repair DNA double-strand breaks. XRCC4 acts as a stabilizer of the DNA ligase LIG4 in the NHEJ process. In humans, XRCC4 pathogenic variants are responsible for a microcephalic primordial dwarfism syndrome (MPD). Currently, 17 patients have been reported with XRCC4-related MPD and we report 7 new patients from 6 different families, including one fetus. The patients present with short stature, severe microcephaly, neurodevelopmental disorder and additional features, such as transient increase in nuchal translucency, congenital glaucoma, thumb anomalies, hepatic steatosis, seizures, essential tremor and oligodontia which have not been previously described. Hyper- and hypopigmented skin macules, dermatofibrosarcoma, mandibular osteoid osteoma and pancytopenia are also new features, reminiscent of cancer susceptibility syndromes. Functional studies were performed on two patients carrying the known pathogenic p.(Trp43Arg) variant in homozygous state, using a fast, cost-effective and non-invasive approach on PBMCs: (1) Survival analyses after ionizing radiation confirm important radiosensitivity. (2) Flow cytometry showed the lack of TCR-Va7+ T-lymphocytes, suggesting recombination defect of V(D)J coding segments. (3) This was confirmed by multiplexed RT-PCR (PROMIDISα biomarker), analyzing the diversity of V(D)J coding segments in a subset of the TCRα repertoire. We therefore extend the phenotype of XRCC4-related MPD and suggest a combination of three functional assays, based on radiosensitivity and V(D)J recombination defect, to improve the interpretation of XRCC4 variants in fast, cost-effective and non-invasive manner. These findings will improve the diagnosis, genetic counselling, follow-up and management of these patients.

Variants in gap junction protein alpha 8 (GJA8), the gene encoding connexin 50 (Cx50), are primarily associated with developmental cataract, although some are associated with severe structural eye anomalies, such as aphakia (absent lens), microphthalmia (small eyes), and sclerocornea. To further define the relationship of GJA8 variants to ocular developmental disorders, we screened four large international cohorts with structural eye anomalies, including anophthalmia, microphthalmia, and coloboma (AMC) or cataracts. We identified 15 new families carrying 14 different heterozygous GJA8 variants (12 missense variants and two 1q21 microdeletions). The missense variants comprised 10 previously reported alterations in cases with eye anomalies [p.(Gly22Ser), p.(Val44Met), p.(Asp67Gly), p.(Arg76Cys), p.(Pro88Leu), p.(Gly94Glu), p.(Gly94Arg), p.(His98Arg), p.(Pro189Ser), and p.(Arg198Trp)] and two not yet linked with disease [p.(Thr39Met) and p.(Tyr66Asp)]. Their associated phenotypes ranged from isolated cataracts to a combination of microphthalmia and cataract with/without sclerocornea. Our study confirms GJA8 variants as an important source of genetic diagnoses for families with structural eye anomalies in addition to cataract and highlights specific mutational hotspots. Furthermore, we confirm an important genotype-phenotype correlation between sclerocornea and the p.(Gly94Arg) variant, and detail intra- and inter-familial phenotypic variability, which is important for clinical assessment and genetic counselling.

Intellectual disability (ID) is the leading cause of patient referral to medical genetic departments in French academic hospitals. Whole genome sequencing (WGS) as a first diagnostic approach is expected to achieve a higher diagnostic yield than the French national reference strategies (RefStrategy) (fragile X expansion testing, chromosomal microarray analysis, and 44 ID genes panel), given its broad and more homogeneous coverage, its ability to identify copy number, structural and intergenic/deep intronic events. DEFIDIAG is a national, prospective pilot investigation, carried out in the framework of the French initiative for genomic medicine (Plan France Médecine Génomique 2025), aimed at comparing the diagnostic yield of WGS trio analysis (WGS-trio) (index case, father, mother) with the RefStrategy in real-life conditions of clinical and laboratory workflows. Both strategies were applied in a blinded fashion in 1239 ID probands (50

Microcephalic osteodysplastic primordial dwarfism type I (MOPDI) is a very rare and severe autosomal recessive disorder characterized by marked intrauterine growth retardation, skeletal dysplasia, microcephaly and brain malformations. MOPDI is caused by biallelic mutations in RNU4ATAC, a non-coding gene involved in U12-type splicing of 1% of the introns in the genome, which are recognized by their specific splicing consensus sequences. Here, we describe a unique observation of immunodeficiency in twin sisters with mild MOPDI, who harbor a novel n.108_126del mutation, encompassing part of the U4atac snRNA 3' stem-loop and Sm protein binding site, and the previously reported n.111G>A mutation. Interestingly, both twin sisters show mild B-cell anomalies, including low naive B-cell counts and increased memory B-cell and plasmablasts counts, suggesting partial and transitory blockage of B-cell maturation and/or excessive activation of naive B-cells. Hence, the localization of a mutation in stem II of U4atac snRNA, as observed in another RNU4ATAC-opathy with immunodeficiency, that is, Roifman syndrome (RFMN), is not required for the occurrence of an immune deficiency. Finally, we emphasize the importance of considering immunodeficiency in MOPDI management to reduce the risk of serious infectious episodes.

PURPOSE:Valproic acid or valproate is an effective antiepileptic drug; however, embryonic exposure to valproate can result in a teratogenic disorder referred to as fetal valproate syndrome (OMIM #609442). Currently there are no diagnostic biomarkers for the condition. This study aims to define an episignature biomarker for teratogenic antenatal exposure to valproate. METHODS:DNA extracted from peripheral blood of individuals with teratogenic antenatal exposure to valproate was processed using DNA methylation microarrays. Subsequently, methylation profiling and construction of support vector machine classifiers were performed in R. RESULTS:Genomic DNA methylation analysis was applied, and a distinct DNA methylation profile was identified in the majority of affected individuals. This profile was used to develop a diagnostic episignature classifier. The valproate exposure episignature exhibited high sensitivity and specificity relative to a large reference data set of unaffected controls and individuals with a wide spectrum of syndromic disorders with episignatures. Gene set enrichment analysis demonstrated an enrichment for terms associated with cell adhesion, including significant overrepresentation of the cadherin superfamily. CONCLUSION:This study provides evidence of a robust peripheral blood-based diagnostic epigenetic biomarker for a prenatal teratogenic disorder.

Taybi-Linder syndrome (TALS) is a rare autosomal recessive disorder characterized by severe microcephaly with abnormal gyral pattern, severe growth retardation and bone abnormalities. It is caused by pathogenic variants in the RNU4ATAC gene. Its transcript, the small nuclear RNA U4atac, is involved in the excision of ~850 minor introns. Here, we report a patient presenting with TALS features but no pathogenic variants were found in RNU4ATAC, instead the homozygous RTTN c.2953A>G variant was detected by whole-exome sequencing. After deciphering the impact of the variant on the RTTN protein function at centrosome in engineered RTTN-depleted RPE1 cells and patient fibroblasts, we analysed neural stem cells (NSC) derived from CRISPR/Cas9-edited induced pluripotent stem cells and revealed major cell cycle and mitotic abnormalities, leading to aneuploidy, cell cycle arrest and cell death. In cortical organoids, we discovered an additional function of RTTN in the self-organisation of NSC into neural rosettes, by observing delayed apico-basal polarization of NSC. Altogether, these defects contributed to a marked delay of rosette formation in RTTN-mutated organoids, thus impeding their overall growth and shedding light on mechanisms leading to microcephaly.

The Houge type of X-linked syndromic intellectual developmental disorder (MRXSHG) encompasses a spectrum of neurodevelopmental disorders characterized by intellectual disability (ID), language/speech delay, attention issues, and epilepsy. These conditions arise from hemizygous or heterozygous deletions, along with point mutations, affecting CNKSR2, a gene located at Xp22.12. CNKSR2, also known as CNK2 or MAGUIN, functions as a synaptic scaffolding molecule within the neuronal postsynaptic density (PSD) of the central nervous system. It acts as a link connecting postsynaptic structural proteins, such as PSD95 and S-SCAM, by employing multiple functional domains crucial for synaptic signaling and protein-protein interactions. Predominantly expressed in dendrites, CNKSR2 is vital for dendritic spine morphogenesis in hippocampal neurons. Its loss-of-function variants result in reduced PSD size and impaired hippocampal development, affecting processes including neuronal proliferation, migration, and synaptogenesis. We present 15 patients including three from the MENA (Middle East and North Africa), a region with no documented mutations in CNKSR2. Each individual displays unique clinical presentations that encompass developmental delay, ID, language/speech delay, epilepsy, and autism. Genetic analyses revealed 14 distinct variants in CNKSR2, comprising five nonsense, three frameshift, two splice, and four missense variants, of which 13 are novel. The ACMG guidelines unanimously interpreted these 14 variants in 15 individuals as pathogenic, highlighting the detrimental impact of these CNKSR2 genetic alterations and confirming the molecular diagnosis of MRXSHG. Importantly, variants Ser767Phe and Ala827Pro may lead to proteasomal degradation or reduced PSD size, contributing to the neurodevelopmental phenotype. Furthermore, these two amino acids, along with another two affected by four missense variants, exhibit complete conservation in nine vertebrate species, illuminating their crucial role in the gene's functionality. Our study revealed unique new digital and brain phenotype, including pointed fingertips (fetal pads of fingertips), syndactyly, tapering fingers, and hippocampal atrophy. These novel clinical features in MRXSHG, combined with 13 novel variants, expand the phenotypic and genotypic spectra of MRXSHG associated with CNKSR2 mutations.

The Taybi-Linder syndrome (TALS) is a rare genetic disorder characterized by a severe microcephaly with abnormal gyral pattern, severe growth retardation, bone abnormalities and a reduced life span for the most severe cases. It is caused by mutations in RNU4ATAC whose transcript, the small nuclear RNA U4atac, is a core component of the minor spliceosome involved in the excision of minor introns spread over ∼750 genes.Here, we report a patient presenting with TALS features but no mutation in RNU4ATAC ; instead, she carries the RTTN c.2953A>G variant at the homozygous state. This variant, already reported in patients with syndromic microcephaly, encodes the missense p.Arg985Gly amino acid change. It is also known to affect RTTN pre-mRNA splicing, with the expression of two forms lacking either exon 23 (in-frame) or exons 22-23 (out-of-frame). By using the engineered RTTN depleted RPE1 cellular model, we analysed independently the impact of the missense and in-frame deletion of exon 23 RTTN isoforms on the localisation and function of the protein at the centrosome, and showed that the pathogenicity of the c.2953A>G variant is mostly due to the latter. In patient fibroblasts, we observed a reduction of the centriole length and an alteration of ciliary function, while the analysis of neuronal stem cells (NSC) derived from CRISPR/Cas9-edited induced pluripotent stem cells revealed major cell cycle and mitotic abnormalities, leading to aneuploidy, cell cycle arrest and increased cell death. Finally, by generating cortical organoids, we discovered a new function of RTTN in the self-organisation of NSC into neural rosettes. We observed a delayed apico-basal polarization of NSC, accompanied with decreased cell division and increased apoptosis. Altogether, these defects lead to a marked decrease of rosette number and size in RTTN -mutated organoids, thus impeding their overall growth.To conclude, our study gives new insights on microcephaly-related pathophysiological mechanisms underlying the only recurrent RTTN mutation, that could also open a path to better understand those involved in RNU4ATAC -associated Taybi-Linder syndrome.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis work was supported by CNRS, Inserm and Universite Lyon 1 through recurrent funding; the Agence Nationale de la Recherche (no. ANR-18CE12-0007; no. ANR-22CE12-0007); the Fondation Jerome Lejeune and the Fondation pour la recherche sur le Cerveau "Espoir en tete" (confocal microscope). J.G. was supported by the Ministere de l'Enseignement Superieur et de la Recherche and by the Fondation pour la Recherche Medicale. T-Y C. was supported by a postdoctoral fellowship from Academia Sinica, Taiwan and T.K.T by the National Science and Technology Council (NSTC 112-2326-B001-010) and Academia Sinica (AS-IA-109-L04), Taiwan. E.B. was supported by an EMBO long-term fellowship (ALTF-284-2019), and V.H. by the Swiss National Foundation (SNSF) 310030_205087. S.T. was supported by Agence Nationale de la Recherche (no. ANR-17-CE16-0003-01).### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:French national ethical committee Comite de Protection des Personnes (number 2021-A01551-40) gave ethical approval for this work.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Humans with monogenic inborn errors responsible for extreme disease phenotypes can reveal essential physiological pathways. We investigated germline mutations in GNAI2, which encodes G(alpha i2), a key component in heterotrimeric G protein signal transduction usually thought to regulate adenylyl cyclase-mediated cyclic adenosine monophosphate (cAMP) production. Patients with activating G(alpha i2) mutations had clinical presentations that included impaired immunity. Mutant G(alpha i2) impaired cell migration and augmented responses to T cell receptor (TCR) stimulation. We found that mutant G(alpha i2) influenced TCR signaling by sequestering the guanosine triphosphatase (GTPase)-activating protein RASA2, thereby promoting RAS activation and increasing downstream extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-AKT S6 signaling to drive cellular growth and proliferation.

BackgroundTatton-Brown-Rahman syndrome (TBRS; OMIM 615879), also known as DNA methyltransferase 3 alpha (DNMT3A)-overgrowth syndrome (DOS), was first described by Tatton-Brown in 2014. This syndrome is characterised by overgrowth, intellectual disability and distinctive facial features and is the consequence of germline loss-of-function variants inDNMT3A, which encodes a DNA methyltransferase involved in epigenetic regulation. Somatic variants ofDNMT3Aare frequently observed in haematological malignancies, including acute myeloid leukaemia (AML). To date, 100 individuals with TBRS with de novo germline variants have been described. We aimed to further characterise this disorder clinically and at the molecular level in a nationwide series of 24 French patients and to investigate the correlation between the severity of intellectual disability and the type of variant.MethodsWe collected genetic and medical information from 24 individuals with TBRS using a questionnaire released through the French National AnDDI-Rares Network.ResultsHere, we describe the first nationwide French cohort of 24 individuals with germline likely pathogenic/pathogenic variants inDNMT3A, including 17 novel variants. We confirmed that the main phenotypic features were intellectual disability (100% of individuals), distinctive facial features (96%) and overgrowth (87%). We highlighted novel clinical features, such as hypertrichosis, and further described the neurological features and EEG results.ConclusionThis study of a nationwide cohort of individuals with TBRS confirms previously published data and provides additional information and clarifies clinical features to facilitate diagnosis and improve care. This study adds value to the growing body of knowledge on TBRS and broadens its clinical and molecular spectrum.

Generation and subsequently accessibility of secondary findings (SF) in diagnostic practice is a subject of debate around the world and particularly in Europe. The French FIND study has been set up to assess patient/parent expectations regarding SF from exome sequencing (ES) and to collect their real-life experience until 1 year after the delivery of results. 340 patients who had ES for undiagnosed developmental disorders were included in this multicenter mixed study (quantitative N = 340; qualitative N = 26). Three groups of actionable SF were rendered: predisposition to late-onset actionable diseases; genetic counseling; pharmacogenomics. Participants expressed strong interest in obtaining SF and a high satisfaction level when a SF is reported. The medical actionability of the SF reinforced parents’ sense of taking action for their child and was seen as an opportunity. While we observed no serious psychological concerns, we showed that these results could have psychological consequences, in particular for late-onset actionable diseases SF, within families already dealing with rare diseases. This study shows that participants remain in favor of accessing SF despite the potential psychological, care, and lifestyle impacts, which are difficult to anticipate. The establishment of a management protocol, including the support of a multidisciplinary team, would be necessary if national policy allows the reporting of these data.

In the human genome, about 750 genes contain one intron excised by the minor spliceosome. This spliceosome comprises its own set of snRNAs, among which U4atac. Its noncoding gene, RNU4ATAC, has been found mutated in Taybi-Linder (TALS/microcephalic osteodysplastic primordial dwarfism type 1), Roifman (RFMN), and Lowry-Wood (LWS) syndromes. These rare developmental disorders, whose physiopathological mechanisms remain unsolved, associate ante- and post-natal growth retardation, microcephaly, skeletal dysplasia, intellectual disability, retinal dystrophy, and immunodeficiency. Here, we report bi-allelic RNU4ATAC mutations in five patients presenting with traits suggestive of the Joubert syndrome (JBTS), a well-characterized ciliopathy. These patients also present with traits typical of TALS/RFMN/LWS, thus widening the clinical spectrum of RNU4ATAC-associated disorders and indicating ciliary dysfunction as a mechanism downstream of minor splicing defects. Intriguingly, all five patients carry the n.16G>A mutation, in the Stem II domain, either at the homozygous or compound heterozygous state. A gene ontology term enrichment analysis on minor intron-containing genes reveals that the cilium assembly process is over-represented, with no less than 86 cilium-related genes containing at least one minor intron, among which there are 23 ciliopathy-related genes. The link between RNU4ATAC mutations and ciliopathy traits is supported by alterations of primary cilium function in TALS and JBTS-like patient fibroblasts, as well as by u4atac zebrafish model, which exhibits ciliopathy-related phenotypes and ciliary defects. These phenotypes could be rescued by WT but not by pathogenic variants-carrying human U4atac. Altogether, our data indicate that alteration of cilium biogenesis is part of the physiopathological mechanisms of TALS/RFMN/LWS, secondarily to defects of minor intron splicing.