Search Results (70)

Pathogenic variants of MYH11, which encode smooth muscle myosin heavy chain 11, have been linked to familial thoracic aortic aneurysms and dissections (FTAAD). However, molecular pathways affected by these mutations have not been well understood. To explore downstream consequences of Myh11 disruption, we analyzed transcriptomic and proteomic profiles of aortas from male Myh11 mice with homozygous deletion of lysine 1256 (K1256) and of wild-type controls. Of 6499 proteins quantified, 1763 were differentially expressed (adjusted p < 0.05), including 942 that were downregulated and 821 that were upregulated in mutant aortas. Enrichment analysis of downregulated genes and proteins revealed a consistent reduction in extracellular matrix-related pathways. Among downregulated proteins, we identified tenascin Xb, transforming growth factor β (Tgfb) 2, and Tgfb receptor 1/2, malfunctions of which are linked to connective tissue diseases, such as Ehlers–Danlos and Loeys–Dietz syndromes. Nevertheless, unlike these syndromic diseases, mice with Myh11 pathogenic variants and patients with FTAAD do not exhibit syndromic features, likely reflecting expression of Myh11 restricted to smooth muscle. These results suggest that loss of Myh11 disrupts maintenance of extracellular matrix by SMCs, the loss of which contributes to aortic fragility without affecting other tissues. Full article

Backgroud: Glutamate dehydrogenase (GDH) is involved in the metabolism of glutamate and ammonia. It is regulated by multiple ligand variants, and hyper-active GDH mutants have been reported for hyperinsulinism hyperammonemia syndrome (HHS). Methods: Here, we constructed the wild-type human GDH and three human GDH454 mutants and investigated their degradation activity and performance under different GDH inhibitors. Results: Protein activity test and SDS-PAGE analysis of the purified proteins showed that the GDH454 mutant from HHS has weaker GDH enzymatic activity but greater resistance to trypsin hydrolysis than the wild type. Interestingly, using the biomolecular interactions technique, it showed that the GDH454 mutant has 10⁹ times weaker affinity for trypsin and 10-fold weaker for epigallocatechin gallate (EGCG) than the wild-type GDH. Subsequently, native-PAGE gel analysis demonstrated that EGCG could break down the GDH hexamer into monomers and form a complex with trypsin to enhance the degradation of both types of GDH. Conclusions: EGCG showed good affinity to both the wild-type and the mutant GDH proteins, promoting protein degradation; this provides a new strategy for the treatment of HHS and other hyper-active GDH-related diseases. Full article

Background and Objectives: PAX2 serves as a critical transcription factor integral to the process of embryogenesis. Variations in the PAX2 gene could result in the aberrant development of numerous organs. Despite the identification of numerous mutations within the PAX2 gene, the correlation between specific genotypes has yet to be fully clarified. The objective of this study was to examine the clinical phenotypes and genotypes associated with PAX2 mutation-induced disorders in pediatric patients of Chinese descent. The aim of our study was to forecast the pathogenic potential of these genetic mutations and to ascertain possible correlations between genotypic variations and the clinical manifestations of disorders linked to PAX2 mutations. Materials and Methods: We recruited 14 pediatric subjects with PAX2 mutations, meticulously examining the clinical characteristics and genetic alterations present in these individuals. Computational techniques were utilized to evaluate the pathogenicity, stability, and biophysical characteristics. A range of computational tools were employed for this assessment, including PredictSNP, MAGPIE, iStable, Align GVGD, ConSurf, and SNP effect. Results: The age at onset ranged from prenatal to 12 years. Five patients progressed to end-stage renal disease. Proteinuria and bilateral renal hypoplasia were observed in 92% of cases. Ocular and auditory abnormalities were also noted. We identified eleven different PAX2 mutations, including five novel variants not previously reported in the literature. We predicted that all mutations, with the exception of p.F27-L33 del and N188S, exhibited high pathogenicity scores. In particular, R117P and R140W are strongly associated with disease pathogenicity and are likely to cause more significant damage than other gene mutants. Conclusions: This study expands the mutational and phenotypic spectrum of PAX2-related disorders in the pediatric population. The identification of five novel variants enhances our understanding of the genetic basis of these conditions. Despite recurrent mutations, marked phenotypic heterogeneity persists, underscoring the need for further research. Full article

Variants in the GBA1 gene, encoding the lysosomal enzyme glucosylceramidase beta 1 (GCase), are linked to Parkinson’s disease (PD) and Gaucher disease (GD). Heterozygous variants increase PD risk, while homozygous variants lead to GD, a lysosomal storage disorder. Some GBA1 variants impair GCase maturation in the endoplasmic reticulum, blocking lysosomal transport and causing glucosylceramide accumulation, which disrupts lysosomal function. This study explores therapeutic strategies to address these dysfunctions. Using Site-directed Enzyme Enhancement Therapy (SEE-Tx^®), two structurally targeted allosteric regulators (STARs), GT-02287 and GT-02329, were developed and tested in GD patient-derived fibroblasts with relevant GCase variants. Treatment with GT-02287 and GT-02329 improved the folding of mutant GCase, protected the GCase_Leu444Pro variant from degradation, and facilitated the delivery of active GCase to lysosomes. This enhanced lysosomal function and reduced cellular stress. These findings validate the STARs’ mechanism of action and highlight their therapeutic potential for GCase-related disorders, including GD, PD, and Dementia with Lewy Bodies. Full article

Maturity-onset diabetes of the young (MODY) is a rare genetic condition that affects children, adolescents, and adults. Studies have shown that genetic changes in the HNF1A gene are associated with MODY-3. However, most of the causative variants and the molecular mechanisms remain underexplored. This study aims to better understand MODY-3 by investigating HNF1A-missense variants with clinical uncertainty. Various bioinformatics tools were utilised to address the clinical uncertainty of missense variants in the HNF1A gene that have not been linked with HNF1A-related conditions, sourced from the Genome Aggregation Database (GnomAD v4.1.0). Among the clinically uncertain 2444 variants, only 138 were classified as missense with clinically uncertain significance. Results show that four variants (Arg168Cys, Glu275Ala, Gly375Asp and Val411Phe) were consistently predicted as pathogenic by all tools. The allele frequency (AF) of the commonly predicted disease-causing variants was very low in the global population. The assessment of the secondary structure of filtered variants indicates that variants (Arg168Cys and Glu275Ala) are located in the helical region of the HNF1A protein. At the same time (Gly375Asp and Val411Phe) are found in the protein’s coil, suggesting structural changes at the site of variations. The prediction of protein stability was conducted using I-Mutant and MuPro. Both tools collectively indicate decreased protein stability for the variants (Arg168Cys, Glu275Ala, Gly375Asp and Val411Phe). Predicting the protein’s 3D structure for the HNF1A wild-type and mutants indicates potential structural damages in Arg168Cys and Gly375Asp. Additionally, results show that the amino acids at the variation sites of the variants (Arg168Cys, Glu275Ala, Gly375Asp and Val411Phe) were highly conserved. To conclude, 4 out of the 138 missense variants labelled as uncertain significance were found to be consistently pathogenic using in silico tools in this study. Our findings aim to support variant interpretation, understand the genotype–phenotype association of diabetes, and provide better healthcare services for patients with diabetes. Full article

Ferroptosis, an iron-dependent form of non-apoptotic cell death, is regulated by a complex network involving lipid metabolism, iron homeostasis, and the oxidative-reductive system, with iron accumulation and lipid peroxidation as key drivers. Mitochondrial dysfunction and ROS overproduction often underlie the pathogenesis of mitochondrial diseases, for which treatment options are limited, emphasizing the need for novel therapies. In this study, we investigated whether polydatin and nicotinamide could reverse ferroptosis-related pathological features in cellular models derived from patients with pathogenic GFM1 variants. Mutant fibroblasts showed increased iron and lipofuscin accumulation, altered expression of iron metabolism-related proteins, elevated lipid peroxidation, and heightened susceptibility to erastin-induced ferroptosis. Treatment with polydatin and nicotinamide effectively corrected these alterations and reduced iron accumulation and lipid peroxidation in induced neurons. Furthermore, chloramphenicol treatment in control cells mimicked the mutant phenotype, suggesting that these pathological changes are linked to the mitochondrial protein synthesis defect characteristic of pathogenic GFM1 variants. Notably, adding vitamin E to the polydatin and nicotinamide co-treatment resulted in a reduction in the minimum effective concentration, suggesting potential benefits of its inclusion. In conclusion, the combination of polydatin, nicotinamide, and vitamin E could represent a promising therapeutic option for patients with mitochondrial disorders caused by pathogenic GFM1 variants. Full article

The unique features of mitochondrial DNA (mtDNA), including its circular and multicopy nature, the possible coexistence of wild-type and mutant molecules (i.e., heteroplasmy) and the presence of nuclear mitochondrial DNA segments (NUMTs), make the diagnosis of mtDNA diseases particularly challenging. The extensive deployment of next-generation sequencing (NGS) technologies has significantly advanced the diagnosis of mtDNA-related diseases. However, the vast amounts and diverse types of sequencing data complicate the interpretation of these variants. From sequence alignment to variant calling, NGS-based mtDNA sequencing requires specialized bioinformatics tools, adapted for the mitochondrial genome. This study presents the use of new bioinformatics approaches, optimized for short- and long-read sequencing data, to enhance the accuracy of mtDNA analysis in diagnostics. Two recent and emerging free bioinformatics tools, Mitopore and MitoSAlt, were evaluated on patients previously diagnosed with single nucleotide variants or large-scale deletions. Analyses were performed in Linux-based environments and web servers implemented in Python, Perl, Java, and R. The results indicated that each tool demonstrated high sensitivity and specific accuracy in identifying and quantifying various types of pathogenic variants. The study suggests that the integrated and parallel use of these tools offers a significant advantage over traditional methods in interpreting mtDNA genetic variants, reducing the computational demands, and provides an accurate diagnostic solution. Full article

Background Hereditary spherocytosis (HS) is an erythrocytic membranopathy that belongs to a group of rare genetic disorders. Mutations in five genes, including ANK1, cause clinical manifestations of the disease. Identified variations in individual families provide a better understanding of the molecular basis of the disease. Methods In this study, we used two sequencing methods, whole exome sequencing (WES) and Sanger sequencing, analyzing gDNA and cDNA as templates, to detect and verify the variants putatively responsible for the clinical symptoms observed in a Polish family diagnosed with HS. Results We detected two variants that occur in cis in the ANK1 gene, a known missense mutation (NP_000028.3:p.V463I) and a novel frameshift mutation (NP_000028.3: p.V1626fs*64) that appears to be crucial for the probands. As shown by transcriptome studies, the mutant allele is not present at a detectable level. Conclusions We conclude that the molecular basis of this case is related to an unstable transcript of the mutant allele and that the direct cause of the HS is a deficiency of erythrocyte ankyrin leading to a disruption of the AE1-erythrocyte ankyrin-spectrin complex in the erythrocyte membrane. Full article

Cytophaga is a genus of Gram-negative bacteria occurring in soil and the gut microbiome. It is closely related to pathogenic Flavobacterium spp. that cause severe diseases in fish. Cytophaga strain L43-1 secretes cytophagalysin (CPL1), a 137 kDa peptidase with reported collagenolytic and gelatinolytic activity. We performed highly-confident structure prediction calculations for CPL1, which identified 11 segments and domains, including a signal peptide for secretion, a prosegment (PS) for latency, a metallopeptidase (MP)-like catalytic domain (CD), and eight immunoglobulin (Ig)-like domains (D3–D10). In addition, two short linkers were found at the D8–D9 and D9–D10 junctions, and the structure would be crosslinked by four disulfide bonds. The CPL1 CD was found closest to ulilysin from Methanosarcina acetivorans, which assigns CPL1 to the lower-pappalysin family within the metzincin clan of MPs. Based on the structure predictions, we aimed to produce constructs spanning the full-length enzyme, as well as PS+CD, PS+CD+D3, and PS+CD+D3+D4. However, we were successful only with the latter three constructs. We could activate recombinant CPL1 by PS removal employing trypsin, and found that both zymogen and mature CPL1 were active in gelatin zymography and against a fluorogenic gelatin variant. This activity was ablated in a mutant, in which the catalytic glutamate described for lower pappalyins and other metzincins was replaced by alanine, and by a broad-spectrum metal chelator. Overall, these results proved that our recombinant CPL1 is a functional active MP, thus supporting the conclusions derived from the structure predictions. Full article

In the current work, we aimed to evaluate the association of clinical data of Cushing’s disease (CD) patients with USP8 mutation status and to study USP8-related molecular mechanisms connected to the regulation of corticotropinoma growth and activity. 35 CD patients were enrolled; the sequencing of exon 14 in USP8 revealed variants in eighteen adenomas, two of which were described for the first time in CD. USP8 variants were more common in women (94% vs. 76%; p = 0.001), and microadenomas and tumor recurrence were prevalent in the USP8-mutant group (44% vs. 29%; p = 0.04 and 44% vs. 22%; p = 0.0015). Preoperative ACTH and serum cortisol did not differ in the USP8-WT and USP8-mutant patients. All USP8-mutant adenomas were SST5-positive, and 73% of them were double-positive (SST5+/SST2+). A total of 50% of USP8-WT adenomas were double-negative (SST5−/SST2−), and 40% of them were SST5-positive. Analysis of transcriptome was performed for nine USP8-mutant and six USP8-WT adenomas and revealed the that the bidirectional dysregulation of Wnt signaling, including both the agonist RSPO2 and antagonist SFRP1, in the USP8-mutant corticotropinomas was downregulated. These alterations may indicate the existence of regulatory connections between USP8 enzyme activity, Wnt signaling, EGFR signaling and somatostatin receptors’ trafficking, which can explain, at least in part, the clinical manifestations of CD in patients with corticotropinomas harboring USP8 variants. Full article

FOXG1 syndrome is a rare neurodevelopmental disorder of the telencephalon, for which there is no cure. Underlying heterozygous pathogenic variants in the Forkhead Box G1 (FOXG1) gene with resulting impaired or loss of FOXG1 function lead to severe neurological impairments. Here, we report a patient with a de novo pathogenic single nucleotide deletion c.946del (p.Leu316Cysfs*10) of the FOXG1 gene that causes a premature protein truncation. To study this variant in vivo, we generated and characterized Foxg1 c946del mice that recapitulate hallmarks of the human disorder. Accordingly, heterozygous Foxg1 c946del mice display neurological symptoms with aberrant neuronal networks and increased seizure susceptibility. Gene expression profiling identified increased oligodendrocyte- and myelination-related gene clusters. Specifically, we showed that expression of the c946del mutant and of other pathogenic FOXG1 variants correlated with overexpression of proteolipid protein 1 (Plp1), a gene linked to white matter disorders. Postnatal administration of Plp1-targeting antisense oligonucleotides (ASOs) in Foxg1 c946del mice improved neurological deficits. Our data suggest Plp1 as a new target for therapeutic strategies mitigating disease phenotypes in FOXG1 syndrome patients. Full article

Background/Objectives: Identifying novel variants in very rare disease genes can be challenging when patients exhibit a complex phenotype that expands the one described, and we provide such an example here. A few terminal truncating variants in KIDINS220 cause spastic paraplegia (SP), intellectual disability (ID), nystagmus, and obesity (SINO, MIM #617296). Prompted by the result of next-generation sequencing on a patient referred for SP associated with complex brain dysmorphisms, we reviewed the phenotype of SINO patients focusing on their brain malformations, mainly described in prenatal age and first years of life, and tried to understand if the predicted effect of the mutant kidins220 may have caused them. Methods: We performed whole exome sequencing (WES) and a literature and mutation databases review. Results: We report a young adult with SP, severe ID, strabismus, and macrocephaly exhibiting brain malformations at follow-up, partially overlapping with those described in TUBB3 tubulinopathy. WES analysis of the proband and parents identified the heterozygous de novo variant (NM_020738.4: c. 4144G > T) p. Glu 1382* in KIDINS220 that was predicted to be causative of SINO. Conclusions: The progression of myelination and the development of brain structures turned out to be crucial for identifying, at follow-up, the whole KIDINS220-related brain malformations. The truncated proteins associated with SINO lack a portion fundamental for the interaction of kidins220 with tubulins and microtubule-associated proteins. The complexity of the brain malformations displayed by our patient, and possibly by other reported SINO patients, could result from an impaired dynamic modulation of the microtubule cytoskeleton during embryogenesis. Brain malformations must be considered as part of the SINO spectrum phenotype. Full article

Amyotrophic lateral sclerosis (ALS) is a fatal disease that causes degeneration of motor neurons (MNs) and paralysis. ALS can be caused by mutations in the gene that encodes copper/zinc superoxide dismutase (SOD1). SOD1 is known mostly as a cytosolic antioxidant protein, but SOD1 is also in the nucleus of non-transgenic (tg) and human SOD1 (hSOD1) tg mouse MNs. SOD1’s nuclear presence in different cell types and subnuclear compartmentations are unknown, as are the nuclear functions of SOD1. We examined hSOD1 nuclear localization and DNA damage in tg mice expressing mutated and wildtype variants of hSOD1 (hSOD1-G93A and hSOD1-wildtype). We also studied ALS patient-derived induced pluripotent stem (iPS) cells to determine the nuclear presence of SOD1 in undifferentiated and differentiated MNs. In hSOD1-G93A and hSOD1-wildtype tg mice, choline acetyltransferase (ChAT)-positive MNs had nuclear hSOD1, but while hSOD1-wildtype mouse MNs also had nuclear ChAT, hSOD1-G93A mouse MNs showed symptom-related loss of nuclear ChAT. The interneurons had preserved parvalbumin nuclear positivity in hSOD1-G93A mice. hSOD1-G93A was seen less commonly in spinal cord astrocytes and, notably, oligodendrocytes, but as the disease emerged, the oligodendrocytes had increased mutant hSOD1 nuclear presence. Brain and spinal cord subcellular fractionation identified mutant hSOD1 in soluble nuclear extracts of the brain and spinal cord, but mutant hSOD1 was concentrated in the chromatin nuclear extract only in the spinal cord. Nuclear extracts from mutant hSOD1 tg mouse spinal cords had altered protein nitration, footprinting peroxynitrite presence, and the intact nuclear extracts had strongly increased superoxide production as well as the active NADPH oxidase marker, p47phox. The comet assay showed that MNs from hSOD1-G93A mice progressively (6–14 weeks of age) accumulated DNA single-strand breaks. Ablation of the NCF1 gene, encoding p47phox, and pharmacological inhibition of NADPH oxidase with systemic treatment of apocynin (10 mg/kg, ip) extended the mean lifespan of hSOD1-G93A mice by about 25% and mitigated genomic DNA damage progression. In human postmortem CNS, SOD1 was found in the nucleus of neurons and glia; nuclear SOD1 was increased in degenerating neurons in ALS cases and formed inclusions. Human iPS cells had nuclear SOD1 during directed differentiation to MNs, but mutant SOD1-expressing cells failed to establish wildtype MN nuclear SOD1 levels. We conclude that SOD1 has a prominent nuclear presence in the central nervous system, perhaps adopting aberrant contexts to participate in ALS pathobiology. Full article

Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours arising from chromaffin cells. Pathogenic variants in the gene succinate dehydrogenase subunit B (SDHB) are associated with malignancy and poor prognosis. When metastases arise, limited treatment options are available. The pathomechanism of SDHB-associated PPGL remains largely unknown, and the lack of suitable models hinders therapy development. Germline heterozygous SDHB pathogenic variants predispose to developing PPGLs with a life-long penetrance of around 50%. To mimic the human disease phenotype, we characterised adult heterozygous sdhb mutant zebrafish as a potential model to study SDHB-related PPGLs. Adult sdhb mutant zebrafish did not develop an obvious tumour phenotype and were anatomically and histologically like their wild-type siblings. However, sdhb mutants showed significantly increased succinate levels, a major hallmark of SDHB-related PPGLs. While basal activity was increased during day periods in mutants, mitochondrial complex activity and catecholamine metabolite levels were not significantly different. In conclusion, we characterised an adult in vivo zebrafish model, genetically resembling human carriers. Adult heterozygous sdhb mutants mimicked their human counterparts, showing systemic elevation of succinate levels despite the absence of a tumour phenotype. This model forms a promising basis for developing a full tumour phenotype and gaining knowledge of the pathomechanism behind SDHB-related PPGLs. Full article

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that affects the motoneurons. More than 40 genes are related with ALS, and amyloidogenic proteins like SOD1 and/or TDP-43 mutants are directly involved in the onset of ALS through the formation of polymorphic fibrillogenic aggregates. However, efficacious therapeutic approaches are still lacking. Notably, heterozygous missense mutations affecting the gene coding for RNase 5, an enzyme also called angiogenin (ANG), were found to favor ALS onset. This is also true for the less-studied but angiogenic RNase 4. This review reports the substrate targets and illustrates the neuroprotective role of native ANG in the neo-vascularization of motoneurons. Then, it discusses the molecular determinants of many pathogenic ANG mutants, which almost always cause loss of function related to ALS, resulting in failures in angiogenesis and motoneuron protection. In addition, ANG mutations are sometimes combined with variants of other factors, thereby potentiating ALS effects. However, the activity of the native ANG enzyme should be finely balanced, and not excessive, to avoid possible harmful effects. Considering the interplay of these angiogenic RNases in many cellular processes, this review aims to stimulate further investigations to better elucidate the consequences of mutations in ANG and/or RNase 4 genes, in order to achieve early diagnosis and, possibly, successful therapies against ALS. Full article