Zn superoxide dismutase (sod1) gene associated with amyotrophic lateral sclerosis

Journal of the Neurological Sciences 254 (2007) 17 – 21 www.elsevier.com/locate/jns

Identification of a novel D109Y mutation in Cu/Zn superoxide dismutase (sod1) gene associated with amyotrophic lateral sclerosis Ali Naini a,⁎, Mahsa Mehrazin a , Jiesheng Lu a , Paul Gordon b , Hiroshi Mitsumoto b a

b

H. Houston Merritt Center, Department of Neurology, Columbia University Medical Center, New York, NY USA The Elenor and Lou Gehrig MDA/ALS Research Center, Department of Neurology, Columbia University Medical Center, New York, NY USA Received 28 August 2006; received in revised form 22 November 2006; accepted 8 December 2006 Available online 25 January 2007

Abstract We report a novel missense mutation (Asp109Tyr) in exon 4 of the Cu/Zn superoxide dismutase (sod1) gene in a woman with apparently sporadic amyotrophic lateral sclerosis (SALS). Signs of motor deficit appeared at the age of 51 years which progressed over the next 6 years to upper and lower motor neuron disease and death occurred by the age of 57 years. In this mutation, the base change of guanine to thymine at codon 109 of sod1 gene leads to the replacement of aspartic by tyrosine in the protein. This amino acid change in the protein however, did not alter the catalytic activity of the SOD1 enzyme as there was no change in the enzymatic activity of purified SOD1 from the patient's erythrocytes compared to control. © 2006 Elsevier B.V. All rights reserved. Keywords: Superoxide dismutase; Amyotrophic lateral sclerosis; SALS

1. Introduction Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive muscle paralysis due to degeneration of motor neurons in the cortex, brainstem, and spinal cord [1]. With a lifetime risk of 1 in 1000, it is the most common motor neuron disease [2]. Despite extensive research, the cause or causes of ALS remain unknown. Most (90–95%) of ALS cases are sporadic (SALS). The remaining 5–10% cases are familial (FALS). Cu/Zn superoxide dismutase (SOD1) mutations are the most commonly known cause of ALS. About 20% of FALS and 4% of SALS cases are associated with the mutation in the sod1 gene [3–6]. SOD1 is a ubiquitously expressed radical oxygen species-scavenging enzyme that catalyzes the dismutation of superoxide radicals into hydrogen peroxide ⁎ Corresponding author. Department of Neurology, Columbia University Medical Center, 630 West 168th Street, P&S 4-448, New York, NY 10032, USA. Tel.: +1 212 305 1476; fax: +1 212 305 3986. E-mail address: [email protected] (A. Naini). 0022-510X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2006.12.009

and molecular oxygen. The resulting hydrogen peroxide is in turn decomposed by the actions of glutathione peroxidase or catalase to water and oxygen [7]. To date, 116 different SOD1 mutations have been associated with ALS (www. alsod.org). However, the mechanism by which mutant SOD1 becomes toxic and leads to the demise of motor neuron is still unknown. Here, we report a novel missense mutation in exon 4 of sod1 causing ALS in a woman who apparently had sporadic ALS. 2. Patient and methods 2.1. Case report and neurological examinations At the age of 51, a left-handed woman first noticed voice change and speech difficulty while giving a talk at a conference. Her speech became “slurred and congested”. She also noticed having difficulty clearing her saliva. Several months later, her left hand had become weak and clumsy while writing. Her initially transient speech difficulty became

18

A. Naini et al. / Journal of the Neurological Sciences 254 (2007) 17–21

constant, and she also developed dysphagia, blurry vision, neck pain, tingling of her left arm, and low back pain. Her past medical history was significant for hypercholesterolemia, a wrist cyst excision, and tonsillectomy. Her medications included atorvastatin and medroxyprogesterone. Review of family history showed no hint of any familial disease. Her father had died at age 62 of metastatic cancer. Her mother and two younger siblings were in good health, as were her three children. She had worked as a manager at a computer company. She smoked one pack of cigarettes a day for 30 years, drank alcohol occasionally, and did not use illicit drugs. Neurological examination showed a pale right optical disc with decreased peripheral vessels. Visual acuity was 20/30 bilaterally and visual fields were full with both pupils reactive to light and accommodation. Tongue was in the midline, without fasciculation or atrophy. Palate elevation was slightly impaired on the right and the uvula deviated to the left. Both gag and jaw reflexes were present. Arms and legs had normal strength and tone, without fasciculation or atrophy. Both finger and foot tapping were impaired on the left, but there was no pronator drift. Finger-to-nose and heel-to-shin maneuvers were normal. Pinprick, temperature, joint position, and vibration sensations were normal. Gait was normal and Romberg sign was negative. Deep tendon reflexes were 3+ on the left side and 2+ on the right. Plantar reflexes were flexor bilaterally. The rest of the physical exam was normal. MRIs of the brain and cervical spine were normal. EMG did not show any sign of denervation or impaired neuromuscular transmission. Lumbar puncture showed normal protein, glucose, cell count, and no oligoclonal bands or myelin basic proteins. By MR spectroscopy, the NAA/Cr ratio was 2.45 on the right and 2.58 on the left, suggesting a borderline abnormality. The visual evoked potential was borderline; brainstem auditory evoked potential and somatosensory evoked potential of median nerve were normal. Anti-Hu antibodies, ACE, anti-GM1 antibodies, VDRL, Lyme test, IFE, and chest X-ray were all negative. There was a mildly increased anti-nuclear antibody (1:80). Two years later, she has constant dysarthria and laughed inappropriately. Her walking had become difficult and she had noticed sudden jerking movements in her left arm, which were triggered by loud noises. Neurological examination showed slow tongue motion, impaired vibratory sensation on the left, spastic catch in right arm, and slight weakness on the left side. She had difficulty walking on her toes and heels, and could hop on the right but not on the left foot. Tendon reflexes were overactive in the arms, more so on the left. Hoffmann sign was present on the left, but not on the right. Alternating movements were grossly impaired in the arms and legs, but more so on the left. There was no cogwheel rigidity, hypomimia, or bradykinesia. There was no focal wasting or fasciculation. Transcranial magnetic stimulation showed that central motor conduction times were prolonged to both legs and to the left arm, but normal to the right arm. Peripheral conductions were normal in both arms and legs. Repeated EMG and nerve conduction studies showed evidence of acute denervation in left arm and leg, left

cervical thoracic paraspinal muscles, and tongue, with mild chronic denervation changes in the distal left arm and leg. These data confirmed the diagnosis of amyotrophic lateral sclerosis with predominant upper motor neuron signs. Five years after the initial presentation, she had weakness of all four limbs. She could not walk, suffered from poor balance and used a wheelchair for mobility. She needed assistance for dressing, bathing, eating and hygiene. She had difficulty swallowing and experienced episodes of choking. The tongue showed atrophy and fasciculation, and its movements were slow and weak. She had dyspnea at rest and used a non-invasive positive pressure ventilator, suction machine, and in/exsufflator. Lower facial weakness was also evident. She died 6 years after her initial symptoms. 2.2. Molecular analysis Genomic DNA was extracted from blood leukocytes using commercial kit reagents (Gentra, Minneapplis, MN). Direct sequencing was carried out on an ABI Prism 310 Genetic Analyzer employing BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Faster City, CA). Sequencing primers and reaction conditions were as described by Yulug et al. [8]. Because the Asp109Tyr mutation eliminates the site for the restriction enzyme BsaI, PCR-RFLP with radiolabeling technique was utilized to confirm the presence of the mutation. A fragment of 274 bp encompassing the mutation site was PCR amplified using 5′AGCTCATGA-ACTACCTTGATG-3′ and 5′-CTTTAGAAACCGCGACTAACAATC-3′ as sense and anti-sense primers respectively. The amplified fragment was digested overnight with the BsaI enzyme and the resulting fragments were resolved on a 12% non-denaturing acryl amide gel and visualized by autoradiography. 2.3. Biochemical analysis The activity of SOD1 in red blood cells was determined spectrophotometrically using a Calbiochem® superoxide dismutase assay kit (EMD Biosciences Inc., USA). Red blood cells were isolated from a 10-mL citrated specimen and washed three times with normal saline. To prepare hemolysates, pellets were resuspended in 1.5 packed-cell volumes of ice-cold water and stored frozen (−20 °C) until analyzed. Purification of SOD1 from hemolysate was carried out as described by Winterbourn et al. [9]. Protein concentration was calculated by the A280 method using the NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Delaware, USA). 3. Results 3.1. Molecular Direct sequencing of five exons and their intron boundaries of the sod1 gene in the patient's DNA revealed

A. Naini et al. / Journal of the Neurological Sciences 254 (2007) 17–21

19

conduction time. Thus, this observation is not specific to SOD1-ALS. Sporadic ALS associated with missense, insertion, and deletion mutation in the sod1 gene has been frequently reported [7]. Recently, Gamez et al. [10], after studying 94 unrelated patients with unaffected relatives in four generations, identified two missense (D90A and N139H) and one silent mutations (A140A) and reported these as SOD1 associated SALS. It must be emphasized that in these studies the ascertainments of sporadic and not familial ALS was reached on the basis of pedigree analysis and family history. In another study [11], a SOD1 missense (H80R) mutation associated with a clinically aggressive form of the ALS was reported in a 24-year-old man as a SALS case, which was confirmed through molecular analysis and paternity testing. In the present study, we identified a novel G- to T-point mutation at nucleotide 1134 in exon4 of Cu/Zn superoxide dismutase gene in a 51-year-old woman with no known family history of motor neuron disease. This transversion leads to an Asp109Tyr substitution in the protein. The age of onset and duration of the disease in the patient was within the range of those reported for other SALS cases. Asp109, which resides in Greek key loop number IV [12], is a moderately conversed amino acid in the SOD1 protein observed in 15 different species (Fig. 2). Our patient's missense mutation, Asp109Tyr, has no adverse effect on the catalytic activity of the enzyme in red

a normal sequence except for a G- to T- transversion at nucleotide 1134 in exon4 (Fig. 1A). The heterozygote transversion was confirmed with PCR-RFLP technique (Fig. 1B). This nucleotide change, which was not seen in the DNA of 100 controls (200 chromosomes), predicts a substitution for aspartic acid with tyrosine at codon 109 in the SOD1 protein. 3.2. Biochemical The enzyme activity of purified SOD1 from the patient's erythrocytes was 1.75 U–525 units/mg protein, which was very similar to that of the five controls (1.79 ± 0.85 U– 25 units/mg protein). 4. Discussion In this study, the diagnosis of ALS was based on progressive upper and lower motor neuron disease. Because there was no known family history of motor neuron disease we conclude that this was a case of sporadic ALS, although the possibility of familial ALS could not be ruled out with absolute certainty. An interesting observation in this case was the finding of prolonged central conduction time shown by transcranial magnetic stimulation, which was consistent with our finding (Misumoto et al., in press) that in more than 70% of sporadic ALS patients there was a prolonged central

Fig. 1.

20

A. Naini et al. / Journal of the Neurological Sciences 254 (2007) 17–21

Fig. 2.

blood cells. Numerous studies of SOD1 mutants have shown that some mutants have normal enzyme activity, and that there is no correlation between SOD1 enzyme activity and age of onset or survival time [7,13]. Mutant SOD1 causing ALS is now thought to invoke an adverse gain of function, independent of the physiologic, antioxidant enzymatic properties of SOD1 [14]. Human SOD1 is a homodimer, where each monomer consists of an eight-stranded beta-barrel with two large loops, called “electrostatic loop”, and a “metal binding” loop [15]. The SOD1 mutants found to this date are scattered throughout the protein, and based on their locations, would be predicted to disrupt, to varying degrees, different aspects of the protein's function. For example, mutations that cause amino acid substitutions at metal-binding ligands or in the functionally important electrostatic loop are more likely to affect enzymatic activity or metal-binding affinity. On the other hand, mutations at the disulfide bond (between residues 57 and 146) or at residues near the dimer interface are thought to influence the protein's stability and structure. However, mutations in remote locations of the critical areas of the protein's structure are predicted to behave more like wild-type SOD1 [13]. Recent biophysical studies of mutant SOD1 proteins suggest dividing them into two groups of metal-binding region (MBR) mutants and wild-type-like (WTL) mutants based on their SOD activities and metal-binding properties [16–18]. Our patient's mutation is not located in a metal binding or catalytically important site, or in any region critical for stabilization of the SOD1 protein structure. This is supported by our finding of no change in SOD1 enzyme activity in our patient's erythrocytes (1.75 U–525 units/mg protein) compare to the 5 controls (1.79 ± 0.85 U–25 units/mg protein). Therefore, our patient's mutation can be categorized as a wild-type-like (WTL) mutant. The mechanism through which SOD1 mutations lead to ALS remains unclear despite extensive research. The two theories that dominate are protein aggregation and specific protein cytotoxicity [7]. Both patients and mice with various SOD1 mutations have shown SOD1-positive intracellular inclusions in neurons and astrocytes of upper motor neuron and lower motor neuron systems, as well as the other parts of the central nervous system [11,19,20]. Experiments have demonstrated that mutant SOD1 is relatively unstable and has

a tendency to form microaggregates consisting of denatured monomers [21]. These microaggregates have been proposed to cause neurodegeneration by interfering with cellular traffic and axonal transport, by blocking the normal function of the proteasome, and by interfering with mitochondrial function [22–24]. Protein aggregation is a common feature of many neurodegenerative diseases, including Alzheimer, and Parkinson disease. It seems likely that impaired axonal transport and cytoskeletal defects caused by mutant SOD1s are key steps in neuronal death in ALS. In conclusion, the D109Y substitution in SOD1 protein confers a toxic gain of function property to the molecule that causes ALS through a mechanism as yet to be elucidated. Because this mutation was found in a sporadic case, it underscores the importance of SOD1 screening in both, familial and sporadic ALS studies. References [1] Rowland LP, Shneider NA. Amyotrophic lateral sclerosis. N Engl J Med May 31 2001;344(22):1688–700. [2] Bruijn LI, Miller TM, Cleveland DW. Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu Rev Neurosci 2004;27:723–49. [3] Siddique T, Deng HX. Genetics of amyotrophic lateral sclerosis. Hum Mol Genet 1996;5:1465–70 [Spec No]. [4] Andersen PM. Genetics of sporadic ALS. Amyotroph Lateral Scler Other Mot Neuron Disord Mar 2001(2 Suppl 1):S37–41. [5] Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 1993;362(6415):59–62. [6] Jones CT, Brock DJ, Chancellor AM, Warlow CP, Swingler RJ. Cu/ Zn superoxide dismutase (SOD1) mutations and sporadic amyotrophic lateral sclerosis. Lancet Oct 23 1993;342(8878):1050–1. [7] Andersen PM. Amyotrophic lateral sclerosis associated with mutations in the CuZn superoxide dismutase gene. Curr Neurol Neurosci Rep Jan 2006;6(1):37–46. [8] Yulug IG, Katsanis N, de Belleroche J, Collinge J, Fisher EM. An improved protocol for the analysis of SOD1 gene mutations, and a new mutation in exon 4. Hum Mol Genet Jun 1995;4(6):1101–4. [9] Winterbourn CC, Hawkins RE, Brian M, Carrell RW. The estimation of red cell superoxide dismutase activity. J Lab Clin Med Feb 1975;85(2):337–41. [10] Gamez J, Corbera-Bellalta M, Nogales G, Raguer N, Garcia-Arumi E, Badia-Canto M, et al. Mutational analysis of the Cu/Zn superoxide dismutase gene in a Catalan ALS population: should all sporadic ALS cases also be screened for SOD1? J Neurol Sci Aug 15 2006;247(1):21–8. [11] Alexander MD, Traynor BJ, Miller N, Corr B, Frost E, McQuaid S, et al. “True” sporadic ALS associated with a novel SOD-1 mutation. Ann Neurol Nov 2002;52(5):680–3. [12] Getzoff ED, Tainer JA, Stempien MM, Bell GI, Hallewell RA. Evolution of CuZn superoxide dismutase and the Greek key betabarrel structural motif. Proteins 1989;5(4):322–36. [13] Valentine JS, Doucette PA, Zittin Potter S. Copper–zinc superoxide dismutase and amyotrophic lateral sclerosis. Ann Rev Biochem 2005;74 (1):563–93. [14] Agar J, Durham H. Relevance of oxidative injury in the pathogenesis of motor neuron diseases. Amyotroph Lateral Scler Other Mot Neuron Disord Dec 2003;4(4):232–42. [15] Tainer JA, Getzoff ED, Beem KM, Richardson JS, Richardson DC. Determination and analysis of the 2 A-structure of copper, zinc superoxide dismutase. J Mol Biol Sep 15 1982;160(2):181–217. [16] Hayward LJ, Rodriguez JA, Kim JW, Tiwari A, Goto JJ, Cabelli DE, et al. Decreased metallation and activity in subsets of mutant

A. Naini et al. / Journal of the Neurological Sciences 254 (2007) 17–21

[17]

[18]

[19]

[20]

superoxide dismutases associated with familial amyotrophic lateral sclerosis. J Biol Chem May 3 2002;277(18):15923–31. Rodriguez JA, Valentine JS, Eggers DK, Roe JA, Tiwari A, Brown Jr RH, et al. Familial amyotrophic lateral sclerosis-associated mutations decrease the thermal stability of distinctly metallated species of human copper/zinc superoxide dismutase. J Biol Chem May 3 2002;277(18):15932–7. Potter SZ, Valentine JS. The perplexing role of copper–zinc superoxide dismutase in amyotrophic lateral sclerosis (Lou Gehrig's disease). J Biol Inorg Chem Apr 2003;8(4):373–80. Andersen PM, Sims KB, Xin WW, Kiely R, O'Neill G, Ravits J, et al. Sixteen novel mutations in the Cu/Zn superoxide dismutase gene in amyotrophic lateral sclerosis: a decade of discoveries, defects and disputes. Amyotroph Lateral Scler Other Mot Neuron Disord Jun 2003;4(2):62–73. Krasnianski A, Deschauer M, Neudecker S, Gellerich FN, Muller T, Schoser BG, et al. Mitochondrial changes in skeletal muscle in amyotrophic lateral sclerosis and other neurogenic atrophies. Brain Aug 2005;128(Pt 8):1870–6.

21

[21] Lindberg MJ, Bystrom R, Boknas N, Andersen PM, Oliveberg M. Systematically perturbed folding patterns of amyotrophic lateral sclerosis (ALS)-associated SOD1 mutants. Proc Natl Acad Sci USA Jul 12 2005;102(28):9754–9. [22] Vlug AS, Jaarsma D. Long term proteasome inhibition does not preferentially afflict motor neurons in organotypical spinal cord cultures. Amyotroph Lateral Scler Other Mot Neuron Disord Mar 2004;5(1):16–21. [23] Menzies FM, Cookson MR, Taylor RW, Turnbull DM, ChrzanowskaLightowlers ZM, Dong L, et al. Mitochondrial dysfunction in a cell culture model of familial amyotrophic lateral sclerosis. Brain Jul 2002;125(Pt 7):1522–33. [24] Pasinelli P, Belford ME, Lennon N, Bacskai BJ, Hyman BT, Trotti D, et al. Amyotrophic lateral sclerosis-associated SOD1 mutant proteins bind and aggregate with Bcl-2 in spinal cord mitochondria. Neuron Jul 8 2004;43 (1):19–30.