Human Molecular Genetics

Very preterm infants (<30 weeks gestation) are at elevated risk for neurodevelopmental and social-behavioral challenges. DNA methylation (DNAm) may provide a biological link between preterm birth and later behavioral outcomes. We examined associations between DNAm profiles at neonatal intensive care unit (NICU) discharge and at age 5 with Social Responsiveness Scale (SRS) scores which measure social communication, social interaction, and repetitive behaviors at age 5, including sex-specific effects, in the Neonatal Neurobehavior and Outcomes in Very Preterm Infants (NOVI) Study. Epigenome-wide buccal DNAm was profiled at NICU discharge (n=218) and at 5 years (n=188). We identified 38 neonatal and 6 age-5 CpG sites associated with SRS scores (all q<0.05) using epigenome-wide association studies (EWAS) at each time point. Several CpGs mapped to genes involved in neurodevelopment including TCF4, KLC4, CAP2, PTDSS1, ADAM12, SENP1, CHN2, SH3D19, and ITGA1, with sex-specific effects observed for CpGs in CAMTA1 and GABBR1. Enriched pathways included neurodevelopment, cytoskeletal regulation, stress-response, and metabolic processes. DNAm patterns during early life, particularly the neonatal period, were associated with social-behavioral development in very preterm children. Findings in key genes such as TCF4 and CAMTA1 highlight potential epigenetic mechanisms linking early-life biology to later behavioral challenges.

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.

ContextThe environmental or other genetic factors might influence the effect of Klotho (KL) on glucose metabolism. ObjectiveWe investigated mitochondrial genetic variants that interact with KL single nucleotide polymorphisms (SNPs) to modulate diabetes risk. MethodsWe used the data from 7,047 non-Hispanic white participants of the Health and Retirement Study, a prospective observational study including adults aged 50 years and older from the United States. First, we performed single gene-wide association scans to identify KL SNPs associated with diabetes. Next, we performed a nuclear-by-mitochondrial interaction test (NuMIT) in which we use an identified KL SNP from the gene-wide scan to evaluate potential interactions with 85 mitochondrial SNPs in relation to diabetes. ResultsWe failed to identify a significant association between diabetes and the KL SNP in our single gene-wide association test. However, we identified a novel variant (KL rs9563121) which showed a trend of increasing klotho mRNA levels with each additional minor allele. A NuMIT analysis identified mitochondrial SNPs, which showed significant interactions with rs9563121 in relation to diabetes risk. MitoG15929A showed significant interactions with rs9563121 in both men and women. MitoG15929A diminished the potential beneficial effect of KL rs9563121 on diabetes risk in women. Among men with the MitoG15929A variant, KL rs9563121 was associated with higher prevalence of diabetes. ConclusionThe NuMIT approach revealed significant interactions between mitochondrial and nuclear DNA variants of KL. Furthermore, MitoG15929A may have a role in the interaction between diabetes and KL in a sex-dependent manner.

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.

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.

Protocadherin-12 (PCDH12), a cell-adhesion protein belonging to the non-clustered protocadherin family, plays a crucial role in the establishment and regulation of neuronal connections and communication. Bi-allelic loss-of-function (LoF) variants in the PCDH12 gene have been associated with several neurodevelopmental disorders (NDDs) such as diencephalic-mesencephalic junction dysplasia syndrome, cerebral palsy, and cerebellar ataxia, often accompanied by ocular abnormalities. However, genotypes exhibit variable expressivity. Affected individuals sharing the same PCDH12 variant presenting differing phenotypic severities have posed major challenges towards identification of the underlying pathogenic mechanisms. Here, we report three affected individuals from two families, each harbouring non-truncating pathogenic missense variants in PCDH12. The patients are compound heterozygous, with each individual carrying one extracellular [c.1742T>G (p.Val581Gly) and c.1861_2del/insCA (p.Ile621His)] and one intracellular variant [c.3370C>T (p.Arg1124Cys) and c.3445G>A (p.Asp1149Asn] on each allele. The children present with a range of phenotypes similar to those associated with LoF variants. One child exhibited microcephaly and seizures, while the two siblings displayed developmental delays and severe behavioral disorders. All three children experienced some degree of visual impairment. The missense variants provided new insights into the neurodevelopmental consequences of compromised PCDH12 function by distinguishing the specific consequences associated with dysfunction in the extracellular versus intracellular domains of PCDH12. All identified missense variants are predicted to be deleterious and destabilizing. The expression of PCDH12 in HEK293T and HeLa cells demonstrated that PCDH12 is expressed effectively, regardless of the presence of missense variants. However, the extracellular variants p.Val581Gly and p.Ile621His compromised the stability of PCDH12s homophilic adhesion. Additionally, we found evidence of an interaction between PCDH12 and the extracellular domain of the epilepsy-associated PCDH19 protein. PCDH12 extracellular missense variants also affect PCDH19 stability. Our study provides evidence that PCDH12 mediates both homophilic and heterophilic interactions. Our findings also highlight the importance of stable PCDH12-mediated adhesion, emphasizing the need to further study the functional consequences of PCDH12 missense variants on brain and visual system development.

Copper is an essential micronutrient required by enzymes that catalyze oxygen-dependent reactions, but toxic in excess. Mutations in the ATP7A and ATP7B copper transporters cause neuropsychiatric symptoms and neurodegeneration by mechanisms that remain to be elucidated. We previously reported that the ATP7A biochemical interactome is enriched in Parkinsons disease (PD) and neurodegeneration associated proteins, yet the functional outcomes of these interactions are unknown. Using Drosophila, we tested genetic interactions between ATP7 mutants that alter copper levels and a subset of these PD and neurodegeneration causative genes and found sex differences with some candidate genes enhancing ATP7 deleterious phenotypes in both sexes, while others were sex specific. Most notably, we found that Lrrk2 (Lrrk), the most commonly mutated gene in familial forms of PD, protects against ATP7 dysfunction in epidermal epithelial cells with a stronger effect in males than females. However, in dopaminergic neurons Lrrk plays a role in intracellular copper induced toxicity in females but not males, supporting context dependent interactions between ATP7A and PD-associated genes to protect against disruptions in copper homeostasis. Summary StatementWe performed a genetic interaction screen to explore the relationship between copper homeostasis and Parkinsons disease and other neurodegeneration associated genes.

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

Females have two X chromosomes, one of which is inactivated early in development with specific regions and genes escaping inactivation. Thus, X chromosome loss putatively results in decreased dosage of X chromosome escapee and pseudoautosomal genes, impacting downstream pathways. Evidence from Turner syndrome indicates that X chromosome monosomy results in consistent neuroanatomical and cognitive phenotypes. However, it remains unclear whether mosaic karyotypes, with mixed proportions of 45X and 46XX cells, attenuate these phenotypes. We examined whether X chromosome mosaicism is predicted by neuroanatomical and cognitive features. Higher proportion of 46XX cells was significantly predicted by structural properties in somatosensory, motor, visual, and language brain areas, and by performance in visuospatial, fine-motor, and language tasks. Thus, mosaicism partially rescues phenotypes linked to full 45X monosomy and may explain the role of the X chromosome not only across heterogeneous phenotypic expression in females, but also in sex differences observed in neuropsychiatric conditions.

Adenylosuccinate synthetase 1 (ADSS1) myopathy is an ultra-rare disease characterized by progressive muscle dysfunction. The objective of this investigation was to employ a non-invasive biomarker approach to phenotype (fine-)motor skills, speech production and cognition in adults with ADSS1 myopathy. Five individuals with ADSS1 myopathy and five age-sex-matched healthy controls (HCs) underwent a comprehensive multimodal evaluation. Assessments included, (i) evaluation of motor performance, (ii) speech production and cognitive test batteries, (iii) patient-reported outcomes, (iv) electrical impedance myography (EIM), (v) musculoskeletal magnetic resonance imaging (MRI) and (vi) plasma proteomics. ADSS1 participants vs. HCs demonstrated reduced performance on the 9-Hole Peg and grip strength tests as well as lower self-reported mobility. Speech production analysis revealed asthenia (p=0.02), lower intelligibility (p=0.008), and worse voice quality during the sustained vowel task (p=0.03) in the ADSS1 cohort. Cognitive functioning remained unaffected in patients with ADSS1. On EIM, ADSS1 participants vs. HCs, demonstrated a pattern of higher resistance and lower reactance and phase across upper- and lower-extremity measurements, indicative of poorer muscle health, with large effect sizes (Cliffs 8=0.5-0.9). MRI revealed intramuscular fat infiltration, particularly in posterior compartments of the upper leg (e.g., biceps femoris). Proteomics indicated reduced (p=0.04) Neurotrophin-3 (NTF3; implicated in neuronal development, survival and differentiation) levels in the ADSS1 cohort relative to HCs. Lower NTF3 levels associated with poorer performance on hand-motor tasks as well as higher resistance and lower reactance and phase on EIM. This study highlighted the value of multimodal phenotyping for quantifying disease expression and advancing monitoring strategies in ADSS1 myopathy. Take-home messageThis multimodal investigation demonstrates that integrating electrical impedance myography with quantitative motor, speech, musculoskeletal imaging, and proteomic assessments provides a sensitive and non-invasive research framework for capturing neuromuscular dysfunction and functional disease burden in patients with ADSS1 myopathy, thereby supporting the current biomarker strategy for refined phenotyping and longitudinal disease monitoring in this ultra-rare condition.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the primary cause of pediatric kidney failure, yet the genetic etiologies remain elusive for most affected individuals. Reanalysis of trio exome sequencing data from 80 Chinese CAKUT patients identified 32 rare, predicted deleterious variants. Replication in unrelated families from a national multicenter database prioritized four novel candidate genes--DOCK11, MIB1, TENM2, and TNS1. These candidates are involved in both well-characterized developmental pathways and more under-explored biological processes relevant to renal and ureteric morphogenesis. CRISPR-Cas9-mediated zebrafish knockout studies were employed to validate the potential association of these genes with kidney abnormalities including significant pericardial edema, malformed renal tubules, and impaired glomerular filtration. These findings offer potential genetic diagnoses for 10% of CAKUT probands, and demonstrate that exome reanalysis can substantially improve diagnostic yield and inform personalized clinical management. Overall, this study expands the known genetic landscape of CAKUT.

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.

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.

The German Twin Family Panel TwinLife is a nationwide longitudinal study of twins and their family members. Primarily focusing on the development of social inequalities over the life course, TwinLife has been collecting data since October 2014 starting with 4,096 twin families (Ntotal = 16,951 individuals). As Germanys largest twin study to date, TwinLife has been surveying four birth cohorts of monozygotic and dizygotic same-sex twin pairs (initially [~]5, 11, 17, and 23 years old) and their families for 11 years. Survey data have been collected through five biennial face-to-face interviews with four computer-assisted telephone interviews in the years between. In addition, saliva samples were collected before the COVID-19 pandemic (2018-2020), during the pandemic (2021), and after (2022-2024). In this Cohort Profile, we describe the curation and initial analyses of molecular genetic and epigenetic data from the two TwinLife satellite projects TwinSNPs and TECS. Together, these projects currently comprise 12,108 processed DNA samples from 6,450 participants, extracted from the first two saliva collections before and during the COVID-19 pandemic. We compared the subsamples with the overall TwinLife sample and provide an overview of derived polygenic scores (PGS), epigenetic clocks and other methylation profile scores (MPS). We found that PGS predicted sample attrition in TwinLife, with small but significant associations between higher PGS for educational attainment and continued participation. Epigenetic clocks derived from saliva were highly correlated with chronological age (r = .71 to r = .94) and were generally more stable over time than other MPS. PGS for epigenetic clocks were associated with the respective clock only during but not before the start of the pandemic. We discuss opportunities of combining prospectively assessed molecular (epi)genetic data in within-family designs such as TwinLife and its implications and avenues for future research.

Spinocerebellar ataxia type 1 (SCA1) 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. These mutants reveal distinct and shared developmental, behavioral, and transcriptomic alterations. All KOs showed reduced early survival and mild larval growth deficits, indicating essential developmental functions. Behavioral assays revealed distinct paralog-specific effects: atxn1a KO larvae exhibited a unique light-dependent locomotor deficit, whereas atxn1b and atxn1l KOs displayed global hypoactivity. Adult behavioral assessment revealed a gradient of phenotypic severity: atxn1a KOs displayed the earliest and most pronounced alterations in vertical tank exploration and the greatest impairment in swim-tunnel performance, followed by atxn1b and then atxn1l mutants. To define molecular mechanisms underlying these phenotypes, we performed RNA-seq at 5 days post-fertilization and identified unique and shared differentially expressed genes across the three KO lines. Shared transcriptomic signatures highlighted suppression of leukotriene-biosynthetic pathways and diminished innate-immune pathways; suggesting that ATXN1-family genes influence neuroimmune signaling during early development. Weighted gene co-expression network analysis identified distinct KO-associated gene modules, including a phototransduction-enriched module strongly correlated with atxn1a KO status, offering a mechanistic link to its light-dependent locomotor phenotype. Together, these findings establish a comprehensive assessment of zebrafish models that reveal both shared core functions and specialized roles of ATXN1-family genes in development, neuroimmune regulation, sensorimotor behavior, and retinal signaling.

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.

The continuous renewal of healthy epidermis depends on the finely regulated proliferation of basal keratinocytes and subsequent differentiation as the newly formed cells move upwards through the different layers of the epidermis. Perturbations in keratinocyte differentiation may lead to cornification disorders. We investigated seven dogs of different breeds belonging to four independent families that showed striking multifocal tree bark-like skin lesions. Histopathologically, lesional skin was characterized by pronounced epidermal and infundibular hyperkeratosis with epidermal and sebaceous gland hyperplasia. We therefore tentatively termed the phenotype phloiokeratosis, derived from the Greek word phloios for tree bark and keratosis indicating abnormal keratinization. Whole genome sequencing of DNA from affected dogs revealed four independent variants in the SUV39H1 gene encoding the SUV39H1 histone lysine methyltransferase, an H3K9 methyltransferase, which is involved in epigenetic silencing of chromatin. Phloiokeratosis is inherited as an X-chromosomal semi-dominant trait. Four of the affected dogs in our study were heterozygous females and had lesion patterns reminiscent of Blaschko lines. In two of them, trio analyses experimentally confirmed de novo mutation events in the SUV39H1 gene. Previously, Suv39h1-/- knockout mice had been reported to have normal skin. So far, no human patients with SUV39H1 loss-of-function variants have been reported. The findings in SUV39H1 mutant dogs with phloiokeratosis for the first time link SUV39H1 deficiency to a heritable skin phenotype. Our study highlights the essential role of SUV39H1-mediated epigenetic silencing during normal keratinocyte differentiation and provides a unique model for further investigations. Author SummaryThe integrity of the skin depends on a balanced equilibrium of keratinocyte proliferation, differentiation, and sloughing of terminally differentiated cells into the environment requiring finely regulated changes in the global transcriptome of differentiating keratinocytes. We investigated seven dogs belonging to four different families with a new disorder of cornification characterized by tree bark-like outgrowths of the epidermis. Histopathological examinations confirmed that the outermost layer of the epidermis was thickened in affected dogs. The genetic analysis yielded four different SUV39H1 loss-of-function variants in the affected dogs from the four families. The SUV39H1 gene encodes an enzyme that is involved in the epigenetic silencing of chromatin. The newly characterized inherited skin disease in dogs is the first clinical phenotype that has been linked to SUV39H1 deficiency. Most likely, SUV39H1 deficiency leads to delayed epigenetic silencing and consequently delayed differentiation of keratinocytes. Dogs with this rare skin disease provide an improved understanding of the essential role of SUV39H1 in the epigenetic control of gene expression in skin.

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.

BackgroundThe transport of transfer RNAs (tRNAs) from the nucleus to the cytoplasm is a crucial step in the regulation of gene expression and protein synthesis. This process is mediated by specialized export molecules, among which XPOT (Exportin-t, XPO3) plays a central role by recognizing and transporting mature tRNAs through the nuclear pore complex. XPOT is not essential in RNA trafficking in the simple organisms, however the potential impact of XPOT deficiency in human health remains unresolved. MethodsWe identified eight patients from five unrelated families with rare biallelic germline variants in XPOT resulting in putative loss-of-function. Functional analyses were carried out in patient-derived fibroblasts, lymphoblastoid cells and zebrafish models. Ex vivo immunohistochemical stainings for Xpot were performed in the mouse cochlea. xpot knockout zebrafish models were generated to assess the morphology and hearing ability. ResultsAll patients presented with a uniform clinical phenotype that included increased susceptibility to infection, bronchiectasis, severe sensorineural hearing loss, developmental delay, and growth retardation. We demonstrated a complete absence of XPOT protein expression in three patient-derived cell lines. XPOT deficiency leads to disruptions in protein synthesis of the cytokine TNF pathway upon cellular stimulation. Additional XPO1 inhibition in XPOT deficient cells had little effect on cellular functions, suggesting alternative tRNA nuclear transporter pathways. Increased XPOT immunoreactivity was observed in type I spiral ganglion neurons and hair cells of the mouse cochlea, with enrichment in stereocilia. xpot knockout zebrafish model showed dysmorphic features, and reduced hearing, recapitulating key patient phenotypes. ConclusionsOur findings establish a direct connection between impaired XPOT-dependent tRNA export and human pathology. It illustrates that perturbations in nuclear export pathways lead to disease. It also raises the possibility that other nuclear transport receptors may play similarly underappreciated roles in human health and disease. The identification of XPOT as a disease-associated gene opens up new research directions and potential targets for therapeutic intervention.