Showing papers by "Anu Suomalainen published in 2012"

Mechanisms of mitochondrial diseases.

Abstract: Mitochondria are essential organelles with multiple functions, the most well known being the production of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS). The mitochondrial diseases are defined by impairment of OXPHOS. They are a diverse group of diseases that can present in virtually any tissue in either adults or children. Here we review the main molecular mechanisms of mitochondrial diseases, as presently known. A number of disease-causing genetic defects, either in the nuclear genome or in the mitochondria's own genome, mitochondrial DNA (mtDNA), have been identified. The most classical genetic defect causing mitochondrial disease is a mutation in a gene encoding a structural OXPHOS subunit. However, mitochondrial diseases can also arise through impaired mtDNA maintenance, defects in mitochondrial translation factors, and various more indirect mechanisms. The putative consequences of mitochondrial dysfunction on a cellular level are discussed.

Mitochondrial phenylalanyl-tRNA synthetase mutations underlie fatal infantile Alpers encephalopathy

Abstract: 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.

Effect of bezafibrate treatment on late-onset mitochondrial myopathy in mice

Abstract: Mitochondrial dysfunction is an important cause of metabolic disorders of children and adults, with no effective therapy options. Recently, induction of mitochondrial biogenesis, by transgenic overexpression of PGC1-alpha [peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1-alpha], was reported to delay progression of early-onset cytochrome-c-oxidase (COX) deficiency in skeletal muscle of two mouse models: a muscle-specific knock-out of COX10 (COX10-mKO) and a constitutive knock-out of Surf1 (Surf1-KO). A pan-PPAR agonist, bezafibrate, could similarly delay myopathy progression in COX10-mKOs, but not in SURF1-KOs. We asked whether bezafibrate affected disease progression in late-onset adult-type mitochondrial myopathy mice. These 'Deletor mice' express a dominant patient mutation in Twinkle-helicase, leading to accumulation of multiple mtDNA deletions and subsequent progressive respiratory chain (RC) deficiency with COX-negative muscle fibers at 12 months of age. The primary and secondary molecular findings in Deletor mice mimic closely those in patients with Twinkle myopathy. We applied 0.5% bezafibrate diet to Deletors for 22 weeks, starting at disease manifestation, mimicking patient treatment after diagnosis. Bezafibrate delayed significantly the accumulation of COX-negative fibers and multiple mtDNA deletions. However, mitochondrial biogenesis was not induced: mitochondrial DNA copy number, transcript and RC protein amounts decreased in both Deletors and wild-type mice. Furthermore, bezafibrate induced severe lipid oxidation effects, with hepatomegaly and loss of adipose tissue, the mechanism involving lipid mobilization by high hepatic expression of FGF21 cytokine. However, as bezafibrate has been tolerated well by humans, the beneficial muscle findings in Deletor mice support consideration of bezafibrate trials on adult patients with mitochondrial myopathy.

Thymidine kinase 2 mutations in autosomal recessive progressive external ophthalmoplegia with multiple mitochondrial DNA deletions

Abstract: Autosomal-inherited progressive external ophthalmoplegia (PEO) is an adult-onset disease characterized by the accumulation of multiple mitochondrial DNA (mtDNA) deletions in post-mitotic tissues. Mutations in six different genes have been described to cause the autosomal dominant form of the disease, but only mutations in the DNA polymerase gamma gene are known to cause autosomal recessive PEO (arPEO), leaving the genetic background of arPEO mostly unknown. Here we used whole-exome sequencing and identified compound heterozygous mutations, leading to two amino acid alterations R225W and a novel T230A in thymidine kinase 2 (TK2) in arPEO patients. TK2 is an enzyme of the mitochondrial nucleotide salvage pathway and its loss-of-function mutations have previously been shown to underlie the early-infantile myopathic form of mtDNA depletion syndrome (MDS). Our TK2 activity measurements of patient fibroblasts and mutant recombinant proteins show that the combination of the identified arPEO variants, R225W and T230A, leads to a significant reduction in TK2 activity, consistent with the late-onset phenotype, whereas homozygosity for R225W, previously associated with MDS, leads to near-total loss of activity. Our finding identifies a new genetic cause of arPEO with multiple mtDNA deletions. Furthermore, MDS and multiple mtDNA deletion disorders are manifestations of the same pathogenic pathways affecting mtDNA replication and repair, indicating that MDS-associated genes should be studied when searching for genetic background of PEO disorders.

New mutation of mitochondrial DNAJC19 causing dilated and noncompaction cardiomyopathy, anemia, ataxia, and male genital anomalies

Abstract: New mutation of mitochondrial DNAJC19 causing dilated and noncompaction cardiomyopathy, anemia, ataxia, and male genital anomalies

Capsid-Modified Adenoviral Vectors for Improved Muscle-Directed Gene Therapy

Abstract: Skeletal muscle represents an attractive target tissue for adenoviral gene therapy to treat muscle disorders and as a production platform for systemic expression of therapeutic proteins. However, adenovirus serotype 5 vectors do not efficiently transduce adult muscle tissue. Here we evaluated whether capsid modifications on adenoviral vectors could improve transduction in mature murine muscle tissue. First-generation and helper-dependent serotype 5 adenoviral vectors featuring the serotype 3 knob (5/3) showed significantly increased transduction of skeletal muscle after intramuscular injection in adult mice. Furthermore, we showed that full-length dystrophin could be more efficiently transferred to muscles of mdx mice using a 5/3-modified helper-dependent adenoviral vector. In contrast to first-generation vectors, helper-dependent adenoviral vectors mediated stable marker gene expression for at least 1 year after intramuscular injection. In conclusion, 5/3 capsid-modified helper-dependent adenoviral vectors show enhanced transduction in adult murine muscle tissue and mediate long-term gene expression, suggesting the suitability of these vectors for muscle-directed gene therapy.

Fatal neonatal lactic acidosis caused by a novel de novo mitochondrial G7453A tRNA-Serine (UCN) mutation

Abstract: Heteroplasmic mitochondrial DNA (mtDNA) mutations are an important cause of childhood disorders, but the role of homoplasmic mtDNA mutations in severe neonatal manifestations is not well understood The following were performed: full mtDNA sequencing for mutation detection, blue-native protein analysis of autopsy-derived tissues to detect respiratory chain (RC) deficiency, light and electron microscopy for morphologic analysis, and northern blot and computational modeling to study the effect of mtDNA mutations on transfer RNA (tRNA) stability We describe data from a patient with fatal neonatal lactic acidosis caused by a novel homoplasmic mutation at a highly conserved nucleotide G7453A within the tRNASer (UCN) in mtDNA The patient’s heart, skeletal muscle, brain, and liver showed severe combined complex I and IV (CI and CIV) deficiencies, accompanied by severe depletion of mature tRNASer (UCN) The mutation was absent in the patient’s mother and in a placental sample from a subsequent pregnancy of the mother, suggesting a de novo mutation We conclude that the G7453A mutation of mtDNA manifests with exceptional severity as compared with other tRNASer (UCN) mutations, typically associated with sensorineural deafness De novo homoplasmic mtDNA tRNA-mutations should be considered as a cause of fatal neonatal lactic acidosis