tapraid5/za3-mus/za3-mus/za300909/za32342d09g knepper1 Sϭ7 7/20/09 11:41 Art: 08-0574 ABSTRACT: The potential pathogenicity of two homoplasmic mtDNA point mu- tations, 9035TϾC and 4452TϾC, found in a family afflicted with maternally trans- mitted cognitive developmental delay, learning disability, and progressive ataxia was evaluated using transmitochondrial cybrids. We confirmed that the 4452TϾC transition in tRNAMet represented a polymorphism; however, 9035TϾC conversion in the ATP6 gene was responsible for a defective F0-ATPase. Accordingly, mutant cybrids had a reduced oligomycin-sensitive ATP hydrolyzing activity. They had less than half of the steady-state content of ATP and nearly an 8-fold higher basal level of reactive oxygen species (ROS). Mutant cybrids were unable to cope with addi- tional insults, i.e., glucose deprivation or tertiary-butyl hydroperoxide, and they succumbed to either apoptotic or necrotic cell death. Both of these outcomes were prevented by the antioxidants CoQ10 and vitamin E, suggesting that the abnormally high levels of ROS were the triggers of cell death. In conclusion, the principal metabolic defects, i.e., energy deficiency and ROS burden, resulted from the 9035TϾC mutation and could be responsible for the development of clinical symp- toms in this family. Furthermore, antioxidant therapy might prove helpful in the management of this disease. Muscle Nerve 39: 000–000, 2009 IDENTIFICATION OF ATAXIA-ASSOCIATED mtDNA MUTATIONS (m.4052T>C and m.9035T>C) AND EVALUATION OF THEIR PATHOGENICITY IN TRANSMITOCHONDRIAL CYBRIDS MARIANNA SIKORSKA, PhD,1 JAGDEEP K. SANDHU, PhD,1 DAVID K. SIMON, MD, PhD,2 VIMUKTHI PATHIRAJA, MD,2 CAROLINE SODJA, MSc,1 YAN LI, MD,1 MARIA RIBECCO-LUTKIEWICZ, PhD,1 PATRICIA LANTHIER, BSc,1 HENRYK BOROWY-BOROWSKI, PhD,1 ADRIAN UPTON, MD, PhD,3 SANDEEP RAHA, PhD,4 STEFAN M. PULST, MD, PhD,5 and MARK A. TARNOPOLSKY, MD, PhD3,4 1 Neurogenesis and Brain Repair Group M54, Institute for Biological Sciences, National Research Council Canada, 1200 Montreal Road, Ottawa, Ontario, K1A 0R6, Canada 2 Department of Neurology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA 3 Department of Medicine (Neurology), McMaster University, Hamilton, Ontario, Canada 4 Department of Pediatrics (Neuromuscular and Neurometabolic Disorders), 1200 Main Street West, Room 2U26, McMaster University, Hamilton, Ontario, L8N 3Z5, Canada 5 Cedars-Sinai Medical Center, Los Angeles, California, USA Accepted 17 February 2009 Maternally inherited mitochondrial diseases are tion/deletions. To date, more than 130 pathogenic linked directly to mutations in the mitochondrial mtDNA mutations have been described. Many of genome, namely, base substitutions and/or inser- them are located in the polypeptide encoding re- gions and impair the functions of respiratory chain complexes, the final common pathway of aerobic Abbreviations: ATP, adenosine triphosphate; CFDA, 5-carboxyfluorescein metabolism. A variety of clinical features have been diacetate; CoQ10, coenzyme Q10; CuZnSOD, copper-zinc superoxide dis- associated with mitochondrial cytopathies, including mutase; ␥GCS, ␥-glutamyl cysteine ligase; GCLC, ␥-glutamyl cysteine ligase catalytic subunit; GCLM, ␥-glutamyl cysteine ligase modulatory subunit; GD, ptosis, ataxia, external ophthalmoplegia, optic atrophy, glucose deprivation; GSH, reduced glutathione; MnSOD, manganese super- pigmentary retinopathy, sensorineural deafness, neu- oxide dismutase; mtDNA, mitochondrial DNA; OXPHOS, oxidative phosphor- ylation; PTS, polyoxyethanyl-alpha-tocopheryl sebacate; ROS, reactive oxy- ropathy, cardiomyopathy, proximal myopathy, and ex- gen species; tBHP, tertiary butyl hydroperoxide ercise intolerance.10,11,28,34,49 Mitochondrial cytopa- Key words: antioxidant; homoplasmic mutations; mitochondrial; oxidative stress; therapy thies often share small clusters of clinical features that Correspondence to: M. Sikorska; e-mail: [email protected] or allow them to be grouped into clinical syndromes, i.e., M.A. Tarnopolsky; e-mail: [email protected] the Kearns–Sayre syndrome (KSS), chronic progressive © 2009 Wiley Periodicals, Inc. 25 Published online in Wiley InterScience (www.interscience. external ophthalmoplegia (CPEO), mitochondrial wiley.com). DOI 10.1002/mus.21355 encephalomyopathy with lactic acidosis and stroke-like Ataxia-Associated mtDNA Mutations MUSCLE & NERVE Month 2009 1 tapraid5/za3-mus/za3-mus/za300909/za32342d09g knepper1 Sϭ7 7/20/09 11:41 Art: 08-0574 episodes (MELAS),7 myoclonic epilepsy with ragged- utilizes the proton motive force generated by the red fibers (MERRF),15 Leigh syndrome (LS),8 or neu- respiratory chain complexes I–IV.30 rogenic weakness with ataxia and retinitis pigmentosa In this study we generated transmitochondrial (NARP).18 cybrids to discern the metabolic defects attributable The majority of pathogenic mtDNA mutations to the homoplasmic base substitutions, 4452TϾC are heteroplasmic, and the manifestation of clinical and 9035TϾC, which was identified in the affected symptoms occurs only after a critical proportion of family. The results revealed a significant decrease in mutant mtDNA in a tissue exceeds the threshold for oligomycin-sensitive ATP hydrolysis, reduction of genotype expression.45 This threshold varies for dif- ATP, and high reactive oxygen species (ROS) levels ferent types of mtDNA mutations. For example, for in the mutant cybrids. All these changes are consis- the 8344AϾG mutation, which causes MERRF syn- tent with alterations in F0-ATPase, which most likely Ͼ drome, the threshold level is about 85% of mutant resulted from the presence of the 9035T C transi- mtDNA,6 whereas for the 8993TϾG mutation, re- tion converting Leu-170 to proline in the A6 subunit. sponsible for the NARP phenotype, the threshold is The data also confirmed the polymorphic nature of Ͼ between 70% and 90%.9,47 Homoplasmic mutations, the 4452T C transition. on the other hand, are mostly regarded as polymor- phisms, and their pathogenic significance is often MATERIALS AND METHODS difficult to demonstrate. However, several homoplas- Subjects/Case Report. The subjects were members mic point mutations have been linked to clinical of a four-generation pedigree, in which 16 out of 17 Ͼ disorders such as deafness, i.e., the 1555A G tran- members were clinically affected by developmental sition in SNHL (nonsyndromic and aminoglycoside- delay, learning disability, and progressive ataxia induced sensorineural hearing loss) or blindness, starting in early to late childhood. The family history i.e., 11778GϾA, 3460AϾG, or 14484TϾC, which are was suggestive of a maternally transmitted mitochon- commonly found in LHON (Leber’s hereditary op- drial cytopathy (Fig. 1). Blood samples for DNA F1 tic neuropathy) patients.4 We and others22,42 have analysis were taken from eight members of the ped- also described a variety of clinical features associated with homoplasmic mutations. Recently, we identified a family in which several members, maternally linked across four generations, were afflicted with developmental delays, learning disabilities, and ataxia. Genetic testing for spinocer- ebellar ataxias 1, 2, 3, 6, 7, 8, 17, Friedreich’s ataxia (FRDA), and the 8993TϾG/C NARP/MILS muta- tions were all negative. Sequencing of the mtDNA revealed that all affected individuals carried two ho- moplasmic basepair (bp) substitutions, 4452TϾCin tRNAMet and 9035TϾC in the ATP6 gene (A6 sub- unit), which might have been an underlying cause of the clinical symptoms. The mitochondrial tRNAMet is one of 22 tRNAs encoded by mtDNA and is utilized in translation of 13 essential subunits of respiratory FIGURE 1. A pedigree diagram. Sixteen of 17 family members, chain complexes (i.e., I, III, IV, and V). This is a maternally linked across four generations (marked in black), have critical gene for mitochondrial protein synthesis, es- been clinically affected with cognitive developmental delay, learn- pecially in the mammalian system, which utilizes a ing disability, and progressive ataxia starting in childhood. The Met single tRNA species not only for two alternative M only person not affected was a 5-year-old boy (III6), born to a codons (AUG and AUA), but also for both initiation male, who showed no evidence of ataxia or developmental is- sues. Arrows point to probands II and III , mother and daughter, 12 1 1 and elongation of translation. The A6 is one of the respectively, who donated blood for the generation of transmito- two mitochondrially encoded subunits of the F0 por- chondrial cybrids. Two cybrid clones from each of these two tion of ATP synthase (complex V). Structurally, ATP mtDNA donors were selected for further studies: CF2B1 and CF2D2 generated from proband II1 and JE1B2 and JE2G1 gen- synthase is comprised of a rotary catalytic F1 portion, erated from proband III1. *Patient a neurological examination a transmembrane F0 portion, and two stalks that link completed by one of the authors (M.T.). Patients IV2 and IV3 were 3,19 F1 and F0. The F1F0-ATP synthase complex is examined by a pediatric neurologist in their home town and were located in the inner mitochondrial membrane and confirmed to have developmental delay and ataxia. 2 Ataxia-Associated mtDNA Mutations MUSCLE & NERVE Month 2009 tapraid5/za3-mus/za3-mus/za300909/za32342d09g knepper1 Sϭ7 7/20/09 11:41 Art: 08-0574 igree across four generations, and muscle biopsies some experiments, cells were pretreated 3ϫ a week and nerve conduction tests were obtained in two for 1 week with a water-soluble