Human Molecular Genetics

Jordans Syndrome (JS) is a rare, neurodevelopmental disorder caused by de novo missense mutations in protein phosphatase 2 regulatory subunit Bdelta (PPP2R5D). JS is characterized by severe neurological impairments starting in early life. PPP2R5D encodes for B56{delta}, one of the regulatory subunits of protein phosphatase 2A (PP2A). PP2A is a heterotrimeric protein serine/threonine phosphatase that is highly expressed in the brain and the liver. Past studies have focused on PP2As role in liver and little is known about the holoenzymes behavior in neuronal cells. Although B56{delta} is known to play an important role in the substrate specificity of PP2A, the identification of validated downstream substrates in JS remains unclear. To better understand how the mutations affect neuronal cells, we developed cerebral cortical-like organoids from an engineered allele series of the most common JS mutations to characterize the physiological changes throughout different stages of neurodevelopment. Organoids were assessed for transcriptomic, protein, and electrophysiological changes utilizing bulk RNA sequencing, immunocytochemistry, Western Blot, and high-density MicroElectrode Array. The results identify differentially expressed genes and translated proteins, potential neuronal substrates, and significant electrophysiological signatures that suggest mutations in B56{delta} lead to variant-specific dysfunction of PP2A. Overexpression of PPP2R5D through AAV transduction of organoids rescued several phenotypes in the variants, suggesting different pathogenetic etiology underneath. Our findings successfully characterized cerebral cortical-like organoids in JS cell lines and demonstrated its potential as a model for studying neurodevelopmental disorder and for screening therapeutic approaches.

Duchenne Muscular Dystrophy (DMD) is a debilitating degenerative condition with complex musculoskeletal and cognitive symptoms. The protein responsible, dystrophin, is expressed in both muscle tissue and within the central nervous system (CNS) where it localizes to inhibitory synapses. Recent work has shown that dystrophin loss in skeletal muscle leads to abnormalities in endocannabinoid signaling, particularly related to Cannabinoid Receptor Type 1 (CB1R) signaling pathways. CB1Rs are highly expressed throughout the CNS, and have been implicated in short- and long-term plasticity mechanisms. Despite this curious overlap, no work examines how dystrophin loss impacts CB1R signaling in the CNS, a mechanism that may contribute to the diverse neurological pathologies seen in DMD patients. To address this, we used a combination of immunofluorescent labeling and ex vivo electrophysiology to examine CB1R signaling at three classes of synapses within the cerebellum. Utilizing DMDmdx mice, a mouse model of DMD, we find that loss of dystrophin significantly impairs CB1R signaling specifically at parallel fiber-Purkinje Cell synapses, a key location for cerebellar learning. We also find that endocannabinoid-mediated long-term depression at these synapses is absent. Loss of endocannabinoid signaling and synaptic plasticity may contribute to cerebellar dysfunction and motor control symptoms in DMD. These data suggest that dystrophin loss may have previously undescribed consequences for CNS function, and that modulation of endocannabinoid signaling may be a therapeutic strategy for symptom management. Significance StatementDuchenne Muscular Dystrophy (DMD) is a degenerative condition with severe CNS deficits in addition to the well-known muscle weakening. However, no effective treatments currently exist for CNS-related aspects of this disease. Given that endocannabinoid signaling is altered in dystrophic muscle and the importance of endocannabinoid signaling in CNS function, we examined endocannabinoid signaling in the cerebellum of DMDmdx mice, a model of DMD. Utilizing immunolabeling and ex vivo electrophysiology, we find a significant decrease in CB1R expression and functionality specifically at parallel fiber synapses, resulting in reduced or abolished short- and long-term synaptic plasticity. These findings demonstrate that changes in endocannabinoid function contribute to CNS deficits in DMD and open the door to new potential therapeutic targets for treatment.

Introduction: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common congenital malformation with complex etiology involving both genetic and environmental factors. Epigenetic mechanisms may mediate environmental contributions, but separating genetic from environmental effects remains challenging. Methods: We present an epigenome-wide association study with 32 monozygotic and 22 dizygotic twin pairs discordant for NSCL/P on blood and saliva samples. Differential methylation analysis was conducted using linear models to identify CpG sites showing significant methylation differences between affected and unaffected twins followed by functional annotation and pathway enrichment analysis. Results: The top-ranked finding is a differentially methylated region comprising two CpG sites at the CYP26A1 locus, cg12110262 (P = 3.21x10-7) and cg15055355 (P = 1.39x10-3). CYP26A1 is essential for retinoic acid catabolism and craniofacial patterning. The chromatin regulator ANKRD11, which causes KBG syndrome featuring cleft palate was the second best hit. Differentially methylated CpG sites showed significant enrichment in craniofacial enhancers and overlap with multiple GWAS-validated cleft genes including VAX1, PVRL1, SMAD3, and PRDM16. Conclusions: Our findings implicate retinoic acid signaling and chromatin regulation in NSCL/P etiology and demonstrate the value of discordant twin designs for distinguishing environmental from genetic epigenetic contributions to complex malformations.

PCDH19-Cluster Epilepsy (PCDH19-CE) is a rare neurological disorder caused by mutations in the PCDH19 (Protocadherin-19) gene and is characterized by early-onset seizures and cognitive impairment. In contrast to most X-linked disorders, PCDH19 mutations predominantly affect heterozygous females, while hemizygous males are largely spared. Although advances have been made to understand the pathological mechanism underlying PCDH19-CE, key downstream targets and compensatory pathways are yet to be elucidated. Using CRISPR/Cas9 technology, we generated both a mouse model of PCDH19-CE and a human embryonic stem cell (ESC) model. Transcriptomic analysis identified genes that were differentially expressed in the brains of heterozygous (Pcdh19WT/mut) female mice compared with wildtype (WT) and homozygous (Pcdh19mut/mut) female mice. Pathway analysis of these differentially expressed genes (DEGs) revealed enrichment in pathways involved in neuronal development, ion channel activity, synaptic development and neuronal signalling. Neurons differentiated from human ESCs carrying a PCDH19 mutation exhibited similar gene expression patterns, with heterozygous neurons displaying a distinct expression pattern compared to both WT and homozygous mutant neurons. In contrast to the molecular phenotype, neurons derived from homozygous mutant cells showed highly elongated neurites while neurons from heterozygous cells showed intermediate neurite elongation. This suggests that neurite morphology correlates directly with levels of WT PCDH19. Overall, our findings indicate that heterozygous PCDH19 mutations are associated with defects in the expression of genes involved in developmental, signalling, and neuronal pathways in both mouse and human disease models, while certain morphological phenotypes appear to depend on the levels of WT PCDH19.

Abstract/SummaryFIG4 deficiency is the cause of Charcot Marie Tooth type 4J, a neurological disorder characterized by enlarged lysosomes. Our CRISPR activation genome wide screen found that upregulation of PIKFYVE rescued the enlarged lysosome phenotype in cultured cells. To assess PIKFYVE upregulation treatment in vivo, we generated Fig4 deficient mice with CRISPR activation of Pikfyve in neurons. Pikfyve was increased 2 fold in whole brain of CRISPR activated mice. Pikfyve upregulation did not extend the 3 week survival of Fig4 deficient mice. Vacuolization of brain was not rescued. The data demonstrates that a 2 fold increase of Pikfyve is not sufficient to treat Fig4 deficiency. Further testing will be required to determine if a higher increase of Pikfyve can ameliorate the effects of FIG4 deficiency in vivo.

TBCK Syndrome is a rare Mendelian disorder caused by variants in the TBCK gene. Although symptoms affect multiple organ systems, hallmark features include intellectual and developmental disability, craniofacial differences, hypotonia, and premature death. At the cellular level, TBCK has been implicated in mTOR signaling, autophagy, mitophagy, and mRNA trafficking; however, the mechanisms underlying disease onset and progression remain unclear. To address this gap, we characterized a mouse model of TBCK Syndrome. These mice lack exon 5 of the TBCK gene, resulting in a whole-body knockout of Tbck, modeling the most severe known variant. We performed a comprehensive battery of developmental assays, along with microcomputed tomography and histological analyses, which revealed systemic alterations consistent with those observed in affected individuals. Notably, phenotypic changes arising from Tbck loss emerge early and are detectable in the brain, indicating a primary neurodevelopmental origin of disease pathology. Rigorous characterization of this Tbck-deficient mouse establishes the first in vivo platform to investigate disease mechanisms and provides a foundation for preclinical evaluation of gene and targeted pharmacological therapy strategies. Summary StatementThis study establishes a rigorously validated animal model recapitulating systemic features of TBCK Syndrome, enabling targeted investigation of disease biology and preclinical assessment of candidate therapies.

Primary genetic mitochondrial diseases (GMDs) are a clinically and genetically diverse group of diseases estimated to impact over 1 in 4,000 individuals. Leigh syndrome (LS) is the most common pediatric presentation of GMD. LS typically presents within the first years of life and is a severe progressive multi-system disorder. Symmetric progressive inflammatory brain lesions are a defining feature of the disease. Patients can also present with seizures, metabolic dysfunction, muscle weakness, and other symptoms. No effective clinical treatments currently exist. Recent data from the Ndufs4(-/-) mouse model shows that peripheral macrophages contribute to brain lesions in LS, that disease is causally driven by innate immune populations, and that depletion of innate immune cells prevents LS disease. However, the precise mechanisms underlying immune activation remain unknown. Certain mitochondrial macromolecules retain bacterial signatures and can act as potent agonists for innate immune pathways. For example, cytoplasmic mitochondrial RNA and mitochondrial DNA are detected by Toll-like receptors (TLRs) 7 and 9, respectively, at the endosome. Accordingly, these are considered strong candidates for mediating innate immune activation in LS. Here, we generated TLR signaling deficient Ndufs4(-/-)/MyD88(-/-) animals to assess whether TLR signaling plays a role in disease onset or progression in LS. Loss of MyD88 in Ndufs4(-/-) animals statistically significantly increased survival and delayed the onset of some symptoms, but the benefits were modest compared to CSF1R inhibition from prior work. We conclude that Myd88-mediated immune signaling is not a primary driver of LS. Notably, prophylactic enrofloxacin treatment, which was necessary for production of innate immune deficient MyD88(-/-) animals, modestly decreased survival and accelerated disease. The impact of enrofloxacin and similar drugs in the context of mitochondrial disease warrants further investigation.

IntroductionNeuromuscular disorders (NMDs) encompass a broad group of conditions that primarily affect the peripheral nervous system. They are often caused by genetic alterations that impair skeletal muscle function and result in debilitating symptoms. Obtaining an accurate molecular diagnosis remains a challenge, potentially because variants in genes that have yet to be identified as causal. We therefore used advanced computational methods to study the genetic architecture of NMDs and to identify key features that distinguish NMD genes from other genes in the broader genome. MethodsCurated genes implicated in NMDs (n = 639; GeneTable of NMDs) were obtained and merged with a comprehensive set of genomic features for human autosomal protein-coding genes. Machine-learning-based feature selection and ranking were performed using Boruta, along with complementary analytical approaches. These analyses were used to identify the most important genic features (n = 134, subcategories: gene complexity, genetic variation, expression patterns, and other general gene traits) for discriminating NMD genes from other genes in the genome ResultsNMD genes exhibit enriched expression in disease-relevant tissues, including skeletal muscle and heart. Additionally, compared with other protein-coding genes, these genes exhibit increased transcriptomic complexity (e.g., longer transcripts and more unique isoforms), contain more short tandem repeats, and show greater variation in conservation across model organisms. ConclusionsThis study identified several key genomic features that may distinguish NMD genes from the rest of the genome. This may enhance the identification of novel causal genes and could ultimately facilitate earlier diagnosis and medical management for affected individuals.

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic dysregulation at the chromosome 4q35 D4Z4 repeat array under specific permissive genetic conditions. Due to the complexity, expense, and general inaccessibility of FSHD genetic testing, many individuals displaying characteristic muscle weakness are never genetically confirmed and at-risk relatives cannot get screened. We previously developed a targeted bisulfite sequencing (BSS) protocol using the Sanger method to determine DNA methylation levels at specific D4Z4 loci relevant to distinguishing forms of FSHD from non-FSHD that can be used with DNA isolated from saliva, thereby reducing cost and increasing accessibility compared to traditional D4Z4 deletion testing that uses DNA isolated from blood. MethodsHere, we adapt the D4Z4 BSS protocol to next-generation sequencing (NGS) to increase sequencing depth and further reduce cost, validate both sequencing technologies against several cohorts of genetically defined samples, and introduce the D4Z4caster software for computing DNA methylation signatures with diagnostic utility from raw sequencing data. ResultsBoth Sanger and NGS BSS methods using D4Z4caster were validated as providing high sensitivity and specificity, with geometric mean of sensitivity and specificity (G-mean) >95% and area-under-the ROC curve (AUC) of 0.99. The NGS method allows for higher throughput and increased read depth, while the Sanger method allows faster processing of individual samples. Importantly, the NGS method could identify FSHD1 cases that are likely mosaic and would otherwise be missed. ConclusionsD4Z4caster methylation signatures can accurately detect contracted FSHD1-permissive chromosome 4q35 alleles, hypomethylation of D4Z4 arrays indicative of FSHD2, and SNPs that are important for diagnostic use. This workflow is amenable to transitioning to clinical settings for an accurate, low-cost FSHD molecular diagnostic test that could be accessible worldwide. What is already known on this topicCurrently accepted genetic diagnostics for FSHD1 are complex and expensive and can mischaracterize certain complex genetic cases. These diagnostics all require high molecular weight genomic DNA typically freshly isolated from blood, highly specialized equipment, and additional testing for FSHD2, making FSHD diagnostics the most expensive among neuromuscular diseases and inaccessible to much of the world. However, the epigenetic status of the 4q35 and 10q26 D4Z4 repeat arrays, as determined by DNA methylation status using our bisulfite sequencing-based protocol, distinguishes genetically FSHD1, FSHD2, and non-FSHD samples. Additionally, since our protocol is PCR-based, it can utilize DNA isolated from multiple sources, including saliva and buccal swabs. What this study addsThis study validates the relevant DNA methylation signatures against several large cohorts of genetically-confirmed FSHD and non-FSHD samples and optimizes the DNA methylation data analysis for the greater accuracy required for diagnostic utility, including the exclusion of nonpathogenic chromosome 10q or 4A166 contractions. In addition, we introduce the D4Z4caster analysis software, which runs in a portable and scalable Docker container, and provides increased quantitative accuracy important for: 1) confirming likely clinical cases of FSHD that do not meet the currently accepted genetic definition of FSHD1 or FSHD2, 2) identifying FSHD1 somatic mosaicism, and 3) potential prognostic applications. How this study might affect research, practice or policyFSHD1 is genetically defined by a D4Z4 array at the 4q35 locus that is contracted to 1-10 repeat units. However, disease penetrance is influenced by repeat number, epigenetic modifications, and genetic background, causing a misalignment of current genetic diagnosis with clinical diagnosis. This study will improve the accuracy of epigenetic analysis for determining cases of genetic FSHD, help broaden the definition of genetic FSHD to more accurately correspond to clinical FSHD, and allow identification of those at risk for developing clinical FSHD in affected families and in large population studies now being performed and proposed. In addition, it will better inform how an individuals epigenetic status is interpreted for potential prognostic value. Overall, this methodology is: 1) significantly less expensive than current clinically-approved FSHD diagnostic technologies, 2) more accessible due to compatibility with DNA isolated from multiple sources including saliva, and 3) compatible with the current sequencing equipment and workflow for DNA isolation used in commercial clinical laboratories. Together, these advantages will help move the technology toward becoming an approved molecular diagnostic test for FSHD in the USA, Europe, and countries currently lacking clear access to testing.

S-acylation is a reversible posttranslational lipid modification important in the nervous system that dynamically regulates protein localization and function. Aberrant S-acylation has been implicated in several neurological conditions. While several de-S-acylases (deacylases hereafter) have been identified, little is known regarding their expression and localization in the brain and in neurons. Here, we characterized the expression, localization, and S-acylation of cytosolic deacylases, including acyl-protein thioesterases APT, APT2, and APT1L and /{beta} hydrolase domain-containing proteins ABHD7, ABHD10, ABHD13, ABHD16A, and ABHD17A-C. Mouse brain RNA sequencing data revealed high expression of Lypla1/APT1, Lypla2/APT2, Ephx4/ABHD7, Abhd16a, and Abhd17A-C in the brain, whereas Lyplal1/APT1L, Abhd10, and Abhd13 were expressed at very low levels. Protein analysis demonstrated region-specific expression, with expression of APT1 and ABHD16A highest in the cerebellum and APT2 highest in the hippocampus, with all three highly expressed in cultured hippocampal neurons. Deacylases were observed distributed throughout neurons on punctate structures, with APT2 and ABHD17C to the Golgi by immunocytochemistry. Finally, all ten cytosolic deacylases are themselves S-acylated. These data characterizing deacylase expression, localization, and S-acylation in neural contexts, provides a foundation for future studies investigating deacylase neuronal functions and potential roles in neurological disease.

Studies of protein quantitative trait loci (pQTLs) provide opportunities to interpret complex trait genetics and identify potential biomarkers and therapeutic targets. Circulating proteins are commonly used in pQTL studies due to the accessibility of blood-based measurements, but their levels may not always reflect regulation in disease-relevant tissues. We assessed colocalization and discordance between plasma and dorsal prefrontal cortex cis-pQTLs using data from four large-scale studies and investigated their implications for downstream analyses. Across the proteins examined, at most 80% of the cis-pQTLs showed evidence of colocalization. Among the colocalized loci, approximately 20% exhibited opposite directions of genetic effects. We characterized tissue-specific gene expression profiles based on data from the Genotype-Tissue Expression project. Proteins with colocalized cis-pQTLs were more likely to have high gene expression levels in systemic tissues and immune cells, whereas the remaining proteins were more likely to have high expression in brain tissues. We conducted Mendelian randomization (MR) analyses using neuroticism as an illustrative outcome to compare effect estimates derived using instruments from different pQTL studies. MR analyses identified 13 proteins significantly associated with neuroticism, including six with opposite effect directions between plasma and dorsal prefrontal cortex, highlighting the importance of tissue context. Overall, circulating pQTLs remain informative for proteins from systemic and immune pathways, while incorporating tissue-specific data may provide additional insight for proteins with more localized expression. Considering multiple tissue contexts may refine the interpretation of protein-trait associations and may improve the prioritization of candidate targets.

Pathogenic variants in the gene KCNT1, which encodes a sodium-activated potassium channel, cause a severe neurodevelopmental disorder with intractable epilepsy. In addition to seizures, affected individuals commonly present with severe respiratory issues and structural heart defects not commonly observed in other genetic pediatric epilepsies, suggesting additional developmental functions for KCNT1 in organs beyond the brain. Here, we characterized the spectrum of clinical diagnoses present in a cohort of 46 individuals with pathogenic variants in KCNT1, ranging from 0 to 19 years of age, by medical record review. We documented the prevalence of diagnoses across organ systems, including dependence on assisted breathing, congenital structural heart defects, urinary dysfunction, and spine deformities, among others. Next, we explored the embryonic expression and function of KCNT1 in diploid frogs (Xenopus tropicalis) and observed expression in developing ciliated tissues such as the brain, heart, kidney, and epidermis. Embryonic perturbation of KCNT1 disrupted developmental signaling pathways and caused ciliogenesis defects in the mucociliary epidermis, a common model for the human airway. Loss of KCNT1 disrupted development of multiciliated cells, reminiscent of recent work on the ion channel Piezo1. Consistently, pharmacological inhibition of Piezo signaling enhanced the ciliogenesis phenotype observed following KCNT1 inhibition, while activation of Piezo1 activity partially rescued ciliogenesis in the context of KCNT1 inhibition. Together, this work establishes that KCNT1 has embryonic functions in Xenopus beyond regulating neuronal activity, specifically in multiciliated cell development, and identifies an interaction with pharmacologically-tractable Piezo channels that may be productive for therapeutic efforts.

Pathogenic RYR1 variants are associated with a set of rare neuromuscular disorders termed RYR1-related disorders (RYR1-RD). Clinical manifestations of RYR1-RD include proximal/axial muscle weakness, delayed motor milestones, impaired mobility, muscle pain, and fatigue. Muscle-specific microRNAs (miRNAs) are mostly expressed in muscle tissue and can be detected peripherally in plasma. Using a digital detection system, here we identified and quantified differential amounts of miRNAs in six adult (four monoallelic and two biallelic) RYR1-RD patient plasma samples compared to controls. Overall, 51 differentially expressed miRNAs were identified and hsa-miR-4454+hsa-miR-7975, in particular, was significantly overexpressed relative to controls (+ 39-fold, P=0.00285). Exploration of these differentially expressed miRNAs warrant further investigation as potential biomarkers of RYR1-RD.

BackgroundType 2 diabetes (T2D) represents a major global health burden, with over 700 GWAS loci identified. Translation to biological mechanisms remains challenging. This study employs systematic post-GWAS functional annotation to characterize the RPTOR locus, encoding Raptor, a scaffold protein critical for mTORC1 signaling and beta-cell function. MethodsWe analyzed 31 GWAS credible sets containing rs12950541 (chr17:80760693 G>A) using Open Targets Platform v24.12, encompassing 20 metabolic traits. L2G scoring, colocalization analysis, and QTL mapping in GTEx v8 were performed. Independent Variant Effect Predictor (VEP) analysis of the linkage disequilibrium (LD) block was conducted to characterize all variants in LD (D [≥] 0.7) with rs12950541. RNA-protein interaction networks were predicted using RNAct/catRAPID for key RPTOR transcripts and functionally enriched using ToppGene. Drug target and novelty analyses were performed using ChEMBL, PubMed, and ClinicalTrials.gov databases. Phenome-wide associations and regulatory annotations were obtained from the T2D Knowledge Portal. ResultsRPTOR was consistently ranked #1 L2G gene across all 31 credible sets (mean score 0.428, range 0.383-0.503). T2D showed strong GWAS-GWAS colocalizations (H4>0.8) with adiposity traits. Skeletal muscle demonstrated strongest QTL evidence with sQTL at P=1.21x10-16 and multiple eQTLs/tuQTLs. Critically, zero GWAS-QTL colocalizations and zero QTL in pancreatic islets, adipose, or liver highlight an "eQTL gap." VEP analysis of 140 LD partners revealed exclusively non-coding variants (100% MODIFIER impact), including 24 regulatory region variants and 2 transcription factor binding site variants. RNAct analysis revealed that the NMD transcript RPTOR-208 shows stronger RNA-protein interactions than the canonical transcript, with predicted binding partners including sulfonylurea receptors (ABCC8/ABCC9), IGF1R, and chromatin remodelers, enriched for glucose-mediated signaling and SWI/SNF complex pathways. ABCC8 is confirmed as the molecular target of sulfonylurea drugs (ChEMBL: CHEMBL2071), and literature analysis confirms that the RPTOR-ABCC8 RNA-protein interaction is completely novel, with no prior publications linking RPTOR transcript biology to sulfonylurea receptor function. T2DKP PheWAS confirmed 78 significant associations across 18 phenotype groups, revealing effects on acute insulin response, insulin sensitivity, HDL cholesterol, hepatic enzymes, and sleep traits, with transcription factor binding analysis showing that rs12950541 directly enhances p300 enhancer marking while reducing CTCF insulator binding. ConclusionsSeven convergent lines of evidence support rs12950541 as a strong candidate regulatory variant at RPTOR. Integration of post-GWAS annotation, VEP characterization, RNA-protein interaction networks, and translational drug target analysis converges on a regulatory mechanism involving splicing, chromatin remodeling, and metabolic signaling pathways. The novel predicted interaction between RPTOR-208 and ABCC8/ABCC9 suggests a previously unrecognized molecular bridge between mTORC1 signaling and KATP channel-mediated insulin secretion, with potential implications for understanding sulfonylurea-mTOR pathway crosstalk in T2D.

Background: Genetic variants impacting red blood cell biology disrupt the relationship between glycaemia and glycated haemoglobin (HbA1c), with implications for diagnosis and management of type 2 diabetes (T2D). Thalassaemia trait is estimated to affect 350 million people globally, but its impact on T2D and related outcomes is not clear. Methods: We explored associations between thalassaemia trait, HbA1c, and T2D diagnosis and complications in 43,088 British Bangladeshi and Pakistani participants in the Genes & Health study with linked multisource England National Health Service (NHS) electronic health record data and whole exome sequencing. Findings: 2,490 participants (5.8%) were heterozygous carriers of ClinVar pathogenic / likely pathogenic thalassaemia variants, however 3 in 4 of these were not diagnosed with thalassaemia in their NHS health records. rs33950507, a common variant causal for HbE thalassaemia, was associated with increased HbA1c (beta=0.13, 95%CI:0.08-0.18, p=7.8x10-8), but not glucose levels (beta=0.01, 95%CI:-0.04-0.06, P=0.72). rs33950507 was associated with increased hazards of prediabetes (HR=1.38, 95%CI:1.26-1.52, p=2.2x10-6) and T2D (HR=1.11, 95%CI:1.01-1.22, p=0.03), and reduced hazards of diabetic eye disease (HR=0.74, 95%CI:0.56-0.96, p=0.02) and cerebrovascular disease (HR=0.44, 95%CI:0.20-0.94, p=0.03). Sensitivity analyses suggested mediation by overdiagnosis and overtreatment of T2D. Interpretation: Alternatives to HbA1c, and/or precision medicine approaches to defining and managing hyperglycaemia, are needed, particularly on a global scale. This may be particularly relevant to individuals from ancestral groups among whom erythrocytic traits are more common but often undiagnosed. Funding: Wellcome Trust, MRC, NIHR, Barts Charity, Genes & Health Industry Consortium

The autosomal dominant p.Ala165Val mutation in LIM Domain Binding Protein 3 (LDB3) causes myofibrillar myopathy marked by Z-disc disruption, accumulation of filamin-C (FLNc) and chaperone proteins, and progressive muscle weakness. We previously showed that this mutation interferes with the LDB3-protein kinase C alpha (PKC)-FLNc mechanosensing axis and impairs chaperone-assisted selective autophagy (CASA), establishing a gain-of-function mechanism. In this study, we examined whether mutant allele-specific knockdown could reverse the disease or mitigate disease progression in-vivo. A single intramuscular-injection of an AAV9-delivered microRNA-based shRNA produced substantial knockdown of mutant Ldb3 transcripts and protein in Ldb3Ala165Val/+ knock-in mice treated either before or after the onset of pathology. Treatment after disease onset reduced filamin-C and CASA protein aggregates and improved muscle strength, whereas early intervention prevented development of molecular and histological features of myopathy. Phosphoproteomic profiling further showed broad remodeling of dysregulated phosphorylation networks, including restoration of PKC-responsive sites and normalization of altered sarcomeric and cytoskeletal signaling observed in Ldb3Ala165Val/+ mice. These findings identify disruption of the LDB3-PKC-FLNc mechanosensing pathway as a central disease driver and suggest that restoring this signaling axis may complement mutant allelespecific RNA interference (RNAi). Overall, our results support RNAi as a promising therapeutic strategy for dominant LDB3-related myofibrillar myopathy.

BackgroundHypertension is a highly heritable cardiovascular disorder and a major determinant of cardiometabolic disease, including diabetes. However, the regulatory genes and tissue-specific mechanisms underlying blood pressure variations remain incompletely understood. MethodsLeveraging a well-characterized prospective population-based cohort comprised of 27,308 participants from the Singapore Chinese Health Study (SCHS), we evaluated genome-wide genetic associations for five blood pressure traits: hypertension status, systolic blood pressure, diastolic blood pressure, mean arterial pressure (MAP) and pulse pressure (PP). Additionally, we conducted a transcriptome-wide association study (TWAS), integrating gene expression data from 49 tissues, followed by colocalization and fine-mapping to prioritize regulatory genes. Association of identified variants with incident diabetes was additionally evaluated in the longitudinal data. ResultsWe validated 10 blood pressure loci (P between 1.64 x 10-20 - 4.10 x 10-8) and identified an East-Asian specific splice donor variant at the COL21A1 gene associated with PP (rs149344559, P = 6.78 x 10-10). Integrative analyses prioritized FGF5 in kidney cortex and ENPEP in pituitary tissue as candidate regulatory genes. The blood pressure-lowering allele at ENPEP (T allele, rs1879056) was associated with reduced risk of incident diabetes. Mediation analysis demonstrated that approximately 21% of the genetic association with diabetes was mediated through MAP (Pindirect-effect = 2 x 10-16). ConclusionThis study refines genetic predispositions for blood pressure among East-Asians. We further delineate tissue-specific regulatory pathways underlying blood pressure variations and identify ENPEP-mediated dysfunctions linking blood pressure genetics to diabetes risk, underscoring integrated disease mechanisms.

Danon disease is a rare disorder caused by mutations in the LAMP2 gene, which encodes a lysosomal membrane protein key to the endolysosomal pathway and autophagy. Affected individuals show multisystemic alterations that include cardiomyopathy, skeletal muscle weakness, visual deficits and cognitive impairment. Here we establish a knockout LAMP2 line in Xenopus tropicalis that reproduces the characteristic cardiac activity, mobility impairments and vision deficits present in the disease. Damaged mitochondria were abundantly found in skeletal muscle fibers. LAMP2 mutant X. tropicalis detected light with a reduced preference for green wavelengths. Visual deficits were consistent with the finding of damaged mitochondria in the inner segment of rods but not in cones. Differences in autophagic flux were found in presynaptic terminals from photoreceptors and olfactory sensory neurons (OSNs), which establish the first synapse processing vision and olfaction, respectively. In wild-type animals autophagic shapes were observed in OSN terminals but were absent from photoreceptor ribbon synapses. In knockout LAMP2 tadpoles, autophagic organelles covered 7% of the OSN presynaptic terminal surface, a three-fold increase compared to photoreceptor terminals. These differences suggest that LAMP2 plays synapse-specific roles that could be an important determinant of the psychiatric manifestations present in Danon disease and support the use of LAMP2 X. tropicalis to shed new light on the pathological bases of this lysosomal storage disorder.

Craniometaphyseal dysplasia (CMD) is a rare genetic disorder characterized by hyperostosis of craniofacial bones and flared metaphyses of long bones. Mutations in ANKH (mouse orthologue ANK), a transmembrane protein mediating ATP and citrate efflux, cause the autosomal dominant form of CMD. How ANK mutations in CMD affect ATP/citrate homeostasis and downstream targets remains unknown. We determined that cellular ATP export, intracellular ATP levels, and plasma citric acid were significantly reduced in ANKF377del knock-in (AnkKI/KI) mice. Enrichment and pathway analyses of the plasma metabolome suggested the involvement of the citric acid cycle. It is known that AMPK is phosphorylated and activated when ATP is low. Phospho-AMPK was significantly upregulated in fusing AnkKI/KI osteoclasts, major contributors to CMD. AMPK inhibitor treatment only during the fusion stage of osteoclasts significantly restored dysfunctional AnkKI/KI osteoclasts, partly by modulating actin structures. Systemic administration of the AMPK inhibitor SBI-0206965 improved the positioning of cervical loops of incisors but failed to correct other skeletal abnormalities in AnkKI/KI mice. Limitations of systemic administration of SBI-0206965 include its off-target effects on other cell types and the inability to inhibit AMPK only on fusing osteoclasts. Nonetheless, this proof-of-principle study reveals an important role of the ATP-AMPK axis in CMD pathogenesis. Take-home messageSuppression of increased activation of AMPK restores the function of osteoclasts, suggesting that abnormal energy metabolism is an integral component of the disease phenotype in CMD.

BackgroundHeterozygous c.283+1G>A and c.283G>A variants in the THRB gene, encoding for thyroid hormone receptor (TR){beta}1 and {beta}2, lead to autosomal dominant macular dystrophy (ADMD). We report the detailed clinical characterization of two first-degree relatives with ADMD, heterozygous for THRB c.283+1G>A, and an unrelated ADMD patient with a novel variant, c.283G>C. The genomic and molecular consequences of both variants were studied. MethodsgDNA and mRNA were obtained from leukocytes. Clinical characterization included biochemistry, bone density and body composition, ECG, echocardiography, ultrasound, audiometry and color-vision. In vitro assays investigated TR function and DNA binding. ResultsThe patients manifested no resistance to thyroid hormone beta (RTH{beta}) and had normal FT4 and TSH. Detailed studies in two patients showed no goiter, tachycardia, hypercholesterinemia or hepatic steatosis. Hearing was not impaired. Both had impaired color vision and reduced bone density. RT-PCR from all three patients revealed skipping of exon 4 exclusive to TR{beta}1, producing a deletion of 87 amino acids in the N-terminal domain (TR{beta}1{Delta}NTD). In vitro, DNA-binding affinity of TR{beta}1{Delta}NTD to DR4-TRE with or without RXR was comparable to TR{beta}1WT. Surprisingly, TR{beta}1{Delta}NTD was transcriptionally twice more active than TR{beta}1WT with a similar EC50 for T3, demonstrating gain-of-function of TR{beta}1{Delta}NTD. THRA expression in leukocytes was increased by 3-fold compared to unrelated controls and different from RTH{beta} patients. ConclusionThese THRB splice site variants produce TR{beta}1 exon 4 skipping, resulting in a gain-of-function mutant, TR{beta}1{Delta}NTD. This explains the dominant ADMD phenotype devoid of RTH{beta} and suggests a TR{beta}1 gain-of-function syndrome.