Leigh syndrome associated with mitochondrial complex I deficiency due to novel mutations In NDUFV1 and NDUFS2

Gene 516 (2013) 162–167

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Short Communication

Leigh syndrome associated with mitochondrial complex I deficiency due to novel mutations In NDUFV1 and NDUFS2☆,☆☆ Samantha E. Marin a, Ronit Mesterman a, Brian Robinson b, Richard J. Rodenburg c, Jan Smeitink c, Mark A. Tarnopolsky a,⁎ a

Department of Pediatrics, McMaster Children's Hospital, Hamilton, Ontario, Canada Metabolism Research Program, Research Institute, Department of Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders, Institute for Genetic and Metabolic Disease, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands

b c

a r t i c l e

i n f o

Article history: Accepted 2 December 2012 Available online 22 December 2012 Keywords: Leigh NDUFV1 NDUFS2 Mitochondria Nicotinamide adenine dinucleotide: ubiquinone oxidoreductase

a b s t r a c t Leigh syndrome (LS) is a progressive neurodegenerative disease caused by either mitochondrial or nuclear DNA mutations resulting in dysfunctional mitochondrial energy metabolism. Mutations in genes encoding for subunits of the respiratory chain or assembly factors of respiratory chain complexes are often documented in LS cases. Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) enzyme deficiencies account for a significant proportion of mitochondrial disorders, including LS. In an attempt to expand the repertoire of known mutations accounting for LS, we describe the clinical, radiological, biochemical and molecular data of six patients with LS found to have novel mutations in two complex I subunits (NDUFV1 and NDUFS2). Two siblings were homozygous for the previously undescribed R386C mutation in NDUFV1, one patient was a compound heterozygote for the R386C mutation in NDUFV1 and a frameshift mutation in the same gene, one patient was a compound heterozygote for the R88G and R199P mutations in NDUFV1, and two siblings were compound heterozygotes for an undescribed E104A mutation in NDUFS2. After the novel mutations were identified, we employed prediction models using protein conservation analysis (SIFT, PolyPhen and UCSC genome browser) to determine pathogenicity. The R386C, R88G, R199P, and E104A mutations were found to be likely pathogenic, and thus presumably account for the LS phenotype. This case series broadens our understanding of the etiology of LS by identifying new molecular defects that can result in complex I deficiency and may assist in targeted diagnostics and/or prenatal diagnosis of LS in the future. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction Abbreviations: A, Alanine; C, Cysteine; CSF, Cerebrospinal fluid; E, Glutamic acid; F, Phenylalanine; G, Glycine; L, Leucine; LS, Leigh syndrome; MRI, Magnetic resonance imaging; MRS, Magnetic resonance spectrometry; mtDNA, Mitochondrial DNA; NAA, N-acetylaspartic acid; NADH, Nicotinamide adenine dinucleotide; nDNA, Nuclear DNA; NDUFA, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex; NDUFS, NADHubiquinone oxidoreductase Fe–S protein; NDUFV, NADH dehydrogenase (ubiquinone) flavoprotein; NDUFS2, NADH dehydrogenase (ubiquinone) Fe–S protein 2; nsSNP, Non-synonymous single nucleotide polymorphism; P, Proline; PDHC, Pyruvate dehydrogenase complex; POLG, Polymerase gamma; R, Arginine; SIFT, Sorting intolerant from tolerant. ☆ Declaration of conflicting interests: The authors declare no conflicts of interest with respect to the research, authorship, and/or publication of this article. ☆☆ Financial disclosure/funding: Financial support for this article was made possible by donations in memory Cilian Barrows. Dr. Tarnopolsky is partially supported from an endowed chair in Neuromuscular and Neurometabolic disorders from McMaster Children's Hospital and Foundation. ⁎ Corresponding author at: Division of Neurology, Department of Pediatrics and Medicine, Head, Neuromuscular and Neurometabolic Disease, McMaster Children's Hospital, 1200 Main St. West, Room 2H26, Hamilton, Ontario, Canada L8N 3Z5. Tel.: +1 905 521 2100x75226; fax: +1 905 577 8380. E-mail address: [email protected] (M.A. Tarnopolsky).

Leigh syndrome (LS) is a progressive neurodegenerative disorder, also known as subacute necrotizing encephalomyelopathy. The syndrome was first described by Leigh (1951) based on the pathological findings of widespread focal, bilaterally symmetrical subacute necrotic lesions extending from the thalamus to the spinal cord in a 7-monthold who presented with rapidly progressive neurological deterioration. The incidence of LS is 1:77,000 live births (Rahman et al., 1996) and the prevalence is estimated at 1:34,000 (Fernandez-Moreira et al., 2007). The incidence of LS is higher in males (male-to-female ratio= 3:2), which is not solely explained by the X-linked inheritance of pyruvate dehydrogenase complex (PDHC) E1 alpha and complex I subunit NDUFA1-associated defects (Fernandez-Moreira et al., 2007; Rahman et al., 1996). The onset of clinical features is typically between 3 and 12 months of age (Rahman et al., 1996; Thorburn and Rahman, 2003); however, a later onset (including onset in adulthood) has been described in 25% of patients (Goldenberg et al., 2003; Huntsman et al., 2005). Initial features may be non-specific, including failure to thrive and/or persistent vomiting. Decompensation during intercurrent illness

0378-1119/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.12.024

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is common, with associated psychomotor regression and the presentation of new neurological features, and often incomplete recovery (Thorburn and Rahman, 2003). The most common neurological features are developmental delay (86%), seizures (79%), and altered level of consciousness (57%) (Huntsman et al., 2005). Other associated neurological features include abnormalities in tone, muscle weakness, movement disorders, ataxia, tremor, peripheral neuropathy, central respiratory disturbance, bulbar symptoms (dysarthria, dysphagia), and abnormalities of thermoregulation (Lee et al., 2009; Rahman et al., 1996; Thorburn and Rahman, 2003). Extraneurologic features include diabetes, short stature, hypertrichosis, anemia, cardiomyopathy (hypertrophic or dilated), hepatomegaly, renal tubulopathy or diffuse glomerulocystic kidney damage, and optic atrophy, retinitis pigmentosa, and ophthalmoplegia to varying degrees (Agapitos et al., 1997; Lee et al., 2009; Leshinsky-Silver et al., 2003; Tay et al., 2005; Yamakawa et al., 2001). The course of illness is one of episodic deterioration interspersed with plateaus, during which development may be staple or even progress. Ultimately, death occurs within early childhood (50% by 3 years of age) due to respiratory insufficiency or cardiac failure (Lee et al., 2009; Rahman et al., 1996; Thorburn and Rahman, 2003). The diagnosis of LS requires a combination of clinical features, serologic and CSF investigations, radiological and pathological features. The criteria for LS have been outlined (Rahman et al., 1996), which require progressive neurologic disease with motor and intellectual developmental delay, signs and symptoms of brainstem and/or basal ganglia disease, raised lactate concentration in blood and/or CSF and one or more of characteristic features on neuroimaging. Neuropathological changes include multiple focal symmetric lesions in the basal ganglia, thalamus, brainstem, dentate nuclei and optic nerves with a spongiform appearance characterized by demyelination, gliosis and vascular proliferation and relative sparing of neurons. Typical MRI findings include bilateral symmetrical T2-weighted imaging hyperintensities in the brainstem and/or basal ganglia, particularly in the dorsal aspects of pons and medulla and putamen (Arii and Tanabe, 2000; Lee et al., 2009). MRS may reveal regional elevations in brain lactate levels. Deficits of respiratory chain complex subunits (complex I, II, IV, and V) and their cofactors (e.g. co-enzyme Q10), mtDNA encoded tRNA or the PDHC are known causes of LS. Greater than 100 mutations have been identified thus far associated with a LS phenotype, both in mitochondrial DNA (30%) and nuclear DNA (Finsterer, 2008). Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest enzymatic complex (45 subunits) of the mitochondrial respiratory chain and deficiencies of complex I account for the majority of mitochondrial disorders, including LS (Smeitink and van den Heuvel, 1999). In recent years, mutations have been described in more than ten nuclear-encoded subunits of complex I, which account for the majority of complex I deficient cases in infancy and childhood with known molecular characterization. We describe the clinical, radiological, biochemical and molecular data of six patients with LS found to have novel mutations in three complex I subunits (NDUFV1 and NDUFS2). Two siblings were homozygous for the previously undescribed R386C mutation in NDUFV1, one patient was a compound heterozygote for a R386C mutation in NDUFV1 and a frameshift mutation (753delCCCC), one patient was a compound heterozygote for R88G and R199P mutations in NDUFV1 and two siblings were compound heterozygotes for the F84L and an undescribed E104A mutation in NDUFS2. After the novel mutations were identified, we employed prediction models using protein conservation analysis (SIFT, PolyPhen and UCSC genome browser) to determine pathogenicity of the novel mutations.

2. Methods A retrospective chart analysis was conducted on six patients from four families with a diagnosis of LS known to have novel mutations in

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complex I subunits who presented to McMaster University Medical Center from 1999 to 2009. All sequencing was completed using PCR amplification and sequencing of exons and 50 bp into the intronic region with dye-labeled primers for the target genes of for the NDUF mutations described below. Sequencing was completed using an ABI Prism 3100 DNA sequencer (Applied Biosystems, Foster City, CA). Protein conservation analysis was performed using the sorting intolerant from tolerant (SIFT) software (Kumar et al., 2009; Ng and Henikoff, 2001, 2002). The SIFT algorithm predicts whether an amino acid substitution in a protein of interest will have a tolerated or deleterious effect on protein function. This is accomplished by aligning similar protein sequences to the sequence in question and calculating normalized probabilities for all possible substitutions from the alignment to determine the evolutionary conservation status of the amino acid of interest. Normalized probabilities less than 0.05 are predicted to be deleterious, while probabilities greater than 0.05 are predicted to be tolerated. The sensitivity and specificity of SIFT in predicting the pathogenicity of single nucleotide polymorphisms associated with disease is 69% and 13%, respectively (Flanagan et al., 2010; Ng and Henikoff, 2002). Polymorphism phenotyping (PolyPhen) (Ramensky et al., 2002; Sunyaev et al., 2000, 2001) was used for further prediction of pathogenicity. PolyPhen is a tool for predicting whether an amino acid substitution in a protein of interest will be benign or damaging. It focuses on non-synonymous single nucleotide polymorphism (nsSNP) effects by applying empirical rules to the sequence, phylogenetic, and structural information characterizing the amino acid substitution. A positionspecific independent counts (PSIC) profile score is calculated that allows for the categorization of amino acid substitutions as benign, possibly damaging or probably damaging. The sensitivity and specificity of PolyPhen in predicting the pathogenicity of single nucleotide polymorphisms associated with disease is 68–82% and 16%, respectively (Flanagan et al., 2010; Ramensky et al., 2002). Amino acid conservation was further sought from the UCSC genome browser (http://genome.ucsc.edu/) (Fujita et al., 2011; Kent et al., 2002). UCSC genome browser gives a pictorial representation of the sequence of the protein of interest in humans and 46 other vertebrate species and leaves the reader to assess whether or not a specific amino acid is conserved but does not itself calculate tolerability. 3. Clinical vignettes 3.1. Case 1 Case 1 is a male who was born at term to non-consanguineous parents of Southeast-Asian descent. Family history was non-contributory. He had an unremarkable medical and developmental history before he presented at 2.8 years with abnormal posturing of his left upper and lower extremity. Over the following 12 months, he developed developmental regression in a step-wise fashion associated with intercurrent illness, left eye esotropia, significant dysphagia requiring gastrostomy tube placement, ataxia, left hemi-body dystonic posturing, generalized spasticity, diffusely brisk reflexes, and extensor plantar responses. As a result of a high suspicion of a mitochondrial disorder, he was started on co-enzyme Q10, creatine monohydrate, riboflavin, thiamine, vitamin E, and vitamin C. Investigations revealed a mildly elevated serum lactate concentration (3.1 mmol/L, NRb 2.2 mmol/L) and a cranial CT scan showed bilateral symmetrical signal hypoattenuation involving the periventricular white matter and basal ganglia. MRI revealed bilateral symmetric hyperintense signal on T2-weighted imaging in the basal ganglia, thalamus, brainstem, corpus callosum and periventricular white matter, associated with cystic necrosis and a high lactate peak in affected areas on MR spectroscopy. A repeat MRI at 4 years showed significant improvement in the T2 hyperintense signal abnormalities and resolution of the lactate peak on

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spectroscopy, but an increase in cystic changes was noted. A muscle biopsy showed non-specific histological changes and muscle enzyme analysis was within normal limits. Fibroblast enzyme analysis showed a borderline low complex I + III activity (0.63 μmol/min/g wet weight; reference control 0.77 ± 0.12 μmol/min/g wet weight). Repeat fibroblast enzyme analysis revealed normal pyruvate dehydrogenase complex activity, normal cellular lactate to pyruvate ratio, deficient complex I+III activity with respect to citrate synthase (0.18; reference control 0.66) and a low pyruvate oxidation rate (0.70±0.02; reference control 1.63 ± 0.31 nmol/h/mg protein). He tested negative for the following mutations: 1. MtDNA: T8993C, T8993G, C11777A, G13513A, T12706C, and G14459A; 2. nDNA: NDUFS1, NDUFS2, NDUFS7, and NDUFS8. He was homozygous for a novel sequence variant in the NDUFV1 gene (NADH dehydrogenase flavoprotein 1) in exon 8 (c.1156C>T; p.Arg386Cys) (see Table 1). SIFT showed that the only amino acid predicted to be tolerated at amino acid 386 was arginine (R), with cysteine (C) predicted to be not tolerated. PolyPhen indicated that the R386C substitution was probably damaging, and UCSC genome browser showed that arginine was conserved at position 386 in all 46 other vertebrate species analyzed; further supporting that the sequence variant would likely be pathogenic. Case 1 is currently 9 years of age. He is wheelchair bound with significant contractures most prominent in knee flexors and hip adductors, despite physiotherapy and pharmacological intervention. He has global muscle atrophy, dystonia, hemiballismus and chorea. He is non-verbal but communicates with facial intonation and eye movements. 3.2. Case 2 Case 2 is a male who was born at 36 weeks gestational age to the parents of Case 1 and was followed from birth due to his brother's diagnosis. At 9 months of age, he developed horizontal nystagmus but his neurological exam was otherwise unremarkable. By 14 months of age, he developed mild titubation, drooling, increased irritability, axial hypotonia, lower extremity hypertonia, diffusely brisk reflexes and an extensor plantar response bilaterally with gait ataxia and frequent falls. He was started on co-enzyme Q10, alpha-lipoic acid, vitamin E, riboflavin and creatine monohydrate. MRI revealed bilateral symmetric hyperintense signal on T2-weighted imaging in his periventricular white matter, centrum semiovale, corpus callosum, substantia nigra and periaqueductal gray associated with cystic necrosis, with a high lactate peak, decreased N-acetylaspartic acid (NAA) peak and increased choline peak in affected areas on spectroscopy with basal ganglia sparing. Mutation analysis confirmed homozygosity for the family mutation in exon 8 of NDUFV1 (c.1156C> T; p.Arg386Cys). Case 2 showed a dramatic improvement with respect to his neurological features on subsequent follow-up, with the exception of intermittent acute decompensation associated with intercurrent illness with full recovery to previous baseline. He is currently 3.6 years of age with mildly spastic tone in the lower extremities and gait instability. 3.3. Case 3 Case 3 is a female who was born at term to non-consanguineous Caucasian parents. Her family history was non-contributory. She had an unremarkable medical and developmental history before she presented at 14.5 months of age with a rapid deterioration in psychomotor functioning (to the point of becoming non-verbal and being unable to sit independently), irritability, horizontal nystagmus, dysphagia, tremor, upper extremity weakness, axial hypotonia with appendicular hypertonia, hyperreflexia, and extensor plantar responses bilaterally after receiving a vaccination. She was started on co-enzyme Q10, alpha-lipoic acid, riboflavin, thiamine, and creatine monohydrate for likely mitochondrial disease, as well as carnitine supplementation. Within a month, she slowly began to make gains in her development such that she was able to fix and follow objects, smile at appropriate

stimuli, speak a few words, transfer objects between her hands and sit independently. Her continued developmental progression was interrupted by acute decompensation secondary to minor falls and head trauma, with incomplete recovery. Investigations revealed an elevated blood lactate concentration (4 mmol/L), low total and free carnitine, and an MRI demonstrated bilateral, confluent signal abnormalities in periventricular, juxtacortical and callosal white matter with a lactate peak in affected areas on spectroscopy that was basal ganglia sparing. Repeat MRIs initially showed an improvement in the white matter signal changes and new areas of restricted diffusion within the frontal lobes but later revealed increases in the abnormal white matter signals with new involvement of the basal ganglia, diffuse cystic change and an elevated lactate peak in the basal ganglia on spectroscopy. Two muscle biopsies revealed non-specific changes. Fibroblast enzyme analysis initially revealed an elevated lactate:pyruvate ratio (51.2 ±13.7; reference control 15.4 ± 1.4 nmol/h/mg protein), a low citrate synthase (51.4; reference control 119.1 nmoles/min/mg protein), low complex I + III activity (20.3; reference control 87.8 nmoles/min/mg protein) and low complex I + III activity with respect to citrate synthase. Repeat enzyme analysis was normal. She tested negative for the following mutations: 1. MtDNA: T8993C, T8993G, C11777A, G13513A, T12706C, and G14459A; 2. nDNA: NDUFA1, NDUFA2, NDUFA4, NDUFA7, NDUFAG2, NDUFS1, NDUFS3, NDUFS4, NDUFS5, NDUFS6, NDUFS7, NDUFS8, NDUFV2, twinkle and POLG. She was a compound heterozygote for two previously undescribed variants in NDUFV1 including the single nucleotide polymorphism in exon 8 (c.1156C>T; p.Arg386Cys; described in Case 1 and 2 above) and a frameshift mutation in exon 6 (c.753delCCCC; p.Ser251SerfsX44). Considering the frameshift mutation resulted in the premature truncation of the protein secondary to the earlier insertion of a stop codon, it was regarded as likely deleterious to protein function. Case 3 is currently 7 years of age. She has decreased visual acuity requiring glasses (20/100 OU), dysphagia and complex partial seizures. She lost the ability to ambulate independently due to severe spasticity, chorea, myoclonus, tremor, and dystonia in her lower extremities. 3.4. Case 4 Case 4 was a male who was born at term to non-consanguineous parents of Scottish, Irish and English descent. Family history was unremarkable. Pregnancy was remarkable for oligohydramnios and intrauterine growth restriction recognized on an ultrasound two days prior to delivery, for which labor was induced. Delivery was complicated by abnormal fetal heart rate prior to delivery, but he subsequently required no resuscitation. He was noted to be microcephalic at birth (head circumference under the third percentile). Soon after birth, he developed lactic acidosis (lactate 20.8 mmol/L) with respiratory compensation, hypoglycemia (0.8 mmol/L; normal range: 4.0–11.1 mmol/L). Physical examination revealed hypotonia, decreased spontaneous movements and hyperreflexia in the left lower extremity. He was placed on biotin, carnitine, and thiamine. His condition improved despite a persistent lactate elevation (6.2 mmol/L). An MRI revealed symmetrical restricted diffusion of the corticospinal tracts and a lactate peak on MRS in the basal ganglia, thalamus and cortex. An electroencephalogram revealed a dysmature background for gestational age and frequent bilateral positive and negative rolandic sharp waves. Plasma amino acids revealed an elevated alanine (1536 μmol/L; normalb 475 μmol/L), urine organic acids revealed an elevated lactate and CSF revealed a mildly elevated glycine compared to plasma (0.07, not sufficient enough to warrant a diagnosis of nonketotic hyperglycinemia), an elevated lactate (18.6 mmol/L; normal b 1.9 mmol/L) and an elevate lactate to pyruvate ratio. A muscle biopsy was inconclusive, but there were no clear changes of a mitochondrial cytopathy. Genetic testing revealed heterozygosity for two novel sequence variants in the NDUFV1 gene (c.262C >G; p.Arg88Gly and c.596G> C, p.Arg199Pro). SIFT showed that the only amino acid

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predicted to be tolerated at amino acid 88 was arginine (R), while glycine (G) was predicted to be not tolerated. At position 199, proline (P) was predicted not to be tolerated. PolyPhen indicated that the R88G substitution was probably damaging while the R199P substitution was probably benign. UCSC genome browser showed that arginine was conserved at position 88 and 199 in all 46 other vertebrate species analyzed; further supporting that these sequence variants would likely be pathogenic. Case 4 expired at the age of 4 months after an acute decompensation with severe lactic acidosis and cerebral edema secondary to an upper respiratory tract infection. 3.5. Case 5 Case 5 was a male who was born at term to non-consanguineous parents of Dutch (paternal) and Italian/Scottish (maternal) descent. Family history was remarkable for a previous miscarriage in his parents, but was otherwise non-contributory. He had an unremarkable developmental and medical history until the age of 6 months, at which point he developed episodes of repeated emesis causing significant weight loss, which improved with smaller feeds. By 3 years of age, he had developed an intention tremor, irritability, multiple apneic episodes requiring tracheostomy placement, left optic atrophy, a left exotropia, gaze-evoked horizontal nystagmus, central hypotonia, diffusely brisk reflexes with clonus, extensor plantar responses bilaterally, an intention tremor, and a spastic-ataxic gait. He was placed on co-enzyme Q10, riboflavin, vitamin C, vitamin E, thiamine and creatine monohydrate for suspected mitochondrial disease. He had a normal serum and CSF lactate. An MRI revealed T2weighted hyperintensities of the substantia nigra and periaqueductal gray matter on the left, bilateral subthalamic nuclei and bilateral thalami. A repeat MRI showed additional signal abnormalities within the pontine tegmentum and progression of the previous abnormalities aforementioned. A muscle biopsy was non-specific. Fibroblast enzyme analysis revealed a borderline low complex I+ III activity (0.89; reference control 0.5–1.9 μmol/min/g wet weight), elevated citrate synthase (16.5; reference control 0.8–5.5 μmol/min/g wet weight), an elevated cellular lactate:pyruvate ratio (51.8; reference control 15±1.2) and a low ratio of complex I +III/citrate synthase 5.3% (N >10%). Repeat fibroblast enzyme analysis revealed low complex I activity (11.4 mU/mg protein; control 18.9–44.2). He tested negative for the following mutations: 1. MtDNA: T8993C, T8993G, A8334G; 2. nDNA: NDUFS1, NDUFS4, and NDUFS7. He was found to be a compound heterozygote for two mutations in the NDUFS2 gene (c.252T> G; p.Phe84Leu and c.311A>G; p.Glu104Ala). Analysis of the p.Glu104Ala mutation through SIFT showed that the only amino acid predicted to be tolerated at amino acid 104 was glutamic acid (E), while alanine (A) was predicted to be not tolerated. PolyPhen indicated that the E104A substitution was predicted to be probably damaging, and UCSC genome browser showed that glutamic acid was conserved at position 104 in all 46 other vertebrate species analyzed; further supporting that the sequence variant would likely be pathogenic. The second mutation (p.Phe84Leu) was not conserved in all species and was deemed likely benign by PolyPhen. At 3.5 years, he died as a result of respiratory failure. 3.6. Case 6 Case 6 is a female born at term to the parents of Case 5. Her medical history prior to presentation was significant for a brief apneic episode at the age of 2 years and recurrent vomiting at the age of 12 months, but she was otherwise healthy and met all of her developmental milestones appropriately. At 5 years of age, she developed decreasing visual acuity and abnormal color vision associated with optic atrophy, horizontal nystagmus, dysarthria with scanning-type speech, dysmetria, ataxia, and right-sided hemiparesis. Considering her brother's course, there was concern that her neurological findings were secondary to similar

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mitochondrial pathology. She was subsequently placed on co-enzyme Q10, alpha-lipoic acid, vitamin E, vitamin C, riboflavin, and creatine monohydrate. A cranial MRI showed bilateral thinning of the optic nerves, atrophic changes of the optic chiasm and mild cerebellar atrophy, but was otherwise unremarkable. She tested negative for mutations associated with Leber's Hereditary Optic Neuropathy but she was found to be a compound heterozygote for the same mutations in the NDUFS2 gene as her brother (c.252T > G; p.Phe84Leu and c.311A> G; p.Glu104Ala). Testing of her parents revealed that their mother carried the E104A allele, while their father carried the F84L allele. As previously noted, their parents were asymptomatic. Case 6 is currently 17 years of age. Her vision has continued to deteriorate. Her current visual acuity is 20/400 bilaterally, which improves to 20/200 with pinhole. Her optic disc pallor and atrophy is unchanged. She has a subtle intension tremor bilaterally and her gait is only mildly ataxic. 4. Discussion Leigh syndrome is a progressive neurodegenerative disorder presenting in early childhood associated with episodic deterioration superimposed on a background of chronic evolution resulting in early demise. Most previous studies have focused on mtDNA abnormalities and have investigated the link between the genetic defect in mtDNA and clinically defined syndromes. Clinical data linked to mutations in nuclear genes have emerged more recently and have been less documented. Complex I catalyzes the proton-motive oxidation of NADH by ubiquinone and consists of 45 subunits, 38 of which are encoded by the nuclear genome (Walker, 1992). Complex I mutations have been implicated in a vast number of mitochondrial disorders, including LS (Smeitink and van den Heuvel, 1999). We have described two novel nDNA mutations within complex I subunits, NDUFV1 and NDUFS2, that are thought to be pathogenic and resulting in a LS phenotype. The first novel mutation described here in exon 8 of NDUFV1 (R386C) was reported in three patients (Cases 1–3) from two unrelated families with different ethic backgrounds. While Cases 1 and 2 are homozygous for this mutation, Case 3 is a compound heterozygote, also carrying a frameshift mutation resulting in protein truncation secondary to the premature insertion of a stop codon. Considering that two different families have exhibited this novel variant and a LS phenotype, there is a stronger impetus to label it pathogenic. Despite all sharing the mutation, all three have differed with respect to age of onset, rapidity of clinical deterioration and MRI features. Interestingly, Case 2, despite having the earliest onset, has had the mildest course. The reasons for this are likely threefold: (1) the identification of his neurological features occurred in a more timely fashion because he was followed closely from birth as a result of the knowledge of his brother's phenotype; (2) the combination of a frameshift mutation causing the insertion of a premature stop codon may have worsened the clinical presentation in Case 3 by producing a non-functional protein; and, although stated with caution as LS patients have a variable disease course, (3) the early implementation of the “mitochondrial cocktail” consisting of coenzyme Q10, creatine monohydrate, vitamin E, riboflavin and alphalipoic acid may have prevented the full expression of his destined clinical phenotype. For a more comprehensive review of the role of the compounds used in the “mitochondrial cocktail” in targeting biochemical pathways implicated in mitochondrial disease and the use of these compounds in mitochondrial disease, the reader is referred to previous studies (Peterson, 1995; Rodriguez et al., 2007; Tarnopolsky, 2008). Novel mutations in the NDUFV1 gene were also found in Case 4 (R88G and R199P). Case 4 presented with a severe phenotype shortly after birth and subsequently expired secondary to decompensation with an upper respiratory infection at only 4 months of life. His persistently elevated plasma, urine and CSF lactate, rapid clinical decompensation

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and lactate peaks on MR spectroscopy were felt to be consistent with LS, and other possible diagnoses (including maple syrup urine disease considering the corticospinal tract signal abnormalities on MRI) were ruled out. On analysis of the mutations identified on genetic testing, the R88G mutation is likely pathogenic considering that arginine was the only amino acid predicted to be tolerated by SIFT, it was conserved across all 46 species examined in UCSC genome browser, and it was found to be likely pathogenic on Polyphen analysis. The R199P mutation, however, was shown to be likely benign by PolyPhen analysis. Considering that a proline substitution was not tolerated according to SIFT analysis and UCSC browser showed arginine preserved at position 199 across all species, it was felt that this mutation likely contributed to his clinical presentation despite the PolyPhen findings. Cases 5 and 6 were compound heterozygotes for the novel F84L and E104A mutations in the NDUFS2 gene. While the F84L mutation was interpreted by SIFT and PolyPhen as likely a benign variant, the E104A mutation was deemed likely pathogenic. Despite having identical genotypes, they had significant differences in clinical phenotypes: Case 5 (male) had a rapidly progressive course with early bulbar and respiratory involvement and MRI signal abnormalities within the brainstem and basal ganglia while Case 6 (female) has a relatively mild disease consisting of primarily optic involvement. While the variance in clinical presentation in mutations in mtDNA can easily be explained by the degree of heteroplasmy in affected tissues, this is less easily explained when there is a mutation in nDNA. With the identification of more patients carrying this mutation, further genotype–phenotype correlations can be made. It is possible that case 5 carries a currently unknown mutation in another subunit of complex I resulting in synergistic heterozygosity and, thus, producing a more severe phenotype. However, considering the gender difference in phenotypic expression (especially considering that their mother, who also carries the pathogenic allele, is asymptomatic), this may highlight is the potential role of estrogen against oxidative stress-mediated neurodegeneration that has been suggested in neurodegenerative diseases, such as Parkinson's disease. Aside from its role in sexual differentiation, studies have shown the role of estrogen, specifically 17β-estradiol (E2), in synaptic plasticity and neuroprotection (Brinton, 2009; Giordano et al., 2011; Numakawa et al., 2011; Simpkins and Dykens, 2008; Simpkins et al., 2009; Zhang et al., 2009). Importantly, estrogen is believed to play a protective role in the maintenance of mitochondrial function under toxic stress (Numakawa et al., 2011; Simpkins et al., 2009). Recently, it has been shown that estrogens ameliorate mitochondrial dysfunction caused by mutations affecting complex I in Leber's Hereditary Optic Neuropathy (LHON), which may account for the previously unexplained male predominance. Giordano et al. (2011) showed the following findings: (1) that E2 rescued LHON cybrid cells from overproduction of reactive oxygen species (leading to an increased apoptotic rate and loss of cell viability); (2) E2 induced an activation of mitochondrial biogenesis and an improvement in energetic competence; and (3) E2-receptor β localizes to the mitochondrial network of human retinal ganglion cells. This supports the notion that further exploration of the role of estrogens in neuroprotection in mitochondrial disease is warranted. Determination of pathogenicity in this current paper involved the use of SIFT and PolyPhen conservation analysis models. As mentioned above, there are inherent limitations to the use of such programs. SIFT correctly predicted only 69% of substitutions associated with disease known to affect protein function (Ng and Henikoff, 2002). The sensitivity and specificity of SIFT in predicting the pathogenicity of single nucleotide polymorphisms associated with disease is 69% and 13%, respectively (Flanagan et al., 2010; Ng and Henikoff, 2002). The sensitivity and specificity of PolyPhen in predicting the pathogenicity of single nucleotide polymorphisms associated with disease is believed to be 68–82% and 16%, respectively (Flanagan et al., 2010; Ramensky et al., 2002). PolyPhen has been associated with high false positive (8%) and false negative (up to 90%) rates by some authors (Sunyaev et al., 2001). Thus, the information provided by these databases must be scrutinized and await

independent confirmation in future cases. However, the certainty with which these mutations are likely to be pathogenic is increased by the fact that each mutation has affected sibling pairs who present with varying degrees of clinical features associated with LS and by the identification of the same novel mutation in separate, unrelated families (as with Cases 1–3). Furthermore, the use of UCSC browser to demonstrate the conservation of the particular mutated amino acid in 46 other vertebrate species also provides support. 5. Conclusions Within the last few decades, the understanding of the genetic underpinnings of LS has expanded. This current paper presents two mutations associated with a LS phenotype not previously described in the literature. With the continued addition of genetic modifications underlying this syndrome, it will potentially allow for a better understanding of the etiology of the disease as well as the potential for genotype–phenotype correlations in the future. Of clinical importance, the knowledge of mutations causing LS may allow for prenatal diagnosis in the near future and assist in earlier implementation of therapies, such as the “mitochondrial cocktail”. Acknowledgments The authors would like to acknowledge Mahmood Akhtar for his diligent work as part of our team. We would also like to acknowledge all those who donated to the Cilian Barrows fund for research into Leigh's disease. The DNA analysis for Patients #3 and 4 was completed by Transgenomic Laboratories, 12325 Emmet Street, Omaha, NE 68164, USA. Appendix A

Table 1 Genetic findings. Gene

Sequence variant

Amino acid substitution

SIFT

Cases NDUFV1 c.1156C > T p.Arg386Cys Not 1–3 (R386C) tolerated Case 4 NDUFV1 c.262C > G p.Arg88Gly Not (R88G) tolerated NDUFV1 c.596G >C p.Arg199Pro Not (R199P) tolerated Cases NDUFS2 c.252T > G p.Phe84Leu Tolerated 5–6 (F84L) NDUFS2 c.311A > G p.Glu104Ala Not (E104A) tolerated

Polyphen

UCSC genome browser

Probably damaging Probably damaging Probably benign Probably benign Probably damaging

Conservation of arginine Conservation of arginine Conservation of arginine Phenylalanine not conserved Conservation of glutamic acid

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