Mutations in the Ky gene are the underlying cause of Myofibrillar Myopathy-7 (MFM-7), a rare progressive muscle weakness disease of childhood onset. A defining characteristic of the KY protein is the presence of a conserved transglutaminase-like domain, but unequivocal evidence of its enzymatic function remains to be established. To investigate the functional relevance of the predicted KY catalytic triad we use here in vitro enzymatic assays, structural modeling and in vivo rescue experiments in ky/ky mice. While structural modelling shows a striking conservation of the catalytic pocket architecture, our results show that recombinant KY proteins showed no detectable enzymatic activity under the assay conditions used. Moreover, while deletion of transglutaminase-like domain prevents phenotype rescue, replacements of the predicted catalytic residues do not impair the protein's ability to rescue fibre size in ky/ky muscle, indicating that the predicted catalytic residues are dispensable for fibre size rescue in these assays. Proteomic analyses identified KY-associated protein complexes involved in protein quality control, including core components of the Chaperone-Assisted Selective Autophagy machinery. In agreement, basal autophagic flux is significantly reduced in both KY-deficient C2C12 cells and ky/ky muscle fibres. Collectively, our data suggests that the TGN/PROT domain facilitates critical molecular associations at the sarcomeric Z-disc through a mechanism independent of catalysis and that impaired autophagic flux may contribute to the muscle phenotype.
Heterozygous deletions in NRXN1, encoding the presynaptic adhesion molecule Neurexin 1, are among the most frequently identified rare variants in schizophrenia and other neuropsychiatric disorders. Patient sequencing has revealed that 3' deletions within NRXN1 generate novel isoforms not produced from the intact locus, yet whether these isoforms are functional, passively non-functional, or actively pathogenic in vivo is still not clear. To address this, we expressed eight human NRXN1 isoforms in C. elegans neurons including four control isoforms and four 3' deletion variant isoforms identified in schizophrenia patient cell lines, and characterized their effects on protein localization and two independent behaviors: a food deprivation response and social feeding behaviors. Most human isoforms showed expression and localization within the nerve ring and neurons similar to the C. elegans ortholog, NRX-1; however, several isoforms, particularly among the 3' deletion variants, displayed aberrant accumulation in neuronal cell bodies or as puncta in neuropil. Functionally, isoforms fell into one of three categories: no effect on nrx-1 loss-of-function behavioral phenotypes, partial rescue, or gain of function, with multiple isoforms showing differences between the behaviors. Strikingly, two 3' deletion isoforms produced gain-of-function behavioral phenotypes more severe than the nrx-1 null mutant, demonstrating that these patient-derived variants can be actively pathogenic. These results establish C. elegans as a tractable in vivo platform for dissecting the isoform-specific functional consequences of NRXN1 variants and suggest that strategies for NRXN1-associated neuropsychiatric diseases must account for both loss-of-function and gain-of-function isoform mechanisms.
Spinocerebellar ataxia type 1 is a progressive neurodegenerative disorder caused by polyglutamine expansion in ATXN1, yet the normal physiological roles of ATXN1 and its paralog ATXN1L remain incompletely understood. To define these roles, we generated the first zebrafish knockouts (KOs) of the three ataxin-1 family genes, atxn1a, atxn1b, and atxn1l, using CRISPR/Cas9 and performed phenotypic and transcriptomic analyses. All KOs exhibited reduced early survival and mild larval growth deficits. Behavioral assays revealed both shared and paralog-specific effects: atxn1a mutants displayed a reproducible light-dependent locomotor deficit, whereas atxn1b and atxn1l mutants showed generalized hypoactivity. Adult behavioral assessment revealed a gradient of phenotypic severity, with atxn1a KOs displaying the earliest and most pronounced alterations in vertical tank exploration and the greatest impairment in swim-tunnel performance. RNA-seq at 5 days post-fertilization identified extensive transcriptional alterations, including both shared and gene-specific differentially expressed genes associated with neural differentiation, microglial/immune cell migration, and immune signaling. Co-expression network analysis further identified distinct KO-associated gene modules, including a phototransduction-enriched module strongly correlated with atxn1a KO status, providing a potential mechanistic link to its light-dependent locomotor phenotype. Together, these findings define conserved and specialized roles of ATXN1-family genes in locomotor behavior, neurodevelopment, retinal function, and immune-related processes.
Retinitis Pigmentosa (RP) is an inherited retinal degenerative disease that affects 1 in 4000 individuals worldwide and can lead to complete blindness. Early stages of RP involve death of rod photoreceptors via apoptosis, causing loss of peripheral and night vision, which is followed by death of cone photoreceptors, leading to loss of central and daytime vision. Mutations in over 300 genes cause RP. Many of these genes encode retina-specific proteins; however, some encode globally expressed proteins, such as pre-mRNA splicing factors. This study is focused on mutations in the SNRNP200 gene encoding a core pre-mRNA splicing factor. The pathological mechanisms of SNRNP200-associated RP are not well understood and treatments are limited. An approach to study pathogenic mechanisms is to utilize model organisms. Therefore, we developed Drosophila models in which RP-causing mutations were introduced into the Drosophila melanogaster orthologue Snrnp200. In addition, we used RNAi to knock-down Snrnp200 in the developing eye. Depletion of Snrnp200 caused an adult rough eye phenotype due to apoptosis of cells in the retina. When human RP-causing mutations were modeled in Drosophila Snrnp200, they resulted in abnormal retinal electrophysiology and defective patterning of photoreceptors. Further analysis of the photoreceptors revealed mitochondrial defects and altered expression of genes related to redox homeostasis. Consistent with these changes, treatment with the antioxidant N-acetylcysteine (NAC) partially suppressed the photoreceptor defects. Taken together, these findings established a new genetic model for studies of splicing-factor associated RP that recapitulates aspects of the human disease and suggests that antioxidants might serve as a treatment for individuals with SNRNP200-associated RP.
Mucolipidosis type II is an autosomal recessive lysosomal storage disease resulting from biallelic variants in the GNPTAB gene encoding the N-acetylglucosamine phosphotransferase α/β subunits. Deficiency of this enzyme disrupts the mannose-6-phosphate (M6P) trafficking pathway, resulting in mistargeting of lysosomal acid hydrolases. Subsequently, lysosomal accumulation of undegraded macromolecules gives rise to a clinical phenotype with developmental/intellectual disability, skeletal deformities, coarse features, and cardiopulmonary dysfunction. Here, we report a cohort of eight patients (7 Egyptians and 1 Yemeni) from seven families with three reported homozygous pathogenic variants and three unreported variants (p.(Asn750Ilefs*4), p.(Val795Glyfs*7), and c.3602 + 2 T > C). In addition, the GNPTAB gene was ablated in the H. sapiens haploid HAP1 cells by CRISPR-Cas9 editing, to investigate the potential of this cell line for the assessment of variant pathogenicity. This included introduction of the recurrent c.3503_3504delTC variant. A phenotype of impaired autophagic flux was recapitulated, suggesting that HAP1 cells are a suitable model for this disorder.
SHINE syndrome is a rare neurodevelopmental disorder caused by mutations in DLG4, which encodes the postsynaptic scaffolding protein PSD-95. Key symptoms include sleep problems, hypotonia, intellectual disability, neurological disorders, and epilepsy, hence the name 'SHINE.' Here, we developed and characterized a mouse model of SHINE syndrome carrying the patient-derived DLG4V692Wfs*12/+ variant associated with a severe form of the disorder. The mutant transcript escapes nonsense-mediated decay but results in reduced PSD-95 protein expression, faithfully reproducing the molecular phenotype observed in the patient. Behavioral analyses revealed that Dlg4V692Wfs*12/+ mice recapitulate several hallmark features of SHINE syndrome, often in a sex-specific manner. Male mutants showed deficits in learning and cognitive flexibility. Dlg4V692Wfs*12/+ mice also demonstrate trends toward altered sensory processing and socialization. Male mutants exhibited an increased proportion of short sleep bouts and compensatory longer average sleep bout length, suggesting sporadic sleep reminiscent of the patient. While spontaneous seizures were not observed, future studies will test susceptibility to provoked seizures. Together, these findings establish Dlg4V692Wfs*12/+ mice as a robust and translationally relevant model that reproduces key molecular and behavioral features of SHINE syndrome. This model provides a valuable resource for elucidating the mechanisms underlying synaptic neurodevelopmental disorders and for identifying potential therapeutic strategies.
Neurodegenerative diseases (NDDs) are clinically and genetically heterogeneous, requiring neuropathology or molecular testing for a definitive diagnosis. Clinical whole genome sequencing (WGS) enables comprehensive variant calling across flexible gene lists that can be tailored to the clinical presentation. By allowing simultaneous detection of single-nucleotide variants, copy-number variants, structural variants, and repeat expansions, WGS has the potential to improve diagnostic yield, facilitate genetic counseling and support clinical trial inclusion. This study assesses the diagnostic performance of WGS in individuals with NDD. WGS in 500 individuals representing a wide spectrum of NDDs identified a disease-causing variant in 61 cases, resulting in a diagnostic yield of 12%. These variants were found in 16 different genes, with C9orf72 being the most prevalent. Repeat expansions represented the largest variant class, accounting for 35 of 61 LP/P cases (57%); most of which were C9orf72 expansions (31/35). In the largest phenotype groups, frontotemporal dementia (FTD) had the highest diagnostic yield (19%) followed by amyotrophic lateral sclerosis (ALS, 13%), whereas an underlying monogenic cause was expectedly low in Alzheimer disease (AD, 4%). A positive family history was present in the majority (74%) of FTD, ALS, combined ALS-FTD and AD cases with an LP/P finding. Clinical WGS provides a clear diagnostic advantage in NDDs marked by substantial clinical and genetic overlap. WGS enables comprehensive variant detection and mapping of genotype-phenotype relationships across the disease continuum. In FTD and ALS, these results support universal access to genetic testing independent of age at onset or family history.
Hypertriglyceridemia (HTG) frequently occurs in population with abnormal glucose metabolism (AGM), exacerbating risk. In our preliminary researches, we have screened hsa_circ_0000973 and MBOAT2 as the possible regulatory elements of HTG in AGM patients. This study enrolled 142 AGM subjects (71 with HTG, 71 with normal triglycerides) to investigate the role and mechanism of the hsa_circ_0000973 and MBOAT2 in AGM-associated HTG. Expression levels of hsa_circ_0000973 and MBOAT2 were measured by RT-qPCR. Diagnostic performance was evaluated via ROC analysis. Functional validation was performed in an insulin-resistant (IR) HepG2 cell model following knockdown of hsa_circ_0000973 or MBOAT2 using siRNA. Cellular triglyceride (TG) content, glucose uptake, proliferation, apoptosis, and expression of lipid metabolism transcription factors (SREBP-1c, PPARα, PPARγ) were assessed. In the AGM population, expression of both hsa_circ_0000973 and MBOAT2 was significantly downregulated in the HTG group and negatively correlated with serum TG levels (P <0 .01). hsa_circ_0000973 exhibited high diagnostic accuracy for HTG in the AGM population (AUC = 0.95). In HepG2-IR cells, knockdown of hsa_circ_0000973 decreased MBOAT2 expression, increased cellular TG accumulation and residual glucose, inhibited proliferation, and promoted apoptosis. Similar phenotypes were observed upon MBOAT2 knockdown. Both knockdowns upregulated SREBP-1c and downregulated PPARα and PPARγ. This study identified that hsa_circ_0000973 may serve as a candidate biomarker for identifying HTG specifically within the AGM population. Our in vitro experiments further suggested that, in the setting of AGM, hsa_circ_0000973 may jointly regulated TG metabolism alongside MBOAT2.
Lymphatic malformations (LMs) can lead to severe clinical complications, including disfigurement and even death. While genomic alterations have been identified in LMs, the genomic landscape of complex LMs remains poorly defined due to their rarity. In this study, we report two novel findings: an NRAS p.Q61R mutation in central conducting lymphatic anomaly (CCLA) and a PPFIBP1::ROS1 fusion in Gorham-Stout disease (GSD), both described for the first time in their respective LM subtypes. The discovery of the PPFIBP1::ROS1 fusion provided a unique opportunity to localize the somatic event to a specific cell type. Using serial tissue sections from the same specimen, we observed that the fusion signal was spatially associated with lymphatic endothelial cells, based on serial section analysis with D2-40 staining. Given that both NRAS mutations and PPFIBP1::ROS1 fusions have also been identified in other LM subtypes, our findings support the hypothesis that LMs may share common molecular mechanisms. We propose that phenotypic diversity among LM subtypes may arise from differences in developmental timing, anatomic location, and microenvironmental context at the time the somatic mutation occurs.
Spondylometaphyseal dysplasia, Kozlowski type (SMDK), is an autosomal dominant skeletal disorder characterized by abnormalities of the spine, metaphyses and epiphyses. It is associated with variants in TRPV4, although the underlying molecular mechanisms remain unclear. A de novo heterozygous TRPV4 variant (c.2479C>G, p.Pro827Ala) was detected in a patient with SMDK by whole-exome sequencing, and transcriptome sequencing was performed in available family members. Expression analyses showed reduced TRPV4 transcript and protein levels associated with the p.Pro827Ala variant. Cellular functional assays further showed decreased intracellular Ca2+ concentrations without detectable changes in plasma membrane localization. In addition, ATP2B1 and PRKCQ were downregulated. To further investigate the pathogenic mechanism, a heterozygous knock-in Trpv4P827A/+ mouse model was generated using CRISPR/Cas9. Trpv4P827A/P827A homozygous mice exhibited significant skeletal developmental delay, and transcriptome profiling revealed dysregulated expression of homeobox, zf_C2H2, and forkhead transcription factor families during early development. Collectively, these findings expand the spectrum of pathogenic TRPV4 variants and provide mechanistic insights into the pathogenic effect of TRPV4 p.Pro827Ala in SMDK, supporting improved clinical diagnosis and future functional studies.
Haploinsufficiency of SKI, PRDM16, RERE, PAX7, and GRHL3 have been implicated in the development of orofacial clefting (OFC) associated with chromosome 1p36 deletions based on human and/or mouse data. Haploinsufficiency of SPEN, a 1p36 gene that encodes a transcriptional repressor, causes Radio-Tartaglia syndrome, a neurodevelopmental syndrome in which high/narrow palates are common, and OFC is occasionally observed. We show that Spen-null embryos have abnormal palatal shelf elevation and extension leading to the development of cleft palate. Mesenchymal cell proliferation in the medial halves of the palatal shelves of Spen-null embryos at E13.5 is significantly reduced. This contributes to the delay of palatal shelf elevation. Tissue specific ablation of Spen in the cranial neural crest cells results in delayed palatal development. This pattern of abnormal palatal development mimics the pattern described in RERE-deficient mice. We show that Rere and Spen are expressed in same cell types, that Rere and Spen interact genetically in the development of the palate, that Spen expression is reduced in the palates of RERE-deficient embryos at E14.5, and that the rate of OFC in individuals with 1p36 deletions involving both RERE and SPEN is higher than those of individuals with RERE or SPEN haploinsufficiency. Our results suggest that SPEN is required for normal mammalian palatal development, that RERE and SPEN interact in a common pathway during palatal development, and that haploinsufficiency of RERE and SPEN are likely to contribute to the development of OFC in individuals with 1p36 deletions.
BACKGROUND:Epigenetic inheritance links fetal chromatin organization and CpG methylation to lifelong tissue phenotypes. We tested the Fetal Chromatin-CpG Developmental Blueprint (FCCD-B) model, proposing that fetal chromatin states shaped by maternal nutrient and transcription factor networks establish regulatory programs preserved in adult traits. METHODS:We integrated 21 970 fetal single-cell transcriptomes with chromatin accessibility data, including 130 myogenic cells. Adult GWAS datasets included 1614 appendicular lean mass (ALM) loci and 445 grip strength loci. Fine-mapping, colocalization, and gene regulatory networks were inferred using GRNBoost2, Enformer, and Basenji, alongside virtual-cell simulations of nutrient and stress perturbations. RESULTS:ALM and grip strength showed strong genetic correlation (rg = 0.49; Z = 26). Fine-mapping identified 1355 high-confidence ALM loci (median PIP = 0.91). A total of 2935 shared GWAS-eQTL variants overlapped fetal chromatin, with 70-88% accessibility preserved in endothelial and cardiomyocyte lineages. A SMAD3-centered TGF-β network (50 targets) was identified. Simulations showed that vitamin D, folate, and oxidative stress increased myofibril index (+0.141), epithelial regeneration (+0.035), and combined effects (+0.147). CONCLUSION:Persistent fetal chromatin and CpG architectures encode regulatory programs linking maternal environment to adult phenotypes, supporting the FCCD-B model as a predictive framework for developmental epigenetics.
Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a rare, inherited genetic disease caused by mutations in the immunoglobulin mu binding protein (IGHMBP2) gene that result in spinal muscular atrophy with respiratory distress (SMARD1) or Charcot-Marie-Tooth Type 2S (CMT2S). SMARD1 clinical symptoms include respiratory failure, progressive muscular weakness, feeding deficiencies, and sensory and autonomic defects. In this paper, we examined respiration in the FVB-Ighmbp2nmd/nmd (FVB-nmd) mouse model that has an average lifespan of twenty days. The previously reported B6.BKS Ighmbp2nmd-2J/J (B6-nmd-2 J) mouse model has a variable lifespan ranging from four weeks to seven months with no respiratory distress noted until end stage; therefore, we wanted to determine whether the reduced lifespan of FVB-nmd mice was attributed to respiratory-associated changes. Our findings demonstrate that FVB-nmd mice showed severe respiratory deficiencies in nearly all parameters quantified by plethysmography. Surprisingly, the innervation status of neuromuscular junctions of respiratory and oral muscles were largely unaffected throughout the lifespan of the FVB-nmd mice. Diaphragm muscle fibers were reduced in size and the phrenic nerve demonstrated changes in fiber size and myelination. The hypoglossal nerve innervating the tongue also showed disease pathology. Assessment of lung tissue also revealed significant pathology. The investigation of the FVB-Ighmbp2nmd/nmd mouse model provides insight on how reduced IGHMBP2 protein alters respiratory function and impacts lifespan.
Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by muscle weakness, thrombocytopenia, spleen anomalies and short stature. They are due to mutations affecting the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1 and leading to aberrant Ca2+ homeostasis. Therapeutic approaches aiming to rebalance intracellular Ca2+ levels largely rescued the multi-systemic phenotype in Stim1R304W/+ mice harboring the most common TAM/STRMK mutation. However, the currently used biomarkers to follow disease progression are costly and inadequate for longitudinal studies. Here, we investigated the suitability of MYOM3 to serve as a robust blood-based biomarker for TAM/STRMK. Using only minimal blood volumes, we detected highly elevated circulating MYOM3 levels in plasma samples from Stim1R304W/+ mice and TAM/STRMK patients with different mutations, and we found that the MYOM3 levels were normalized in Stim1R304W/+ mice undergoing efficient therapies. We also identified skeletal muscle as the primary source of circulating MYOM3, a structural protein of the contractile unit in myofibers, and uncovered that MYOM3 is primarily expressed in regenerating muscle fibers and in fast-twitch type IIa fibers. Overall, this work emphasizes the utility of MYOM3 as a minimally-invasive biomarker for disorders involving myofiber degeneration, and highlights the ability of MYOM3 to detect early muscle dysfunction in TAM/STRMK and evaluate therapeutic efficiency.
Dystrophin links the actin cytoskeleton to the extracellular matrix through the dystrophin-glycoprotein complex (DGC), providing structural stability to muscle fibers. In mdx mice, which lack dystrophin, neuromuscular junctions (NMJs) remain largely structurally and functionally intact despite extensive muscle pathology. Here, using single- and double-mutant mice deficient in dystrophin and α-syntrophin (α-syn), we investigated how utrophin upregulation contributes to NMJ maintenance in dystrophic muscle. During early postnatal development, when dystrophic muscles are transiently resistant to degeneration, the DGC proteins α-dystrobrevin, α-syn, and β-dystroglycan are broadly distributed along both synaptic and extra-synaptic regions of the sarcolemma, although their overall levels are reduced compared with wild-type (WT) muscle. In contrast, in WT mice, utrophin is restricted to NMJs, whereas in dystrophic muscles from both mutants, it is distributed along both synaptic and extra-synaptic regions of the sarcolemma, particularly within the innervated zone of muscle fibers. As muscles mature and degeneration begins, these DGC components become highly restricted to NMJs while being markedly reduced or absent from the extra-synaptic sarcolemma in many muscle cells. Although utrophin remains highly enriched at synaptic sites in mdx:α-syn-/- muscles, their NMJs display severe structural abnormalities compared with those of mdx mice. These include a dramatic reduction in synaptic fold depth and density, a decrease in presynaptic vesicle density, retraction of nerve terminals, and axonal thinning. These findings demonstrate that utrophin localization at the NMJ is not sufficient to preserve synaptic integrity and that functional interactions between utrophin and α-syn are required to maintain the NMJ in dystrophic muscle.
Numerous actin-related proteins (ARPs) deficiencies have been confirmed to cause sperm acrosomal structural abnormalities, representing significant genetic factors in male infertility. We identified a patient with a missense mutation in ACTRT3, who presented with teratozoospermia and experienced embryonic developmental arrest following intracytoplasmic sperm injection (ICSI). Further analysis revealed ultrastructural abnormalities in the sperm acrosome. To validate these findings, we generated a conditional knockout transgenic mouse model. Male homozygous mutant mice recapitulated the human phenotype, exhibiting consistent acrosomal ultrastructural defects, infertility, and embryonic developmental arrest. To overcome this limitation, a dual-agonist artificial oocyte activation (AOA) protocol combining ionomycin and SrCl2 was developed. This strategy successfully rescued blastocyst formation in Actrt3-deficient mouse models and enabled embryos from the affected couple to develop to the blastocyst stage after ICSI, culminating in a successful pregnancy post-transfer. The combinatorial AOA approach outperformed monotherapy by synergistically restoring PLCZ1-associated calcium signaling defects, providing a tailored solution for infertility linked to actin-related protein deficiencies.
Cystic fibrosis (CF) is a life-limiting autosomal recessive disorder caused by pathogenic variants in the CF transmembrane conductance regulator (CFTR) gene that impair epithelial chloride ion transport, leading to progressive lung dysfunction among other systemic manifestations. Small molecule CFTR modulators have dramatically improved outcomes for people with CF (pwCF) by promoting the trafficking of defective CFTR protein to the cell surface and enhancing its activity. However, a substantial proportion of pwCF harbor genotypes which remain refractory to CFTR modulators and thus lack approved disease-modifying treatments, most commonly nonsense and splicing variants that disrupt CFTR expression at the transcript or translation level. This review delineates the molecular mechanisms underlying modulator-refractory CFTR genotypes, focusing on premature termination codons, nonsense-mediated mRNA decay (NMD), and aberrant pre-mRNA splicing. We examine how these biological phenomena determine and constrain modulator efficacy while simultaneously defining therapeutic entry points. We evaluate emerging DNA-targeted strategies, such as gene replacement and gene editing, alongside RNA-directed approaches, including mRNA replacement, antisense oligonucleotides, CFTR amplifiers, translational readthrough agents and NMD inhibition. Variant-specific sensitivity, reported clinical and preclinical evidence, and practical considerations for implementation are critically assessed. By aligning molecular defect with therapeutic modality, this framework enables rational prioritization of intervention strategies for modulator-refractory CFTR genotypes.
Nemaline Myopathy type 6 (NEM6) is a congenital myopathy caused by variants in Kelch-repeat-and-BTB-(POZ)-Domain-Containing-13 (KBTBD13). The majority of the NEM6 patients harbor the Dutch founding variant KBTBD13R408C (c.1222C > T, p.Arg408Cys) and experience skeletal muscle weakness and sarcomere-based hypercontractility. Histological characterization of NEM6 patient biopsies by NADH staining shows the presence of cores, suggesting mitochondrial dysfunction. We aimed to elucidate the role of mitochondrial dysfunction in NEM6 pathology and tested the ability of the NAD+ precursor nicotinamide riboside (NR) to improve mitochondrial performance. We performed a natural history study in homozygous Kbtbd13R408C-knockin mice (NEM6 mouse model) to investigate the onset and progression of mitochondrial dysfunction in NEM6. We performed high-resolution respirometry, metabolic treadmill experiments and histoenzymatic NADH and SDH stainings on cryosections. Additionally, we used multi-omics analyses to investigate impacted pathways and metabolite dysregulation and performed NR supplementation for eight weeks to prevent the onset of mitochondrial dysfunction in NEM6 mice. Throughout disease progression, NEM6 mice display decreased mitochondrial respiration, impaired metabolic performance and the presence of cores with histoenzymatic reactions. Multi-omics studies revealed that the TCA cycle is heavily impacted and that NAD+ levels are decreased throughout disease progression. We aimed to restore NAD+ levels by supplementation of NR. Remarkably, NR treatment in 1-months-old NEM6 mice, prevented the onset of mitochondrial dysfunction. In conclusion, these results provide insight in the onset and progression of mitochondrial dysfunction in NEM6 and offer proof-of-concept for NR as a therapeutic strategy.
Orofacial clefting (OFC) is among the most common birth defects and can occur either as part of a syndrome or in isolation (nonsyndromic, ns). Cleft palate only (CPO) is an OFC subtype. Here, we searched for novel nsCPO risk genes carrying homozygous and compound-heterozygous variants, by analyzing exome data from six sibling pairs with nsCPO born to unaffected parents. After stringent quality control and filtering, we identified 6 homozygous variants and 32 compound-heterozygous variants in 5 and 16 candidate genes, respectively. We prioritized DDR2, a collagen-activated receptor-tyrosine-kinase influencing extracellular matrix composition, as our top candidate for functional follow-up, since variants in this gene can cause Warburg-Cinotti syndrome, the phenotypic spectrum of which includes palatal abnormalities. Knock-down and knock-out of DDR2-orthologs in zebrafish caused craniofacial abnormalities resembling CPO in humans. Zebrafish immunostaining indicated that DDR2-orthologs were expressed in mature head muscle cells, while murine single-cell RNA-Sequencing data detected Ddr2 expression only in head muscle progenitor cells; the latter finding was confirmed in human embryo sections stained for DDR2. DDR2-expressing head muscle progenitor cells may influence extracellular matrix composition through DDR2-mediated signaling, thereby affecting outgrowth, elevation, and fusion of the palatal shelves, a previously postulated mechanism involved in palatogenesis. Most established OFC genes (e.g. CDH1, CTNND1, IRF6, and GRHL3) act via mechanisms related to epithelial integrity and periderm differentiation, whereas our data provide evidence supporting DDR2 as a risk gene for nsOFC that functions by influencing extracellular matrix composition.
