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SOD1 gene mutations in Italian patients with Sporadic Amyotrophic Lateral Sclerosis (ALS)
Neuromuscular Disorders 16 (2006) 800–804 www.elsevier.com/locate/nmd
SOD1 gene mutations in Italian patients with Sporadic Amyotrophic Lateral Sclerosis (ALS) L. Corrado
b
a,b,*
, S. D’Alfonso a,b, L. Bergamaschi a,b, L. Testa c, M. Leone N. Nasuelli c, P. Momigliano-Richiardi a,b, L. Mazzini c
b,c
,
a Department of Medical Sciences, University of Eastern Piedmont, Novara, Italy Interdisciplinary Research Center of Autoimmune Diseases (IRCAD), University of Eastern Piedmont, Novara, Italy c Department of Neurology, University of Eastern Piedmont, Novara, Italy
Received 7 March 2006; received in revised form 4 July 2006; accepted 10 July 2006
Abstract Mutations in the SOD1 gene exons and exon/intron boundaries were searched in 66 sporadic and 4 familial Italian ALS cases consecutively referred to our centre from different Italian regions. A mutation was found in three sporadic cases (4.5%): a new nonsense mutation in exon 5 (K136X) in a patient with a rapid and severe disease course and two previously described missense nucleotide substitutions (N65S and A95T) in two patients with a mild disease course. Comparison of the clinical characteristics with previously reported patients carrying the same or similar mutations showed a remarkable genotype–phenotype correlation. No association was found with intronic sequence variations by comparing their frequency in the patients and in 181 matched controls. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Amyotrophic Lateral Sclerosis; SOD1; Genotype–phenotype correlation
1. Introduction Amyotrophic Lateral Sclerosis (ALS) is one of the most common adult-onset neurodegenerative diseases affecting the motor neuron population. The loss of motorneurons results in neuromuscular failure and death, usually from compromised respiratory function, over a time course of approximately 3–5 years from symptom onset. The annual incidence in Italy is 1.7– 2.5 per 100,000 [1–3]. Five to 10% of all cases of ALS are familial (FALS), almost always autosomal dominant and 14–24% of them have mutations in the gene encoding for Cu/Zn superoxide dismutase [4–7] (SOD1; also see www.alsod1.iop.kcl.ac.uk/). The other 90% are considered to be sporadic (SALS), as they have no family history of the disease, but 3–7% of them carry SOD1 *
Corresponding author. Tel.: +39 321 660606; fax: +39 321 620421. E-mail address: [email protected] (L. Corrado).
0960-8966/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2006.07.004
mutations too [4,8,9]. In Italy one study found no mutations in sporadic cases, but another found them in 6% [10,11]. We here describe the SOD1 mutations detected in a third large independent Italian sample. 2. Patients The 70 Italian ALS patients included in this study consecutively referred to our ALS centre from different Italian regions (36 from Northern Italy, 16 from the Center and 18 from the South). Diagnosis was performed according to the El Escorial revised criteria [12]. ALS was classified as familial when at least one further member was reported to be clinically affected in the same pedigree. On this basis, 66 patients (42M and 26F; mean age: 48 years. Range: 20–78) were classified as sporadic cases, while 4 unrelated patients (2M, 2F Mean age: 65 years. Range: 32–65) were classified as FALS.
L. Corrado et al. / Neuromuscular Disorders 16 (2006) 800–804
All patients were examined by an ALS expert multidisciplinary team that collected the case history and clinical data. The case history included personal data (age, sex, area of residence and area of birth, type of job), anamnestic data (date of onset of the first symptom, date of diagnosis, first symptom, progression, associated diseases, exposure to toxic agents) and full pedigree data (family history of ALS or other neurological diseases, age and health status, or age and cause of death of parents, grandparents, sibs, offspring). The clinical report included signs of upper (spasticity, hyperreflexia, Hoffmann’s and Babinski signs, loss of dexterity, emotional lability, snout and jaw reflexes) and lower (muscle atrophy and weakness, hyporeflexia, hypotonia and fasciculations) motor neuron impairment both at the spinal and bulbar levels. Lower motor neuron involvement was also tested by electromyography of the four limbs and in the doubtful cases also of the bulbar and paraspinal muscles. Functional impairment was scored using the ALS-Functional Rating Scale [13]. The Forced Vital Capacity (FVC) was also assessed. Clinical data were updated every three months to determine the rate of progression. The slope of progression of ALS-FRS score and FVC were considered as the main endpoints of the severity of the disease [14,15]. The study was approved by the local hospital Ethic Committee. A blood sample of the patients was drawn after informed consent to genetic analysis only for research purpose. 3. Methods 3.1. DNA analysis Testing for SOD1 mutations was performed on six amplified fragments including the five exons and about 60 nucleotides of each intronic flanking region. PCR products were then screened for sequence variations by DHPLC (Denaturing High Performance Liquid Chromatography; Wave, Transgenomic, Santa Clara, CA) as previously described [16]. The PCR products displaying an heteroduplex peak were sequenced on an Applied Biosystems (ABI) 3100 Genetic Analyzer. Exon 4 was always directly sequenced in order not to miss the previously described mutation D90A that can be associated to a recessive phenotype [17] and thus is not detectable by DHPLC. Intronic variations detected in the patients were also analysed in a panel of 181 unrelated healthy Italian controls. 3.2. Messenger RNA analysis Total RNA was isolated from PHA activated peripheral blood lymphocytes using the RNAeasy kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions. cDNA, obtained by mRNA reverse transcription (Thermoscript, Invitrogen), was amplified with
801
primers: 5 0 -CTGGTTTGCGTCGTAGTCTC-3 0 ; 5 0 -TG GCAAAATACAGGTCATTG-3 0 . 4. Results An heterozygous mutation in the SOD1 gene was found in three cases. There was no recorded history consistent with, or family knowledge of, neurological disease suggestive of motor neuron disease (MND) in the relatives of these three patients that were, therefore, considered sporadic. However, none of the relatives had been seen by a neurologist. Two mutations, namely N65S and A95T, had been previously described (Table 1). The third one (K136X) is a newly described substitution from A to T in the first nucleotide of codon 136 in exon 5, introducing a TAG premature stop codon. DNA samples from other family members were not available for molecular analysis of the SOD1 gene; therefore we were not able to define whether the mutations were inherited or de novo. 4.1. K136X mutation It was detected in a 46-year-old male with onset at age 45. His parents (both died at 68 years), grandparents (died at >80 years), sibs (47, 43, 38 years old, healthy) had no reported signs of MND. The clinical phenotype was characterized by a rapidly progressive muscle weakness and wasting of the left lower limb with hyperreflexia in the four limbs leading to an ALS-FRS score = 37 and FVC = 80% 12 months after diagnosis. The muscular weakness and wasting continued to worsen and extended to both upper and lower limbs and three months later ALS-FRS score was 30 and FVC = 52%. Twelve months later he developed quadriplegia with severe bulbar palsy. Artificial ventilation and enteral nutrition were necessary. 4.2. N65S mutation This mutation in exon 3 was detected in a 50-year-old female patient. Her parents (mother, 70 years old, healthy; father died at 65 years of infarction) and grandparents (died at >70 years) had no signs of MND. She reported the disease onset at the age of 40 with dropped foot. At the time of genetic examination, 10 years after onset, she showed a mild muscle weakness and wasting with prevalent involvement of the distal muscles of both lower limbs. Deep tendon reflexes were decreased in the lower limbs but Babinski sign was positive. EMG examination showed a diffuse impairment of the lower motor neuron in both lower limbs. The patient reported a mild functional impairment (ALS-FRS = 34). Respiratory function was normal. Twelve months later the clinical and functional status mildly worsened in the lower limbs
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L. Corrado et al. / Neuromuscular Disorders 16 (2006) 800–804
Table 1 Comparison of clinical characteristics of patients carrying the same missense SOD1 mutation Mutation
Patient
Age at onset (years)
Other affected family members
Durationa (years)
Major source of disabilitya
N65S N65S N65S N65S A95T A95T
Our case [19] [25] Alsod1db Our case [11]
40 38 44 34 45 26
No Yes No No No No
10 14 18 11 20 2b
LMN LMN LMN LMN LMN LMN
a b
At last reported examination. No information is available after that present in Ref. [11].
but she was able to walk with aid of a cane (ALSFRS = 33). 4.3. A95T mutation This mutation in exon 4 was identified in a 64-yearold woman. Her parents (father died at 66 years of pulmonary disease; mother died at 72 years of colon cancer), grandparents (died at 60–80 years), sibs (two healthy, 63, 64 years old and one died at 66 of pneumonia) had no signs of MND. The patient refers the onset of the disease at the age of 45 with left foot drop. Seven years later she reported right foot drop and in the following years she experienced diffuse fasciculations and extension of the muscle weakness to the upper limbs. Twenty-years after onset she is still alive showing a severe tetraparesis with muscle weakness and wasting with hyperreflexia and Babinski signs without bulbar or pseudobulbar signs (ALSFRS = 27; FVC = 81%). 4.4. Intronic variations Five sequence variations in the noncoding regions were detected in the patients. Three of these were also found in normal subjects. The already described polymorphisms IVS3 + 34 A/C and IVS1 108T/A were detected in 8/ 70 patients and 11/181 controls and were in perfect linkage disequilibrium. A deletion of 7 nucleotides present in intron 2 of the gene (IVS2 + 51 del7nt) was detected in two patients, one of these carrying in trans the mutation A95T, but also in 3/181 normal individuals. The frequencies of these intronic variations were not significantly different in the patients and controls. Two other variants (IVS1 + 48C/T and IVS3 + 24G/A) were identified each in one SALS patient. The functional relevance of the detected intronic variants for the splicing mechanism, was assessed by RT-PCR analysis of SOD1 mRNA in peripheral blood lymphocytes. No aberrantly spliced transcript was detected (data not shown). 5. Discussion SOD1 mutations were detected in 4.5% (3/66) sporadic Italian ALS patients, similarly to what reported
by Gellera et al. [11] who found mutations in 6% (3/ 48) Italian SALS patients and to what observed in Northern Europe [4,8,9]. Of the three detected mutations, K136X is a new nonsense mutation in the last exon. This mutation falls in the protein region contributing to the active-site loop (amino acids 121–144) analogously to the previously reported L126X and G141X (database, [18,19]). Besides these two nonsense mutations, five frameshift mutations generating a stop codon in the last exon and a truncated protein have been reported in ALS patients [9,20,21,4,18,22]. Five of these seven truncating mutations were associated with a rapid clinical course as in our patient. All these truncated polypeptides lack cysteine 146 (cys146), an essential residue for the formation of an intrasubunit disulphide bond, which stabilizes the SOD1 monomer and allows the formation of an active SOD1 dimer. Thus, the absence of cys146 leads to the formation of inactive misfolded SOD1 proteins [23]. Aggregates of inactive misfolded SOD1 proteins, accumulating in the spinal cord, were detected in a patient with the G127insTGGG mutation [24,23] and in MND transgenic mice overexpressing human SOD1 truncated proteins (G127insTGGG, L126X) [23]. By analogy with molecularly similar mutations we presume that K136X is a causative sequence variation, although this is not proved by formal or functional data. The N65S and A95T are already described missense mutations. Three other cases presenting the same N65S mutation have been reported (Table 1): (1) a Caucasoid male from Zimbabwe with age at onset of 34, a duration of the disease of almost 11 years and apparently no family history (www.alsod1.iop.kcl.ac.uk/); (2) a Caucasoid male from USA of Spanish origin and age at onset of 38. He had a slowly progressive form and was still alive 14 years after onset. His grandmother presented MND; thus he represents a familial case with a probably reduced penetrance [19]. (3) A female from Spain [25], with age at onset of 44 and still alive after 18 years. She has two unaffected siblings with the same genetic lesion, confirming the reduced penetrance of this mutation. The A95T sequence was previously described in an Italian male patient [11]. He presented the first symptoms at the age of 26 and his disease was described as
L. Corrado et al. / Neuromuscular Disorders 16 (2006) 800–804
a slowly progressive form, at least in the first two years after onset for which information is available. The mutation was present also in his unaffected mother and siblings but was not observed in 83 normal individuals and 120 patients with MND [11]. Our case represents the second detection of the A95T mutation in an ALS patient, thus increasing the likelihood that it is a disease-associated mutation. The two patients are likely unrelated since both are unaware of other affected relatives. Notably, the N65S mutation has been found only in Spanish or Italian people (‘‘Mediterranean’’) and the A95T only in two Italians. This concentration, although too few for reliable generalization, may be comparable to the predominance of the A4V mutation in the United States and the D90A mutation in Scandinavia. Similarly to what reported for other SOD1 mutations [26–28], a remarkable phenotype–genotype correlation seems to emerge when comparing the clinical characteristics of the patients carrying the same or similar mutations. The nonsense mutation in exon 5 was present in a patient with a severe and rapid clinical course, analogous to what found for most SOD1 mutations leading to a truncated protein [29]. Conversely, N65S and A95T were both associated to a slowly, or very slowly, progressive course of the disease (Table 1) similarly to other SOD1 mutations (H46R, D76V, I113T, L144P, G93V, I151T, D90A, A89T) detected in patients with a disease duration >10 years [[19], alsod1db]. In addition, N65S seems to be strictly correlated to a prevalent involvement of the lower motor neurons and only at the spinal level. If confirmed in a larger patient panel, these findings might help to improve prognostic evaluation.
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
Acknowledgements We are grateful to the patients for their collaboration.
[18]
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dismutase in amyotrophic lateral sclerosis. Ann Neurol 1997;1(2):210–21. Shaw CE, Enayat E, Chioza BA, Al-Chalabi A, Radunovic A, Powell JF, et al. Mutations in all five exons of SOD-1 may cause ALS. Ann Neurol. 1998(3):390–4. Boukaftane Y, Khoris J, Moulard B, Salachas F, Meininger V, Malafosse A, et al. Identification of six novel SD1 gene mutations in familial amyotrophic lateral sclerosis. Can J Neuro Sci 1998:192–6. Jones CT, Swingler RJ, Simpson SA, Brock DJH. Superoxide dismutase mutations in an unselected cohort of Scottish amyotrophic lateral sclerosis patients. J Med Genet 1995;32:290–2. Jackson M, Al-Chalabi A, Enayat ZE, Choza B, Leigh PN, Morrison KE. Copper/Zinc superoxide dismutase 1 and sporadic amyotrophic lateral sclerosis: analysis of 155 cases and identification of a novel insertion mutation. Ann Neurol 1997;42:803–7. Battistini S, Giannini F, Greco G, Bibbo G, Ferrera L, Marini V, et al. SOD1 mutations in amyotrophic lateral sclerosis. Results from a multicenter Italian study. J Neurol 2005;252(7):782–8. Gellera C, Castellotti B, Riggio MC, Silani V, Morandi L, Testa D, et al. Superoxide dismutase gene mutations in Italian patients with familial and sporadic amyotrophic lateral sclerosis: identification of three novel missense mutations. Neuromuscul Disord 2001:404–10. Brooks BR, Miller RG, Swash M, Munsat TL. World Federation of Neurology Research Group on MNDs. El Escorial revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord 2000;1:293–9. The ALS-CNTF treatment Study (ACTS) Phase I-II study group. The Amyotrophic Lateral Sclersis functional rating scale: assessment of activities of daily living in patients with amyotrophic lateral sclerosis. Arch Neurol 1996;53:141–7. Kimura F, Fujimura C, Ishida S, Nakajima H, Furutama D, Uehara H, et al. Progression rate of ALSFRS-R at time of diagnosis predicts survival time in ALS. Neurology 2006(2):265–7. Czaplinski A, Yen AA, Appel SH. Forced vital capacity (FVC) as an indicator of survival and disease progression in an ALS clinic population. J Neurol Neurosurg Psychiatry 2006;77(3):390–2. Mellai M, Giordano M, D’Alfonso S, Marchini M, Scorza R, Giovanna Danieli M, et al. Prolactin and prolactin receptor gene polymorphisms in multiple sclerosis and systemic lupus erythematosus. Hum Immunol 2003;64(2):274–84. Andersen PM, Nilsson P, Ala-Hurula V, Keranen ML, Tarvainen I, Haltia T, et al. Amyotrophic lateral sclerosis associated with homozygosity for an Asp90Ala mutation in CuZn-superoxide dismutase. Nat Genet 1995(1):61–6. Zu JS, Deng HX, Lo TP, Mitsumoto H, Ahmed MS, Hung WY, et al. Exon 5 encoded domain is not required for the toxic function of mutant SOD1 but essential for the dismutase activity: identification and characterization of two new SOD1 mutations associated with familial amyotrophic lateral sclerosis. Neurogenetics 1997(1):65–71. 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 Motor Neuron Disord. 2003(2):62–73. Pramatarova A, Goto J, Nanba E, Nakashima K, Takahashi K, Takagi A, et al. A two basepair deletion in the SOD 1 gene causes familial amyotrophic lateral sclerosis. Hum Mol Genet 1994(11): 2061–2. Nakashima K, Watanabe Y, Kuno N, Nanba E, Takahashi K. Abnormality of Cu/Zn superoxide dismutase (SOD1) activity in Japanese familial amyotrophic lateral sclerosis with a two base pair deletion in the SOD1 gene. Neurology 1995;45:1019–20. Orrell RW, Habgood JJ, Gardiner I, King AW, Bowe FA, Hallewell RA, et al. Clinical and functional investigation of 10
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missense mutations and a novel frameshift insertion mutation of the gene for copper-zinc superoxide dismutase in UK families with amyotrophic lateral sclerosis. Neurology 1997(3):746–51. [23] Furukawa Y, Fu R, Deng HX, Siddique T, O’Halloran TV. Disulfide crosslinked protein represent a significant fraction of ALS-associated Cu, Znsuperoxide dismutase aggregates in spinal cords of model mice. Proc Natl Acad Sci USA 2006;18:7148–53. [24] Jonsson PA, Ernhill K, Andersen PM, Bergemalm D, Brannstrom T, Gredal O, et al. Minute quantities of misfolded mutant superoxide dismutase-1 cause amyotrophic lateral sclerosis. Brain 2003;127:73–88. [25] Garcia-Redondo A, Bustos F, Juan Y Seva B, Del Hoyo P, Jimenez S, Campos Y, et al. Molecular analysis of the superoxide dismutase 1 gene in Spanish patients with sporadic or familial amyotrophic lateral sclerosis. Muscle Nerve 2002(2):274–8.
[26] Andersen PM. Amyotrophic lateral sclerosis associated with mutations in the CuZn superoxide dismutase gene. Curr Neurol Neurosci Rep 2006;6(1):37–46. [27] Regal L, Vanopdenbosch L, Tilkin P, Van den Bosch L, Thijs V, Sciot R, et al. The G93C mutation in superoxide dismutase 1: clinicopathologic phenotype and prognosis. Arch Neurol 2006:262–7. [28] Rosen DR, Bowling AC, Patterson D, Usdin TB, Sapp P, Mezey E, et al. A frequent ala 4 to val superoxide dismutase-1 mutation is associated with a rapidly progressive familial amyotrophic lateral sclerosis. Hum Mol Genet 1994(6):981–7. [29] Watanabe Y, Adachi Y, Nakashima K. Japanese familial amyotrophic lateral sclerosis family with a two-base deletion in the superoxide dismutase-1 gene. Neuropathology 2001;21: 61–6.
SOD1 gene mutations in Italian patients with Sporadic Amyotrophic Lateral Sclerosis (ALS) L. Corrado
b
a,b,*
, S. D’Alfonso a,b, L. Bergamaschi a,b, L. Testa c, M. Leone N. Nasuelli c, P. Momigliano-Richiardi a,b, L. Mazzini c
b,c
,
a Department of Medical Sciences, University of Eastern Piedmont, Novara, Italy Interdisciplinary Research Center of Autoimmune Diseases (IRCAD), University of Eastern Piedmont, Novara, Italy c Department of Neurology, University of Eastern Piedmont, Novara, Italy
Received 7 March 2006; received in revised form 4 July 2006; accepted 10 July 2006
Abstract Mutations in the SOD1 gene exons and exon/intron boundaries were searched in 66 sporadic and 4 familial Italian ALS cases consecutively referred to our centre from different Italian regions. A mutation was found in three sporadic cases (4.5%): a new nonsense mutation in exon 5 (K136X) in a patient with a rapid and severe disease course and two previously described missense nucleotide substitutions (N65S and A95T) in two patients with a mild disease course. Comparison of the clinical characteristics with previously reported patients carrying the same or similar mutations showed a remarkable genotype–phenotype correlation. No association was found with intronic sequence variations by comparing their frequency in the patients and in 181 matched controls. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Amyotrophic Lateral Sclerosis; SOD1; Genotype–phenotype correlation
1. Introduction Amyotrophic Lateral Sclerosis (ALS) is one of the most common adult-onset neurodegenerative diseases affecting the motor neuron population. The loss of motorneurons results in neuromuscular failure and death, usually from compromised respiratory function, over a time course of approximately 3–5 years from symptom onset. The annual incidence in Italy is 1.7– 2.5 per 100,000 [1–3]. Five to 10% of all cases of ALS are familial (FALS), almost always autosomal dominant and 14–24% of them have mutations in the gene encoding for Cu/Zn superoxide dismutase [4–7] (SOD1; also see www.alsod1.iop.kcl.ac.uk/). The other 90% are considered to be sporadic (SALS), as they have no family history of the disease, but 3–7% of them carry SOD1 *
Corresponding author. Tel.: +39 321 660606; fax: +39 321 620421. E-mail address: [email protected] (L. Corrado).
0960-8966/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2006.07.004
mutations too [4,8,9]. In Italy one study found no mutations in sporadic cases, but another found them in 6% [10,11]. We here describe the SOD1 mutations detected in a third large independent Italian sample. 2. Patients The 70 Italian ALS patients included in this study consecutively referred to our ALS centre from different Italian regions (36 from Northern Italy, 16 from the Center and 18 from the South). Diagnosis was performed according to the El Escorial revised criteria [12]. ALS was classified as familial when at least one further member was reported to be clinically affected in the same pedigree. On this basis, 66 patients (42M and 26F; mean age: 48 years. Range: 20–78) were classified as sporadic cases, while 4 unrelated patients (2M, 2F Mean age: 65 years. Range: 32–65) were classified as FALS.
L. Corrado et al. / Neuromuscular Disorders 16 (2006) 800–804
All patients were examined by an ALS expert multidisciplinary team that collected the case history and clinical data. The case history included personal data (age, sex, area of residence and area of birth, type of job), anamnestic data (date of onset of the first symptom, date of diagnosis, first symptom, progression, associated diseases, exposure to toxic agents) and full pedigree data (family history of ALS or other neurological diseases, age and health status, or age and cause of death of parents, grandparents, sibs, offspring). The clinical report included signs of upper (spasticity, hyperreflexia, Hoffmann’s and Babinski signs, loss of dexterity, emotional lability, snout and jaw reflexes) and lower (muscle atrophy and weakness, hyporeflexia, hypotonia and fasciculations) motor neuron impairment both at the spinal and bulbar levels. Lower motor neuron involvement was also tested by electromyography of the four limbs and in the doubtful cases also of the bulbar and paraspinal muscles. Functional impairment was scored using the ALS-Functional Rating Scale [13]. The Forced Vital Capacity (FVC) was also assessed. Clinical data were updated every three months to determine the rate of progression. The slope of progression of ALS-FRS score and FVC were considered as the main endpoints of the severity of the disease [14,15]. The study was approved by the local hospital Ethic Committee. A blood sample of the patients was drawn after informed consent to genetic analysis only for research purpose. 3. Methods 3.1. DNA analysis Testing for SOD1 mutations was performed on six amplified fragments including the five exons and about 60 nucleotides of each intronic flanking region. PCR products were then screened for sequence variations by DHPLC (Denaturing High Performance Liquid Chromatography; Wave, Transgenomic, Santa Clara, CA) as previously described [16]. The PCR products displaying an heteroduplex peak were sequenced on an Applied Biosystems (ABI) 3100 Genetic Analyzer. Exon 4 was always directly sequenced in order not to miss the previously described mutation D90A that can be associated to a recessive phenotype [17] and thus is not detectable by DHPLC. Intronic variations detected in the patients were also analysed in a panel of 181 unrelated healthy Italian controls. 3.2. Messenger RNA analysis Total RNA was isolated from PHA activated peripheral blood lymphocytes using the RNAeasy kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions. cDNA, obtained by mRNA reverse transcription (Thermoscript, Invitrogen), was amplified with
801
primers: 5 0 -CTGGTTTGCGTCGTAGTCTC-3 0 ; 5 0 -TG GCAAAATACAGGTCATTG-3 0 . 4. Results An heterozygous mutation in the SOD1 gene was found in three cases. There was no recorded history consistent with, or family knowledge of, neurological disease suggestive of motor neuron disease (MND) in the relatives of these three patients that were, therefore, considered sporadic. However, none of the relatives had been seen by a neurologist. Two mutations, namely N65S and A95T, had been previously described (Table 1). The third one (K136X) is a newly described substitution from A to T in the first nucleotide of codon 136 in exon 5, introducing a TAG premature stop codon. DNA samples from other family members were not available for molecular analysis of the SOD1 gene; therefore we were not able to define whether the mutations were inherited or de novo. 4.1. K136X mutation It was detected in a 46-year-old male with onset at age 45. His parents (both died at 68 years), grandparents (died at >80 years), sibs (47, 43, 38 years old, healthy) had no reported signs of MND. The clinical phenotype was characterized by a rapidly progressive muscle weakness and wasting of the left lower limb with hyperreflexia in the four limbs leading to an ALS-FRS score = 37 and FVC = 80% 12 months after diagnosis. The muscular weakness and wasting continued to worsen and extended to both upper and lower limbs and three months later ALS-FRS score was 30 and FVC = 52%. Twelve months later he developed quadriplegia with severe bulbar palsy. Artificial ventilation and enteral nutrition were necessary. 4.2. N65S mutation This mutation in exon 3 was detected in a 50-year-old female patient. Her parents (mother, 70 years old, healthy; father died at 65 years of infarction) and grandparents (died at >70 years) had no signs of MND. She reported the disease onset at the age of 40 with dropped foot. At the time of genetic examination, 10 years after onset, she showed a mild muscle weakness and wasting with prevalent involvement of the distal muscles of both lower limbs. Deep tendon reflexes were decreased in the lower limbs but Babinski sign was positive. EMG examination showed a diffuse impairment of the lower motor neuron in both lower limbs. The patient reported a mild functional impairment (ALS-FRS = 34). Respiratory function was normal. Twelve months later the clinical and functional status mildly worsened in the lower limbs
802
L. Corrado et al. / Neuromuscular Disorders 16 (2006) 800–804
Table 1 Comparison of clinical characteristics of patients carrying the same missense SOD1 mutation Mutation
Patient
Age at onset (years)
Other affected family members
Durationa (years)
Major source of disabilitya
N65S N65S N65S N65S A95T A95T
Our case [19] [25] Alsod1db Our case [11]
40 38 44 34 45 26
No Yes No No No No
10 14 18 11 20 2b
LMN LMN LMN LMN LMN LMN
a b
At last reported examination. No information is available after that present in Ref. [11].
but she was able to walk with aid of a cane (ALSFRS = 33). 4.3. A95T mutation This mutation in exon 4 was identified in a 64-yearold woman. Her parents (father died at 66 years of pulmonary disease; mother died at 72 years of colon cancer), grandparents (died at 60–80 years), sibs (two healthy, 63, 64 years old and one died at 66 of pneumonia) had no signs of MND. The patient refers the onset of the disease at the age of 45 with left foot drop. Seven years later she reported right foot drop and in the following years she experienced diffuse fasciculations and extension of the muscle weakness to the upper limbs. Twenty-years after onset she is still alive showing a severe tetraparesis with muscle weakness and wasting with hyperreflexia and Babinski signs without bulbar or pseudobulbar signs (ALSFRS = 27; FVC = 81%). 4.4. Intronic variations Five sequence variations in the noncoding regions were detected in the patients. Three of these were also found in normal subjects. The already described polymorphisms IVS3 + 34 A/C and IVS1 108T/A were detected in 8/ 70 patients and 11/181 controls and were in perfect linkage disequilibrium. A deletion of 7 nucleotides present in intron 2 of the gene (IVS2 + 51 del7nt) was detected in two patients, one of these carrying in trans the mutation A95T, but also in 3/181 normal individuals. The frequencies of these intronic variations were not significantly different in the patients and controls. Two other variants (IVS1 + 48C/T and IVS3 + 24G/A) were identified each in one SALS patient. The functional relevance of the detected intronic variants for the splicing mechanism, was assessed by RT-PCR analysis of SOD1 mRNA in peripheral blood lymphocytes. No aberrantly spliced transcript was detected (data not shown). 5. Discussion SOD1 mutations were detected in 4.5% (3/66) sporadic Italian ALS patients, similarly to what reported
by Gellera et al. [11] who found mutations in 6% (3/ 48) Italian SALS patients and to what observed in Northern Europe [4,8,9]. Of the three detected mutations, K136X is a new nonsense mutation in the last exon. This mutation falls in the protein region contributing to the active-site loop (amino acids 121–144) analogously to the previously reported L126X and G141X (database, [18,19]). Besides these two nonsense mutations, five frameshift mutations generating a stop codon in the last exon and a truncated protein have been reported in ALS patients [9,20,21,4,18,22]. Five of these seven truncating mutations were associated with a rapid clinical course as in our patient. All these truncated polypeptides lack cysteine 146 (cys146), an essential residue for the formation of an intrasubunit disulphide bond, which stabilizes the SOD1 monomer and allows the formation of an active SOD1 dimer. Thus, the absence of cys146 leads to the formation of inactive misfolded SOD1 proteins [23]. Aggregates of inactive misfolded SOD1 proteins, accumulating in the spinal cord, were detected in a patient with the G127insTGGG mutation [24,23] and in MND transgenic mice overexpressing human SOD1 truncated proteins (G127insTGGG, L126X) [23]. By analogy with molecularly similar mutations we presume that K136X is a causative sequence variation, although this is not proved by formal or functional data. The N65S and A95T are already described missense mutations. Three other cases presenting the same N65S mutation have been reported (Table 1): (1) a Caucasoid male from Zimbabwe with age at onset of 34, a duration of the disease of almost 11 years and apparently no family history (www.alsod1.iop.kcl.ac.uk/); (2) a Caucasoid male from USA of Spanish origin and age at onset of 38. He had a slowly progressive form and was still alive 14 years after onset. His grandmother presented MND; thus he represents a familial case with a probably reduced penetrance [19]. (3) A female from Spain [25], with age at onset of 44 and still alive after 18 years. She has two unaffected siblings with the same genetic lesion, confirming the reduced penetrance of this mutation. The A95T sequence was previously described in an Italian male patient [11]. He presented the first symptoms at the age of 26 and his disease was described as
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a slowly progressive form, at least in the first two years after onset for which information is available. The mutation was present also in his unaffected mother and siblings but was not observed in 83 normal individuals and 120 patients with MND [11]. Our case represents the second detection of the A95T mutation in an ALS patient, thus increasing the likelihood that it is a disease-associated mutation. The two patients are likely unrelated since both are unaware of other affected relatives. Notably, the N65S mutation has been found only in Spanish or Italian people (‘‘Mediterranean’’) and the A95T only in two Italians. This concentration, although too few for reliable generalization, may be comparable to the predominance of the A4V mutation in the United States and the D90A mutation in Scandinavia. Similarly to what reported for other SOD1 mutations [26–28], a remarkable phenotype–genotype correlation seems to emerge when comparing the clinical characteristics of the patients carrying the same or similar mutations. The nonsense mutation in exon 5 was present in a patient with a severe and rapid clinical course, analogous to what found for most SOD1 mutations leading to a truncated protein [29]. Conversely, N65S and A95T were both associated to a slowly, or very slowly, progressive course of the disease (Table 1) similarly to other SOD1 mutations (H46R, D76V, I113T, L144P, G93V, I151T, D90A, A89T) detected in patients with a disease duration >10 years [[19], alsod1db]. In addition, N65S seems to be strictly correlated to a prevalent involvement of the lower motor neurons and only at the spinal level. If confirmed in a larger patient panel, these findings might help to improve prognostic evaluation.
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
Acknowledgements We are grateful to the patients for their collaboration.
[18]
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