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.

Oxidative stress-induced apoptosis in peripheral blood lymphocytes from patients with POLG-related disorders.

BACKGROUND: POLG-related disorders are a group of heterogeneous diseases characterized by an overlapping clinical presentations and associated with mutations in the POLG gene. POLG codes for the catalytic subunit of mitochondrial polymerase gamma (POLG), essential for mitochondrial DNA (mtDNA) replication and repair. Studies on mutator POLG mice showed an increase in oxidative stress and apoptosis. In this regard we analysed the involvement of POLG mutations in the apoptotic regulation, evaluating apoptosis in peripheral blood lymphocytes (PBLs) from patients with POLG-related diseases. METHODS: Cells were cultured under basal conditions and with 2-deoxy-d-ribose (dRib), a reducing sugar that induces apoptosis by oxidative stress. Apoptosis rate was assessed by flow cytometry. Phosphatidylserine translocation, mitochondrial membrane depolarization and caspase 3 activation were also analysed. RESULTS: Our data showed higher percentages of apoptosis after dRib treatment in patients with POLG mutations than in controls, while under basal culture conditions, apoptosis levels were similar in the two groups. CONCLUSIONS: Cells with POLG mutations are more sensitive than control cells to oxidative stress-induced apoptosis, confirming that mtDNA mutations may have a role in mitochondrial apoptosis pathway. We also suggest that redox state homeostasis may play a crucial role in phenotypic expression of POLG-related diseases.

Sporadic PEO caused by a novel POLG variation and a Twinkle mutation: digenic inheritance?

Progressive external ophthalmoplegia (PEO) with multiple deletions of mitochondrial DNA (mtDNA) is associated with several mutations in nuclear genes. They include POLG, POLG2, ANT1, C10orf2/Twinkle, and OPA1. However, digenic inheritance in mitochondrial disorders has been documented in a few cases over the years. Here we describe an 80-year-old man with sporadic PEO associated with mtDNA deletions. Sequencing of the POLG revealed a novel heterozygous mutation (c.2831A>G; p.Glu944Gly), predicted in silico as damaging, in the patient who also carried a heterozygous mutation in C10orf2/Twinkle (c.1142T>C; p.Leu381Pro). This case provides a second report of a PEO with different mutations in the POLG and C10orf2/Twinkle genes, supporting the hypothesis that the PEO phenotype can be determined by the co-existence of two abnormalities in separate genes, both involved in the maintenance and stability of mtDNA. Finally, this study expands the spectrum of POLG mutations and highlights the need to sequence the whole set of nuclear genes associated with PEO and multiple mtDNA deletions.

Redefining phenotypes associated with mitochondrial DNA single deletion.

Progressive external ophthalmoplegia (PEO), Kearns-Sayre syndrome (KSS) and Pearson syndrome are the three sporadic clinical syndromes classically associated with single large-scale deletions of mitochondrial DNA (mtDNA). PEO plus is a term frequently utilized in the clinical setting to identify patients with PEO and some degree of multisystem involvement, but a precise definition is not available. The purpose of the present study is to better define the clinical phenotypes associated with a single mtDNA deletion, by a retrospective study on a large cohort of 228 patients from the database of the "Nation-wide Italian Collaborative Network of Mitochondrial Diseases". In our database, single deletions account for about a third of all patients with mtDNA-related disease, more than previously recognized. We elaborated new criteria for the definition of PEO and "KSS spectrum" (a category of which classic KSS represents the most severe extreme). The criteria for "KSS spectrum" include the resulting multisystem clinical features associated with the KSS features, and which therefore can predict their presence or subsequent development. With the new criteria, we were able to classify nearly all our single-deletion patients: 64.5% PEO, 31.6% KSS spectrum (including classic KSS 6.6%) and 2.6% Pearson syndrome. The deletion length was greater in KSS spectrum than in PEO, whereas heteroplasmy was inversely related with age at onset. We believe that the new phenotype definitions implemented here may contribute to a more homogeneous patient categorization, which will be useful in future cohort studies of natural history and clinical trials.

A case of 3243A>G mutation in mtDNA presenting as apparently idiopathic hyperCKemia.

The 3243A>G mutation of mtDNA usually is associated with MELAS syndrome. Here we report a patient with the 3243A>G mutation presenting only recurrent muscle fatigue and elevated levels of serum creatine kinase (CK). The mother of the proband was referred to us for type 2 diabetes mellitus, muscle pain and sensorineural hearing loss. The percentage of mutation load in different tissues was similar in both subjects, except in the urinary epithelium. The mutation load in the son's urinary epithelial cells (UEC) was consistently higher (nearly 50%) than in his muscle (nearly 20%). We conclude that a correlation between the proportion of the UEC mutation load and the severity of the disease was lacking in this pedigree. The use of UEC as the tissue of choice in the noninvasive diagnosis of the 3243A>G mutation offers a very attractive alternative to muscle biopsy. Finally, our data expand the clinical spectrum of the 3243A>G mutation.

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.

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 Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's 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.

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).

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. Proband's 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 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.

Alu-element insertion in an OPA1 intron sequence associated with autosomal dominant optic atrophy.

PURPOSE: Autosomal dominant optic atrophy (ADOA) is the most common form of hereditary optic neuropathy caused by mutations in the optic atrophy 1 (OPA1) gene. It is characterized by insidious onset with a selective degeneration of retinal ganglion cells, variable loss of visual acuity, temporal optic nerve pallor, tritanopia, and development of central, paracentral, or cecocentral scotomas. Here we describe the clinical and molecular findings in a large Italian family with ADOA. METHODS: Routine ophthalmologic examination and direct sequencing of all coding regions of the OPA1 gene were performed. Further characterization of a new OPA1 gene insertion was performed by reverse transcription-PCR (RT-PCR) of RNA from patients and control subjects. RESULTS: We identified an Alu-element insertion located in intron 7 of OPA1 causing an in-frame deletion of exon 8 in 18 family members. CONCLUSIONS: The predicted consequence of this mutation is the loss of the guanosine triphosphatase (GTPase) activity of OPA1. Alu insertions have been reported in the literature as causing human genetic disease. However, this is the first report of a pathogenic OPA1 gene mutation resulting from an Alu insertion.

A novel mutation in the mitochondrial tRNA(Pro) gene associated with late-onset ataxia, retinitis pigmentosa, deafness, leukoencephalopathy and complex I deficiency.

We present a patient with ataxia, retinitis pigmentosa, dysarthria, neurosensorial deafness, nystagmus and leukoencephalopathy. A novel heteroplasmic G to A transition at nucleotide 15 975 was found, affecting the T arm of the mitochondrial (mt) tRNA(Pro) gene. A biochemical analysis of respiratory chain enzymes in muscle revealed isolated complex I deficiency. This is the fourth pathogenic tRNA(Pro) point mutation to be associated with an mt disorder. The result highlights the importance of molecular dissection of mtDNA in patients with defined mt disorder and confirms the clinical and biochemical heterogeneity associated with tRNA(Pro) mutations.

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

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 tRNA ser(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.