Abelardo Solano

Cerebral folate deficiency and leukoencephalopathy caused by a mitochondrial DNA deletion.

Our aim was to describe a child with an incomplete form of Kearns–Sayre syndrome who presented profound cerebrospinal fluid (CSF) folate deficiency and his response to folinic acid supplementation

Bilateral striatal necrosis associated with a novel mutation in the mitochondrial ND6 gene

We report the molecular findings in two independent patients presenting with progressive generalized dystonia and bilateral striatal necrosis in whom we have identified a mutation (T14487C) in the mitochondrial ND6 gene. The mutation is heteroplasmic in all samples analyzed, and it fulfills all accepted criteria of pathogenicity. Transmitochondrial cell lines harboring 100% mutant mitochondrial DNA showed a marked decrease in the activity of complex I of the respiratory chain supporting the pathogenic role of T14487C.

Liver failure caused by herpes simplex virus thymidine kinase plus ganciclovir therapy is associated with mitochondrial dysfunction and mitochondrial DNA depletion.

Herpes simplex virus thymidine kinase (HSV-tk) converts ganciclovir (GCV) into an active compound, which can be incorporated into DNA molecules and terminate DNA synthesis. Gene transfer of HSV-tk followed by GCV administration has been used with success to treat experimental cancer and this strategy has entered into clinical trials. Although it is thought that the cytotoxic effect occurs mainly in tumoral dividing cells, where mitotic activity favors integration of the genotoxic compound into nuclear DNA, there are concerns of potential damage to normal nondividing cells. In the present work we have explored the mechanisms of HSV-tk/GCV toxicity and in particular whether this therapy may cause lesions of mitochondrial DNA (mtDNA) and mitochondrial dysfunction. We found that the administration of GCV to rats injected with adenovirus encoding HSV-tk induced hepatocellular damage characterized by the presence of apoptotic bodies, ballooning of hepatocytes, and severe hepatic steatosis with mitochondria enlargement and cristae dissolution at the ultrastructural level. Remarkably, Southern blot analysis showed substantial reduction in the amount of mtDNA in the liver. Using radiolabeled GCV we could demonstrate incorporation of this compound into both nuclear and mtDNA in HSV-tk-transduced rat hepatocytic cell line MCA-RH7777 and subsequent alteration of mitochondrial function. Our observations confirm that GCV can damage both nuclear and mtDNA in cells transduced with HSV-tk and that this effect could be responsible for severe mitochondrial dysfunction and toxicity in normal nondividing cells. These data are relevant for the design of clinical trials using adenoviral vectors encoding HSV-tk.

NARP syndrome in a patient harbouring an insertion in the MT-ATP6 gene that results in a truncated protein

Background: Neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) syndrome have been associated to m.8993T>G/C mutations in the subunit 6 of the ATP synthase (p.MT-ATP6). Methods: We have performed a mutational screening of the mitochondrial DNA gene encoding for this protein in 62 patients with the disease, that do not carry any of the common mutations described to date. Results: We report clinical and molecular data in one patient who harbours a de novo insertion in the MT-ATP6 gene that results in a truncated subunit. The mutation was heteroplasmic (85%) in muscle DNA and the BN-PAGE analysis showed a clear decrease in the amount of ATP synthase. Conclusion: Molecular analysis of NARP patients cannot be limited to the search of the m.8993T>G/C and either the ATP6 or the whole mtDNA should be sequenced.

Free radicals-mediated damage in transmitochondrial cells harboring the T14487C mutation in the ND6 gene of mtDNA

We have studied the production of reactive oxygen species (ROS) in transmitochondrial cells, harboring homoplasmic levels of the T14487C mtDNA mutation in the ND6 gene of mitochondrial DNA (mtDNA). Previous work has shown that this mutation causes complex I deficiency. Here, we show that this mutation causes an overproduction of ROS leading to an increase in the oxidation of lipids and mtDNA without modification of antioxidant enzyme activities. We suggest that mutations in mtDNA affecting complex I activity may result in oxidative cellular damage, and reinforce the possible role of ROS‐mediated mechanisms participating in some mtDNA‐related disorders.

Enfermedades genéticas del ADN mitocondrial humano

Mitochondrial diseases are a group of disorders produced by defects in the oxidative phosphorylation system (Oxphos system), the final pathway of the mitochondrial energetic metabolism, resulting in a deficiency of the biosynthesis of ATP. Part of the polypeptide subunits involved in the Oxphos system are codified by the mitochondrial DNA. In the last years, mutations in this genetic system have been described and associated to well defined clinical syndromes. The clinical features of these disorders are very heterogeneous affecting, in most cases, to different organs and tissues and their correct diagnosis require precise clinical, morphological, biochemical and genetic data. The peculiar genetic characteristics of the mitochondrial DNA (maternal inheritance, polyplasmia and mitotic segregation) give to these disorders very distinctive properties. The English version of this paper is available at: http://www.insp.mx/salud/ index.html

Exercise intolerance resulting from a muscle-restricted mutation in the mitochondrial tRNALeu (CUN) gene

BACKGROUND. Some patients presenting with isolated lifelong exercise intolerance and ragged-red fibres, harbour skeletal-muscle restricted mutations in their mitochondrial DNA. AIM. To identify the molecular defect in a patient presenting with lifelong exercise intolerance, ragged-red fibres and deficiencies of complexes III and IV in skeletal muscle. METHODS. The muscle biopsy was studied for activities of the respiratory chain, histochemical stains, and sequencing the tRNA genes of mitochondrial DNA. RESULTS. The patient had a heteroplasmic mutation in the tRNALeu(CUN) gene of mitochondrial DNA (G12334A). Clinical and morphological data as well as restriction fragment length polymorphism (RFLP) and single-fibre polymerase chain reaction (PCR) analyses strongly indicate that this molecular defect is the primary cause of the myopathy. CONCLUSION. Mutations in any mitochondrial gene should be considered in the differential diagnosis of patients with lifelong exercise intolerance, even when the neurological examination is normal.

Characterisation of repeat and palindrome elements in patients harbouring single deletions of mitochondrial DNA

Single deletions of mitochondrial DNA (mtDNA) were the first pathogenic mutations to be identified in human mtDNA. In a seminal paper, Holt et al 1 reported the presence of single deletions of the mitochondrial genome in patients presenting with mitochondrial myopathies, and since then, the field has experienced enormous progress. To date, 97 different deletions have been reported in MITOMAP, the main international database for mtDNA related disorders (www.mitomap.org), and most of these deletions are associated with two clinical presentations: chronic progressive external ophthalmoplegia (CPEO) and Kearns-Sayre syndrome (KSS). Here, we report the molecular characterisation of a series of 18 patients in whom we have identified single deletions of mtDNA. These patients were, as expected, diagnosed with CPEO and KSS. Deletions were identified and measured by Southern blot analysis and were mapped by long polymerase chain reaction (PCR) to locate the deletion breakpoint. We report nine novel deletions and defined their characteristics in terms of sequence of the tandem repeat, presence of palindrome sequences, length of the deleted molecule, and heteroplasmy level in muscle. ### Patients The 18 patients included in this study were clinically diagnosed with CPEO (10 patients) and KSS (eight patients), and there was no evidence of maternal inheritance. CPEO was defined by the presence of ophthalmoplegia, ptosis, and proximal limb weakness. Patients with KSS presented with the invariant triad of: (1) onset before 20 years, (2) progressive external ophthalmoplegia; and (3) pigmentary retinopathy, plus at least one of heart block, cerebellar syndrome, or a protein concentration in cerebrospinal fluid above 100 mg/ml. Muscle biopsies were performed after informed consent. ### Molecular genetic studies Blood DNA was extracted from peripheral blood cells by conventional methods, and muscle DNA was obtained from 10 mg of muscle after treatment with proteinase K and extraction with phenol/chloroform/isoamyl alcohol. Southern blot analysis was performed using 5 μg …

Expanding the clinical phenotypes of MT-ATP6 mutations

Mitochondrial DNA mutations at MT-ATP6 gene are relatively common in individuals suffering from striatal necrosis syndromes. These patients usually do not show apparent histochemical and/or biochemical signs of oxidative phosphorylation dysfunction. Because of this, MT-ATP6 is not typically analyzed in many other mitochondrial disorders that have not been previously associated to mutations in this gene. To correct this bias, we have performed a screening of the MT-ATP6 gene in a large collection of patients suspected of suffering different mitochondrial DNA (mtDNA) disorders. In three cases, biochemical, molecular-genetics and other analyses in patient tissues and cybrids were also carried out. We found three new pathologic mutations. Two of them in patients showing phenotypes that have not been commonly associated to mutations in the MT-ATP6 gene. These results remark the importance of sequencing the MT-ATP6 gene in patients with striatal necrosis syndromes, but also within other mitochondrial pathologies. This gene should be sequenced at least in all those patients suspected of suffering an mtDNA disorder disclosing normal results for histochemical and biochemical analyses of respiratory chain.

Peripheral neuropathy with ataxia in childhood as a result of the G8363A mutation in mitochondrial DNA.

Peripheral neuropathy has been identified in children with mitochondrial encephalomyopathies but not as a main clinical landmark. Here we report the clinical, electrophysiologic, biochemical, and genetic findings in a family who harbors the G8363A mutation in the tRNALys gene of mitochondrial DNA. Affected individuals presented with peripheral neuropathy and ataxia as the main clinical sign. Additional involvement included muscle weakness and multiple lipomatosis. Other common clinical characteristics associated with the G8363A mutation, such as cardiomyopathy and myoclonus epilepsy, were not observed. These findings suggest that a mitochondrial disease should be considered in the differential diagnosis of children with here-doataxic syndrome and peripheral neuropathy of unknown origin.