Disease Models & Mechanisms

Cytoplasmic Dynein-2 or IFT-dynein is the only known retrograde motor for intraflagellar transport, enabling protein trafficking from the ciliary tip to the base. Dysfunction of WDR34 and WDR60, the two intermediate chains of this complex, causes Short Rib Thoracic Dystrophy (SRTD), human skeletal chondrodysplasias with high lethality. Complete loss of function of WDR34 or WDR60 is lethal in vertebrates and individuals with SRTD carry at least one putative hypomorphic missense allele. Gene knockout is therefore not suitable to study the effect of these human missense disease alleles. Using CRISPR single base editors, we recreated three different patient missense alleles in cilia-APEX-IMCD3 cells. Consistent with previous findings in dynein-2 full loss of function models and patient fibroblasts, mutant cell lines showed hedgehog signaling defects as well as disturbed retrograde IFT. Transcriptomics analysis revealed differentially regulated expression of genes associated with various biological processes, including G-protein-coupled receptor signaling as well extracellular matrix composition, endochondral bone growth and chondrocyte development. Further, we also observed differential regulation of genes associated with Golgi intracellular transport, including downregulation of Rab6b, a GTPase involved in Golgi-ER retrograde protein trafficking and interacting with components of cytoplasmic dynein-1, in mutant ciliated and non-ciliated clones compared to controls. In addition to providing cellular model systems enabling investigations of the effect of human SRTD disease alleles, our findings indicate non-ciliary functions for WDR34 and WDR60 in addition to the established roles as components of the retrograde IFT motor complex in cilia.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease for which there is no cure. There is a critical need for additional therapeutics. Human genome-wide association studies (GWAS) have identified candidate DMD genetic modifiers that could serve as therapeutic targets. Because many GWAS-identified single nucleotide polymorphisms (SNPs) lie in noncoding, putative regulatory regions, it can be challenging to identify which gene(s) are regulated by these SNPs and how gene expression is altered to modify disease severity even with extensive in silico modeling. We analyzed expression of zebrafish orthologs of putative DMD modifiers and showed almost all are comparably expressed in wild-type and dmd mutant zebrafish at three different stages of disease. To model decreased expression of candidate modifiers, we pursued a zebrafish CRISPR-based screening approach, which we validated by testing zebrafish orthologs of two extensively studied DMD modifiers, LTBP4 and THBS1. We then tested candidates from the most recent GWAS and demonstrate that galntl6, man1a1, etaa1a;etaa1b, and adamts17 are bona fide DMD modifiers. Our findings demonstrate the utility of zebrafish for DMD genetic modifier screening and characterizing modifier function. Summary StatementZebrafish CRISPR-based screening approach validates new genetic modifiers of Duchenne muscular dystrophy.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes that complete the first step of protein translation: ligation of amino acids to cognate tRNAs. Genes encoding ARSs have been implicated in myriad dominant and recessive phenotypes, the latter often affecting multiple tissues but with frequent involvement of the central and peripheral nervous system, liver, and lungs. Threonyl-tRNA synthetase (TARS1) encodes the enzyme that ligates threonine to tRNATHR in the cytoplasm. To date, TARS1 variants have been implicated in a recessive brittle hair phenotype. To better understand TARS1-related recessive phenotypes, we engineered three TARS1 missense mutations predicted to cause a loss-of-function effect and studied these variants in yeast and worm models. This revealed two loss-of-function mutations, including one hypomorphic allele (R433H). We next used R433H to study the effects of partial loss of TARS1 function in a compound heterozygous mouse model (R433H/null). This model presents with phenotypes reminiscent of patients with TARS1 variants and with distinct lung and skin defects. This study expands the potential clinical heterogeneity of TARS1-related recessive disease, which should guide future clinical and genetic evaluations of patient populations. SUMMARY STATEMENTThis study leverages an engineered, hypomorphic variant of threonyl-tRNA synthetase (TARS1) to capture TARS1-associated recessive phenotypes. This strategy revealed both known and previously unappreciated phenotypes, expanding the clinical heterogeneity associated with TARS1 and informing future genetic and clinical evaluations of patient populations.

Type A and B Niemann Pick (NPD) is an inherited multisystem lysosomal storage disorder caused by mutations in the SMPD1 gene. Respiratory dysfunction is a key hallmark of NPD, although the precise mechanisms underlying these pathologies is underexplored. Here we present a Drosophila model of Smpd1 loss-of-function that displays significant respiratory defects. Smpd1 is expressed in the late-embryonic fly respiratory network, the trachea, and is secreted into the tracheal lumen. Loss of Smpd1 results in embryonic lethality, and although tracheal morphology appears normal, trachea fail to fill with gas prior to eclosion. We demonstrate that clearance of luminal constituents through endocytosis prior to gas-filling is defective in Smpd1 mutants. This is coincident with autophagic, but not lysosomal defects. Finally, we show that although bulk sphingolipids are unchanged, dietary loss of lipids in combination with genetic and pharmacological block of ceramide synthesis is sufficient to rescue gas-filling defects. In summary, we present a novel NPD model amenable to genetic and pharmacological screens, and highlight myriocin, an inhibitor of ceramide synthesis, as a potential therapeutic drug for the treatment of NPD.

There are thousands of Mendelian diseases with more being discovered weekly and the majority have no approved treatments. To address this need, we require scalable approaches that are relatively inexpensive compared to traditional drug development. In the absence of a validated drug target, phenotypic screening in model organisms provides a route for identifying candidate treatments. Success requires a screenable phenotype, however the right phenotype and assay may not be obvious for pleiotropic neuromuscular disorders. Here we show that high-throughput imaging and quantitative phenotyping can be conducted systematically on a panel of C. elegans disease model strains. We used CRISPR genome-editing to create 25 worm models of human Mendelian diseases and phenotyped them using a single standardised assay. All but two strains were significantly different from wild-type controls in at least one feature. The observed phenotypes were diverse, but mutations of genes predicted to have related functions in their human orthologs led to similar behavioural differences in worms. As a proof-of-concept, we performed a drug repurposing screen of an FDA approved compound library, and identified two compounds that rescued the behavioural phenotype of a model of UNC80 deficiency. Our results show that a single assay to measure multiple phenotypes can be applied systematically to diverse Mendelian disease models. The relatively short time and low cost associated with creating and phenotyping multiple strains suggests that high-throughput worm tracking could provide a scalable approach to drug repurposing commensurate with the number of Mendelian diseases.

Adverse pregnancy outcomes, such as sporadic and recurrent miscarriages and stillbirths, are significant medical concerns, impacting up to 15% of clinically recognised pregnancies. These outcomes are highly complex and multifactorial, with up to 50% of cases classified as idiopathic, highlighting a substantial gap in our understanding of their biological basis. Along with external risk factors, polygenic variability contributes to idiopathic pregnancy loss, suggesting that large-scale genetic studies could offer insights into its mechanisms, reveal novel drug targets, and lead to new treatments. This study assesses current knowledge from genome-wide association studies (GWAS) using genotyping arrays, whole-genome imputation, and sequencing for variant discovery, emphasising genetic predisposition to adverse pregnancy outcomes. We summarise existing efforts identifying 30 genetic loci associated with pregnancy loss and related endophenotypes, integrating them into a polygenic score (PGS) and conducting a phenome-wide PGS association analysis of 280 ICD-10 outcomes in nearly 500,000 UK Biobank participants. We report associations between pregnancy loss PGS and an increased risk for diaphragmatic hernia (OR[95%CI]=1.02[1.01-1.03], P=9.15x10-), eosinophilic esophagitis (OR[95%CI]=1.05[1.03-1.06], P=1.44x10-), and asthma with exacerbation (OR[95%CI]=1.02[1.01-1.03], P=1.71x10-), significant after correction for multiple testing and suggesting new mechanistic pathophysiology in pregnancy loss susceptibility. Additionally, Mendelian Randomisation (MR) studies identified higher BMI and smoking as risk factors for pregnancy loss, while the roles of caffeine and alcohol intake, maternal age, and family history of miscarriage warrant further investigation through adequately powered MR analyses. Well-designed and comprehensive GWAS studies, particularly across diverse ancestry groups, are urgently needed for idiopathic recurrent pregnancy loss. Such studies should overcome issues with identification of women suffering for this condition and related pregnancy losses to support better care and timely interventions, aiming for healthy live birth outcomes.

Kabuki syndrome (KS) is a rare cause of intellectual disability resulting from heterozygous pathogenic variants in the gene encoding the histone methyltransferase KMT2D. A previously established loss-of-function mouse model of KS exhibits key phenotypic features, and therapeutic trials in this mouse model suggest postnatal malleability of neurological symptoms. However, 15-30% of individuals with KS, carry missense variants. To investigate whether missense variants lead to similar phenotypic presentation in mice, we used CRISPR-Cas9 to introduce the KS patient variant R5230H into C57BL/6NTac. Computational and in vitro testing suggests that the R5230H variant does not impair protein stability or loss of enzyme function of KMT2D. Despite a distinct mechanistic basis, our new mouse model (Kmt2d+/R5230H) recapitulates most phenotypes of our prior loss-of-function model, including growth deficiency, craniofacial anomalies, and IgA deficiency but not altered neurological function. Kmt2d+/R5230Hmice show perinatal lethality and a high frequency of unilateral kidney agenesis, a novel phenotype in KS mouse models. Kmt2d+/R5230H mice provide a unique opportunity to understand the impact of missense variants on KMT2D function and uncover developmental and perinatal abnormalities in KS. Summary statementA novel Kabuki syndrome missense mouse model with intact KMT2D enzymatic function shares most features with prior KS models, except disruption of adult neurogenesis, and exhibits novel unilateral kidney loss.

The zebrafish is an important animal system for modelling human diseases. This includes kidney dysfunction as the embryonic kidney (pronephros) shares considerable molecular and morphological homology with the human nephron. Zebrafish also have a high fecundity, with females capable of laying 200-300 eggs per week, thereby facilitating chemical and mutation screening. A key clinical indicator of kidney disease is proteinuria, but a high-throughput readout of proteinuria in the zebrafish is lacking. Coupling the advantages of the zebrafish system with a tool to measure proteinuria will advance the scope for testing the efficacy of drugs to treat kidney diseases. Here, we generated a stable transgenic zebrafish line using the l-fabp10 liver-specific promoter to over-express a nanoluciferase molecule fused with the D3 domain of Receptor-Associated-Protein (RAP) to create NL-D3. In the healthy state, NL-D3 is excreted, but when embryos were treated with chemicals that affected either proximal tubular reabsorption (cisplatin, gentamicin) or glomerular filtration (angiotensin II, Hanks Balanced Salt Solution, Bovine Serum Albumin), NL-D3 presence in the urine increased. Similarly, depletion of several gene products associated with kidney disease (nphs1, nphs2, lrp2a, ocrl, col4a3, col4a4, and col4a5) also induced NL-D3 proteinuria. Furthermore, we found that treating col4a4 depleted zebrafish larvae (a model of Alport syndrome) with captopril reduced proteinuria. Our findings confirm the use of the NL-D3 transgenic zebrafish as a robust and quantifiable proteinuria reporter. Given the feasibility of high-throughput assays in zebrafish, this novel reporter will permit screening for drugs that ameliorate proteinuria and thereby prioritise candidates for further translational studies. Significance StatementThe zebrafish has become an important system for modelling kidney disease. However, proteinuria, an important clinical indicator of kidney dysfunction, is not easily detected in zebrafish. Here, we describe a transgenic line that uses a nanoluciferase reporter to enable detection of proteinuria in multiple models of glomerular and proximal tubular kidney disease in the zebrafish. In this system proteinuria can be accurately measured in a high-throughput manner and will enable the screening of drugs that affect glomerular filtration or protein re-uptake in the proximal tubule.

Becker Muscular Dystrophy (BMD) is a rare X-linked recessive neuromuscular disorder caused by in-frame deletions in the DMD gene that result in the production of a truncated, yet functional, dystrophin protein. BMD is often considered a milder form of Duchenne Muscular Dystrophy, in which mutations typically result in the disruption of the reading frame and the malfunction or loss of dystrophin. The consequences of BMD-causing in-frame deletions on the organism are more difficult to predict, especially in regard to long-term prognosis. Here, we employed CRISPR-Cas9 technology to generate a new Dmd del52-55 mouse model by deleting exons 52-55, resulting in a typical BMD-like in-frame deletion. To delineate the long-term effects of this deletion, we studied these mice over 52 weeks. Our results suggest that a truncated dystrophin is sufficient to maintain wildtype-like muscle and heart functions in young mice. However, the truncated protein appears insufficient to maintain normal muscle homeostasis and protect against exercise-induced damage at 52 weeks. To further delineate the effects of the exons 52-55 in-frame deletion, we performed RNA-Seq pre- and post-exercise and identified several differentially expressed pathways that could explain the abnormal muscle phenotype observed at 52 weeks in the BMD model. Summary StatementWe generated and characterized the long-term effects of a Becker Muscular Dystrophy-like in-frame deletion of exon 52 to 55 in mice.

Alport syndrome is a hereditary chronic kidney disease, attributed to rare pathogenic variants in either of three collagen genes (COL4A3/4/5) with most localized in COL4A5. Trimeric type IV Collagen 345 is essential for the glomerular basement membrane that forms the kidney filtration barrier. A means to functionally assess the many candidate variants and determine pathogenicity is urgently needed. We used Drosophila, an established model for kidney disease, and identify Col4a1 as the functional homolog of human COL4A5 in the fly nephrocyte (equivalent of human podocyte). Fly nephrocytes deficient for Col4a1 showed an irregular and thickened basement membrane and significantly reduced nephrocyte filtration function. This phenotype was restored by expressing human reference (wildtype) COL4A5, but not by COL4A5 carrying any of three established pathogenic patient-derived variants. We then screened seven additional patient COL4A5 variants; their ClinVar classification was either likely pathogenic or of uncertain significance. The findings support pathogenicity for four of these variants; the three others were found benign. Thus, demonstrating the effectiveness of this Drosophila in vivo kidney platform in providing the urgently needed variant-level functional validation. SUMMARY STATEMENTDrosophila, an established model of kidney disease, was used to develop an in vivo functional screen to determine causation for COL4A5 genetic variants linked to Alport syndrome, a progressive nephropathy.

Rare genetic disease discovery efforts typically lead to the identification of new disease genes. PreMIER (Precision Medicine Integrated Experimental Resources) is a collaborative platform designed to facilitate functional evaluation of human genetic variants in model systems, and to date the PreMIER Consortium has evaluated over 50 variants in patients with genetic disorders. To understand if Drosophila could be used to identify pathogenic disease loci as part of the PreMIER Consortium, we used tissue-specific gene knockdown in the fly as a proof of principle experiment. Tissue-specific knockdown of seven conserved disease genes caused significant changes in viability, longevity, behavior, motor function, and neuronal survival arguing a set of defined assays can be used to determine if a gene of uncertain significance (GUS) regulates specific physiological processes. This study highlights the utility of a tissue-specific knockdown platform in Drosophila to characterize GUS, which may provide the first genephenotype correlations for patients with idiopathic genetic disorders

Biallelic loss of expression/function variants in MTMR5 cause the inherited peripheral neuropathy Charcot-Marie-Tooth (CMT) Type 4B3. There is an incomplete understanding of the disease pathomechanism(s) underlying CMT4B3, and despite its severe clinical presentation, currently no disease modifying therapies. A key barrier to the study of CMT4B3 is the lack of pre-clinical models that recapitulate the clinical and pathologic features of the disease. To address this barrier, we generated a zebrafish CRISPR/Cas9 mutant line with a full gene deletion of mtmr5. Resulting homozygous deletion zebrafish are born at normal Mendelian ratios and have preserved motor function. However, starting by 14 day-post-fertilization, mutant zebrafish develop obvious morphometric changes in head size and brain volume. These changes are accompanied at the pathological level by abnormal axon outgrowths and by the presence of dysmyelination, changes reminiscent of the nerve pathology in human CMT4B3. Overall, our mtmr5 zebrafish mirror genetic, clinical, and pathologic features of human CMT4B3. As such, it represents a first pre-clinical model to phenocopy the disease, and an ideal tool for future studies on disease pathomechanism(s) and therapy development. Summary StatementWe created a novel zebrafish mtmr5/sbf1 mutant model of Charcot-Marie-Tooth Type 4B3 that recapitulates key features of the human disorder and provides the first in vivo model for therapy development.

SHP2, a protein tyrosine phosphatase (PTP) crucial in Ras-MAPK signaling, is associated with various human congenital diseases and cancers. Here, we show that the catalytically inactive Shp2C459S mutation results in communicating hydrocephalus, similar to the catalytically activating Shp2E76K and Mek1DD mutants. Unlike previous mutants, however, Shp2C459S/+ mutation uniquely affects ciliary development rather than neurogenesis, leading to reduced cilia density and impaired ciliary motility. Differential scanning fluorimetry revealed that SHP2C459S, SHP2E76K and SHP2C459S/E76K mutations all induce an open SHP2 conformation, but only SHP2C459S leads to aberrant GAB1 phosphorylation in cells expressing wild-type SHP2. This distinctive signaling pattern correlates with our observations in brain ventricular tissues of Shp2C459S/+ mice, where Erk and Stat3 activities remain normal but Gab1 phosphorylation is elevated. Critically, we show that the hydrocephalus phenotype in Shp2C459S mice can be mitigated by allosteric inhibition of Shp2. These findings suggest that Shp2-associated hydrocephalus is driven by conformational changes rather than altered catalytic activity. Our results underscore the therapeutic potential of conformation-specific allosteric inhibitors in targeting both catalytically active and inactive SHP2 mutants.

Spinocerebellar Ataxia Type 17 (SCA17) is the most recently identified member of the polyglutamine (polyQ) family of disorders, resulting from abnormal CAG/CAA expansion of TATA box binding protein (TBP), an initiation factor essential for of all eukaryotic transcription. A largely autosomal dominant inherited disease, SCA17 is unique in both its heterogeneous clinical presentation and low incidence of genetic anticipation, the phenomenon in which subsequent generations inherit longer polyQ expansions that yield earlier and more severe symptom onset. Like other polyQ disease family members, SCA17 patients experience progressive ataxia and dementia, and treatments are limited to preventing symptoms and increasing quality of life. Here, we report two new Drosophila models that express human TBP with polyQ repeats in either wild-type or SCA17 patient range. We find that TBP expression has age- and tissue-specific effects on neurodegeneration, with polyQ expanded SCA17 protein expression generally having more severe effects. In addition, SCA17 model flies accumulate more aggregation prone TBP, with a greater proportion localizing to the nucleus. These new lines provide a new resource for the biochemical characterization of SCA17 pathology and the future identification of therapeutic targets.

BackgroundPERK (EIF2AK3) is an endoplasmic reticulum stress kinase whose loss of function disturbs human development, leading to skeletal dysplasia and permanent neonatal diabetes, as observed in the Wolcott-Rallison Syndrome (WRS). The lack of effective, less invasive therapies for developmental diseases highlights the need for animal models that replicate complex pathological phenotypes, while allowing scalable drug screening. Zebrafish, with their high fecundity and rapid development, facilitate efficient in vivo drug testing. MethodsWe aimed to assess the potential of zebrafish for studying PERK function and its pharmacological modulation, particularly as a model for developmental diseases like WRS. Bioinformatic analyses assessed the similarity between human and zebrafish PERK. Increasing concentrations of GSK2606414 were used to inhibit PERK. A combination of behavioural and functional assays evaluated the effects of GSK2606414 on zebrafish skeletal, neuromuscular, and cardiac development. Fluorescence microscopy in transgenic zebrafish expressing fluorescent pancreatic markers and a glucose probe assessed the diabetic-like phenotype. ResultsWe found high similarity between human and zebrafish PERK, along with bioactivity of the PERK inhibitor GSK2606414 in zebrafish. PERK inhibition evoked defects in WRS relevant parameters, such as growth and skeletal development, as well as neuromuscular and cardiac deficiencies, whereas parameters not associated with WRS like otolith area and eye/body ratio remained unaffected. Moreover, PERK inhibition decreased pancreatic ! cell mass and disrupted glucose homeostasis, indicating a diabetic phenotype. ConclusionThese findings evidence zebrafishs potential for studying PERK function and its pharmacological modulation in developmental disorders like WRS, aiding research on pathophysiology and experimental treatments.

The RAS oncogene and upregulation of the RAS signalling pathway is highly prevalent in human cancer, and therefore, therapeutically targeting the RAS pathway is a common treatment in cancer. However, RAS pathway upregulation is not sufficient to drive malignant cancer, since senescence mechanisms prevent cancer progression. Thus, additional mutations, such as mutations that prevent senescence or alter the tissue architecture (cell polarity), are required for RAS-driven tumour progression. Moreover, targeting RAS-driven cancers with RAS pathway inhibitors can often lead to undesirable side-effects and to drug resistance. Thus, identifying compounds that synergise with RAS-pathway inhibitors would enable lower doses of the RAS pathway inhibitors to be used and also decrease the acquisition of drug resistance. Here, in a boutique chemical screen using a Drosophila model of Ras-driven cell polarity-impaired cancer, we have identified compounds that reduce tumour burden by synergising with subtherapeutic doses of the RAS pathway inhibitor, Trametinib, which inhibits mitogen-activated kinase kinase (MEK). Analysis of one of the hits from the screen, Ritanserin, which targets serotonin receptors and diacy glycerol kinase alpha (DGK), revealed that DGK was the critical target in its synergism with Trametinib. We show that human mammary epithelial cells harbouring the H-RAS oncogene and knockdown of the cell polarity gene, SCRIB, are also sensitive to treatment with low doses of Trametinib and DGK inhibition. Mechanistically, DGK inhibition synergises with Trametinib by inhibiting MEK and mTOR activity. Altogether, our results provide evidence that targeting RAS-driven human cancers with RAS pathway and DGK inhibitors will be an effective combination therapy.

Mutations of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) lead to Cystic Fibrosis (CF), but the substantial phenotypic variations are determined by non-CFTR allelic diversity. To map novel disease phenotypes in a CF mouse model, we used Collaborative Cross (CC) mice, a highly genetically diverse mouse resource population.{Delta} F508-Cftr homozygosity produced a fully penetrant lethal phenotype by eight weeks in two CC lines. The lethality of CC006{Delta}F508/{Delta}F508 was fully prenatal while CC037{Delta}F508/{Delta}F508 showed either prenatal or postnatal lethality. Novel phenotypes of CC037{Delta}F508/{Delta}F508 were revealed early in life including respiratory and systemic inflammatory profiles, and blood, bone marrow, pancreas, heart, and reproductive tract pathologies. Severe intestinal blockage was observed as common in other CF mouse models. These results suggest that the exploration of CF disease phenotypes in a mouse population with diverse genetic profiles is needed to map the genetic origin of currently unidentified disease traits and their potential translation to humans.

The most frequent and unique features of Down syndrome (DS) are learning disability and ucraniofacial (CF) dysmorphism. The DS-specific CF features are an overall reduction in head dimensions (microcephaly), relatively wide neurocranium (brachycephaly), reduced mediolaterally orbital region, reduced bizygomatic breadth, small maxilla, small mandible, and increased individual variability. Until now, the cellular and molecular mechanisms underlying the specific craniofacial phenotype have remained poorly understood. Investigating a new panel of DS mouse models with different segmental duplications on mouse chromosome 16 in the region homologous to human chromosome 21, we identified new regions and the role of two candidate gene for DS-specific CF phenotypes. First, we confirmed the role of Dyrk1a in the neurocranium brachycephaly. Then, we identified the role of the transcription factor Ripply3 overdosage in midface shortening through the downregulation of Tbx1, another transcription factor involved in the CF midface phenotype encountered in DiGeorge syndrome. This last effect occurs during branchial arches development through a reduction in cell proliferation. Our findings define a new dosage-sensitive gene responsible for the DS craniofacial features and propose new models for rescuing all aspects of DS CF phenotypes. This data may also provide insights into specific brain and cardiovascular phenotypes observed in DiGeorge and DS models, opening avenues for potential targeted treatment to soften craniofacial dysmorphism in Down syndrome.

BackgroundPatients affected by Rett syndrome (RTT) and MECP2 duplication syndrome (MDS) experience disabling muscle weakness and gastrointestinal dysmotility of unclear origin. Whether these defects arise cell-autonomously, rather than secondarily to neural dysfunction, and which developmental windows are most vulnerable to MeCP2 disfunction remains unresolved. MeCP2 is a dosage-sensitive transcriptional regulator, whose functions are tightly linked to chromatin states. Because short-chain fatty acids (SCFAs) are known to inhibit histone deacetylases (HDACs), a tractable in vivo model is needed to test the effect of HDAC modulation on muscle defects. MethodsWe misexpressed human MECP2 in the Drosophila melanogaster mesoderm that gives rise to skeletal and visceral muscles. We analyzed quantitatively their morphology and function. To assess the effects of SCFA supplementation, we also supplemented diets with sodium butyrate (NaB), Lalbaay(R), a NaB-containing supplement, acetate (AcOH), and valproate (VPA). FindingsMECP2 misexpression caused pre-eclosion lethality, thinning of larval skeletal fibers with nuclear mispositioning and altered mitochondria. Functionally, it reduced locomotion, decreased food transit and gut peristalsis. Phenotypes were strongest when expression began during development. NaB and VPA supplementation rescue most of these phenotypes, consistent with their histone-deacetylase (HDAC) activity. Defects were not observed upon comparable misexpression of an RTT-associated MeCP2 loss-of-function variant, indicating that they might be relevant to pathogenesis of MECP2-related disorders. InterpretationOur genetic in vivo analysis models peripheral effects of MeCP2 dysregulation and their amelioration, supporting the possibility of HDAC-targeted strategies for MECP2-related muscle and gastrointestinal dysfunction.

The principal component of the protein homeostasis network is the ubiquitin-proteasome system. Ubiquitination is mediated by an enzymatic cascade involving, i.e., E3 ubiquitin ligases, many of which belong to the cullin-RING ligases family. Genetic defects in the ubiquitin-proteasome system components, including cullin-RING ligases, are known causes of neurodevelopmental disorders. Using exome sequencing to diagnose a pediatric patient with developmental delay, pyramidal signs, and limb ataxia, we identified a de novo missense variant c.376G>C; p.(Asp126His) in the FEM1C gene encoding a cullin-RING ligase substrate receptor. This variant alters a conserved amino acid located within a highly constrained coding region and is predicted as pathogenic by most in silico tools. In addition, a de novo FEM1C mutation of the same residue p.(Asp126Val) was associated with an undiagnosed developmental disorder, and the relevant variant (FEM1CAsp126Ala) was found to be functionally compromised in vitro. Our computational analysis showed that FEM1CAsp126His hampers protein substrate binding. To further assess its pathogenicity, we used the nematode Caenorhabditis elegans. We found that the FEM-1Asp133His animals (expressing variant homologous to the FEM1C p.(Asp126Val)) had normal muscle architecture yet impaired mobility. Mutant worms were sensitive to the acetylcholinesterase inhibitor aldicarb but not levamisole (acetylcholine receptor agonist), showing that their disabled locomotion is caused by synaptic abnormalities and not muscle dysfunction. In conclusion, we provide the first evidence from an animal model suggesting that a mutation in the evolutionarily conserved FEM1C Asp126 position causes a neurodevelopmental disorder in humans.