POLG Variants Research Articles

Evidence for sodium valproate toxicity in mitochondrial diseases: a systematic analysis

BackgroundWe aimed to determine whether sodium valproate (VPA) should be contraindicated in all mitochondrial diseases, due to known VPA-induced severe hepatotoxicity in some mitochondrial diseases.MethodsWe systematically reviewed the published literature...

Cells expressing POLG loss-of-function variants can decrease mtDNA copy number and increased growth rates in neighbouring A7r5 and HeLa cells

Mitochondrial disfunction promotes vascular disease, and mitochondrial diseases offer valuable insights into the pathological mechanisms of mitochondrial dysfunction. We previously described a patient cohort with variants in the mitochondrial DNA gamma polymerase (POLG) that were prone hypertension. We hypothesized that the POLG variants promote hypertrophy or hyperplasia of vascular smooth muscle cells in a way that stiffens or thickens blood vessels to explain the high rate of resistant hypertension in this cohort. The objective of this study was to assess the cellular phenotype of cells over-expressing POLG variants: pathogenic Y955C and variants of unknown significance; R964C, I1098N, Y1138C. We transiently expressed human FLAG-tagged POLG variants in rat A7r5 and HeLa cells for 2-4 days. Transfection effciency ranged from 20-30% in A7r5 and >60% in HeLa cells. POLG variants increased cell density by nuclear counting (20-60%) and growth rates measured by 48-72 h of live-cell imaging (Y955C > R964C > I1089N » Y1138C). Compared to wild-type, Y955C and R964C reduced mtDNA copy number (mCN) measured by droplet digital PCR by 10-20%. Imaging mtDNA nucleoids (anti-dsDNA) and the FLAG-tag showed that reduced mCN was not restricted to transfected cells. Addition of a P2A-T2A-nuclear-EGFP to the POLG constructs demonstrated that faster growth rates were not specific to transfected cells. Resting and oligomycin-stimulated mitochondrial membrane potential imaged with TMRM were unaffected by Y955C and R964C. Mitochondrial ROS (mitoSox) was similar between cultures transfected with Wild-type versus POLG variants and lower than levels stimulated by antimycin-A’s inhibition of complex III. Notably, we found a linear relationship between mCN and cell density that was not simply due to smaller cell size at higher densities. Using Hoescht-33342 staining to assess cell cycle, no clear difference in cell-cycle distribution was evident in unsynchronized cultures. We conclude that modest reduction of mCN induces a mitogenic effect, likely via a diffusible messenger that further reduces mCN in a cell population. The signalling mechanism of this phenomenon remains to be identified, but our results confirm that POLG malfunction can alter smooth muscle physiology in a way that could support pathogenic vascular remodelling and could promote enhanced growth in cancerous cells. Funded by the Natural Sciences and Research Council of Canada. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Linking pathogenic and likely pathogenic gene variants to long-COVID symptoms.

Long-COVID is a clinical syndrome characterized by the presence of symptoms related to SARS-CoV-2 infection that persist for at least four weeks after recovery from COVID-19. Genetics have been proposed to play an important role in long-COVID syndrome onset. This study aimed to identify genetic pathogenetic and likely pathogenetic causative variants of Mendelian genetic diseases in patients with Long-COVID syndrome. Additionally, we aimed to establish an association between these genetic variants and the clinical symptoms manifested during long-COVID syndrome. 95 patients affected by long-COVID syndrome were analyzed with a Next-Generation Sequencing (NGS) panel comprising 494 genes. The analyzed genes and the symptoms of the patients collected with an ad-hoc questionnaire were divided into four groups (cardiological, respiratory, immunological, and neurological). Finally, a statistical analysis comprising descriptive statistics, classification based on reported symptoms, and comparative analysis against a control group of healthy individuals was conducted. 12 patients resulted positive for genetic testing with an autosomal dominance (8) or autosomal recessive (4) inheritance, showing a higher prevalence of cardiovascular genetic diseases (9) in the analyzed cohort compared to the normal population. Moreover, the onset of the long-COVID syndrome and its cardiovascular manifestations was compliant with the onset reported in the literature for the identified genetic diseases, suggesting that COVID-19 could manifest late-onset genetic diseases associated with their appearance. Apart from the 12 positive patients, 57 were healthy carriers of genetic diseases. Analyzing the whole cohort, a statistical correlation between prevalent symptomatology and the gene class was established, suggesting an association between the genetic susceptibility of an individual and the possibility of developing specific long-COVID syndrome symptoms, especially cardiovascular symptoms. Furthermore, 17 genetic variants were identified in CFTR. Finally, we identified genetic variants in IFNAR2 and POLG, supporting their respective involvement in inflammation and mitochondria mechanisms, correlated with long-COVID syndrome according to literature data. This study proposed COVID-19 to act as a manifest of underlying late-onset genetic diseases Mendelian associated with carrier status. Moreover, according to our results, mutations in cardiological genes are more present in patients who show cardiological symptoms during the syndrome. This underscores the necessity for cardiological investigation and genetic screening in long-COVID patients to address existing or potential clinical implications.

A novel mouse model of mitochondrial disease exhibits juvenile-onset severe neurological impairment due to parvalbumin cell mitochondrial dysfunction

Mitochondrial diseases comprise a common group of neurometabolic disorders resulting from OXPHOS defects, that may manifest with neurological impairments, for which there are currently no disease-modifying therapies. Previous studies suggest inhibitory interneuron susceptibility to mitochondrial impairment, especially of parvalbumin-expressing interneurons (PV+). We have developed a mouse model of mitochondrial dysfunction specifically in PV+ cells via conditional Tfam knockout, that exhibited a juvenile-onset progressive phenotype characterised by cognitive deficits, anxiety-like behaviour, head-nodding, stargazing, ataxia, and reduced lifespan. A brain region-dependent decrease of OXPHOS complexes I and IV in PV+ neurons was detected, with Purkinje neurons being most affected. We validated these findings in a neuropathological study of patients with pathogenic mtDNA and POLG variants showing PV+ interneuron loss and deficiencies in complexes I and IV. This mouse model offers a drug screening platform to propel the discovery of therapeutics to treat severe neurological impairment due to mitochondrial dysfunction.

DNA polymerase gamma variants and hepatotoxicity during maintenance therapy of childhood acute lymphoblastic leukemia: is there a causal relationship?

Hepatotoxicity is a frequent complication during maintenance therapy of acute lymphoblastic leukemia (ALL) with 6-mercaptopurine and methotrexate. Elevated levels of methylated 6-mercaptopurine metabolites (MeMP) are associated with hepatotoxicity. However, not all mechanisms are known that lead to liver failure in patients with ALL. Variants in the POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase gamma (POLG1), have been related to drug-induced hepatotoxicity, for example, by sodium valproate. The association of common POLG variants with hepatotoxicity during maintenance therapy was studied in 34 patients with childhood ALL. Of the screened POLG variants, four different variants were detected in 12 patients. One patient developed severe hepatotoxicity without elevated MeMP levels and harbored a heterozygous POLG p.G517V variant, which was not found in the other patients.

POLG-Related Ataxia and Ophthalmoplegia: A Case Report (P9-11.008)

<h3>Objective:</h3> Not applicable <h3>Background:</h3> Genetic variants of the POLG gene result in mitochondrial DNA defects and account for a large proportion of mitochondrial diseases. These disorders encompass a spectrum of phenotypes, with onset ranging from infancy to late adulthood. In addition to hepatic dysfunction and myopathy in younger populations, and hormone derangements with cataracts in older patients, patients may also present with prominent neurologic symptoms, including ataxia, parkinsonism, epilepsy, and ophthalmoplegia. More than 300 pathogenic mutations of POLG have been reported and there are no clear genotype-phenotype correlations. Given the rarity of these syndromes and wide range of clinical phenotypes, there is often delay in diagnosis. <h3>Design/Methods:</h3> Not applicable <h3>Results:</h3> We present a 47-year old woman with symptoms of difficulty speaking and development of tremor, vision loss, dizziness, and gait difficulty progressing over several months. Initial exam was significant for ataxia and choreiform movements. Extraocular movement (EOM) testing revealed delayed initiation of saccades and slow, saccadic pursuits with nystagmus. Bloodwork, which included extensive workup for causes of chorea and ataxia, electromyography/nerve conduction studies, and imaging (MRI brain and malignancy work-up) were unremarkable. She continued to decline with development of ophthalmoplegia, ptosis, numbness, weakness, and pain, severely limiting her functional capacity. Whole exome sequencing was performed and showed a heterozygous variant of uncertain significance (VUS) in the POLG gene, which may explain her presentation. <h3>Conclusions:</h3> This case adds to a growing collection of case reports describing the various phenotypes of POLG-related disorders. Early detection of POLG variants and phenotype/genotype characterization may help guide development of future treatments for patients with similar conditions. <b>Disclosure:</b> Dr. Peabody has nothing to disclose. An immediate family member of Dr. Shpiner has received personal compensation for serving as an employee of University of Miami.

Fever related super-refractory status epilepticus: An adulthood presentation of a novel POLG variant: A case report

Mitochondrial DNA depletion syndromes caused by POLG mutations clinically constitute a heterogeneous group and can involve any part of the neuraxis [1]. Non-syndromic presentations that do not conform with the recognised classical POLG mutation syndromes are infrequently encountered, and given their protean manifestations, require a high index of suspicion to delineate from other more common aetiologies. Here we expand the FIRES (febrile infection-related status epilepticus)- like presentation of a novel POLG mutation in a twenty-eight-year-old lady, who had a complex medical history that included intellectual disability, behavioural changes, and initially well-controlled seizures.

Clinical and molecular spectrum associated with Polymerase-γ related disorders.

POLG pathogenic variants are the commonest single-gene cause of inherited mitochondrial disease. However, the data on clinicogenetic associations in POLG-related disorders are sparse. This study maps the clinicogenetic spectrum of POLG-related disorders in the pediatric population. Individuals were recruited across 6 centers in India. Children diagnosed between January 2015 and August 2020 with pathogenic or likely pathogenic POLG variants and age of onset <15 years were eligible. Phenotypically, patients were categorized into Alpers-Huttenlocher syndrome; myocerebrohepatopathy syndrome; myoclonic epilepsy, myopathy, and sensory ataxia; ataxia-neuropathy spectrum; Leigh disease; and autosomal dominant / recessive progressive external ophthalmoplegia. A total of 3729 genetic reports and 4256 hospital records were screened. Twenty-two patients with pathogenic variants were included. Phenotypically, patients were classifiable into Alpers-Huttenlocher syndrome (8/22; 36.4%), progressive external ophthalmoplegia (8/22; 36.4%), Leigh disease (2/22; 9.1%), ataxia-neuropathy spectrum (2/22; 9.1%), and unclassified (2/22; 9.1%). The prominent clinical manifestations included developmental delay (n = 14; 63.7%), neuroregression (n = 14; 63.7%), encephalopathy (n = 11; 50%), epilepsy (n = 11; 50%), ophthalmoplegia (n = 8; 36.4%), and liver dysfunction (n = 8; 36.4%). Forty-four pathogenic variants were identified at 13 loci, and these were clustered at exonuclease (18/44; 40.9%), linker (13/44; 29.5%), polymerase (10/44; 22.7%), and N-terminal domains (3/44; 6.8%). Genotype-phenotype analysis suggested that serious outcomes including neuroregression (odds ratio [OR] 11, 95% CI 2.5, 41), epilepsy (OR 9, 95% CI 2.4, 39), encephalopathy (OR 5.7, 95% CI 1.4, 19), and hepatic dysfunction (OR 4.6, 95% CI 21.3, 15) were associated with at least 1 variant involving linker or polymerase domain. We describe the clinical subgroups and their associations with different POLG domains. These can aid in the development of follow-up and management strategies of presymptomatic individuals.

Forecasting stroke-like episodes and outcomes in mitochondrial disease.

In this retrospective, multicentre, observational cohort study, we sought to determine the clinical, radiological, EEG, genetics and neuropathological characteristics of mitochondrial stroke-like episodes and to identify associated risk predictors.Between January 1998 and June 2018, we identified 111 patients with genetically determined mitochondrial disease who developed stroke-like episodes. Post-mortem cases of mitochondrial disease (n = 26) were identified from Newcastle Brain Tissue Resource. The primary outcome was to interrogate the clinico-radiopathological correlates and prognostic indicators of stroke-like episode in patients with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). The secondary objective was to develop a multivariable prediction model to forecast stroke-like episode risk.The most common genetic cause of stroke-like episodes was the m.3243A>G variant in MT-TL1 (n = 66), followed by recessive pathogenic POLG variants (n = 22), and 11 other rarer pathogenic mitochondrial DNA variants (n = 23). The age of first stroke-like episode was available for 105 patients [mean (SD) age: 31.8 (16.1)]; a total of 35 patients (32%) presented with their first stroke-like episode ≥40 years of age. The median interval (interquartile range) between first and second stroke-like episodes was 1.33 (2.86) years; 43% of patients developed recurrent stroke-like episodes within 12 months. Clinico-radiological, electrophysiological and neuropathological findings of stroke-like episodes were consistent with the hallmarks of medically refractory epilepsy. Patients with POLG-related stroke-like episodes demonstrated more fulminant disease trajectories than cases of m.3243A>G and other mitochondrial DNA pathogenic variants, in terms of the frequency of refractory status epilepticus, rapidity of progression and overall mortality. In multivariate analysis, baseline factors of body mass index, age-adjusted blood m.3243A>G heteroplasmy, sensorineural hearing loss and serum lactate were significantly associated with risk of stroke-like episodes in patients with the m.3243A>G variant. These factors informed the development of a prediction model to assess the risk of developing stroke-like episodes that demonstrated good overall discrimination (area under the curve = 0.87, 95% CI 0.82–0.93; c-statistic = 0.89). Significant radiological and pathological features of neurodegeneration were more evident in patients harbouring pathogenic mtDNA variants compared with POLG: brain atrophy on cranial MRI (90% versus 44%, P < 0.001) and reduced mean brain weight (SD) [1044 g (148) versus 1304 g (142), P = 0.005].Our findings highlight the often idiosyncratic clinical, radiological and EEG characteristics of mitochondrial stroke-like episodes. Early recognition of seizures and aggressive instigation of treatment may help circumvent or slow neuronal loss and abate increasing disease burden. The risk-prediction model for the m.3243A>G variant can help inform more tailored genetic counselling and prognostication in routine clinical practice.

Adult-onset mitochondrial movement disorders: a national picture from the Italian Network

IntroductionBoth prevalence and clinical features of the various movement disorders in adults with primary mitochondrial diseases are unknown.MethodsBased on the database of the “Nation-wide Italian Collaborative Network of Mitochondrial Diseases”, we reviewed the clinical, genetic, neuroimaging and neurophysiological data of adult patients with primary mitochondrial diseases (n = 764) where ataxia, myoclonus or other movement disorders were part of the clinical phenotype.ResultsAtaxia, myoclonus and movement disorders were present in 105/764 adults (13.7%), with the onset coinciding or preceding the diagnosis of the mitochondrial disease in 49/105 (46.7%). Ataxia and parkinsonism were the most represented, with an overall prevalence at last follow-up of 59.1% and 30.5%, respectively. Hyperkinetic movement disorders were reported in 15.3% at last follow-up, being the less common reported movement disorders. The pathogenic m.8344A > G and POLG variants were always associated with a movement disorder, while LHON variants and mtDNA single deletions were more commonly found in the subjects who did not present a movement disorder. The most common neuroimaging features were cortical and/or cerebellar atrophy, white matter hyperintensities, basal ganglia abnormalities and nigro-striatal degeneration. Almost 70% of patients with parkinsonism responded to dopaminergic therapy, mainly levodopa, and 50% with myoclonus were successfully treated with levetiracetam.ConclusionMovement disorders, mainly ataxia and parkinsonism, are important findings in adult primary mitochondrial diseases. This study underlies the importance of looking for a mitochondrial etiology in the diagnostic flowchart of a movement disorder and may help direct genetic screening in daily practice.

Novel biallelic mutations in POLG gene: large deletion and missense variant associated with PEO.

Mitochondrial disorders are clinically heterogeneous diseases associated with impaired oxidative phosphorylation (OXPHOS) activity. POLG, which encodes the DNA polymerase-γ (Polγ) catalytic subunit, is the most commonly mutated nuclear gene associated with mitochondrial disorders. We carried out whole-exome sequencing (WES) to identify the gene associated with progressive external ophthalmoplegia (PEO). We then performed histopathological analyses, assessed mitochondrial biology, and executed functional studies to evaluate the potential pathogenicity of the identified genetic mutations. Novel biallelic POLG mutations, including a large deletion mutation (exons 7-21) and a missense variant c.1796C>T (p.Thr599Ile) were detected in the proband. Histopathological analysis of a biopsied muscle sample from this patient revealed the presence of approximately 20% COX-negative fibers. Bioinformatics analyses confirmed that the detected mutations were pathogenic. Furthermore, levels of mitochondrial complex I, II, and IV subunit protein expressions were found to be decreased in the proband, and marked impairment of mitochondrial respiration was evident in cells harboring these mutations. This study expands the spectrum of known POLG variants associated with PEO and advances current understanding regarding the structural and functional impacts of these mutations.

The impact of mitochondria-related POLG and TFAM variants on breast cancer pathomorphological characteristics and patient outcomes

Purpose Breast cancer is the most frequent female cancer, leading to relapse with distant metastasis of approximately one-third of patients. Cancer is usually considered a genetic disease involving mutations in nuclear DNA. However, genes, coding for mitochondrial proteins or regulatory molecules, are rarely under consideration. This study aimed to analyse 10 single nucleotide variants in POLG and TFAM genes and assess their association with tumour phenotype and disease outcome. Materials and methods A total of 234 breast cancer patients were included in this study. Variations were determined with Real-Time PCR using TaqMan® probes. Results We found that patients with POLG rs2307441 TT and CT genotypes had a lower probability for vascular invasion than those with CC genotype (p = 0.001). Patients with POLG rs2072267 AG genotype were predisposed for progression compared with GG genotype (p = 0.015). TFAM rs3900887 TT genotype was associated with a higher probability for positive oestrogen receptors (p = 0.003) and lymphatic invasion (p = 0.001) in comparison to AA genotype, patients with TT (p = 0.000) were more likely to have positive lymph nodes. Conclusions Our data suggest that variations in POLG and TFAM genes are important determinacies of tumour phenotype and disease outcome in breast cancer patients.

Ultrasensitive deletion detection links mitochondrial DNA replication, disease, and aging

BackgroundAcquired human mitochondrial genome (mtDNA) deletions are symptoms and drivers of focal mitochondrial respiratory deficiency, a pathological hallmark of aging and late-onset mitochondrial disease.ResultsTo decipher connections between these processes, we create LostArc, an ultrasensitive method for quantifying deletions in circular mtDNA molecules. LostArc reveals 35 million deletions (~ 470,000 unique spans) in skeletal muscle from 22 individuals with and 19 individuals without pathogenic variants in POLG. This nuclear gene encodes the catalytic subunit of replicative mitochondrial DNA polymerase γ. Ablation, the deleted mtDNA fraction, suffices to explain skeletal muscle phenotypes of aging and POLG-derived disease. Unsupervised bioinformatic analyses reveal distinct age- and disease-correlated deletion patterns.ConclusionsThese patterns implicate replication by DNA polymerase γ as the deletion driver and suggest little purifying selection against mtDNA deletions by mitophagy in postmitotic muscle fibers. Observed deletion patterns are best modeled as mtDNA deletions initiated by replication fork stalling during strand displacement mtDNA synthesis.

High rate of hypertension in patients with m.3243A>G MELAS mutations and POLG variants

Animal studies suggest that decreased vascular mitochondrial DNA copy number can promote hypertension. We conducted a chart review of blood pressure and hemodynamics in patients with either mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS, n = 36) or individuals with variants in the mitochondrial DNA polymerase gamma (POLG, n = 26). The latter included both pathogenic variants and variants of unknown significance (VUS). Hypertension rates (MELAS 50%, POLG 50%) were elevated relative to Canadian norms in 20–39 (MELAS) and 40–59 (MELAS and POLG) years of age groups. Peripheral resistance was high in the hypertensive versus normotensive patients, potentially indicative of microvascular disease. Despite antihypertensive treatment, systolic blood pressure remained elevated in the POLG versus MELAS group. The risk of hypertension was not associated with MELAS heteroplasmy. Hypertension rates were not different between individuals with known pathogenic POLG variants and those with VUS, including common variants. Hypertension (HT) also did not differ between patients with POLG variants with (n = 17) and without chronic progressive external opthalmoplegia (n = 9) (CPEO). HT was associated with variants in all three functional domains of POLG. These findings suggest that both pathogenic variants and several VUS in the POLG gene may promote human hypertension and extend our past reports that increased risk of HT is associated with MELAS.

The frequency of mitochondrial polymerase gamma related disorders in a large Polish population cohort

Diseases related to DNA polymerase gamma dysfunction comprise of heterogeneous clinical presentations with variable severity and age of onset. Molecular screening for the common POLG variants: p.Ala467Thr, p.Trp748Ser, p.Gly848Ser, and p.Tre251Ile has been conducted in a large population cohort (n = 3123) and in a clinically heterogeneous group of 1289 patients. Recessive pathogenic variants, including six novel ones were revealed in 22/26 patients. Infantile Alpers-Huttenlocher syndrome and adulthood ataxia spectrum were the most common found in our group. Distinct molecular profile identified in the Polish patients with significant predominance of p.Trp748Ser variant (50% of mutant alleles) reflected strikingly low population frequency of the three remaining variants and slightly higher p.Trp748Ser allele frequency in the general Polish population as compared to the non-Finish European population.

Frequent genes in rare diseases: panel-based next generation sequencing to disclose causal mutations in hereditary neuropathies.

Hereditary neuropathies comprise a wide variety of chronic diseases associated to more than 80 genes identified to date. We herein examined 612 index patients with either a Charcot-Marie-Tooth phenotype, hereditary sensory neuropathy, familial amyloid neuropathy, or small fiber neuropathy using a customized multigene panel based on the next generation sequencing technique. In 121 cases (19.8%), we identified at least one putative pathogenic mutation. Of these, 54.4% showed an autosomal dominant, 33.9% an autosomal recessive, and 11.6% an X-linked inheritance. The most frequently affected genes were PMP22 (16.4%), GJB1 (10.7%), MPZ, and SH3TC2 (both 9.9%), and MFN2 (8.3%). We further detected likely or known pathogenic variants in HINT1, HSPB1, NEFL, PRX, IGHMBP2, NDRG1, TTR, EGR2, FIG4, GDAP1, LMNA, LRSAM1, POLG, TRPV4, AARS, BIC2, DHTKD1, FGD4, HK1, INF2, KIF5A, PDK3, REEP1, SBF1, SBF2, SCN9A, and SPTLC2 with a declining frequency. Thirty-four novel variants were considered likely pathogenic not having previously been described in association with any disorder in the literature. In one patient, two homozygous mutations in HK1 were detected in the multigene panel, but not by whole exome sequencing. A novel missense mutation in KIF5A was considered pathogenic because of the highly compatible phenotype. In one patient, the plasma sphingolipid profile could functionally prove the pathogenicity of a mutation in SPTLC2. One pathogenic mutation in MPZ was identified after being previously missed by Sanger sequencing. We conclude that panel based next generation sequencing is a useful, time- and cost-effective approach to assist clinicians in identifying the correct diagnosis and enable causative treatment considerations.

Novel POLG variants associated with late-onset de novo status epilepticus and progressive ataxia.

Mitochondrial disease is phenotypically and genetically heterogeneous with an estimated prevalence of 1 in 4,300.1 Mutations in the POLG gene, encoding the catalytic subunit of DNA polymerase gamma, are an important cause of mitochondrial disease. The spectrum of clinical manifestations in POLG-related mitochondrial disease is variable,2 with disease onset ranging from adulthood-onset dominant or recessive progressive external ophthalmoplegia (chronic progressive external ophthalmoplegia), ataxia-neuropathy spectrum, myoclonic epilepsy, myopathy, and sensory ataxia to childhood-onset Alpers syndrome, which is characterized by intractable seizures, psychomotor regression, and hepatic impairment. Epilepsy is a poor prognostic factor in POLG mutations,3 and the onset of epilepsy often clusters in childhood (<5 years) and teenage.4 However, late-onset epileptic encephalopathy is uncommon.4,5 Herein, we describe a patient who died of de novo, late-onset refractory status epilepticus with the identification of 2 novel variants in the POLG gene.

A.01 Targeted analysis of whole exome sequencing and genotype-phenotype correlation in epileptic encephalopathies

Background: Epileptic encephalopathy (EE) is a severe condition in which epileptic activity itself may contribute to severe cognitive and behavioural impairments above and beyond what might be expected from the underlying pathology alone. Next generation sequencing technologies such as whole exome sequencing (WES) can detect underlying genetic causes of in EE. Methods: This report describes genotype-phenotype correlation of 29 subjects with unexplained epileptic encephalopathy, in whom WES, targeting a list of 557 epilepsy-associated genes was performed. Epilepsy phenotyping was done according to current ILAE recommendations. Results: Median age at seizure onset was 14 months (range 1-48). Electroclinical syndromes were applicable for 16/29, 8/16 had a definite/likely diagnosis. 6/8 subjects with West syndrome had variants in ALG13, STXBP1, PAFAH1B1, SLC35A2, CDKL5 and ADSL. 2 patients with Dravet syndrome had variants in SCN1A and PCDH19 respectively. 4/29 had unspecified EE and definite/likely diagnosis due to STXBP1, POLG, and KCNQ2 (2) variants. 4/29 had a possible diagnosis involving GABRB3, ARHGEF9, PCDH19 and SCN3A variants. Conclusions: The high diagnostic yield (definite/likely diagnosis in 11/29 = 38%), involving a broad variety of epilepsy-associated genes in different electroclinical syndromes justifies the diagnostic approach of early onset EE by next generation sequencing.

Mitochondrial pathology in inclusion body myositis

Inclusion body myositis (IBM) is usually associated with a large number of cytochrome c oxidase (COX)-deficient muscle fibers and acquired mitochondrial DNA (mtDNA) deletions.We studied the number of COX-deficient fibers and the amount of mtDNA deletions, and if variants in nuclear genes involved in mtDNA maintenance may contribute to the occurrence of mtDNA deletions in IBM muscle.Twenty-six IBM patients were included. COX-deficient fibers were assayed by morphometry and mtDNA deletions by qPCR. POLG was analyzed in all patients by Sanger sequencing and C10orf2 (Twinkle), DNA2, MGME1, OPA1, POLG2, RRM2B, SLC25A4 and TYMP in six patients by next generation sequencing.Patients with many COX-deficient muscle fibers had a significantly higher proportion of mtDNA deletions than patients with few COX-deficient fibers. We found previously unreported variants in POLG and C10orf2 and IBM patients had a significantly higher frequency of an RRM2B variant than controls. POLG variants appeared more common in IBM patients with many COX-deficient fibers, but the difference was not statistically significant.We conclude that COX-deficient fibers in inclusion body myositis are associated with multiple mtDNA deletions. In IBM patients we found novel and also previously reported variants in genes of importance for mtDNA maintenance that warrants further studies.

52.: Patient derived stem cell models to investigate POLG related disease

POLG is a gene that encodes the catalytic subunit of DNA polymerase gamma (polγ). Polγ is responsible for mtDNA replication, and aberrant replication can lead to mitochondrial dysfunction and decreased adenosine triphosphate (ATP) production. Neuronal cell models were generated from a patient harbouring pathogenic POLG variants, p.T851A and novel p.P163S, to determine disturbances in mitochondrial function. Olfactory neurospheres (ONS) were generated from the patient, and mitochondrial function was analysed including mtDNA visualised using fluorescence microscopy, mtDNA/nDNA ratio (real time quantitative reverse transcription polymerase chain reaction) and ATP levels. Inducible pluripotent stem cell (iPSC) derived neurons were generated from the patient to see if a similar phenotype to the ONS cell model was expressed. POLG mutant ONS showed a mosaic pattern of mtDNA depletion over successive passages. Progressive mtDNA depletion was associated with reduced mitochondrial function. In control iPSC, mtDNA increased during iPSC differentiation into neurons, presumably to meet the need for the high metabolic demand of the neurons. However, the POLG mutant showed significantly decreased mtDNA repopulation levels during neural differentiation. This lagging of mtDNA levels seen in mutant versus control iPCS may be due to the pathogenic POLG variants affecting mtDNA replication, which was also shown in the ONS cell model. The POLG mutant ONS and iPSC derived neuronal cell models showed different mtDNA replication defects, given that ONS are actively dividing while the iPSC derived neurons are post-mitotic and do not divide. These data indicate that the pathogenic effects of the POLG variants p.T851A and p.P163S are related to mtDNA depletion and a failure of mtDNA replication during times of high metabolic demand. Non-dividing tissue such as neurons are vulnerable to mtDNA depletion and can be used for assessing POLG related disease.