Patient Fibroblasts Research Articles

ADGRB3-High and POSTN-High Fibroblasts Are Markers of Endotypic Traits in Chronic Rhinosinusitis

Background: Chronic rhinosinusitis (CRS) is a disease characterized by persistent sinonasal mucosal inflammation. Fibroblasts play a crucial role in extracellular matrix production and inflammation. We investigated the heterogeneity of fibroblasts in patients with CRS. Methods: Fibroblasts were isolated from nasal polyp tissues. RNA sequencing was then performed. We also analyzed the GSE136825 dataset obtained from the Gene Expression Omnibus database. Alternatively, fibroblasts were stimulated in vitro. Results: Hierarchical clustering of samples indicated ADGRB3-high and POSTN-high fibroblasts. A Gene Set Enrichment Analysis (GSEA) revealed that cytotoxic immune responses were enriched in ADGRB3-high fibroblasts, while cell cycle pathways were enriched in POSTN-high fibroblasts. Similar GSEA results were observed in the GSE136825 dataset. Additionally, type 1 and type 3 inflammation-related genes were highly expressed in ADGRB3-high samples, whereas type 2-related genes were highly expressed in POSTN-high samples. In vitro, ADGRB3 expression increased in fibroblasts stimulated with IFN-γ, while POSTN increased in those stimulated with IL-4 and IL-13. Conclusions: Our study demonstrates that type 1 inflammation induces ADGRB3-high fibroblasts, associated with the cytotoxic immune response, while type 2 inflammation induces POSTN-high fibroblasts, linked to CRS progression via an elevated cell cycle. The further characterization of fibroblasts could provide insights into the stromal networks in the CRS microenvironment.

SURF1 Deficiency: Expanding on Disease Phenotype and Assessing Disease Burden by Describing Clinical and Biochemical Phenotype.

Leigh syndrome, a severe neurological disorder is commonly caused by homozygous or bi-allelic pathogenic variants in the SURF1 gene. SURF1 deficiency leads to dysfunction of Cytochrome C Oxidase (COX) activity, which is crucial for mitochondrial oxidative phosphorylation. Understanding COX activity's correlation with disease severity is essential for developing SURF1 Leigh Syndrome biomarkers. This study assesses the disease burden in SURF1 Leigh Syndrome and evaluates COX activity as a treatment biomarker. We reviewed records and questionnaires from 17 individuals, classifying them into phenotypic and genotypic groups. We compared COX activity assays in patient fibroblasts to age-matched controls, clinical data, and neuroimaging findings. Patient COX activity was at most 50% of controls, averaging 32% (p < 0.001). Common clinical features included brainstem abnormalities (93.3%), motor regression (92.3%), bi-allelic heterozygous SURF1 variants (88.2%), and delayed growth/development (35.7%). Homozygous and heterozygous nonsense/frameshift variants showed more severe phenotypes (p = 0.008) and more MRI abnormalities (p = 0.005). Significant COX activity reduction is linked to SURF1 Leigh Syndrome, with genotype influencing disease severity. Clinical and neuroimaging correlations show potential for prognostic indicators. This study lays the groundwork for future research and clinical application of COX activity as a SURF1 Leigh Syndrome biomarker.

Missense variants in the TRPMr7 α-kinase domain are associated with recurrent pediatric acute liver failure

Background: Pediatric acute liver failure (PALF) is a rare and life-threatening condition. In up to 50% of PALF cases, the underlying etiology remains unknown during routine clinical testing. This lack of knowledge complicates clinical management and liver transplantation decisions. Recently, whole-exome sequencing has identified genetic disorders in a large number of cases without specific laboratory biomarkers or metabolic fingerprints. Methods: We describe how further analysis of whole-exome sequencing data combined with proteomic analyses in 5 previously unsolved PALF patients, where no pathogenic variants in genes previously associated with acute liver failure were identified, revealed rare biallelic variants in transient receptor potential cation channel subfamily M member 7 (TRPM7). Results: We establishe TRPM7 as a novel disease gene for PALF. Yet, the cation channel kinase TRPM7 has not been associated with any Mendelian disorder. No homozygous loss-of-function variants were found in in-house exomes or publicly available databases. Rare biallelic TRPM7-variants were significantly enriched in the PALF cohort compared with a pediatric control cohort. Viral infections preceded the majority of PALF episodes. Recurrent PALF episodes characterized the disease course with rapid progression, leading to early death in 3 cases. Proteomic analyses of patient fibroblasts unveiled significantly reduced TRPM7 protein levels, indicative of functional impairment. Severely reduced Mg2+ levels in one individual with a mutation in the channel domain suggests a potential interaction between disturbed Mg2+ homeostasis and PALF. The consistent presence of mutations in the TRPM7 protein-kinase-domain across all patients suggests its specific relevance in PALF. Conclusions: Our data extend the genetic spectrum of recurrent PALF and prompt consideration of TRPM7 in children with unexplained liver failure.

Variants in MICOS10 Identified by Whole Genome Sequencing and RNA Sequencing in a New Type of Hepatocerebral Mitochondrial DNA Depletion Syndrome.

The mitochondrial contact site and cristae organising system (MICOS) complex is required for cristae formation and is composed of seven proteins. Among the genes of MICOS complex, variants of MICOS13, IMMT and APOO have been reported to cause diseases. We report a case in which whole genome sequencing identified a variant of the MICOS10 gene associated with mitochondrial hepatopathy along with mitochondrial DNA depletion. We identified the deletion g.19596826_19601303del and the single nucleotide variant c.173G>C (p.Cys58Ser). The deletion including exon 1 might have caused complete loss of gene expression, indicating monoallelic expression from RNA sequencing. MIC10 was lost at the protein level in the patient's fibroblasts, and mitochondrial oxygen consumption was impaired. These were restored by overexpression of MICOS10 in the patient's fibroblasts. Taken together, these findings indicate that MICOS10 is a causative gene for hepatopathy and neuropathy, a disease very similar to that associated with MICOS13.

Restored Collagen VI Microfilaments Network in the Extracellular Matrix of CRISPR-Edited Ullrich Congenital Muscular Dystrophy Fibroblasts.

Collagen VI is an essential component of the extracellular matrix (ECM) composed by α1, α2 and α3 chains and encoded by COL6A1, COL6A2 and COL6A3 genes. Dominant negative pathogenic variants in COL6A genes result in defects in collagen VI protein and are implicated in the pathogenesis of muscular diseases, including Ullrich congenital muscular dystrophy (UCMD). Here, we designed a CRISPR genome editing strategy to tackle a dominant heterozygous deletion c.824_838del in exon 9 of the COL6A1 gene, causing a lack of secreted collagen VI in a patient's dermal fibroblasts. The evaluation of efficiency and specificity of gene editing in treating patient's fibroblasts revealed the 32% efficiency of editing the mutated allele but negligible editing of the wild-type allele. CRISPR-treated UCMD skin fibroblasts rescued the secretion of collagen VI in the ECM, which restored the ultrastructure of the collagen VI microfibril network. By using normal melanocytes as surrogates of muscle cells, we found that collagen VI secreted by the corrected patient's skin fibroblasts recovered the anchorage to the cell surface, pointing to a functional improvement of the protein properties. These results support the application of the CRISPR editing approach to knock out COL6A1 mutated alleles and rescue the UCMD phenotype in patient-derived fibroblasts.

Homozygous synonymous FAM111A variant underlies an autosomal recessive form of Kenny-Caffey syndrome.

FAM111A (family with sequence similarity 111 member A) is a serine protease and removes covalent DNA-protein cross-links during DNA replication. Heterozygous gain-of-function variants in FAM111A cause skeletal dysplasias, such as the perinatal lethal osteocraniostenosis and the milder Kenny-Caffey syndrome (KCS). We report two siblings born to consanguineous parents with dysmorphic craniofacial features, postnatal growth retardation, ophthalmologic manifestations, hair and nail anomalies, and skeletal abnormalities such as thickened cortex and stenosis of the medullary cavity of the long bones suggestive of KCS. Using exome sequencing, a homozygous synonymous FAM111A variant, NM_001312909.2:c.81 G > A; p.Pro27=, that affects the last base of the exon and is predicted to alter FAM111A pre-mRNA splicing, was identified in both siblings. We identified aberrantly spliced FAM111A transcripts, reduced FAM111A mRNA levels, and near-complete absence of FAM111A protein in fibroblasts of both patients. After treatment of patient and control fibroblasts with different concentrations of camptothecin that induces covalent DNA-protein cross-links, we observed a tendency towards a reduced proportion of metabolically active cells in patient compared to control fibroblasts. However, under these culture conditions, we did not find consistent and statistically significant differences in cell cycle progression and apoptotic cell death between patient and control cells. Our findings show that FAM111A deficiency underlies an autosomal recessive form of FAM111A-related KCS. Based on our results and published data, we hypothesize that loss of FAM111A and FAM111A protease hyperactivity, as observed for gain-of-function patient-variant proteins, may converge on a similar pathomechanism underlying skeletal dysplasias.

Heterozygous de novo variants in HSPD1 cause hypomyelinating leukodystrophy through impaired HSP60 oligomerisation

IntroductionHypomyelinating leukodystrophies are a group of genetic disorders, characterised by severe permanent myelin deficiency. Their clinical features include developmental delay with or without neuroregression, nystagmus, central hypotonia, progressing to spasticity...

Salirasib Inhibits the Expression of Genes Involved in Fibrosis in Fibroblasts of Systemic Sclerosis Patients.

Fibrosis is a principal sign of systemic sclerosis (SSc) which can affect several organs including the lung, heart, and dermis. Dermal fibroblasts of SSc patients are characterized by persistent and activated Ras and ERK1/2 signaling which stimulates extreme collagen and extracellular matrix synthesis. Salirasib is a Ras inhibitor that competitively prevents the adherence of GTP-bound Ras to the plasma membrane, that inhibits Ras signaling. This study intended to clarify whether salirasib can influence fibrotic mediators in SSc fibroblasts. Dermal fibroblasts from 10 SSc patients were treated with salirasib in the presence of TGF-β1, and mRNA levels of H-Ras and genes related to fibrosis, such as COL1A1, COL1A2, CTGF, TGF-β1, fibronectin, ACTA2, and MMP1 was measured by real-time PCR. The α-SMA protein expression was analyzed by immunofluorescence staining. In dermal fibroblasts of SSc patients, salirasib treatment, markedly downregulated the H-Ras gene expression. In addition, the protein expression of α-SMA and gene expression of ACTA2 were inhibited upon salirasib treatment. Salirasib also significantly reduced the expression of COL1A1, and COL1A2 genes and augmented the gene expression of MMP1. The mRNA levels of other genes related to fibrosis such as FN1, CTGF, and TGF-β1 were significantly decreased upon salirasib treatment. Considering salirasib significantly reduced the expression of genes related to the fibrosis process and α-SMA gene and protein expression, and given significant upregulation of MMP1 by salirasib, it can be considered as a new curative strategy for fibrotic diseases like SSc.

Generation of an induced pluripotent stem cell line (TRNDi042-A) from a Mucopolysaccharidosis type IIIB patient with homozygous p. R626X (c. 1876C > T) mutation in the NAGLU gene

Mucopolysaccharidosis type IIIB (MPS IIIB), also known as Sanfilippo syndrome, is an autosomal recessive lysosomal storage disorder caused by mutations in the NAGLU gene. It is characterized by progressive neurodegeneration, behavioral problems, and motor function difficulties. A human induced pluripotent stem cell (iPSC) TRNDi042-A line was generated from fibroblasts of a male patient with a homozygous p. R626X (c.1876C > T) in the NAGLU gene producing N-acetyl-glucosaminidase. This iPSC line is a useful resource to study disease pathophysiology and to develop therapeutics treatments. The cell line has a normal karyotype, is free of plasmid integration, and expresses high levels of pluripotency-associated markers.

Deciphering Immune-related Gene Signatures in Diabetic Retinopathy: Insights from In silico Analysis and In vitro Experiment.

Diabetes retinopathy (DR) is one of the most common microvascular consequences of diabetes, and the economic burden is increasing. Our aim is to decipher the relevant mechanisms of immune-related gene features in DR and explore biomarkers targeting DR. Provide a basis for the treatment and prevention of DR. The immune infiltration enrichment score of DR patients was evaluated from the single- cell RNA sequencing dataset, and the samples were divided into low immune subgroups and high immune subgroups based on this result. Through weighted gene correlation network analysis, differentially expressed genes (DEGs) between two subgroups were identified and crossed with genes with the strongest immune association, resulting in significant key genes. Then divide the DR individuals into two immune related differentially expressed gene (IDEG) clusters, A and B. Submit cross DEGs between two clusters through Gene Set Enrichment Analysis (GSEA) to further explore their functions. A protein-protein interaction (PPI) network of IDEG was established to further identify central genes associated with DR. Use the discovered central genes to predict the regulatory network involved in the pathogenesis of DR. Then, the role of the identified hub gene in the pathogenesis of DR was further studied through in vitro experiments. We found that the immune scores of DR and control groups were different, and 27 IDEGs were found in the DR subgroup. Compared with cluster A, the proportion of cytotoxic lymphocytes, B lineage, monocyte lineage, and fibroblasts in DR patients in cluster B is significantly enriched. GSEA indicates that these genes are associated with T cell activation, regulation of immune response processes, lymphocyte-mediated immunity, TNF signaling pathway, and other signaling pathways. The PPI network subsequently identified 10 hub genes in DR, including SIGLEC10, RGS10, PENK, FGD2, LILRA6, CIITA, EGR2, SIGLEC7, LILRB1, and CD300LB. The upstream regulatory network and lncRNA miRNA mRNA ceRNA network of these hub genes were ultimately constructed. The discovery and identification of these genes will provide biomarkers for targeted prediction and treatment of DR. By integrating bioinformatics analysis and in vitro experiments, we have identified a set of central genes, indicating that these genes can serve as potential biomarkers for DR, which may be promising targets for future DR immunotherapy interventions.

A New Case of Mitochondrial RNA Helicase SUPV3L1-Associated Neurodegenerative Disease: Ataxia, Spasticity, Optic Atrophy, and Skin Hypopigmentation (ASOASH).

The SUPV3L1 gene encodes ATP-dependent RNA helicase SUPV3L1, which is a part of the mitochondrial degradosome complex or SUV3. SUPV3L1 unwinds secondary structures of mitochondrial RNA (mtRNA) and facilitates the degradation of mtRNA molecules. A nonsense homozygous variant in the SUPV3L1 gene was recently associated with mitochondrial disease. Our study presents the second documented case of SUPV3L1 pathology in humans. Whole-genome sequencing was performed on the NovaSeq 6000 platform using pair-end reading. Data analysis was performed with an in-house developed pipeline. The 17-year-old female patient exhibited a diverse array of symptoms, including ataxia, spastic paraparesis, cognitive deficit, optic atrophy, and horizontal gaze-evoked nystagmus. Early onset of symptoms, such as ataxic gait and nystagmus, was noted, with subsequent progression of neurological manifestations. At the time of the observation, the proband had extensive regions of hypopigmented skin patches on the body and extremities, which have progressed over time. Whole-genome sequencing revealed compound heterozygous variants in the SUPV3L1 gene: c.272-2A>G and c.1924A>C; p.(Ser642Arg). RNA analysis demonstrated splicing changes attributable to the c.272-2A>G variant. ELISA assay showed increased Complex I content in the patient's fibroblasts. This case underscores the phenotypic diversity associated with SUPV3L1 mutations, emphasizing the importance of considering mitochondrial RNA helicase dysfunction in the differential diagnosis of neurodegenerative disorders. Further elucidation of the molecular mechanisms underlying SUPV3L1-associated pathology may provide valuable insights into targeted therapeutic interventions.

Nifuroxazide rescues the deleterious effects due to CHCHD10-associated MICOS defects in disease models.

The identification of a point mutation (p.Ser59Leu) in the CHCHD10 gene was the first genetic evidence that mitochondrial dysfunction can trigger motor neuron disease. Since then, we have shown that this mutation leads to the disorganization of the MItochondrial contact site and Cristae Organizing System (MICOS) complex that maintains the mitochondrial cristae structure. Here, we generated yeast mutant strains mimicking MICOS instability and used them to test the ability of more than 1600 compounds from 2 repurposed libraries to rescue the growth defect of those cells. Among the hits identified, we selected nifuroxazide, a broad-spectrum antibacterial molecule. We show that nifuroxazide rescues mitochondrial network fragmentation and cristae abnormalities in CHCHD10S59L/+ patient fibroblasts. This molecule also decreases caspase-dependent death of human CHCHD10S59L/+ iPSC-derived motor neurons. Its benefits involve KIF5B-mediated mitochondrial transport enhancement, evidenced by increased axonal movement and syntaphilin degradation in patient-derived motor neurons. Our findings strengthen the MICOS-mitochondrial transport connection. Nifuroxazide and analogues emerge as potential therapeutics for MICOS-related disorders like motor neuron disease. Its impact on syntaphilin hints at broader neurological disorder applicability for nifuroxazide.

Increased cholesterol synthesis drives neurotoxicity in patient stem cell-derived model of multiple sclerosis

Senescent neural progenitor cells have been identified in brain lesions of people with progressive multiple sclerosis (PMS). However, their role in disease pathobiology and contribution to the lesion environment remains unclear. By establishing directly induced neural stem/progenitor cell (iNSC) lines from PMS patient fibroblasts, we studied their senescent phenotype invitro. Senescence was strongly associated with inflammatory signaling, hypermetabolism, and the senescence-associated secretory phenotype (SASP). PMS-derived iNSCs displayed increased glucose-dependent fatty acid and cholesterol synthesis, which resulted in the accumulation of lipid droplets. A 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase (HMGCR)-mediated lipogenic state was found to induce a SASP in PMS iNSCs via cholesterol-dependent transcription factors. SASP from PMS iNSC lines induced neurotoxicity in mature neurons, and treatment with the HMGCR inhibitor simvastatin altered the PMS iNSC SASP, promoting cytoprotective qualities and reducing neurotoxicity. Our findings suggest a disease-associated, cholesterol-related, hypermetabolic phenotype of PMS iNSCs that leads to neurotoxic signaling and is rescuable pharmacologically.

Mutual Amplification of GLI2/Hedgehog and Transcription Factor JUN/AP-1 Signaling in Fibroblasts in Systemic Sclerosis: Potential Implications for Combined Therapies.

Deregulation of the cJUN/AP-1 and hedgehog/GLI2 signaling pathways has been implicated in fibroblast activation in systemic sclerosis (SSc). However, the consequences of their concomitant up-regulation are unknown. Here, we tested the hypothesis that mutual amplification of both pathways might drive persistent fibroblast activation. Cultured fibroblasts and skin sections of patients with diffuse SSc and healthy volunteers were analyzed. cJUN/AP-1 signaling and hedgehog/GLI2 signaling were inhibited using knockdown and pharmacologic approaches. Hedgehog signaling was activated in mice by fibroblast-specific overexpression of constitutively active Smoothened. cJUN and GLI2 are concomitantly up-regulated and colocalize in fibroblasts of patients with SSc compared to healthy controls. Activation of hedgehog/GLI2 signaling induces the expression of cJUN in vitro and in vivo, whereas inactivation of GLI2 inhibits cJUN expression. Likewise, inactivation of cJUN impairs the expression of GLI2. This mutual regulation occurs at the level of transcription with binding of cJUN and GLI2 to specific binding motifs. Interference with this mutual amplification of cJUN signaling and GLI2 signaling inhibits fibroblast activation and collagen release: Inhibition of cJUN/AP-1 signaling ameliorates hedgehog-induced fibroblast activation and skin fibrosis in SmoACT mice with a reduction of skin thickness of 103% (P = 0.0043) in the treatment group compared to the fibrotic control group. Moreover, combined pharmacologic inhibition of cJUN/AP-1 and hedgehog/GLI2 exerts additive antifibrotic effects in a model of TGFβ-driven experimental fibrosis (TBRACT mice). The transcription factors cJUN and GLI2 reinforce each other's activity to promote fibroblast activation in SSc. Interruption of this crosstalk by combined inhibition of both pathways exerts additive antifibrotic effects at well-tolerated doses.

NLRP3 inflammasome-mitochondrion loop in autism spectrum disorder

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication and the presence of restricted interests and repetitive behavior. To date, no single cause has been demonstrated but both genetic and environmental factors are believed to be involved in abnormal brain development. In recent years, immunological and mitochondrial dysfunctions acquired particular interest in the study of the molecular mechanisms underlying the pathophysiology of ASD. For this reason, our study focused on evaluating the mitochondrial component and activation of the NLRP3 inflammasome, a critical player of the innate immune system. The assembly of NLRP3 with ASC mediates activation of Caspase-1, which in turn, by proteolytic cleavage, activates Gasdermin D and the proinflammatory cytokines IL-1β/IL-18 with their subsequent secretion. Using primary fibroblasts of autistic and control patients we studied basal and stimulated conditions. Specifically, LPS and ATP were used to activate the NLRP3 inflammasome and MCC950 for its inhibition. In addition, FCCP was used as a mitochondrial stressor and MitoTEMPO as a scavenger of mitochondrial ROS. Our results showed a hyperactivation of NLRP3 inflammasome in ASDs, as evidenced by the co-localization of the two main components, NLRP3 and ASC, by the higher levels of ASC specks, oligomers and dimers and by the increased amounts of active Caspase-1 and IL-1β. In addition, increased mitochondrial superoxide anion and reduced mitochondrial membrane potential were detected in ASD cells. These data are in accordance with the abnormal mitochondrial morphology evidenced by transmission electron microscopy analysis. Interestingly, NLRP3 inflammasome inhibition with MCC950 improved mitochondrial parameters, while the use of MitoTEMPO, in addition to decrease mitochondrial ROS production, was able to prevent NLRP3 inflammasome activation suggesting for the first time an abnormal bidirectional crosstalk between mitochondria and NLRP3 inflammasome in ASD.

Multiple outcomes of the germline p16INK4a mutation affecting senescence and immunity in human skin.

The integrated behaviour of multiple senescent cell types within a single human tissue leading to the development of malignancy is unclear. Patients with Familial Melanoma Syndrome (FMS) have heterozygous germline defects in the CDKN2A gene coding for the cyclin inhibitor p16INK4a. Melanocytes within skin biopsies from FMS patients express significantly less p16INK4a but express higher levels of the DNA-damage protein 𝛾H2AX a than fibroblastic cells. However, patient fibroblasts also exhibit defects since senescent cells do not increase in the skin during ageing and fibroblasts isolated from the skin of patients have increased replicative capacity compared to control fibroblasts invitro, culminating in abnormal nuclear morphology. Patient derived fibroblasts also secreted less SASP than control cells. Predisposition of FMS patients to melanoma may therefore result from integrated dysregulation of senescence in multiple cell types invivo. The inherently greater levels of DNA damage and the overdependence of melanocytes on p16 for cell cycle inhibition after DNA damage makes them exquisitely susceptible to malignant transformation. This may be accentuated by senescence-related defects in fibroblasts, in particular reduced SASP secretion that hinders recruitment of T cells in the steady state and thus reduces cutaneous immunosurveillance invivo.

Altered tubulin detyrosination due to SVBP malfunction induces cytokinesis failure and senescence, underlying a complex hereditary spastic paraplegia.

Senescence, marked by permanent cell cycle arrest may contribute to the decline in regenerative potential and neuronal function, thereby promoting neurodegenerative disorders. In this study, we employed whole exome sequencing to identify a previously unreported biallelic missense variant in SVBP (p.Leu49Pro) in six patients from three unrelated families. These affected individuals present with a complex hereditary spastic paraplegia (HSP), peripheral neuropathy, verbal apraxia, and intellectual disability, exhibiting a milder phenotype compared to patients with nonsense SVBP mutations described previously. Consistent with SVBP's primary role as a chaperone necessary for VASH-mediated tubulin detyrosination, both patient fibroblasts with the p.Leu49Pro mutation, and HeLa cells harboring an SVBP knockdown exhibit microtubule dynamic instability and alterations in pericentriolar material (PCM) component trafficking and centrosome cohesion. In patient fibroblasts, structural abnormalities in the centrosome trigger mitotic errors and cellular senescence. Notably, premature senescence characterized by elevated levels of p16INK4, was also observed in patient peripheral blood mononuclear cells (PBMCs). Taken together, our findings underscore the critical role of SVBP in the development and maintenance of the central nervous system, providing novel insights associating cytokinesis failure with cortical motor neuron disease and intellectual disability.

The clinical spectrum and pathogenesis associated with KMT2B variants in Chinese pediatric patients

ObjectiveTo evaluate the clinical spectrum and pathogenesis associated with KMT2B variants in Chinese children with dystonia or developmental delay. MethodsWe reported twenty-seven (fourteen males and thirteen females) pediatric patients with KMT2B variants identified via next-generation sequencing from a single Chinese center. Moreover, transcriptomics and proteomics assays were performed on fibroblasts from patients with different genotypes to investigate the pathogenic mechanisms involved. ResultsTwenty-six patients had dystonia including generalized dystonia (n = 19), multifocal dystonia (n = 6), and segmental dystonia (n = 1), and one patient had nondystonic severe-developmental delay (DD). All the twenty-six patients had complex dystonia compounded with other manifestations of movement disorders (tremor (n = 6), myoclonus (n = 5), status dystonicus (n = 2), and tic (n = 1)) or dysmorphic features and developmental delay. The onset of dystonia was between 1 month and 13 years 8 months (median 4 years 4 months). Dystonia was aggravated by fever (n = 11), and diurnal and climate fluctuations (n = 4). Eleven patients underwent deep brain stimulation and experienced significant improvements in motor function and disability. We identified twenty-six intragenic heterozygous KMT2B pathogenic variants and one Chr:19q13.12 contiguous gene deletion. Sixteen variants were novel. Differentially expressed genes induced by KMT2B variants were significantly enriched for mitochondria-related biological processes in patient fibroblasts. As a result, mitochondrial morphology of mitochondria was altered, and aerobic respiration was impaired. ConclusionOur study reports the pediatric cases of KMT2B-related disorder from a single center in China. Additionally, our study highlights the role of KMT2B variants in mitochondrial dysfunction.

Valvular and Myocardial Fibroblast Activation in Aortic Stenosis

Activated fibroblasts drive leaflet thickening and left ventricular decompensation in aortic stenosis. Gallium-68 Fibroblast Activation Protein Inhibitor (68Ga-FAPI) binds to these key effector cells, and provides a readout of fibroblast activation. We aimed to describe the role of activated fibroblasts in patients with aortic stenosis in vivo using 68Ga-FAPI. In a prospective observational study, patients with aortic stenosis and control subjects underwent echocardiography, 68Ga-FAPI PET, CT, and MRI. Valvular and myocardial 68Ga-FAPI uptake was quantified using maximal standardised uptake values (SUVmax), and target-to-background ratio (TBRmax). Aortic stenosis severity was measured by peak velocity on echocardiography and the CT calcium score. Myocardial fibrosis was quantified by late gadolinium enhancement (LGE) on MRI. 86 patients with aortic valve disease (72±11 years, 68% male) plus 9 matched control subjects (72±9 years, 67%% male) participated. Increased 68Ga-FAPI uptake was observed in the aortic valves of patients with aortic stenosis compared with controls (p&lt;0.001). 68Ga-FAPI TBRmax correlated with peak velocity (r=0.532, p&lt;0.0010) and calcium score (r = 0.577, p&lt;0.001). 54 patients (69%) had myocardial 68Ga-FAPI uptake, of whom 30 (38%) also had LGE corresponding to the region of 68Ga-FAPI uptake. Myocardial 68Ga-FAPI uptake correlated with increased indexed left ventricular mass (r=0.429, p&lt;0.001) and indexed Extracellular Volume (r=0.404, p&lt;0.001). For the first time, we have described valvular and myocardial fibroblast activation in patients with aortic stenosis in vivo. Valvular fibroblast activation is increased in patients with aortic stenosis, correlating with increased disease severity. Myocardial fibroblast activation is seen in the majority of patients with aortic stenosis in areas with and without established fibrosis, and is associated with adverse left ventricular remodelling. 68Ga-FAPI PET can visualise the key effector cell driving aortic valve disease, and may have a role in monitoring disease modifying treatments, as well as identifying patients at high risk of myocardial decompensation. Please click on the 'PDF' for the full abstract!

Site-blocking antisense oligonucleotides as a mechanism to fine-tune MeCP2 expression.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by loss-of-function mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Despite its severe phenotypes, studies in mouse models suggest that restoring MeCP2 levels can reverse RTT symptomology. Nevertheless, traditional gene therapy approaches are hindered by MeCP2's narrow therapeutic window, complicating the safe delivery of viral constructs without overshooting the threshold for toxicity. The 3' untranslated region (3' UTR) plays a key role in gene regulation, where factors like miRNAs bind to pre-mRNA and fine-tune expression. Given that each miRNA's contribution is modest, blocking miRNA binding may represent a potential therapeutic strategy for diseases with high dosage sensitivity, like RTT. Here, we present a series of site-blocking antisense oligonucleotides (sbASOs) designed to outcompete repressive miRNA binding at the MECP2 3' UTR. This strategy aims to increase MeCP2 levels in patients with missense or late-truncating mutations, where the hypomorphic nature of the protein can be offset by enhanced abundance. Our results demonstrate that sbASOs can elevate MeCP2 levels in a dose-dependent manner in SH-SY5Y and patient fibroblast cell lines, plateauing at levels projected to be safe. Confirming in vivo functionality, sbASO administration in wild-type mice led to significant Mecp2 upregulation and the emergence of phenotypes associated with Mecp2 overexpression. In a T158M neural stem cell model of RTT, sbASO treatment significantly increased MeCP2 expression and levels of the downstream effector protein brain-derived neurotrophic factor (BDNF). These findings highlight the potential of sbASO-based therapies for MeCP2-related disorders and advocate for their continued development.