Johanna Uusimaa

FGF-21 as a biomarker for muscle-manifesting mitochondrial respiratory chain deficiencies: a diagnostic study

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 Jusélius 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 Ylppö Foundation.

Impaired complex I assembly in a Leigh syndrome patient with a novel missense mutation in the ND6 gene.

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 patients 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 patients fibroblasts and cybrids, suggesting that the mutation has a profound effect on complex I assembly and/or stability.

Prevalence, segregation, and phenotype of the mitochondrial DNA 3243A>G mutation in children

We studied the prevalence, segregation, and phenotype of the mitochondrial DNA 3243A>G mutation in children in a defined population in Northern Ostrobothnia, Finland.

Childhood Encephalopathies and Myopathies: A Prospective Study in a Defined Population to Assess the Frequency of Mitochondrial Disorders

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.

Antipyretic agents for preventing recurrences of febrile seizures: randomized controlled trial.

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 degrees C vs 38.9 degrees C; difference, 0.7 degrees C; 95% confidence interval, -0.9 degrees C to -0.6 degrees C; P < .001), and this phenomenon was independent of the medication given. 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.

Homozygous W748S mutation in the POLG1 gene in patients with juvenile-onset Alpers syndrome and status epilepticus.

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.

SIRT5 is under the control of PGC-1α and AMPK and is involved in regulation of mitochondrial energy metabolism

The sirtuins (SIRTs; SIRT1–7) are a family of NAD+‐dependent enzymes that dynamically regulate cellular physiology. Apart from SIRT1, the functions and regulatory mechanisms of the SIRTs are poorly defined. We explored regulation of the SIRT family by 2 energy metabolism–controlling factors: peroxisome proliferator‐activated receptor γ coactivator 1‐α (PGC‐1α) and AMP‐activated protein kinase (AMPK). Overexpression of PGC‐1α in mouse primary hepatocytes increased SIRT5 mRNA expression 4‐fold and also the protein in a peroxisome proliferator‐activated receptor α (PPARα)‐ and estrogen‐related receptor α (ERRα)‐dependent manner. Furthermore, food withdrawal increased SIRT5 mRNA 1.3‐fold in rat liver. Overexpression of AMPK in mouse hepatocytes increased expression of SIRT1, SIRT2, SIRT3, and SIRT6 <2‐fold. In contrast, SIRT5 mRNA was down‐regulated by 58%. The antidiabetes drug metformin (1 mM), an established AMPK activator, reduced the mouse SIRT5 protein level by 44% in cultured hepatocytes and by 31% in liver in vivo (300 mg/kg, 7 d). Metformin also induced hypersuccinylation of mitochondrial proteins. Moreover, SIRT5 overexpression increased ATP synthesis and oxygen consumption in HepG2 cells, but did not affect mitochondrial biogenesis. In summary, our results identified SIRT5 as a novel factor that controls mitochondrial function. Moreover, SIRT5 levels are regulated by PGC‐1α and AMPK, which have opposite effects on its expression.—Buler, M., Aatsinki, S.‐M., Izzi, V., Uusimaa, J., Hakkola, J. SIRT5 is under the control of PGC‐1α and AMPK and is involved in regulation of mitochondrial energy metabolism. FASEB J. 28, 3225–3237 (2014). www.fasebj.org

POLG1 manifestations in childhood

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.

Reversible infantile respiratory chain deficiency is a unique, genetically heterogenous mitochondrial disease

Objectives Homoplasmic maternally inherited, m.14674T>C or m. 14674T>G mt-tRNAGlu 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-tRNAGlu 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-tRNAGlu 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.

Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts.