Search Results (31)

Impaired function of Polymerase-γ (Pol-γ) results in impaired replication of the mitochondrial genome (mtDNA). Pathogenic mutations in the POLG gene cause dysfunctional Pol-γ and dysfunctional mitochondria and are associated with a spectrum of neurogenetic disorders referred to as POLG spectrum disorders (POLG-SDs), which are characterized by neurologic dysfunction and premature death. Pathomechanistic studies and human cell models of these diseases are scarce. SH-SY5Y cells (SHC) are an easy-to-handle and low-cost human-derived neuronal cell model commonly used in neuroscientific research. Here, we aimed to study the effect of reduced Pol-γ function using stable lentivirus-based shRNA-mediated knockdown of POLG in SHC, in both the proliferating cells and SHC-derived neurons. POLG knockdown resulted in approximately 50% reductions in POLG mRNA and protein levels in naïve SHC, mimicking the residual Pol-γ activity observed in patients with common pathogenic POLG mutations. Knockdown cells exhibited decreased mtDNA content, reduced levels of mitochondrial-encoded proteins, and altered mitochondrial morphology and distribution. Notably, while chemical induction of mtDNA depletion via ddC could be rescued by the mitochondrial biosynthesis stimulators AICAR, cilostazol and resveratrol (but not MitoQ and formoterol) in control cells, POLG-knockdown cells were resistant to mitochondrial biosynthesis-mediated induction of mtDNA increase, highlighting the specificity of the model, and pathomechanistically hinting towards inefficiency of mitochondrial stimulation without sufficient Pol-γ activity. In differentiated SHC-derived human neurons, POLG-knockdown cells showed impaired neuronal differentiation capacity, disrupted cytoskeletal organization, and abnormal perinuclear clustering of mitochondria. In sum, our model not only recapitulates key features of POLG-SDs such as impaired mtDNA content, which cannot be rescued by mitochondrial biosynthesis stimulation, but also reduced ATP production, perinuclear clustering of mitochondria and impaired neuronal differentiation. It also offers a simple, cost-effective and human (and, as such, disease-relevant) platform for investigating disease mechanisms, one with screening potential for therapeutic approaches for POLG-related mitochondrial dysfunction in human neurons. Full article

Background: The nuclear-encoded enzyme polymerase gamma (Pol-γ) is crucial in the replication of the mitochondrial genome (mtDNA), which in turn is vital for mitochondria and hence numerous metabolic processes and energy production in eukaryotic cells. Variants in the POLG gene, which encodes the catalytic subunit of Pol-γ, can significantly impair Pol-γ enzyme function. Pol-γ-associated disorders are referred to as POLG-spectrum disorders (POLG-SDs) and are mainly autosomal-recessively inherited. Clinical manifestations include muscle weakness and fatigue, and severe forms of the disease can lead to premature death in infancy, childhood, and early adulthood, often associated with seizures, liver failure, or intractable epilepsy. Here, we analyzed fibroblasts from a compound heterozygous patient with the established pathogenic variant c.2419C>T; p.(Arg807Cys) and a previously undescribed variant c.678G>C; p.(Gln226His) with a clinical manifestation compatible with POLG-SDs, sensory ataxic neuropathy, and infantile muscular atrophy. We conducted a battery of functional studies for Pol-γ and mitochondrial dysfunction on the patient’s fibroblasts, to test whether the novel variant c.678G>C; p.(Gln226His) may be causative in human disease. Aims/Methods: We analyzed skin-derived fibroblasts in comparison to a first-degree relative (the mother of the patient), an asymptomatic carrier harboring only the established c.2419C>T; p.(Arg807Cys) mutation. Assessments of mitochondrial function included measurements of mtDNA content, mRNA levels of mitochondrial genes, mitochondrial mass, and mitochondrial morphology. Case Presentation and Results: A 13-year-old male presented with symptoms starting at three years of age, including muscle weakness and atrophy in the lower extremities and facial muscles, which later extended to the upper limbs, voice, and back muscles, without further progression. The patient also reported fatigue and muscle pain after physical activity, with no sensory deficits. Extensive diagnostic tests such as electromyography, nerve conduction studies, muscle biopsy, and MRI were unremarkable. Exome sequencing revealed that he carried the compound heterozygous variants in POLG c.678G>C; p.(Gln226His) and c.2419C>T; p.(Arg807Cys), but no other potential genetic pathogenic causes. In comparison to a first-degree relative (his mother) who only carried the c.2419C>T; p.(Arg807Cys) pathogenic mutation, in vitro analyses revealed a significant reduction in mtDNA content (~50%) and mRNA levels of mtDNA-encoded proteins. Mitochondrial mass was reduced by approximately 20%, and mitochondrial interconnectivity within cells was impaired, as determined by fluorescence microscopy and mitochondrial staining. Conclusions: Our findings suggest that the c.678G>C; p.(Gln226His) variant, in conjunction with the c.2419C>T; p.(Arg807Cys) mutation, may compromise mtDNA replication and mitochondrial function and could result in clinically significant mitochondriopathy. As this study is based on one patient compared to a first-degree relative (but with an identical mitochondrial genome), the pathogenicity of c.678G>C; p.(Gln226His) of POLG should be confirmed in future studies, in particular, in conjunction with other POLG-variants. Full article

Background and Objectives: The long-term prognosis of acute myeloid leukemia (AML) is challenging due to limited understanding of the molecular markers involved in its development. This study investigates the role of DNA polymerases in AML to offer new insights for diagnosis and treatment. Materials and Methods: A retrospective study on pediatric AML patients with POL gene family mutations from 2021 to 2024 was conducted. Patients were categorized based on risk stratification criteria, and the DAH regimen was used for induction chemotherapy. Bioinformatics analysis integrated data from various databases to identify key genes and develop survival analysis plots and AUC curves. Results: The study included 59 pediatric AML patients, revealing no significant differences in demographic or clinical characteristics between those with and without POL family gene mutations. However, patients with POL gene mutations showed higher complete remission rates after initial DAH chemotherapy (91.67% vs. 59.57%, p = 0.03607), indicating a potential treatment benefit. High expression of four POL genes (POLD1, POLE, POLG, and POLQ) in bone marrow and immune cells suggests their crucial role in hematopoiesis and immune response. Survival analysis across different datasets indicated that AML patients with overexpressed POL family genes had significantly worse outcomes, proposing these genes as potential prognostic biomarkers for AML. Conclusions: This study on pediatric AML demonstrates that POL gene family mutations are associated with higher remission rates post-chemotherapy, indicating their potential as prognostic markers. Bioinformatics analysis emphasizes the significance of these mutations in AML, highlighting their impact on disease prognosis. Full article

Background: The etiology of most mesenchymal tumors is unknown, and knowledge about syndromes with an increased risk of tumors in bone or soft tissue is sparse. Methods: We present a prospective germline analysis of 312 patients with tumors suspected of being sarcomas at a tertiary sarcoma center. Germline and tumor whole genome sequencing, tumor transcriptome, and methylome analyses were performed. Results: Germline pathogenic or likely pathogenic variants associated with an increased risk of tumors were detected in 24 patients (8%), of which 11 (4%) harbored a detectable second hit in the tumor. Second hits were confirmed in genes with (NF1, RB1, TP53, EXT2, and SDHC) and without (ATM, CDC73, MLH1, MSH6, POLG, and KCNQ1) known association with mesenchymal tumor predisposition. Sarcomas from two Lynch syndrome patients showed mismatch repair deficiency, predicting a treatment response to immune checkpoint inhibitors (Level 1 biomarker according to the FDA (Federal Drug Administration) and ESMO (European Society for Medical Oncology)). None of the three CHEK2 carriers had a second hit in the tumor, suggesting a weak link to sarcoma. Conclusions: We conclude that second-hit analyses can be used in standard of care to identify syndrome-related tumors. This approach can help distinguish true manifestations of tumor syndromes from unrelated germline findings and enhance the understanding of germline predisposition in soft tissue tumors. Prospective screening using germline whole genome sequencing should be considered when comprehensive somatic sequencing is introduced into clinical practice. Full article

Diminished ovarian reserve (DOR) and primary ovarian insufficiency (POI) are major causes of female infertility. We recently found a monogenic etiology in 29.3% of POI, leading to personalized medicine. The genetic landscape of DOR is unknown. A prospective study (2018–2023) of an international cohort of 120 patients with unexplained DOR was performed using a large custom targeted next-generation sequencing panel including all known POI-causing genes. The diagnostic yield, based on the American College of Medical Genetics, was 24, 2%. Genes belong to different pathways: metabolism and mitochondria (29.7%), follicular growth (24.3%), DNA repair/meiosis (18.9%), aging (16.2%), ovarian development (8.1%), and autophagy (2.7%). Five genes were recurrently found: LMNA, ERCC6, SOX8, POLG, and BMPR1B. Six genes identified in single families with POI were involved in DOR, GNAS, TGFBR3, XPNPEP2, EXO1, BNC1, ATG, highlighting their role in maintaining ovarian reserve. In our cohort, 26 pregnancies were recorded, but no pregnancy was observed when meiosis/DNA repair genes were involved, suggesting severely impaired oocyte quality. Additional studies should confirm these preliminary results. This study with a large NGS panel defines the genetic landscape of a large cohort of DOR. It supports routine genetic diagnosis. Genetics could be a biomarker predicting infertility and progression to POI. Full article

Using Chlamydomonas as a model organism, we attempted to eliminate mitochondrial DNA (mtDNA) similar to rho⁰ or rho⁻ cells (completely or partially mtDNA-eliminated cells) in yeast. We successfully generated partially mtDNA-eliminated cells named as crm- cells, causing the inactivation of mitochondrial activity. We used three different chemicals to eliminate mtDNA including acriflavine (AF), ethidium bromide (EB) and dideoxycytidine (ddC) which prevents replication, inhibits POLG (DNA polymerase gamma) and terminates the mtDNA chain, respectively. The qPCR method was used to detect the mtDNA copy number and the selected rrnL6 gene for the detection of mitochondria, as well as the selected Chlamydomonas CC-124 strain. A reduction in the mitochondrial copy number led to a higher expression of AOX1, UCP1, PGRL1 and ICL1, which indicates the disturbance of the mitochondria–chloroplast ATP and NADPH balance. We selected AOX genes to further study this family and carried out a genome-wide search to identify AOX genes in green algae (C. reinhardtii). Our results revealed that C. reinhardtii contains four AOX genes, i.e., CrAOX1, CrAOX2, CrAOX3 and CrAOX4, which are distributed on Chr 3, Chr7 and Chr9. All CrAOX genes were predicted to localize in mitochondria using bioinformatics tools. Phylogenetic analysis suggests that these CrAOXs are subdivided into four groups and genes existing in the same group could perform identical functions. Collinearity analysis describes the strong evolutionary relationships of AOXs between the unicellular green algae Chlamydomonas reinhardtii and the multicellular green algae Volvox carteri. GO (gene ontology) annotation analysis predicted that CrAOXs played an integral part in carrying out alternate oxidative and respirative activities. Three putative miRNAs, cre-miR1162-3p, cre-miR1171 and cre-miR914, targeting the CrAOX2 gene were identified. Our studies have laid a foundation for the further use of partially mtDNA-eliminated cells and elucidating the functional characteristics of the AOX gene family. Full article

Systematic evaluation of 80 history and 40 history findings diagnosed 1261 patients with Ehlers–Danlos syndrome (EDS) by direct or online interaction, and 60 key findings were selected for their relation to clinical mechanisms and/or management. Genomic testing results in 566 of these patients supported EDS relevance by their differences from those in 82 developmental disability patients and by their association with general rather than type-specific EDS findings. The 437 nuclear and 79 mitochondrial DNA changes included 71 impacting joint matrix (49 COL5), 39 bone (30 COL1/2/9/11), 22 vessel (12 COL3/8VWF), 43 vessel–heart (17FBN1/11TGFB/BR), 59 muscle (28 COL6/12), 56 neural (16 SCN9A/10A/11A), and 74 autonomic (13 POLG/25porphyria related). These genes were distributed over all chromosomes but the Y, a network analogized to an ‘entome’ where DNA change disrupts truncal mechanisms (skin constraint, neuromuscular support, joint vessel flexibility) and produces a mirroring cascade of articular and autonomic symptoms. The implied sequences of genes from nodal proteins to hypermobility to branching tissue laxity or dysautonomia symptoms would be ideal for large language/artificial intelligence analyses. Full article

Neurofibromatosis type 1 (NF1) stands as a prevalent neurocutaneous disorder. Approximately a quarter of NF1 patients experience the development of plexiform neurofibromas, potentially progressing into malignant peripheral nerve sheath tumors (MPNST). FT895, an HDAC11 inhibitor, exhibits potent anti-tumor effects on MPNST cells and enhances the cytotoxicity of cordycepin against MPNST. The study aims to investigate the molecular mechanism underlying FT895’s efficacy against MPNST cells. Initially, our study unveiled that FT895 disrupts mitochondrial biogenesis and function. Post-FT895 treatment, reactive oxygen species (ROS) in MPNST notably increased, while mitochondrial DNA copy numbers decreased significantly. Seahorse analysis indicated a considerable decrease in basal, maximal, and ATP-production-coupled respiration following FT895 treatment. Immunostaining highlighted FT895’s role in promoting mitochondrial aggregation without triggering mitophagy, possibly due to reduced levels of XBP1, Parkin, and PINK1 proteins. Moreover, the study using CHIP-qPCR analysis revealed a significant reduction in the copy numbers of promoters of the MPV17L2, POLG, TFAM, PINK1, and Parkin genes. The RNA-seq analysis underscored the prominent role of the HIF-1α signaling pathway post-FT895 treatment, aligning with the observed impairment in mitochondrial respiration. In summary, the study pioneers the revelation that FT895 induces mitochondrial respiratory damage in MPNST cells. Full article

Parkinson’s disease (PD) is a multisystem and multifactorial disorder and, therefore, the application of modern genetic techniques may assist in unraveling its complex pathophysiology. We conducted a clinical–demographic evaluation of 126 patients with PD, all of whom were Caucasian and of Sicilian ancestry. DNA was extracted from the peripheral blood for each patient, followed by sequencing using a Next-Generation Sequencing system. This system was based on a custom gene panel comprising 162 genes. The sample underwent further filtering, taking into account the allele frequencies of genetic variants, their presence in the Human Gene Mutation Database, and their association in the literature with PD or other movement/neurodegenerative disorders. The largest number of variants was identified in the leucine-rich repeat kinase 2 (LRRK2) gene. However, variants in other genes, such as acid beta-glucosidase (GBA), DNA polymerase gamma catalytic subunit (POLG), and parkin RBR E3 ubiquitin protein ligase (PRKN), were also discovered. Interestingly, some of these variants had not been previously associated with PD. Enhancing our understanding of the genetic basis of PD and identifying new variants possibly linked to the disease will contribute to improved diagnostic accuracy, therapeutic developments, and prognostic insights for affected individuals. Full article

One of the key features of major depressive disorder (MDD, depression) is increased oxidative stress manifested by elevated levels of mtROS, a hallmark of mitochondrial dysfunction, which can arise from mitochondrial DNA (mtDNA) damage. Thus, the current study explores possibility that the single-nucleotide polymorphisms (SNPs) of genes encoding the three enzymes that are thought to be implicated in the replication, repair or degradation of mtDNA, i.e., POLG, ENDOG and EXOG, have an impact on the occurrence, onset, severity and treatment of MDD. Five SNPs were selected: EXOG c.-188T > G (rs9838614), EXOG c.*627G > A (rs1065800), POLG c.-1370T > A (rs1054875), ENDOG c.-394T > C (rs2977998) and ENDOG c.-220C > T (rs2997922), while genotyping was performed on 538 DNA samples (277 cases and 261 controls) using TaqMan probes. All SNPs of EXOG and ENDOG modulated the risk of depression, but the strongest effect was observed for rs1065800, while rs9838614 and rs2977998 indicate that they might influence the severity of symptoms, and, to a lesser extent, treatment effectiveness. Although the SNP located in POLG did not affect occurrence of the disease, the result suggests that it may influence the onset and treatment outcome. These findings further support the hypothesis that mtDNA damage and impairment in its metabolism play a crucial role not only in the development, but also in the treatment of depression. Full article

Mitochondrial diseases (MDs) are heterogeneous genetic disorders characterized by mitochondrial DNA (mtDNA) defects, involving tissues highly dependent on oxidative metabolism: the inner ear, brain, eye, skeletal muscle, and heart. We describe adult patients with genetically defined MDs, characterizing hearing function and neuroimaging results. We enrolled 34 patients (mean age: 50.02 ± 15 years, range: 18–75 years; 20 females and 14 males) classified in four groups: MELAS, MIDD, PEO, and Encephalopathy/Polyneuropathy. Audiological evaluations included psychoacoustical tests (pure-tone and speech audiometry), electrophysiological tests (Auditory Brainstem Responses, ABRs), and Impedenzometry. Neuroimaging evaluations considered global MRI abnormalities or structural brain changes. In total, 19/34 patients carried the m.3243A > G mutation (6 affected by MELAS, 12 affected by MIDD, and 1 affected by PEO); 11 had an mtDNA deletion (all affected by PEO); 3 had nuclear genes associated with MDs (POLG1 and OPA1); and 1 patient had an mtDNA deletion without an identified nuclear gene defect (affected by PEO). Sensory neural, bilateral, and symmetrical hearing loss was present in 25 patients (73.5%) to different degrees: 9 mild, 9 moderate, 5 severe, and 2 profound. The severe/profound and mild hearing losses were associated with pantonal and high-frequency audiograms, respectively. Instead, moderate hearing losses were associated with both high-frequency (five cases) and pantonal (five cases) audiogram shapes. In addition, 21/25 patients showed a cochlear site of lesion (84%), and 4/25 (16%) showed a retrocochlear site. We found global MRI abnormalities or structural brain changes in 26/30 subjects (86.6%): 21 had white matter abnormalities, 15 had cortical atrophy, 10 had subcortical atrophy, 8 had basal nuclei involvement or cerebellar atrophy, 4 had stroke-like lesions or laminar necrosis, and 1 had cysts or vacuolated lesions. We concluded that genetic alterations are associated with different clinical presentations for both auditory function and neuroradiological findings. There is no fixed relationship between genotype and phenotype for the clinical conditions analyzed. Full article

A substantial fraction of the 15% with double-jointedness or hypermobility have the traditionally ascertained joint-skeletal, cutaneous, and cardiovascular symptoms of connective tissue dysplasia and its particular manifestation as Ehlers–Danlos syndrome (EDS). The holistic ascertainment of 120 findings in 1261 EDS patients added neuro-autonomic symptoms like headaches, muscle weakness, brain fog, chronic fatigue, dyspnea, and bowel irregularity to those of arthralgia and skin laxity, 15 of these symptoms shared with those of post-infectious SARS-CoV-2 (long COVID-19). Underlying articulo-autonomic mechanisms guided a clinical qualification protocol that qualified DNA variants in 317 genes as having diagnostic utility for EDS, six of them identical (F2-LIFR-NLRP3-STAT1-T1CAM1-TNFRSF13B) and eighteen similar to those modifying COVID-19 severity/EDS, including ADAMTS13/ADAMTS2-C3/C1R-IKBKG/IKBKAP-PIK3C3/PIK3R1-POLD4/POLG-TMPRSS2/TMPRSS6-WNT3/WNT10A. Also, contributing to EDS and COVID-19 severity were forty and three genes, respectively, impacting mitochondrial functions as well as parts of an overlapping gene network, or entome, that are hypothesized to mediate the cognitive–behavioral, neuro-autonomic, and immune-inflammatory alterations of connective tissue in these conditions. The further characterization of long COVID-19 natural history and genetic predisposition will be necessary before these parallels to EDS can be carefully delineated and translated into therapies. Full article

Background: The POLG gene encodes the catalytic subunit of DNA polymerase γ, which is crucial for mitochondrial DNA (mtDNA) repair and replication. Gene mutation alters the stability of mtDNA and is associated with several clinical presentations, such as dysarthria and ophthalmoplegia (SANDO), progressive external ophthalmoplegia (PEO), spinocerebellar ataxia and epilepsy (SCAE), Alpers syndrome, and sensory ataxic neuropathy. Recent evidence has also indicated that POLG mutations may be involved in some neurodegenerative disorders, although systematic screening is currently lacking. Methods: To investigate the frequency of POLG gene mutations in neurodegenerative disorders, we screened a group of 33 patients affected by neurodegenerative diseases, including Parkinson’s disease, some atypical parkinsonisms, and dementia of different types. Results: Mutational analysis revealed the presence of the heterozygous Y831C mutation in two patients, one with frontotemporal dementia and one with Lewy body dementia. The allele frequency of this mutation reported by the 1000 Genomes Project in the healthy population is 0.22%, while in our group of patients, it was 3.03%, thus showing a statistically significant difference between the two groups. Conclusions: Our results may expand the genotype-phenotype spectrum associated with mutations in the POLG gene and strengthen the hypothesis of a pathogenic role of the Y831C mutation in neurodegeneration. Full article

In 2020, 878,348 newly reported cases and 444,347 deaths related to head and neck cancer were reported. These numbers suggest that there is still a need for molecular biomarkers for the diagnosis and prognosis of the disease. In this study, we aimed to analyze mitochondria-related mitochondrial transcription factor A (TFAM) and DNA polymerase γ (POLG) single-nucleotide polymorphisms (SNPs) in the head and neck cancer patient group and evaluate associations between SNPs, disease characteristics, and patient outcomes. Genotyping was performed using TaqMan probes with Real-Time polymerase chain reaction. We found associations between TFAM gene SNPs rs11006129 and rs3900887 and patient survival status. We found that patients with the TFAM rs11006129 CC genotype and non-carriers of the T allele had longer survival times than those with the CT genotype or T-allele carriers. Additionally, patients with the TFAM rs3900887 A allele tended to have shorter survival times than non-carriers of the A allele. Our findings suggest that variants in the TFAM gene may play an important role in head and neck cancer patient survival and could be considered and further evaluated as prognostic biomarkers. However, due to the limited sample size (n = 115), further studies in larger and more diverse cohorts are needed to confirm these findings. Full article

The formation of embryoid bodies (EBs) from human pluripotent stem cells resembles the early stages of human embryo development, mimicking the organization of three germ layers. In our study, EBs were tested for their vulnerability to chronic exposure to low doses of MeHgCl (1 nM) under atmospheric (21%O₂) and physioxia (5%O₂) conditions. Significant differences were observed in the relative expression of genes associated with DNA repair and mitophagy between the tested oxygen conditions in nontreated EBs. When compared to physioxia conditions, the significant differences recorded in EBs cultured at 21% O₂ included: (1) lower expression of genes associated with DNA repair (ATM, OGG1, PARP1, POLG1) and mitophagy (PARK2); (2) higher level of mtDNA copy number; and (3) higher expression of the neuroectodermal gene (NES). Chronic exposure to a low dose of MeHgCl (1 nM) disrupted the development of EBs under both oxygen conditions. However, only EBs exposed to MeHgCl at 21% O₂ revealed downregulation of mtDNA copy number, increased oxidative DNA damage and DNA fragmentation, as well as disturbances in SOX17 (endoderm) and TBXT (mesoderm) genes expression. Our data revealed that physioxia conditions protected EBs genome integrity and their further differentiation. Full article