Alessandra Torraco

Progressive cavitating leukoencephalopathy associated with respiratory chain complex I deficiency and a novel mutation in NDUFS1

We present clinical, neuroimaging, and molecular data on the identification of a new homozygous c.1783A>G (p.Thr595Ala) mutation in NDUFS1 in two inbred siblings with isolated complex I deficiency associated to a progressive cavitating leukoencephalopathy, a clinical and neuroradiological entity originally related to unknown defects of the mitochondrial energy metabolism. In both sibs, the muscle biopsy showed severe reduction of complex I enzyme activity, which was not obvious in fibroblasts. We also observed complex I dysfunction in a Neurospora crassa model of the disease, obtained by insertional mutagenesis, and in patient fibroblasts grown in galactose. Altogether, these results indicate that the NDUFS1 mutation is responsible for the disease and complex I deficiency. Clinical presentation of complex I defect is heterogeneous and includes an ample array of clinical phenotypes. Expanding the number of allelic variants in NDUFS1, our findings also contribute to a better understanding on the function of complex I.

Novel TTC19 mutation in a family with severe psychiatric manifestations and complex III deficiency

Complex III of the mitochondrial respiratory chain (CIII) catalyzes transfer of electrons from reduced coenzyme Q to cytochrome c. Low biochemical activity of CIII is not a frequent etiology in disorders of oxidative metabolism and is genetically heterogeneous. Recently, mutations in the human tetratricopeptide 19 gene (TTC19) have been involved in the etiology of CIII deficiency through impaired assembly of the holocomplex. We investigated a consanguineous Portuguese family where four siblings had reduced enzymatic activity of CIII in muscle and harbored a novel homozygous mutation in TTC19. The clinical phenotype in the four sibs was consistent with severe olivo–ponto–cerebellar atrophy, although their age at onset differed slightly. Interestingly, three patients also presented progressive psychosis. The mutation resulted in almost complete absence of TTC19 protein, defective assembly of CIII in muscle, and enhanced production of reactive oxygen species in cultured skin fibroblasts. Our findings add to the array of mutations in TTC19, corroborate the notion of genotype/phenotype variability in mitochondrial encephalomyopathies even within a single family, and indicate that psychiatric manifestations are a further presentation of low CIII.

Mutations in APOPT1, encoding a mitochondrial protein, cause cavitating leukoencephalopathy with cytochrome c oxidase deficiency

Cytochrome c oxidase (COX) deficiency is a frequent biochemical abnormality in mitochondrial disorders, but a large fraction of cases remains genetically undetermined. Whole-exome sequencing led to the identification of APOPT1 mutations in two Italian sisters and in a third Turkish individual presenting severe COX deficiency. All three subjects presented a distinctive brain MRI pattern characterized by cavitating leukodystrophy, predominantly in the posterior region of the cerebral hemispheres. We then found APOPT1 mutations in three additional unrelated children, selected on the basis of these particular MRI features. All identified mutations predicted the synthesis of severely damaged protein variants. The clinical features of the six subjects varied widely from acute neurometabolic decompensation in late infancy to subtle neurological signs, which appeared in adolescence; all presented a chronic, long-surviving clinical course. We showed that APOPT1 is targeted to and localized within mitochondria by an N-terminal mitochondrial targeting sequence that is eventually cleaved off from the mature protein. We then showed that APOPT1 is virtually absent in fibroblasts cultured in standard conditions, but its levels increase by inhibiting the proteasome or after oxidative challenge. Mutant fibroblasts showed reduced amount of COX holocomplex and higher levels of reactive oxygen species, which both shifted toward control values by expressing a recombinant, wild-type APOPT1 cDNA. The shRNA-mediated knockdown of APOPT1 in myoblasts and fibroblasts caused dramatic decrease in cell viability. APOPT1 mutations are responsible for infantile or childhood-onset mitochondrial disease, hallmarked by the combination of profound COX deficiency with a distinctive neuroimaging presentation.

A new locus on 3p23-p25 for an autosomal-dominant limb-girdle muscular dystrophy, LGMD1H.

Limb-girdle muscular dystrophies (LGMDs) are a genetically heterogeneous group of neuromuscular disorders with a selective or predominant involvement of shoulder and pelvic girdles. We clinically examined 19 members in a four-generation Italian family with autosomal-dominant LGMD. A total of 11 subjects were affected. Clinical findings showed variable expressivity in terms of age at onset and disease severity. Five subjects presented with a slowly progressive proximal muscle weakness, in both upper and lower limbs, with onset during the fourth–fifth decade of life, which fulfilled the consensus diagnostic criteria for LGMD. Earlier onset of the disease was observed in a group of patients presenting with muscle weakness and/or calf hypertrophy, and/or occasionally high CK and lactate serum levels. Two muscle biopsies showed morphological findings compatible with MD associated with subsarcolemmal accumulation of mitochondria and the presence of multiple mitochondrial DNA deletions. A genome-wide scan performed using microsatellite markers mapped the disease on chromosome 3p23–p25.1 locus in a 25-cM region between markers D3S1263 and D3S3685. The highest two-point LOD score was 3.26 (θ=0) at marker D3S1286 and D3S3613, whereas non-parametric analysis reached a P-value=0.0004. Four candidate genes within the refined region were analysed but did not reveal any mutations. Our findings further expand the clinical and genetic heterogeneity of LGMDs.

Atypical Leigh syndrome associated with the D393N mutation in the mitochondrial ND5 subunit

Leigh syndrome (LS) is a neurodegenerative disorder usually starting before 1 year of age and leading to death within months or years.1 In most patients, LS is caused by defects of mitochondrial oxidative phosphorylation (OXPHOS), the most common involving pyruvate dehydrogenase complex (PDH), cytochrome c oxidase (complex IV), and NADH-ubiquinone oxidoreductase (complex I).1 Complex I consists of at least 45 subunits,2 7 of which are encoded by the mitochondrial genome, and is the largest and the most complex in terms of function and structure among the OXPHOS complexes. Recent focus on the culprit role of nuclear genes encoding the majority of the structural subunits of complex I was the effect of the discovery of autosomal recessive mutations in several patients with LS or Leigh-like phenotypes. More recently, the 12706T>C and the 13513G>A mutations in the ND5 gene, one of seven subunits of complex I encoded by mitochondrial genome (mtDNA), were identified in patients with LS.3,4⇓ Thus, both nuclear gene defects and mtDNA mutations are possible in patients with LS with complex I deficiency. Here we report on an additional child with LS harboring the 13513G→A mutation. A 4-year-old Italian boy, born to unrelated parents, was the product of uncomplicated pregnancy and delivery. He …

Mitochondrial diseases part I: Mouse models of OXPHOS deficiencies caused by defects in respiratory complex subunits or assembly factors

Mitochondrial disorders are the most common inborn errors of metabolism affecting the oxidative phosphorylation system (OXPHOS). Because of the poor knowledge of the pathogenic mechanisms, a cure for these disorders is still unavailable and all the treatments currently in use are supportive more than curative. Therefore, in the past decade a great variety of mouse models have been developed to assess the in vivo function of several mitochondrial proteins involved in human diseases. Due to the genetic and physiological similarity to humans, mice represent reliable models to study the pathogenic mechanisms of mitochondrial disorders and are precious to test new therapeutic approaches. Here we summarize the features of several mouse models of mitochondrial diseases directly related to defects in subunits of the OXPHOS complexes or in assembly factors. We discuss how these models recapitulate many human conditions and how they have contributed to the understanding of mitochondrial function in health and disease.

Effects of levosimendan on mitochondrial function in patients with septic shock: A randomized trial

Mitochondrial dysfunction is key feature of septic shock and contributes to the development of sepsis related organ dysfunction. It is characterized by a variable reduction of the respiratory chain (RC) activities, altered mitochondrial morphology and reactive oxygen species production. Recent data have reported the efficacy of levosimendan, a calcium sensitizer, in improving heart performance and organ perfusion in critically ill patients. Moreover, it has been demonstrated that Levosimendan has antioxidant properties. Nevertheless, the effects of levosimendan on mitochondrial function are not fully elucidated. The objective of this study was therefore to evaluate the effect of levosimendan on mitochondria performance. Five mitochondrial parameters were screened: the redox status; the amount of scavenging enzymes; the activities of the RC complexes; the mitochondrial content; the steady state levels of the RC subunits; the mitochondrial biogenesis. Our results show that patients treated with levosimendan had a significant reduction of glutathionylated proteins and an increase in the amount of the antioxidant enzyme MnSOD, underlining its antioxidant properties. The activities of the RC complexes I, II and III were unchanged in the mitochondria of patients treated with levosimendan compared to controls whereas the mitochondrial content was significantly higher in levosimendan vs. control patients. Finally, evaluation of mitochondrial biogenesis did not show any significant difference in the two groups, although an overall increase in the amount of the RC subunits was observed in the levosimendan group. In conclusion, our study demonstrated that in septic shock patients, Levosimendan exerts antioxidant action by increasing antioxidant defense and lowering oxidative damage.

Mutations in the NDUFS4 gene of mitochondrial complex I alter stability of the splice variants

The effect on the stability of alternative transcripts of different mutations of the NDUFS4 gene in patients with Leigh syndrome with complex I deficiency is presented. Normally, two NDUFS4 splice variants are degraded by nonsense mediated mRNA decay (NMD) while a third form does not trigger NMD degradation. In a patient with a premature termination codon in exon 1, all the three splice variants are up‐regulated. The present is the first case of a nonsense mutation leading to the abrogation of NMD, which can represent an additional event to be considered in the evaluation of clinically relevant mutations.

MITOCHONDRIAL DISEASES PART II: MOUSE MODELS OF OXPHOS DEFICIENCIES CAUSED BY DEFECTS IN REGULATORY FACTORS AND OTHER COMPONENTS REQUIRED FOR MITOCHONDRIAL FUNCTION

Mitochondrial disorders are defined as defects that affect the oxidative phosphorylation system (OXPHOS). They are characterized by a heterogeneous array of clinical presentations due in part to a wide variety of factors required for proper function of the components of the OXPHOS system. There is no cure for these disorders owing to our poor knowledge of the pathogenic mechanisms of disease. To understand the mechanisms of human disease numerous mouse models have been developed in recent years. Here we summarize the features of several mouse models of mitochondrial diseases directly related to those factors affecting mtDNA maintenance, replication, transcription, translation as well as other proteins that are involved in mitochondrial dynamics and quality control which affect mitochondrial OXPHOS function without being intrinsic components of the system. We discuss how these models have contributed to our understanding of mitochondrial diseases and their pathogenic mechanisms.

Novel mutations in IBA57 are associated with leukodystrophy and variable clinical phenotypes

Defects of the Fe/S cluster biosynthesis represent a subgroup of diseases affecting the mitochondrial energy metabolism. In the last years, mutations in four genes (NFU1, BOLA3, ISCA2 and IBA57) have been related to a new group of multiple mitochondrial dysfunction syndromes characterized by lactic acidosis, hyperglycinemia, multiple defects of the respiratory chain complexes, and impairment of four lipoic acid-dependent enzymes: α-ketoglutarate dehydrogenase complex, pyruvic dehydrogenase, branched-chain α-keto acid dehydrogenase complex and the H protein of the glycine cleavage system. Few patients have been reported with mutations in IBA57 and with variable clinical phenotype. Herein, we describe four unrelated patients carrying novel mutations in IBA57. All patients presented with combined or isolated defect of complex I and II. Clinical features varied widely, ranging from fatal infantile onset of the disease to acute and severe psychomotor regression after the first year of life. Brain MRI was characterized by cavitating leukodystrophy. The identified mutations were never reported previously and all had a dramatic effect on IBA57 stability. Our study contributes to expand the array of the genotypic variation of IBA57 and delineates the leukodystrophic pattern of IBA57 deficient patients.