Paola Da Pozzo

Mitochondria, oxidative stress and neurodegeneration

Mitochondria are involved in ATP supply to cells through oxidative phosphorylation (OXPHOS), synthesis of key molecules and response to oxidative stress, as well as in apoptosis. They contain many redox enzymes and naturally occurring inefficiencies of oxidative phosphorylation generate reactive oxygen species (ROS). CNS functions depend heavily on efficient mitochondrial function, since brain tissue has a high energy demand. Mutations in mitochondrial DNA (mtDNA), generation and presence of ROS and environmental factors may contribute to energy failure and lead to neurodegenerative diseases. Many rare metabolic disorders have been associated with mitochondrial dysfunction. More than 300 pathogenic mtDNA mutations involve proteins that regulate OXPHOS and mitochondrial structural integrity, and have also been described in neurodegenerative diseases with autosomal inheritance. Mitochondria may have an important role in ageing-related neurodegenerative disorders like Parkinsons disease (PD), Alzheimers disease (AD), Huntingtons disease (HD) and amyotrophic lateral sclerosis (ALS). In primary mitochondrial and neurodegenerative disorders, there is strong evidence that mitochondrial dysfunction occurs early and has a primary role in pathogenesis. In the present review, we discuss several mitochondrial diseases as models of neurodegeneration.

A novel heteroplasmic tRNASer(UCN) mtDNA point mutation associated with progressive external ophthalmoplegia and hearing loss

Abstract We sequenced all mitochondrial tRNA genes from a patient with sporadic external ophthalmoplegia (PEO) and 5% COX-negative fibers in muscle biopsy, who had no detectable large mtDNA deletions. Direct sequencing showed a heteroplasmic mutation at nucleotide 7506 in the dihydrouridine stem of the tRNA Ser(UCN) gene. RFLP analysis confirmed that 30% of muscle and 20% of urinary epithelium mtDNA harbored the mutation, which was absent in other tissues of the proband as well as in mtDNA of his mother and 100 patients with various encephalomyopathies. Several point mutations on mitochondrial tRNA genes have been reported in PEO patients without large-scale rearrangements of mtDNA but no point mutations have hitherto been found in the gene coding for tRNA Ser(UCN) .

Rapidly progressive neurodegeneration in a case with the 7472insC mutation and the A7472C polymorphism in the mtDNA tRNAser(UCN) gene

The authors report the clinical, neuroimaging, muscle biopsy and mtDNA findings in a patient affected by bilateral hearing loss and mental retardation since infancy, presenting at age 31 years with a rapid deterioration of mental status and ataxia leading to vegetative condition and death at the age of 32 years. Clinical and genetic studies have been also performed in the mother, affected by neurosensorial hearing loss. Muscle biopsy showed severe mitochondrial alterations in the propositus and evidence of mitochondrial alterations in his mother. Direct mtDNA sequencing in all family members revealed the known 7472insC mutation and the recently described A7472C sequence variation in the tRNA(Ser(UCN))gene. RFLP-PCR confirmed the heteroplasmic nature of the two mutations and failed to find the second transversion in 200 controls. The percentage of mutant genomes harbouring 7472insC ranged from 3 to 7% in asymptomatic family members to 70% in the proband and his mother, whereas the percentage of A7472C mutant genomes was about 90% in all maternal relatives except the proband (56%) and his sister (5%). In conclusion, this is the first report of a rapidly progressive encephalopathy in association with the 7472insC mutation in mtDNA, combined with an A>C variation at the same nucleotide with a possible suppression effect on the pathogenic mutation.

A second MNGIE patient without typical mitochondrial skeletal muscle involvement

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease caused by mutations in the gene encoding thymidine phosphorylase (TYMP). Clinically, MNGIE is characterized by gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy and leukoencephalopathy. Most MNGIE patients have signs of mitochondrial dysfunction in skeletal muscle at morphological and enzyme level, as well as mitochondrial DNA depletion, multiple deletions and point mutations. A case without mitochondrial skeletal muscle involvement and with a TYMP splice-acceptor site mutation (c. 215–1 G>C) has been reported. Here, we describe an Italian patient with the same mutation and without mitochondrial skeletal muscle involvement, suggesting a possible genotype–phenotype correlation.

Leber Hereditary Optic Neuropathy in 2 of 4 Siblings with 11778 mtDNA Mutation: Clinical Variability or Effect of Toxic Environmental Exposure?

Although mitochondrial (mt) DNA mutation at nucleotide position 11778 accounts for most cases of Leber’s hereditary optic neuropathy (LHON), the phenotypic expression may vary greatly even in different members of the same family. The possible influence of exogenous toxicity on phenotypic expression is still debated in LHON. Here we describe 4 siblings carrying the 11778 mtDNA mutation with a different phenotype. The index case developed an atypical optic neuropathy at the age of 60 years after a long history of occupational exposure to polycyclic aromatic hydrocarbons (PAHs). This report underlines a number of unanswered questions about phenotypic variability of LHON including the possible influence of PAH toxicity.

A novel point mutation in the mitochondrial tRNA((Trp)) gene produces late-onset encephalomyopathy, plus additional features.

BACKGROUND Mitochondrial diseases due to mitochondrial tRNA genes mutations are usually multisystem disorders with infantile or adult onset. OBJECTIVE To identify the molecular defect underlying a mitochondrial encephalomyopathy. METHODS/PATIENTS Case report of a 51year-old woman presenting with late-onset myoclonic epilepsy plus additional features. Probands mother presented hypothyroidism and diabetes. RESULTS Muscle biopsy showed mitochondrial changes. Respiratory chain activities were reduced. The novel G5538A mutation was identified in different tissues DNAs from the proband and from her mother. CONCLUSION We were able to identify a novel mtDNA tRNA((Trp)) gene pathogenic mutation.

A new thymidine phosphorylase mutation causing elongation of the protein underlies mitochondrial neurogastrointestinal encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE; OMIM: #603041) is an uncommon, autosomal recessive multisystem disorder, caused by loss-of-function mutations in the nuclear TYMP gene (OMIM: *131222) encoding thymidine phosphorylase (TP). MNGIE should be suspected in young patients presenting with gastrointestinal tract symptoms (abdominal pain, diarrhoea and repeated episodes of pseudo-occlusion) leading to relevant weight loss, and associated with neurological involvement including muscle weakness, ptosis, peripheral neuropathy and leukoencephalopathy. Parental consanguinity has been reported in nearly half of all cases [1, 2]. Clinical diagnosis is confirmed by determination of TP activity in buffy coat samples, which in MNGIE patients is \10% of the mean activity in controls. The lack of TP activity leads to a general increase in its substrates, deoxythymidine (dThd) and deoxyuridine (dUrd), in urine and serum [3]. Different mutations in the TYMP gene have been identified but no predominant mutation has yet been reported. While most are missense mutations, splice-site mutations, deletions, insertions and nonsense mutations have also been reported (http://www.hgmd.org, http://www.genetests.org/). In this study, we report the clinical and genetic findings of a 21-year-old patient with a mild MNGIE phenotype carrying a novel homozygous 1-bp insertion mutation in the TYMP gene. A 21-year-old Italian female, born of healthy consanguineous parents, came to our attention for cervicobrachialgia and paresthesia presenting after a road accident. The patient’s clinical history disclosed repeated episodes of abdominal pain, recurrent vomiting and diarrhoea since the age of 15 years, previously thought to be Campylobacter jejuni infection. Physical examination revealed short stature (150 cm) and thinness (weight 37 kg), with a body mass index (BMI) of 16.4 kg/m. Neurological examination showed mild limb weakness, absent deep tendon reflexes and mild palpebral ptosis. Routine blood chemistry was normal. Serum lactate was significantly increased (2.3 mmol/l; normal value: 0.3–1.3 mmol/l). Peripheral nerve conduction velocity analysis were consistent with a demyelinating sensory motor neuropathy. A diagnosis of MNGIE was suspected. TP activity was absent in the buffy coat (normal range 75–850 nmol/h/mg) and deoxythymidine and deoxyuridine plasma levels were elevated, at 9.0 and 16.0 lmol/L, respectively (normally not detected). Brain magnetic resonance imaging (MRI), showed bilateral periventricular white matter hyperintensities in T2-W and FLAIR images (Fig. 1a). Having obtained informed consent, genomic DNA was extracted from blood samples of the proband, her parents E. Cardaioli F. Sicurelli M. A. Carluccio G. N. Gallus P. Da Pozzo A. Federico M. T. Dotti (&) Department of Neurological, Neurosurgical and Behavioural Sciences, University of Siena, Siena, Italy e-mail: dotti@unisi.it

Novel POLG mutations and variable clinical phenotypes in 13 Italian patients

POLG gene encodes the catalytic subunit of DNA polymerase gamma, essential for mitochondrial DNA (mtDNA) replication and repair. Mutations in POLG have been linked to a spectrum of clinical phenotypes, resulting in autosomal recessive or dominant mitochondrial diseases. These mutations have been associated with heterogeneous phenotypes, presenting with varying severity and at different ages of onset, ranging from the neonatal period to late adult life. We screened 13 patients for POLG mutations. All patients underwent a complete neurological examination, and in most of cases, muscle biopsy was performed. We detected 15 different variations in 13 unrelated Italian patients. Two mutations were novel and mapped in the pol domain (p.Thr989dup and p.Ala847Thr) of the enzyme. We also report new cases carrying controversial variations previously described as incompletely penetrant or a variant of unknown significance. Our study increases the range of clinical presentations associated with mutations in POLG gene, underlining some peculiar clinical features, such as PEO associated with corneal edema, and epilepsy, severe neuropathy with achalasia. The addition of two new substitutions, including the second report of an in-frame duplication, to the growing list of defects increases the value of POLG genetic diagnosis in a range of neurological presentations.

Progressive mitochondrial myopathy, deafness, and sporadic seizures associated with a novel mutation in the mitochondrial tRNASer (AGY) gene

We sequenced the mitochondrial genome from a patient with progressive mitochondrial myopathy associated with deafness, sporadic seizures, and histological and biochemical features of mitochondrial respiratory chain dysfunction. Direct sequencing showed a heteroplasmic mutation at nucleotide 12262 in the tRNASer(AGY) gene. RFLP analysis confirmed that 63% of muscle mtDNA harboured the mutation, while it was absent in all the other tissues. The mutation is predicted to influence the functional behaviour of the aminoacyl acceptor stem of the tRNA. Several point mutations on mitochondrial tRNA genes have been reported in patients affected by encephalomyopathies, but between them only four were reported for tRNASer(AGY).

Sporadic myopathy, myoclonus, leukoencephalopathy, neurosensory deafness, hypertrophic cardiomyopathy and insulin resistance associated with the mitochondrial 8306 T>C MTTK mutation.

We report a new T8306C transition in the D-stem of the MTTK gene of a 67-year-old man who manifested severe adult onset myopathy, myoclonus, leukoencephalopathy, neurosensory hypoacusis, hypertrophic cardiomyopathy and insulin resistance. No other family member was affected, suggesting that our patient was a sporadic case. The T8306C mutation was heteroplasmic in several tissues of the proband, while it was absent from his asymptomatic siblings. Single fibre analysis confirmed the segregation of higher mutational load in cytochrome c oxidase-deficient fibres. The mutation T8306C is predicted to disrupt a highly conserved base pair and was not found in more than 120 controls. This finding broadens the phenotypic and molecular spectrum of mitochondrial tRNA(Lys) associated disorders.