Search

AIM To examine the epidemiological, clinical, and genetic characteristics of paediatric patients with genetic white matter disorders (GWMDs) in Northern Finland. METHOD A longitudinal population-based cohort study was conducted in the tertiary catchment area of Oulu University Hospital from 1990 to 2019. Patients were identified retrospectively by International Statistical Classification of Diseases and Related Health Problems codes in hospital records and prospectively by attending physicians. Inclusion criteria were children younger than 18 years with defined GWMDs or genetic disorders associated with white matter abnormalities (WMAs) on brain magnetic resonance imaging. RESULTS Eighty patients (mean age [SD] at the end of the study 11y [8y 6mo], range 0-35y; 45 males, 35 females) were diagnosed with a defined GWMD. The cumulative childhood incidence was 30 per 100 000 live births. Regarding those patients with 49 distinct GWMDs, 20% had classic leukodystrophies and 80% had genetic leukoencephalopathies. The most common leukodystrophies were cerebral adrenoleukodystrophy, Krabbe disease, and Salla disease. Additionally, 29 patients (36%) had genetic aetiologies not previously associated with brain WMAs or they had recently characterised GWMDs, including SAMD9L- and NHLRC2-related neurological disorders. Aetiology was mitochondrial in 21% of patients. The most common clinical findings were motor developmental delay, intellectual disability, hypotonia, and spasticity. INTERPRETATION The cumulative childhood incidence of childhood-onset GWMDs was higher than previously described. Comprehensive epidemiological and natural history data are needed before future clinical trials are undertaken. What this paper adds Forty-nine distinct genetic white matter disorders (GWMDs) were identified, with 20% of cases being classic leukodystrophies. The cumulative childhood incidence of GWMDs was higher than described previously. A considerable proportion (36%) of GWMDs were previously undefined or recently characterised GWMDs. Mitochondrial aetiology was more common (21%) than previously reported.

Objective To study the impact of gender, puberty, and pregnancy on the expression of POLG disease, one of the most common mitochondrial diseases known. Methods Clinical, laboratory, and genetic data were collected retrospectively from 155 patients with genetically confirmed POLG disease recruited from seven European countries. We used the available data to study the impact of gender, puberty, and pregnancy on disease onset and deterioration. Results We found that disease onset early in life was common in both sexes but there was also a second peak in females around the time of puberty. Further, pregnancy had a negative impact with 10 of 14 women (71%) experiencing disease onset or deterioration during pregnancy. Interpretation Gender clearly influences the expression of POLG disease. While onset very early in life was common in both males and females, puberty in females appeared associated both with disease onset and increased disease activity. Further, both disease onset and deterioration, including seizure aggravation and status epilepticus, appeared to be associated with pregnancy. Thus, whereas disease activity appears maximal early in life with no subsequent peaks in males, both menarche and pregnancy appear associated with disease onset or worsening in females. This suggests that hormonal changes may be a modulating factor. © 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) deficiency due to the homozygous PCK1 variant has recently been associated with childhood-onset hypoglycemia with a recognizable pattern of abnormal urine organic acids. In this study 21 children and three adult patients with genetically confirmed PEPCK-C deficiency were diagnosed during the years 2016-2019 and the available biochemical and clinical data were collected. All patients were ethnic Finns. Most patients (22 out of 24) had a previously published homozygous PCK1 variant c.925G>A. Two patients had a novel compound heterozygous PCK1 variant c.925G>A and c.716C>T. The laboratory results showed abnormal urine organic acid profile with increased tricarboxylic acid cycle intermediates and inadequate ketone body production during hypoglycemia. The hypoglycemic episodes manifested predominantly in the morning. Infections, fasting or poor food intake, heavy exercise, alcohol consumption and breastfeeding were identified as triggering factors. Five patients presented with neonatal hypoglycemia. Hypoglycemic seizures occurred in half of the patients (12 out of 24). The first hypoglycemic episode often occurred at the age of 1-2 years, but it sometimes presented at a later age, and could re-occur during school-age or adulthood. This study adds to the laboratory data on PEPCK-C deficiency, confirming the recognizable urine organic acid pattern and identifying deficient ketogenesis as a novel laboratory finding. The phenotype is expanded suggesting that the risk of hypoglycemia may continue into adulthood if predisposing factors are present. This article is protected by copyright. All rights reserved.

Purpose Next-generation sequencing (NGS) has made genetic testing of patients with epileptic encephalopathies easier – novel variants are discovered and new phenotypes described. Variants in the same gene – even the same variant – can cause different types of epilepsy and neurodevelopmental disorders. Our aim was to identify the genetic causes of epileptic encephalopathies in paediatric patients with complex phenotypes. Methods NGS was carried out for three patients with epileptic encephalopathies. Detailed clinical features, brain magnetic resonance imaging and electroencephalography were analysed. We searched the Human Gene Mutation Database for the published GABRG2 variants with clinical description of patients and composed a summary of the variants and their phenotypic features. Results We identified two novel de novo GABRG2 variants, p.P282T and p.S306F, with new phenotypes including neuroradiological evidence of neurodegeneration and epilepsy of infancy with migrating focal seizures (EIMFS). One patient carried previously reported p.P83S variant with autism spectrum disorder (ASD) phenotype that has not yet been described related to GABRG2 disorders and a more severe epilepsy phenotype than reported earlier. In all, the literature search yielded twenty-two articles describing 27 different variants that were divided into two categories: those with self-limiting epilepsies and febrile seizures and those with more severe drug-resistant epileptic encephalopathies. Conclusion This study further expands the genotypic and phenotypic spectrum of epilepsies associated with GABRG2 variants. More knowledge is still needed about the influence of the environment, genetic background and other epilepsy susceptibility genes on the phenotype of the specific GABRG2 variants.

BackgroundTranscriptomic and proteomic profiling of human brain tissue is hindered by availability of fresh samples from living patients. Postmortem samples usually represent the advanced disease stage of the patient. Furthermore, the postmortem interval affects the observed transcriptomic and proteomic profiles. Therefore, access to fresh brain tissue samples from living patients is valuable resource to obtain information on metabolically intact tissue. The implantation of deep brain stimulation (DBS) electrodes into the human brain is a neurosurgical treatment for, e.g., movement disorders. Here, we describe an improved approach to collect brain tissue from surgical instruments used in implantation of DBS device for transcriptomics and proteomics analyses.MethodsSamples were extracted from guide tubes and recording electrodes used in routine DBS implantation procedure that was carried out to treat patients with Parkinson’s disease, genetic dystonia and tremor. RNA sequencing was carried out to tissue extracted from the recording microelectrodes and liquid chromatography-mass spectrometry was carried out to analyze tissue from guide tubes. To assess the performance of the current approach, obtained datasets were compared with previously published datasets representing brain tissue.ResultsIn RNA sequencing, altogether 32,034 transcripts representing unique Ensembl gene identifiers were detected from eight samples representing both hemispheres of four patients. By using liquid chromatography-mass spectrometry, we identified 734 unique proteins from 31 samples collected from 14 patients. Comparison with previously published brain derived data indicated that both of our datasets reflected the expected brain tissue specific features. The datasets are available via BioStudies database (accession number S-BSST667).ConclusionsSurgical instruments used in DBS installation retain enough brain material for protein and gene expression studies. Analysis of the datasets indicated that hemisphere-specific expression data can be obtained from individual patients without any sample pooling and without any modifications to the standard surgical protocol. Comparison with previously published datasets obtained with similar approach proved the robustness and reproducibility of the current improved protocol. This approach overcomes the issues that arise from using postmortem tissue, such as effect of postmortem interval, on proteomic and transcriptomic landscape of the brain and can be used for studying molecular aspects of DBS-treatable diseases.

PURPOSE: To compare the value of serum biomarkers, FGF21 and GDF15 with histological analysis of muscle in the diagnosis of mitochondrial disease. METHODS: We collected 194 serum samples from patients with a suspected or known mitochondrial disease. Biomarkers were analyzed blinded using enzyme labelled immunosorbent assay (ELISA). Clinical data was collected using a structured questionnaire. RESULTS: Only 39% of patients with genetically verified mitochondrial disease had mitochondrial pathology in their muscle histology. In contrast, biomarkers were elevated in 62% of patients with genetically verified mitochondrial disease. Those with both biomarkers elevated had a muscle manifesting disorder and a defect affecting mitochondrial DNA expression. If at least one of the biomarkers was induced and the patient had a myopathic disease, a mitochondrial DNA expression disease was the cause with 94% probability. Among patients with biomarker analysis and muscle biopsy taken

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6‐months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt‐tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease., Heterozygous mutations in nuclear genes interacting with mt‐tRNAGlu induce the integrated stress response and metabolic rearrangements, reducing penetrance and promoting spontaneous recovery in a rare mitochondrial myopathy.

Background Variants in POLG are one of the most common causes of inherited mitochondrial disease. Phenotypic classification of POLG disease has evolved haphazardly making it complicated and difficult to implement in everyday clinical practise. The aim of our study was to simplify the classification and facilitate better clinical recognition. Methods A multinational, retrospective study using data from 155 patients with POLG variants recruited from seven European countries. Results We describe the spectrum of clinical features associated with POLG variants in the largest known cohort of patients. While clinical features clearly form a continuum, stratifying patients simply according to age of onset—onset prior to age 12 years; onset between 12 and 40 years and onset after the age of 40 years, permitted us to identify clear phenotypic and prognostic differences. Prior to 12 years of age, liver involvement (87%), seizures (84%), and feeding difficulties (84%) were the major features. For those with onset between 12 and 40 years, ataxia (90%), peripheral neuropathy (84%), and seizures (71%) predominated, while for those with onset over 40 years, ptosis (95%), progressive external ophthalmoplegia (89%), and ataxia (58%) were the major clinical features. The earlier the onset the worse the prognosis. Patients with epilepsy and those with compound heterozygous variants carried significantly worse prognosis. Conclusion Based on our data, we propose a simplified POLG disease classification, which can be used to guide diagnostic investigations and predict disease course. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Background FINCA disease is a pediatric cerebropulmonary disease caused by variants in the NHL repeat-containing 2 (NHLRC2) gene. Neurological symptoms are among the first manifestations of FINCA disease, but the consequences of NHLRC2 deficiency in the central nervous system are currently unexplored. Methods The orthologous mouse gene is essential for development, and its complete loss leads to early embryonic lethality. In the current study, we used CRISPR/Cas9 to generate an Nhlrc2 knockin (KI) mouse line, harboring the FINCA patient missense mutation (c.442G > T, p.Asp148Tyr). A FINCA mouse model, resembling the compound heterozygote genotype of FINCA patients, was obtained by crossing the KI and Nhlrc2 knockout mouse lines. To reveal NHLRC2-interacting proteins in developing neurons, we compared cortical neuronal precursor cells of E13.5 FINCA and wild-type mouse embryos by two-dimensional difference gel electrophoresis. Results Despite the significant decrease in NHLRC2, the mice did not develop severe early onset multiorgan disease in either sex. We discovered 19 altered proteins in FINCA neuronal precursor cells; several of which are involved in vesicular transport pathways and actin dynamics which have been previously reported in other cell types including human to have an association with dysfunctional NHLRC2. Interestingly, isoform C2 of hnRNP C1/C2 was significantly increased in both developing neurons and the hippocampus of adult female FINCA mice, connecting NHLRC2 dysfunction with accumulation of RNA binding protein. Conclusions We describe here the first NHLRC2-deficient mouse model to overcome embryonic lethality, enabling further studies on predisposing and causative mechanisms behind FINCA disease. Our novel findings suggest that disrupted RNA metabolism may contribute to the neurodegeneration observed in FINCA patients.

Objective Previous studies have suggested that heterozygous variants p.Q1236H and p.E1143G in mitochondrial DNA polymerase gamma (POLG1) increase the risk for liver injury for patients on valproate (VPA) therapy. We assessed the prevalence of these common variants and seven other pathogenic mutations in POLG1 and determined the occurrence of VPA-induced hepatotoxicity (VHT) or pancreatic toxicity in a cohort of patients with epilepsy. Methods Patients with epilepsy (N = 367) were retrospectively identified from medical record files and screened for mutations in POLG1. Patients who had received VPA monotherapy and carried either of the two variants, p.Q1236H or p.E1143G, without other pathogenic mutations in POLG1 (n = 33, variant group) and patients without these variants (n = 28, nonvariant group) were included in the study. Clinical data on epilepsy, characteristics of VPA treatment, risk factors for VHT, laboratory data on liver and pancreas functions, and adverse effects were collected. Results A total of 122 patients had either the POLG1 p.Q1236H (n = 99) or p.E1143G (n = 24) variant in the heterozygous or homozygous state. Transient liver dysfunction was identified in three (n = 33, 9.1%) variant group patients and in one (n = 28, 3.6%) nonvariant group patient (P = 0.62). Mild to moderate elevations in liver enzymes were encountered in both groups. Furthermore, two patients on VPA polytherapy developed acute pancreatitis, and two pediatric patients with heterozygous p.Q1236H variants and mutations in IQSEC2 and GLDC, respectively, had elevated levels of VPA metabolites in urine, elevated plasma glycine, and/or increased acylglycine excretion. Significance POLG1 p.Q1236H and p.E1143G variants could not be identified as statistically significant risk factors for VHT or pancreatic toxicity. We suggest that VPA treatment could be suitable for patients who harbor these common variants in the absence of other pathogenic mutations in POLG1.

BackgroundLeigh syndrome is a phenotypically and genetically heterogeneous mitochondrial disorder. While some genetic defects are associated with well-described phenotypes, phenotype-genotype correlations in Leigh syndrome are not fully explored.ObjectiveWe aimed to identify phenotype-genotype correlations in Leigh syndrome in a large cohort of systematically evaluated patients.MethodsWe studied 96 patients with genetically confirmed Leigh syndrome diagnosed and followed in eight European centres specialising in mitochondrial diseases.ResultsWe found that ataxia, ophthalmoplegia and cardiomyopathy were more prevalent among patients with mitochondrial DNA defects. Patients with mutations in MT-ND and NDUF genes with complex I deficiency shared common phenotypic features, such as early development of central nervous system disease, followed by high occurrence of cardiac and ocular manifestations. The cerebral cortex was affected in patients with NDUF mutations significantly more often than the rest of the cohort. Patients with the m.8993T>G mutation in MT-ATP6 gene had more severe clinical and radiological manifestations and poorer disease outcome compared with patients with the m.8993T>C mutation.ConclusionOur study provides new insights into phenotype-genotype correlations in Leigh syndrome and particularly in patients with complex I deficiency and with defects in the mitochondrial ATP synthase.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare inborn error of metabolism affecting both fatty acid and amino acid oxidation. It can manifest at any age, but riboflavin-responsiveness has mainly been described in less severely affected patients. We describe an infant with severe MADD presenting with profound hypotonia and hepatomegaly. Treatment with riboflavin improved his muscle strength, liver size, and biochemical markers. A homozygous mutation of electron transfer flavoprotein dehydrogenase (ETFDH) was found. His motor skills continued to progress until a fatal infection-triggered deterioration at the age of 34 months. We show changes in brain magnetic resonance imaging over the course of the disease, with profound white matter abnormalities during the deterioration phase. Aggregates of mitochondria with abnormal cristae in muscle electron microscopy were noticed already in infancy. An unusual lactate dehydrogenase (LDH) isoenzyme pattern with LDH-1 predominance was additionally observed. This case demonstrates riboflavin-responsiveness in a severely affected infant with both muscular and extramuscular involvement and further underlines the variable nature of this disease.

Alexander disease (AxD) is a genetic leukodystrophy caused by GFAP mutations leading to astrocyte dysfunction. Neonatal AxD is a rare phenotype with onset in the first month of life. The proband, belonging to a large pedigree with dominantly inherited benign familial neonatal epilepsy (BFNE), had a phenotype distinct from the rest of the family, with hypotonia and macrocephaly in addition to drug-resistant neonatal seizures. The patient deteriorated and passed away at 6 weeks of age. The pathological and neuroimaging data were consistent with the diagnosis of AxD. Genetic analysis of the proband identified a novel de novo GFAP missense mutation and a KCNQ2 splice site mutation segregating with the BFNE phenotype in the family. The GFAP mutation was located in the coil 2B region of GFAP protein, similar to most neonatal-onset AxD cases with an early death. The clinical and neuroradiological features of the previously published neonatal AxD patients are presented. This study further supports the classification of neonatal-onset AxD as a distinct phenotype based on the age of onset.

The development of tissue fibrosis is complex and at the present time, not fully understood. Fibrosis, neurodegeneration and cerebral angiomatosis (FINCA disease) have been described in patients with mutations in NHL repeat-containing protein 2 (NHLRC2). However, the molecular functions of NHLRC2 are uncharacterized. Herein, we identified putative interacting partners for NHLRC2 using proximity-labeling mass spectrometry. We also investigated the function of NHLRC2 using immortalized cells cultured from skin biopsies of FINCA patients and normal fibroblasts with NHLRC2 knock-down and NHLRC2 overexpressing gene modifications. Transmission electron microscopy analysis of immortalized cell cultures from three FINCA patients demonstrated multilamellar bodies and distinctly organized vimentin filaments. Additionally, two of three cultures derived from patient skin biopsies contained cells that exhibited features characteristic of myofibroblasts. Altogether, the data presented in this study show for the first time that NHLRC2 is involved in cellular organization through regulation of the cytoskeleton and vesicle transport. We conclude that compound heterozygous p.Asp148Tyr and p.Arg201GlyfsTer6 mutations in NHLRC2 lead to severe tissue fibrosis in humans by enhancing the differentiation of fibroblasts to myofibroblasts.

A novel multi-organ disease that is fatal in early childhood was identified in three patients from two non-consanguineous families. These children were born asymptomatic but at the age of 2 months they manifested progressive multi-organ symptoms resembling no previously known disease. The main clinical features included progressive cerebropulmonary symptoms, malabsorption, progressive growth failure, recurrent infections, chronic haemolytic anaemia and transient liver dysfunction. In the affected children, neuropathology revealed increased angiomatosis-like leptomeningeal, cortical and superficial white matter vascularisation and congestion, vacuolar degeneration and myelin loss in white matter, as well as neuronal degeneration. Interstitial fibrosis and previously undescribed granuloma-like lesions were observed in the lungs. Hepatomegaly, steatosis and collagen accumulation were detected in the liver. A whole-exome sequencing of the two unrelated families with the affected children revealed the transmission of two heterozygous variants in the NHL repeat-containing protein 2 (NHLRC2); an amino acid substitution p.Asp148Tyr and a frameshift 2-bp deletion p.Arg201GlyfsTer6. NHLRC2 is highly conserved and expressed in multiple organs and its function is unknown. It contains a thioredoxin-like domain; however, an insulin turbidity assay on human recombinant NHLRC2 showed no thioredoxin activity. In patient-derived fibroblasts, NHLRC2 levels were low, and only p.Asp148Tyr was expressed. Therefore, the allele with the frameshift deletion is likely non-functional. Development of the Nhlrc2 null mouse strain stalled before the morula stage. Morpholino knockdown of nhlrc2 in zebrafish embryos affected the integrity of cells in the midbrain region. This is the first description of a fatal, early-onset disease; we have named it FINCA disease based on the combination of pathological features that include fibrosis, neurodegeneration, and cerebral angiomatosis.

Background Methionine synthase deficiency is a rare inborn error of intracellular cobalamin metabolism caused by mutations in the MTR (5-methyltetrahydrofolate-homocysteine S-methyltransferase) gene, resulting in megaloblastic anemia and neurologic symptoms. Methods and Results We describe for the first time a homozygous MTR gene c.3518C > T (p.P1173L) mutation in a patient with severe megaloblastic anemia, developmental delay, and drug-resistant seizures associated with hyperhomocysteinemia and hypomethioninemia. Methionine synthase activity was only 9% of the reference value, and MTR protein expression was decreased in the fibroblasts of the patient. The clinical features of our patient are similar to previously published patients with the complementation type G disorder of methionine synthase deficiency with the exception of drug-resistant seizures. However, intramuscular injections of hydroxocobalamin (OHCbl) in conjunction with betaine and folic acid provided verified clinical and electrophysiological treatment response. Conclusion This study emphasizes the importance of early diagnosis of patients having neurologic symptoms due to methionine synthase deficiency where early treatment has significant effects on the clinical outcome of the patients. Elevated level of plasma homocysteine together with low methionine in plasma amino acid analysis should raise a suspicion of remethylation disorder.

Clinical and laboratory data were collected from three Finnish patients including a sibling pair and another unrelated child with unexplained childhood hypoglycemia. Transient elevation of alanine transaminase, lactate and tricarboxylic acid cycle intermediates, especially fumarate, were noticed in urine organic acid analysis. Exome sequencing was performed for the patients and their parents. A novel homozygous PCK1 c.925G>A (p.G309R) mutation was detected in all affected individuals. COS-1 cells transfected with mutant PCK1 transcripts were used to study the pathogenic nature of the detected variant. The COS-1 transfected cells showed the mutant gene to be incapable of producing a normally functioning cytosolic phosphoenolpyruvate carboxykinase (PEPCK) enzyme. This report further delineates the clinical phenotype of isolated cytosolic PEPCK deficiency and offers a metabolic pattern helping to recognize these patients. Cytosolic PEPCK deficiency should be considered in the differential diagnosis of children presenting with hypoglycemia, hepatic dysfunction and elevated tricarboxylic acid intermediates in urinary organic acid analysis.

Patients with mutations in the POLG1 gene encoding mitochondrial DNA polymerase gamma have an increased risk of valproate-induced liver failure. POLG1 mutations are common, and these patients often suffer from intractable seizures. The role of liver transplantation in the treatment of patients with mitochondrial diseases has been controversial. We studied valproate-induced liver failure associated with POLG1 mutations and the prognosis for these patients after liver transplantation. POLG1 was analyzed in blood DNA, mitochondrial DNA (mtDNA) was quantified in liver samples, and clinical data were collected. Five patients with valproate-induced liver failure associated with POLG1 mutations were retrospectively identified. Three patients were previously suspected to have Wilson's disease. Four patients with homozygous p.W748S and p.E1143G mutations had mtDNA depletion in the liver. One of these patients died before anticipated transplantation; the other 3 patients with liver transplantation have survived 4 to 19 years. Two patients have presented with occasional epileptic seizures, and 1 patient has been seizure-free for 11 years. One patient with a heterozygous p.Q1236H mutation (but without mtDNA depletion in the liver) died suddenly 2 years after liver transplantation. In conclusion, the POLG1 mutation status and the age at presentation of valproate-induced liver failure can affect the prognosis after liver transplantation. A heterozygous POLG1 p.Q1236H mutation was related to valproate-induced liver failure without mtDNA depletion, whereas patients homozygous for POLG1 p.W748S and p.E1143G mutations had mtDNA depletion. An analysis of the POLG1 gene should be performed for all patients with suspected mitochondrial disease before the introduction of valproate therapy, and treatment with valproic acid should be avoided in these patients. Liver Transpl 20:1402–1412, 2014. © 2014 AASLD.

Epilepsy has both genetic and environmental causes. Recently, Ottman reported on more than 20 genes thought to be associated with idiopathic epilepsy (Ottman et al., 2010), but still these are relevant for only a small proportion of patients, and few patients have access to DNA diagnostics. This is unfortunate because genetic testing may clarify a diagnosis and allow better genetic counseling, help to optimize medication-avoiding side effects, and reduce the need for further investigation (Delgado-Escueta & Bourgeois, 2008). The term “intractable” epilepsy has been generally used when epilepsy is difficult to treat and there has been failure of two or more first-line antiseizure medications. Instead of “intractable epilepsy,” the ILAE (International League Against Epilepsy) has recently recommended the phrase “drug resistant epilepsy” to describe the condition where two tolerated and appropriately chosen antiepileptic drugs have failed to achieve sustained freedom from seizures, with no mention of the frequency of seizures (Kwan et al., 2010). Intractable epilepsy can be associated with genetic and chromosomal abnormalities, cortical malformations, congenital and acquired central nervous system (CNS) infections, inborn errors of metabolism, hypoxic-ischemic injury, and neoplasm but in many cases it is of unknown etiology. Several mitochondrial diseases caused by mutations in either mitochondrial or nuclear genes are characterized by “intractable” epilepsy as one of the presenting features. Epilepsy associated with mitochondrial diseases can manifest as infantile spasms; astatic convulsions; myoclonic, focal, or generalized seizures; or as epilepsia partialis continua (Canafoglia et al., 2001). It has clearly been shown that POLG encoding the catalytic α-subunit of mitochondrial DNA polymerase gamma, is one of the genes causing epilepsy. Patients carrying this gene frequently have epilepsy in addition to numerous other neurologic manifestations including ophthalmoplegia and ataxia (Rantamaki et al., 2001; Van Goethem et al., 2001; Lamantea et al., 2002; Van Goethem et al., 2003; Hakonen et al., 2005) and Alpers' disease (OMIM 203700), which is characterized by intractable seizures, episodic progression of neurologic symptoms, liver failure, and pharmacogenetic sensitivity to valproic acid (VPA) toxicity (Harding et al., 1995; Ferrari et al., 2005; Nguyen et al., 2005; Tzoulis et al., 2006; Uusimaa et al., 2008). The most common mutations in the POLG gene are p. A467T, p.W748S, and p.G848S with carrier frequencies up to 1% in some populations (Hakonen et al., 2005; Winterthun et al., 2005; Kollberg et al., 2006). It has been shown that VPA should not to be used to treat patients with mitochondrial disease, particularly because patients with POLG mutations are at increased risk for VPA-induced liver failure (Ferrari et al., 2005; Gordon, 2006; Uusimaa et al., 2008; Stewart et al., 2009). The aim of this study was to assess prospectively the prevalence of the three most common POLG mutations in a defined population of children with nonsyndromic intractable epilepsy, but without liver manifestation typical for Alpers disease at the presentation of their epilepsy. We have previously reported POLG findings in a group of children, most of whom had typical clinical features of Alpers disease (Ashley et al., 2008). We also retrospectively reviewed the notes of children with a presentation of intractable epilepsy and one of the common POLG mutations who did not have classic Alpers syndrome, because liver dysfunction was absent, and report on the clinical features and laboratory findings of these patients. This study is significant for understanding and treating patients with epilepsy, because POLG gene mutations have a high prevalence and are hence a potentially important cause of severe intractable epilepsy. Our findings further expand the clinical phenotypes associated with POLG mutations. This is very important for clinical diagnostics, genetic counseling, and treatment decisions because of the increased risk for VPA-induced liver failure in patients with POLG mutations.

Several monogenic causes of familial myelodysplastic syndrome (MDS) have recently been identified. We studied 2 families with cytopenia, predisposition to MDS with chromosome 7 aberrations, immunodeficiency, and progressive cerebellar dysfunction. Genetic studies uncovered heterozygous missense mutations in SAMD9L, a tumor suppressor gene located on chromosome arm 7q. Consistent with a gain-of-function effect, ectopic expression of the 2 identified SAMD9L mutants decreased cell proliferation relative to wild-type protein. Of the 10 individuals identified who were heterozygous for either SAMD9L mutation, 3 developed MDS upon loss of the mutated SAMD9L allele following intracellular infections associated with myeloid, B-, and natural killer (NK)-cell deficiency. Five other individuals, 3 with spontaneously resolved cytopenic episodes in infancy, harbored hematopoietic revertant mosaicism by uniparental disomy of 7q, with loss of the mutated allele or additional in cisSAMD9L truncating mutations. Examination of 1 individual indicated that somatic reversions were postnatally selected. Somatic mutations were tracked to CD34+ hematopoietic progenitor cell populations, being further enriched in B and NK cells. Stimulation of these cell types with interferon (IFN)-α or IFN-γ induced SAMD9L expression. Clinically, revertant mosaicism was associated with milder disease, yet neurological manifestations persisted in 3 individuals. Two carriers also harbored a rare, in trans germ line SAMD9L missense loss-of-function variant, potentially counteracting the SAMD9L mutation. Our results demonstrate that gain-of-function mutations in the tumor suppressor SAMD9L cause cytopenia, immunodeficiency, variable neurological presentation, and predisposition to MDS with -7/del(7q), whereas hematopoietic revertant mosaicism commonly ameliorated clinical manifestations. The findings suggest a role for SAMD9L in regulating IFN-driven, demand-adapted hematopoiesis.

Deficits in the basal ganglia pathways modulating cortical motor activity underlie both Parkinson disease (PD) and Huntington disease (HD). Phosphodiesterase 10A (PDE10A) is enriched in the striatum, and animal data suggest that it is a key regulator of this circuitry. Here, we report on germline PDE10A mutations in eight individuals from two families affected by a hyperkinetic movement disorder due to homozygous mutations c.320A>G (p.Tyr107Cys) and c.346G>C (p.Ala116Pro). Both mutations lead to a reduction in PDE10A levels in recombinant cellular systems, and critically, positron-emission-tomography (PET) studies with a specific PDE10A ligand confirmed that the p.Tyr107Cys variant also reduced striatal PDE10A levels in one of the affected individuals. A knock-in mouse model carrying the homologous p.Tyr97Cys variant had decreased striatal PDE10A and also displayed motor abnormalities. Striatal preparations from this animal had an impaired capacity to degrade cyclic adenosine monophosphate (cAMP) and a blunted pharmacological response to PDE10A inhibitors. These observations highlight the critical role of PDE10A in motor control across species.

Purpose: Leber hereditary optic neuropathy (LHON) is regarded as the most common mitochondrial disease. We have previously reported comprehensive population-based epidemiological data on common mitochondrial DNA (mtDNA) mutations including m.3243A>G, m.8344A>G and large-scale mtDNA deletions in Northern Finland. Our aim was to investigate the prevalence of primary LHON mutations and mutations in the four mtDNA genes considered hot spots for LHON in the same population. Methods: The study population consisted of 42 adult patients with an aetiologically undefined bilateral optic atrophy. The major LHON mutations m.3460G>A, m.11778G>A and m.14484T>C were analysed by restriction fragment length polymorphism (RFLP), and MTND1, MTND6 and MTATP6 genes were sequenced. MTND5 gene was analysed by conformation-sensitive gel electrophoresis (CSGE). Results: No major LHON mutations were found in the population of the province of Northern Ostrobothnia giving the prevalence of these mutations 0–1.36:100 000 (95% CI). However, two main mutations were found elsewhere in Northern Finland, homoplasmic m.11778G>A from Kainuu and heteroplasmic m.3460G>A from Central Ostrobothnia. Furthermore, tobacco-alcohol amblyopia was diagnosed in five patients in the study population and one of them had m.11778G>A. Conclusion: The prevalence of the three major LHON mutations is lower in Northern Finland than elsewhere in Finland or in Western Europe. As LHON and tobacco-alcohol amblyopia have a similar phenotype, we recommend analysing the known LHON-associated mutations before setting tobacco-alcohol amblyopia diagnosis.

Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding the mitochondrial phenylalanyl transfer RNA (tRNA) synthetase (mtPheRS) in two patients with fatal epileptic mitochondrial encephalopathy. The mutations affected highly conserved amino acids, p.I329T and p.D391V. Recently, a homozygous FARS2 variant p.Y144C was reported in a Saudi girl with mitochondrial encephalopathy, but the pathogenic role of the variant remained open. Clinical features, including postnatal onset, catastrophic epilepsy, lactic acidemia, early lethality and neuroimaging findings of the patients with FARS2 variants, resembled each other closely, and neuropathology was consistent with Alpers syndrome. Our structural analysis of mtPheRS predicted that p.I329T weakened ATP binding in the aminoacylation domain, and in vitro studies with recombinant mutant protein showed decreased affinity of this variant to ATP. Furthermore, p.D391V and p.Y144C were predicted to disrupt synthetase function by interrupting the rotation of the tRNA anticodon stem-binding domain from a closed to an open form. In vitro characterization indicated reduced affinity of p.D391V mutant protein to phenylalanine, whereas p.Y144C disrupted tRNA binding. The stability of p.I329T and p.D391V mutants in a refolding assay was impaired. Our results imply that the three FARS2 mutations directly impair aminoacylation function and stability of mtPheRS, leading to a decrease in overall tRNA charging capacity. This study establishes a new genetic cause of infantile mitochondrial Alpers encephalopathy and reports a new mitochondrial aminoacyl-tRNA synthetase as a cause of mitochondrial disease.

Defects in complex I due to mutations in mitochondrial DNA are associated with clinical features ranging from single organ manifestation like Leber hereditary optic neuropathy (LHON) to multiorgan disorders like mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Specific mutations cause overlap syndromes combining several phenotypes, but the mechanisms of their biochemical effects are largely unknown. The m.3376G>A transition leading to p.E24K substitution in ND1 with LHON/MELAS phenotype was modeled here in a homologous position (NuoH-E36K) in the Escherichia coli enzyme and it almost totally abolished complex I activity. The more conservative mutation NuoH-E36Q resulted in higher apparent Km for ubiquinone and diminished inhibitor sensitivity. A NuoH homolog of the m.3865A>G transition, which has been found concomitantly in the overlap syndrome patient with the m.3376G>A, had only a minor effect. Consequences of a primary LHON-mutation m.3460G>A affecting the same extramembrane loop as the m.3376G>A substitution were also studied in the E. coli model and were found to be mild. The results indicate that the overlap syndrome-associated m.3376G>A transition in MTND1 is the pathogenic mutation and m.3865A>G transition has minor, if any, effect on presentation of the disease. The kinetic effects of the NuoH-E36Q mutation suggest its proximity to the putative ubiquinone binding domain in 49kD/PSST subunits. In all, m.3376G>A perturbs ubiquinone binding, a phenomenon found in LHON, and decreases the activity of fully assembled complex I as in MELAS.

Objective: Mitochondrial DNA polymerase γ (POLG1) mutations in children often manifest as Alpers syndrome, whereas in adults, a common manifestation is mitochondrial recessive ataxia syndrome (MIRAS) with severe epilepsy. Because some patients with MIRAS have presented with ataxia or epilepsy already in childhood, we searched for POLG1 mutations in neurologic manifestations in childhood. Methods: We investigated POLG1 in 136 children, all clinically suspected to have mitochondrial disease, with one or more of the following: ataxia, axonal neuropathy, severe epilepsy without known epilepsy syndrome, epileptic encephalopathy, encephalohepatopathy, or neuropathologically verified Alpers syndrome. Results: Seven patients had POLG1 mutations, and all of them had severe encephalopathy with intractable epilepsy. Four patients had died after exposure to sodium valproate. Brain MRI showed parieto-occipital or thalamic hyperintense lesions, white matter abnormality, and atrophy. Muscle histology and mitochondrial biochemistry results were normal in all. Conclusions: POLG1 analysis should belong to the first-line DNA diagnostic tests for children with an encephalitis-like presentation evolving into epileptic encephalopathy with liver involvement (Alpers syndrome), even if brain MRI and morphology, respiratory chain activities, and the amount of mitochondrial DNA in the skeletal muscle are normal. POLG1 analysis should precede valproate therapy in pediatric patients with a typical phenotype. However, POLG1 is not a common cause of isolated epilepsy or ataxia in childhood.

Contains fulltext : 95893.pdf (Publisher’s version ) (Closed access) BACKGROUND: Muscle biopsy is the gold standard for diagnosis of mitochondrial disorders because of the lack of sensitive biomarkers in serum. Fibroblast growth factor 21 (FGF-21) is a growth factor with regulatory roles in lipid metabolism and the starvation response, and concentrations are raised in skeletal muscle and serum in mice with mitochondrial respiratory chain deficiencies. We investigated in a retrospective diagnostic study whether FGF-21 could be a biomarker for human mitochondrial disorders. METHODS: We assessed samples from adults and children with mitochondrial disorders or non-mitochondrial neurological disorders (disease controls) from seven study centres in Europe and the USA, and recruited healthy volunteers (healthy controls), matched for age where possible, from the same centres. We used ELISA to measure FGF-21 concentrations in serum or plasma samples (abnormal values were defined as >200 pg/mL). We compared these concentrations with values for lactate, pyruvate, lactate-to-pyruvate ratio, and creatine kinase in serum or plasma and calculated sensitivity, specificity, and positive and negative predictive values for all biomarkers. FINDINGS: We analysed serum or plasma from 67 patients (41 adults and 26 children) with mitochondrial disorders, 34 disease controls (22 adults and 12 children), and 74 healthy controls. Mean FGF-21 concentrations in serum were 820 (SD 1151) pg/mL in adult and 1983 (1550) pg/mL in child patients with respiratory chain deficiencies and 76 (58) pg/mL in healthy controls. FGF-21 concentrations were high in patients with mitochondrial disorders affecting skeletal muscle but not in disease controls, including those with dystrophies. In patients with abnormal FGF-21 concentrations in serum, the odds ratio of having a muscle-manifesting mitochondrial disease was 132.0 (95% CI 38.7-450.3). For the identification of muscle-manifesting mitochondrial disease, the sensitivity was 92.3% (95% CI 81.5-97.9%) and specificity was 91.7% (84.8-96.1%). The positive and negative predictive values for FGF-21 were 84.2% (95% CI 72.1-92.5%) and 96.1 (90.4-98.9%). The accuracy of FGF-21 to correctly identify muscle-manifesting respiratory chain disorders was better than that for all conventional biomarkers. The area under the receiver-operating-characteristic curve for FGF-21 was 0.95; by comparison, the values for other biomarkers were 0.83 lactate (p=0.037, 0.83 for pyruvate (p=0.015), 0.72 for the lactate-to-pyruvate ratio (p=0.0002), and 0.77 for creatine kinase (p=0.013). INTERPRETATION: Measurement of FGF-21 concentrations in serum identified primary muscle-manifesting respiratory chain deficiencies in adults and children and might be feasible as a first-line diagnostic test for these disorders to reduce the need for muscle biopsy. FUNDING: Sigrid Juselius Foundation, Jane and Aatos Erkko Foundation, Molecular Medicine Institute of Finland, University of Helsinki, Helsinki University Central Hospital, Academy of Finland, Novo Nordisk, Arvo and Lea Ylppo Foundation. 01 september 2011

Summary Purpose: Polymerase gamma (POLG) is the sole enzyme in the replication of mitochondrial DNA (mtDNA). Numerous mutations in the POLG1 gene have been detected recently in patients with various phenotypes including a classic infantile-onset Alpers-Huttenlocher syndrome (AHS). Here we studied the molecular etiology of juvenile-onset AHS manifesting with status epilepticus and liver disease in three teenagers. Patients and Methods: We examined 14- and 17-year-old female siblings (patients 1 and 2) and an unrelated 15-year-old girl (patient 3) with juvenile-onset AHS, sequenced POLG1, and the entire mtDNA, examined mtDNA deletions by amplification of the full-length mtDNA with the long PCR method and used real-time PCR to quantify mtDNA in the tissue samples. Results: The initial manifestations were migraine-like headache and epilepsy, and the terminal manifestations status epilepticus and hepatic failure. A homozygous W748S mutation in POLG1 was detected in the three patients. No deletions or pathogenic point mutations were found in mtDNA, but all three patients had mtDNA depletion. Conclusions: POLG mutations should be considered in cases of teenagers and young adults with a sudden onset of intractable seizures or status epilepticus, and acute liver failure. The W748S POLG1 mutation seems to lead to tissue-specific, partial mtDNA depletion in patients with juvenile-onset Alpers syndrome. Valproic acid should be avoided in the treatment of epileptic seizures in these patients.

Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity.

The ND1 subunit gene of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a hot spot for mutations causing Leber hereditary optic neuropathy and several mutations causing the mitochondrial encephalopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). We have used Escherichia coli and Paracoccus denitrificans as model systems to study the effect of mutations 3946 and 3949, which change conserved residues in ND1 and cause MELAS. The vicinity of these mutations was also explored with a series of mutations in charged residues. The 3946 mutation results in E214K substitution in human ND1. Replacement of the equivalent residue in E. coli with lysine or glutamine detracted from enzyme assembly and the assembled enzyme was inactive. However, the equivalent E234Q mutant enzyme in P. denitrificans failed to assemble completely (or was rapidly degraded). Also the corresponding substitution with aspartate decreased the enzyme activity in P. denitrificans and E. coli. The 3949-equivalent substitution, Y229H in E. coli, lowered the catalytic activity by 30%. In addition, an activation of the enzyme during catalytic turnover was seen in this bacterial NDH-1, something that was even more pronounced in another mutant in the same loop, D213E. Several other mutations in this region decreased the enzyme activity. The studied MELAS mutations are situated in a matrix-side loop, which appears to be highly sensitive to structural perturbations. The results provide new information on the function of the region affected by the MELAS mutations 3946 and 3949 that is not obtainable from patient samples or current eukaryote models.

Objective:We describe the neurologic, neuroradiologic, and ophthalmologic phenotype of 1 Swedish and 1 Finnish family with autosomal dominant ataxia-pancytopenia (ATXPC) syndrome and SAMD9L mutations.Methods:Members of these families with germline SAMD9L c.2956C>T, p.Arg986Cys, or c.2672T>C, p.Ile891Thr mutations underwent structured interviews and neurologic and ophthalmologic examinations. Neuroimaging was performed, and medical records were reviewed. Previous publications on SAMD9L-ATXPC were reviewed.Results:Twelve individuals in both families were affected clinically. All mutation carriers examined had balance impairment, although severity was very variable. All but 1 had nystagmus, and all but 1 had pyramidal tract signs. Neurologic features were generally present from childhood on and progressed slowly. Two adult patients, who experienced increasing clumsiness, glare, and difficulties with gaze fixation, had paracentral retinal dysfunction verified by multifocal electroretinography. Brain MRI showed early, marked cerebellar atrophy in most carriers and variable cerebral periventricular white matter T2 hyperintensities. Two children were treated with hematopoietic stem cell transplantation for hematologic malignancies, and the neurologic symptoms of one of these worsened after treatment. Three affected individuals had attention deficit hyperactivity disorder or cognitive problems. Retinal dysfunction was not previously reported in individuals with ATXPC.Conclusions:The neurologic phenotype of this syndrome is defined by balance or gait impairment, nystagmus, hyperreflexia in the lower limbs and, frequently, marked cerebellar atrophy. Paracentral retinal dysfunction may contribute to glare, reading problems, and clumsiness. Timely diagnosis of ATXPC is important to address the risk for severe hemorrhage, infection, and hematologic malignancies inherent in this syndrome; regular hematologic follow-up might be beneficial.

Purpose To investigate the association of mutations in the mitochondrial DNA (mtDNA) or nuclear candidate genes with mitochondrial disease-related ophthalmic manifestations (nystagmus, ptosis, ophthalmoplegia, optic neuropathy and retinopathy) in children. Methods A retrospective cohort of children (n = 98) was identified from the medical record files of a tertiary care hospital. The entire mtDNA and nuclear genes POLG1, OPA1 and PEO1 were analysed from the available DNA samples (n = 38). Furthermore, some nuclear candidate genes were investigated based on family history and phenotype. Rare mtDNA mutations were evaluated using in silico predictors and sequence alignment. Results Three patients had previously identified mutations in mtDNA that are associated with optic neuropathy (in MT-ND6 and MT-ND1) and nystagmus (in tRNA Arg). Nine rare mutations in MT-ATP6 were identified in seven patients, of whom four manifested with retinopathy and three had clusters of MT-ATP6 mutations. Nuclear PEO1 and OPA1 were unchanged in all samples, but a patient with nystagmus had a heterozygous POLG1 mutation. The analysis of nuclear candidate genes revealed mutations in NDUF8 (patient with nystagmus), TULP1 (patient with optic neuropathy, nystagmus and retinopathy) and RP2 (patient with retinopathy) genes. Conclusions Children with retinopathy, nystagmus or optic neuropathy, especially together with developmental delay or positive family history, should be considered for mitochondrial disease. MT-ATP6 should be taken into account for children with retinopathy of unknown aetiology.

To study the clinical manifestations and occurrence of mtDNA depletion and deletions in paediatric patients with neuromuscular diseases and to identify novel clinical phenotypes associated with mtDNA depletion or deletions.Muscle DNA samples from patients presenting with undefined encephalomyopathies or myopathies were analysed for mtDNA content by quantitative real-time PCR and for deletions by long-range PCR. Direct sequencing of mtDNA maintenance genes and whole-exome sequencing were used to study the genetic aetiologies of the diseases. Clinical and laboratory findings were collected.Muscle samples were obtained from 104 paediatric patients with neuromuscular diseases. mtDNA depletion was found in three patients with severe early-onset encephalomyopathy or myopathy. Two of these patients presented with novel types of mitochondrial DNA depletion syndromes associated with increased serum creatine kinase (CK) and multiorgan disease without mutations in any of the known mtDNA maintenance genes; one patient had pathologic endoplasmic reticulum (ER) membranes in muscle. The third patient with mtDNA depletion was diagnosed with merosine-deficient muscular dystrophy caused by a homozygous mutation in the LAMA2 gene. Two patients with an early-onset Kearns-Sayre/Pearson-like phenotype harboured a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content.Novel encephalomyopathic mtDNA depletion syndrome with structural alterations in muscle ER was identified. mtDNA depletion may also refer to secondary mitochondrial changes related to muscular dystrophy. We suggest that a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content associated with Kearns-Sayre/Pearson syndromes may be secondary changes caused by mutations in an unknown nuclear gene.

Background: Leigh syndrome is a progressive neurodegenerative disorder, associated with primary or secondary dysfunction of the mitochondrial oxidative phosphorylation. Despite the fact that Leigh syndrome is the most common phenotype of mitochondrial disorders in children, longitudinal natural history data is missing. This study was undertaken to assess the phenotypic and genotypic spectrum of patients with Leigh syndrome, characterise the clinical course and identify predictors of survival in a large cohort of patients. Methods: This is a retrospective study of patients with Leigh syndrome that have been followed at eight centers specialising in mitochondrial diseases in Europe; Gothenburg, Rotterdam, Helsinki, Copenhagen, Stockholm, Brussels, Bergen and Oulu. Results: A total of 130 patients were included (78 males; 52 females), of whom 77 patients had identified pathogenic mutations. The median age of disease onset was 7 months, with 80.8% of patients presenting by the age of 2 years. The most common clinical features were abnormal motor findings, followed by abnormal ocular findings. Epileptic seizures were reported in 40% of patients. Approximately 44% of patients experienced acute exacerbations requiring hospitalisation during the previous year, mainly due to infections. The presence of pathological signs at birth and a history of epileptic seizures were associated with higher occurrence of acute exacerbations and/or relapses. Increased lactate in the cerebrospinal fluid was significantly correlated to a more severe disease course, characterised by early onset before 6 months of age, acute exacerbations and/or relapses, as well as brainstem involvement. 39% of patients had died by the age of 21 years, at a median age of 2.4 years. Disease onset before 6 months of age, failure to thrive, brainstem lesions on neuroimaging and intensive care treatment were significantly associated with poorer survival. Conclusions: This is a multicenter study performed in a large cohort of patients with Leigh syndrome. Our data help define the natural history of Leigh syndrome and identify novel predictors of disease severity and long-term prognosis. publishedVersion

Objectives. To assess the frequency of mitochondrial abnormalities in muscle histology, defects in respiratory chain enzyme activities, and mutations in mitochondrial DNA (mtDNA) in children with unexplained psychomotor retardation in the population of Northern Finland. Background. The frequency of mitochondrial diseases among patients with childhood encephalopathies and myopathies is not known. Frequencies are difficult to estimate because the clinical presentation of these disorders is variable. Methods. A total of 116 consecutive patients with undefined encephalopathies and myopathies were enrolled during a 7-year period in a hospital serving as the only neurologic unit for a pediatric population of 97 609 and as the only tertiary level neurologic unit for a pediatric population of 48 873. Biochemical and morphologic investigations were performed on muscle biopsy material, including oximetric and spectrophotometric analyses of oxidative phosphorylation, histochemistry, electron microscopy, and molecular analysis of mtDNA. Results. Ultrastructural changes in the mitochondria were the most common finding in the muscle biopsies (71%). Ragged-red fibers were found in 4 cases. An oxidative phosphorylation defect was found in 26 children (28%), complex I (n = 15) and complex IV (n = 13) defects being the most common. Fifteen percent of patients (n = 17/116) with unexplained encephalomyopathy or myopathy had a probable mitochondrial disease. Common pathogenic mutations were found in the mtDNA of only 1 patient (.9%). Conclusions. The common known mutations in mtDNA are rarely causes of childhood encephalomyopathies, which is in contrast to the considerable frequency of the common MELAS mutation observed among adults in the same geographical area. Biochemically and morphologically verified mitochondrial disorders were nevertheless common among the children, making the analysis of a muscle biopsy very important for clinical diagnostic purposes.

Parkinsonism has been described in patients with mutations in POLG1 gene. The W748S mutation is one of the most common mutations in this gene and it has been found to be a frequent cause of autosomal recessive ataxia in adults and the Alpers syndrome in children. We found the W748S mutation in a 65-year-old man with a late-onset syndrome consisting of ataxia, parkinsonism, ophthalmoplegia, peripheral neuropathy, and sensorineural hearing loss. Parkinsonism is one of the phenotypic features associated also with the W748S mutation.

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic diseases characterized by impaired hematopoiesis and progression to acute myeloid leukemia (AML). Although rare, several monogenic causes of familial MDS/AML have recently been molecularly defined. We studied two families with variable manifestation of cytopenia, MDS with cytogenetic aberrations involving chromosome 7, immunodeficiency, and neurologic disease consistent with ataxia-pancytopenia syndrome. Genetic studies uncovered heterozygous missense variants (p.Arg986Cys and p.Ile891Thr) in SAMD9L, a tumor suppressor gene located on chromosome 7. Consistent with a gain-of-function effect, transfection and over-expression of both SAMD9L variants decreased cell proliferation relative to wild-type protein. In the two families, a total of 10 individuals heterozygous for either SAMD9L mutation were identified. Three individuals developed MDS, with monosomy 7 or der(1;7)(q10;p10) as cytogenetic aberrations that encompassed the mutant SAMD9L locus. In an additional five individuals, three of which experienced a spontaneously resolved cytopenic episodes in infancy, we detected mosaic copy-neutral loss of heterozygosity of 7q by uniparental disomy, with loss of the mutated allele, or mosaic cis SAMD9L mutations. By digital PCR, we identified these events in hematopoietic progenitor cell populations, which were further enriched in B and NK cell lineages. Absent in non hematological tissues, these mutations thus represented somatic revertant mosaicism. Clinically, revertant mosaicism was associated with reduced disease severity, although in two individuals neurological manifestations persisted. Of note, two unaffected carriers without revertant mosaicism harbored an additional rare in trans germline SAMD9L p.Thr233Asn missense variant. In cellular assays, the SAMD9L p.Thr233Asn variant increased proliferation, indicating a loss-of-function effect that potentially compensates for the SAMD9L p.Arg986Cys mutation. Together, our results demonstrate that gain-of-function mutations in the tumor suppressor SAMD9L cause a disease with cytopenia, immunodeficiency, variable neurological presentation, and predisposition to MDS with chromosome 7 aberrations, where hematopoietic revertant mosaicism is common and ameliorates the clinical presentation. Disclosures Fioretos: Cantargia: Equity Ownership.

OBJECTIVES: Homoplasmic maternally inherited, m.14674T>C or m. 14674T>G mt-tRNA(Glu) mutations have recently been identified in reversible infantile cytochrome c oxidase deficiency (or 'benign COX deficiency'). This study sought other genetic defects that may give rise to similar presentations. PATIENTS: Eight patients from seven families with clinicopathological features of infantile reversible cytochrome c oxidase deficiency were investigated. METHODS: The study reviewed the diagnostic features and performed molecular genetic analyses of mitochondrial DNA and nuclear encoded candidate genes. RESULTS: Patients presented with subacute onset of profound hypotonia, feeding difficulties and lactic acidosis within the first months of life. Although recovery was remarkable, a mild myopathy persisted into adulthood. Histopathological findings in muscle included increased lipid and/or glycogen content, ragged-red and COX negative fibres. Biochemical studies suggested more generalised abnormalities than pure COX deficiency. Clinical improvement was reflected by normalisation of lactic acidosis and histopathological abnormalities. The m.14674T>C mt-tRNA(Glu) mutation was identified in four families, but none had the m. 14674T>G mutation. Furthermore, in two families pathogenic mutations were also found in the nuclear TRMU gene which has not previously been associated with this phenotype. In one family, the genetic aetiology still remains unknown. CONCLUSIONS: Benign COX deficiency is better described as 'reversible infantile respiratory chain deficiency'. It is genetically heterogeneous, and patients not carrying the m.14674T>C or T>G mt-tRNA(Glu) mutations may have mutations in the TRMU gene. Diagnosing this disorder at the molecular level is a significant advance for paediatric neurologists and intensive care paediatricians, enabling them to select children with an excellent prognosis for continuing respiratory support from those with severe mitochondrial presentation in infancy.

Contains fulltext : 88043.pdf (Publisher’s version ) (Closed access) The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1-NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are "ciliopathies". Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.

Objective To evaluate the efficacy of different antipyretic agents and their highest recommended doses for preventing febrile seizures. Design Randomized, placebo-controlled, double-blind trial. Setting Five hospitals, each working as the only pediatric hospital in its region. Participants A total of 231 children who experienced their first febrile seizure between January 1, 1997, and December 31, 2003. The children were observed for 2 years. Interventions All febrile episodes during follow-up were treated first with either rectal diclofenac or placebo. After 8 hours, treatment was continued with oral ibuprofen, acetaminophen, or placebo. Main Outcome Measure Recurrence of febrile seizures. Results The children experienced 851 febrile episodes, and 89 of these included a febrile seizure. Febrile seizure recurrences occurred in 54 of the 231 children (23.4%). There were no significant differences between the groups in the main measure of effect, and the effect estimates were similar, as the rate was 23.4% (46 of 197) in those receiving antipyretic agents and 23.5% (8 of 34) in those receiving placebo (difference, 0.2; 95% confidence interval, −12.8 to 17.6; P = .99). Fever was significantly higher during the episodes with seizure than in those without seizure (39.7°C vs 38.9°C; difference, 0.7°C; 95% confidence interval, −0.9°C to −0.6°C; P Conclusions Antipyretic agents are ineffective for the prevention of recurrences of febrile seizures and for the lowering of body temperature in patients with a febrile episode that leads to a recurrent febrile seizure. Trial Registration clinicaltrials.gov Identifier:NCT00568217

The clinical features of severe myoclonic epilepsy of infancy (SMEI) resemble those of mitochondrial diseases, although most patients have the sodium channel (SCN1A) mutation. We describe a patient with SMEI and enlarged muscle mitochondria associated with mutations in mitochondrial polymerase gamma 1 (POLG1) and SCN1A. Due to increased risk of valproate-induced liver failure in patients with POLG1 mutations, we recommend POLG1 gene analysis for SMEI patients before valproate administration.

Contains fulltext : 53614.pdf (Publisher’s version ) (Closed access) We describe a novel mitochondrial ND2 mutation (T4681C) in a patient presenting with Leigh Syndrome. Biochemical analyses revealed a low isolated complex I activity in patient's fibroblasts, blood and skeletal muscle. Mutant transmitochondrial cybrid clones retained the specific complex I defect, demonstrating the mitochondrial genetic origin of the disease. The mutation leads to a L71P substitution at an evolutionary conserved amino acid stretch. By two-dimensional blue native electrophoresis (2D-BN-SDS-PAGE), decreased complex I levels were observed together with an accumulation of specific assembly intermediates, suggesting that the mutation disturbs the complex I assembly pathway.

Objective To study the clinical manifestations and occurrence of mtDNA depletion and deletions in paediatric patients with neuromuscular diseases, in order to estimate the role of mtDNA rearrangements in pathogenesis of these diseases and to identify novel clinical phenotypes associated with mtDNA depletion or deletions. Methods Muscle DNA samples from patients presenting with undefined encephalomyopathies or myopathies were analysed for mtDNA content by quantitative real-time PCR and for deletions by long-range PCR. Direct sequencing of mtDNA maintenance genes and whole-exome sequencing were used to study the genetic aetiologies of diseases. Clinical and laboratory findings were collected. Results Muscle samples were obtained from 104 paediatric patients with neuromuscular diseases. MtDNA depletion was found in three patients with severe early-onset encephalomyopathy or myopathy Two of these patients presented with novel types of mitochondrial DNA depletion syndromes associated with increased serum creatine kinase and multiorgan disease without mutations in any of the known mtDNA maintenance genes; muscle electron micrograph of one patient revealed disordered myofibrillar structure, pathological endoplasmic reticulum membranes and accumulation of glycogen and extracellular collagen fibres. The third patient with mtDNA depletion and severe myopathy was diagnosed with merosine-deficient muscular dystrophy caused by a homozygous mutation in the LAMA2 gene. Two patients with an early-onset Kearns-Sayre/Pearson-like phenotype harboured a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content (4.9 and 6.9-fold increase relative to the median of age-matched controls). Conclusion We describe two novel early-onset phenotypes associated with mtDNA depletion, one of them presenting with pathologic endoplasmic reticulum membranes. Furthermore, mtDNA depletion can be a secondary finding in hereditary muscular dystrophy. We suggest that a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content associated with Kearns-Sayre/Pearson syndromes may be secondary changes caused by mutations in an unknown nuclear gene.

Congenital nephrotic syndrome of the Finnish type (NPHS1, CNF) is an autosomal recessively inherited disease occurring due to mutations in the nephrin gene (NPHS1). Two main Finnish mutations exist: Fin-major and minor, which both cause a lack of nephrin and absence of the slit diaphragm between the podocytes. This leads to severe proteinuria, nephrotic syndrome and infections, and without dialysis or renal transplantation, death in infancy. Between 1984 and 2003, six (8.6%) of the 70 NPHS1 patients diagnosed at our institution had, in addition to their renal disease, similar neurological symptoms. All six showed a severe dyskinetic cerebral palsy-like syndrome with dystonic features, athetosis and a hearing defect. The neurological symptoms became apparent during their 1st year of life and were diagnosed before 11 months of age. MRI showed increased signal intensity in T2-weighted images in the globus pallidus area. No mitochondrial gene mutations explaining the neurological symptoms were found, nor did external neurological complications explain them when compared with 29 NPHS1 control patients. Four children died at an early age: two during dialysis and two shortly after renal transplantation. Two are still alive with a functioning graft. Both have severe motor defects, but are mentally active and social.

Objectives. Many heteroplasmic point mutations in tRNA genes of mitochondrial DNA (mtDNA) have been associated with human diseases. We recently reported on a prospective 7-year study in which we enrolled 116 consecutive children with undefined encephalomyopathy. Seventeen of them were found to have both a defect in the mitochondrial respiratory chain and abnormal ultrastructure of muscle mitochondria, suggesting a clinically probable mitochondrial encephalopathy. Methods. We determined the frequency of mtDNA mutations in these 17 children by analyzing the entire sequence of mtDNA by conformation-sensitive gel electrophoresis and sequencing. Results. Three heteroplasmic tRNA mutations that were considered to be pathogenic were detected. Two of the mutations were novel transitions, 10438A>G in the tRNAArg gene and 14696A>G in the tRNAGlu gene, whereas the third one was 3243A>G, the common MELAS mutation. The mutant load was very high in the blood and skeletal muscle of the patients and markedly lower in the blood of asymptomatic maternal relatives. The 10438A>G mutation changes the nucleotide flanking the anticodon, whereas 14696A>G changes a nucleotide in the stem of the pseudouridine loop, creating a novel base pair and reducing the wobble. Conclusions. Our results emphasize that the analysis of the entire sequence of mtDNA is worthwhile in the diagnostic evaluation of patients with clinically probable mitochondrial encephalomyopathy. The frequency of pathogenic mtDNA mutations was found to be 18% among children with biochemically and histologically defined mitochondrial disease, suggesting that the likelihood of nuclear DNA mutations in such a group is several times higher than that of mtDNA mutations.

Item does not contain fulltext We describe a novel mutation in the ND6 gene (T14487C) in a patient with Leigh syndrome. Biochemical analyses indicated a low complex I activity in the patient's fibroblasts but normal values in muscle and liver. Cybrid clones showed a specific complex I defect that correlates with the mutant heteroplasmy levels. Additionally, we demonstrate an altered mobility and a decrease in the levels of fully assembled complex I in the patient's fibroblasts and cybrids, suggesting that the mutation has a profound effect on complex I assembly and/or stability.