Lack of SOD1 gene mutations and activity alterations in two Italian families with amyotrophic lateral sclerosis

Neuroscience Letters 289 (2000) 157±160

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Lack of SOD1 gene mutations and activity alterations in two Italian families with amyotrophic lateral sclerosis Donella Gestri a, Cristina Cecchi b, Andrea Tedde c, Stefania Latorraca a, Antonio Orlacchio d, Enrico Grassi a, Anna Maria Massaro a, Gianfranco Liguri b, Peter H. St. George-Hyslop d, Sandro Sorbi a,* a

Department of Neurological and Psychiatric Sciences, University of Florence, Florence, Italy b Department of Biochemical Sciences, University of Florence, Florence, Italy c Department of Biochemical Sciences and Molecular Biotechnologies, University of Perugia, 06100 Perugia, Italy d Centre for Research in Neurodegenerative Diseases, Department of Medicine, University of Toronto, Toronto, Canada Received 21 March 2000; received in revised form 9 June 2000; accepted 12 June 2000

Abstract Amyotrophic lateral sclerosis (ALS) is a progressive fatal disorder, which results from the degeneration of motor neurons in the brain and spinal cord. Approximately 20% of the inherited autosomal dominant cases are due to mutations within the gene coding for Cu/Zn superoxide dismutase 1 (SOD1), a cytosolic homodimeric enzyme that catalyzes the dismutation of toxic superoxide anion. We investigated the presence of SOD1 gene mutations and activity alterations in two unrelated families of ALS patients from Elba, an island of central Italy. No mutation in SOD1 exon 1 to 5 and no activity alteration were observed in all members of the two analyzed ALS families (FALS). These data show an apparent heterogeneous distribution of ALS patients with SOD1 gene mutations among different populations and suggest that another genetic locus could be involved in the disease. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Familial Amyotrophic Lateral Sclerosis; Cu/Zn Superoxide Dismutase 1; Gene mutation

Amyotrophic lateral sclerosis (ALS) is a progressive paralytic fatal disorder, which results from the degeneration of motor neurons in the brain and spinal cord with a selective damage to the neural system that mediates voluntary movements. Approximately 10% of cases are familial (FALS) [7]. Mutations of SOD1 are present in around 20% of FALS world-wide. SOD1 gene mutations have been widely reported among American, British and Japanese families [1,4,5,12]. Only few SOD1 gene mutations have been detected in the continental European ALS populations [2,17]. Only three families harbouring a SOD1 gene mutation have been reported in Italy [3,13,14] showing an apparent heterogeneous distribution of genetic abnormalities in ALS patients among different ethnic groups [9]. The inheritance is usually autosomal dominant with age dependent penetrance, but in regions of Sweden and Finland mutations with autosomal recessive inheritance have been identi®ed [17]. It is widely believed that FALS patients * Corresponding author. Tel.: 139-55-4298465; fax: 139-554271380. E-mail address: [email protected]®.it (S. Sorbi).

differ from sporadic ALS having a younger age of onset and a generally more severe course of disease, considering that these patients are heterogeneous in clinic, genetic and biochemical features [8]. It is not clear how many patients with apparently sporadic ALS actually have new mutations in the SOD1 gene or incomplete penetrance of known familial SOD1 mutations, but such cases are increasingly being recognized [18]. In contrast to sporadic ALS, men are no more likely to develop FALS than women. Known genetic abnormalities in FALS consist of mutations within the gene coding for Cu/Zn superoxide dismutase 1 (SOD1) mapped to chromosome 21q22.1 [15]. The gene encoding the SOD1 protein consists of 5 exons and 153 codons [16]. Pathological mutations have been demonstrated in all ®ve exons affecting 43 of the 153SOD1 residues and show little regional clustering in a computer-generated reconstruction of the protein. Nearly 60 different SOD1 missense mutations have been identi®ed in more than 250 ALS pedigrees [7,14]. Human SOD1 is a cytosolic homodimeric Cu/Zn binding metalloenzyme that catalyzes the dismutation of toxic superoxide anion radical O22 to O2 and H2O2. Moreover, it functions to remove toxic free radicals generated by normal

0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 27 3- 8

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Fig. 1. Pedigree of ALS1. Male family members are represented by squares, females by circles, and deceased with diagonals; affected members are represented by solid symbols.

cellular processes, that are implicated in a number of neurodegenerative disorders. Whether SOD activity is altered in mutated FALS patients is still controversial: initial studies of SOD activity in red cells of FALS patients carrying mutations at the SOD1 locus demonstrated reduced SOD dismutation activity [20]; other studies found that not in all mutated FALS patients SOD activity is altered [10]. Given these ®ndings, we investigated the presence of SOD1 mutations and enzyme activity alterations in two central Italy families of ALS patients. We have included in this study three living affected members of two unrelated ALS Italian families from Elba Island, conformed to the diagnostic criteria for ALS as outlined by El Escorial classi®cation [19]. All patients gave informed consent and had their blood sampled. The

clinical data were available from the ongoing follow-up and retrospectively from the analysis of medical records. In the ®rst family (ALS1; see Fig. 1) the disease was inherited as an autosomal dominant trait. The onset was in the ®fth decade and the disease's duration was about 2 years. In the second family (ALS2; see Fig. 2) the disease was inherited as an autosomal dominant trait too. The relative lack of bulbar and respiratory muscle weakness distinguishes this family from the other family. The onset was in fourth decade and the mean time from onset of symptoms to death (15 ^ 5) is much longer than in ALS1. Nine subjects at risk belonging to ALS1 and ALS2 and ten unrelated health controls were included in this study. All controls were sex and age matched to ALS patients. DNA was extracted from the peripheral blood leuko-

Fig. 2. Pedigree of ALS2. Male family members are represented by squares, females by circles, and deceased with diagonals; affected members are represented by solid symbols.

D. Gestri et al. / Neuroscience Letters 289 (2000) 157±160

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Table 1 Cu/Zn SOD1 erythrocyte activity in the patients from two FALS families is compared to the non-affected FALS and unrelated normal control subjects a

Affected family members Non-affected family members Unrelated control subjects a

Age (years) mean

Sex (M/F)

Number of subjects

Speci®c activity (U-525/mg red cell protein) Mean ^ SD

48 39 50

1/2 7/2 6/4

3 9 10

121.80 ^ 23.18 123.89 ^ 15.47 111.03 ^ 11.64

Statistical analysis was performed using Student's t-test.

cytes. Exons 1 through 5 of the SOD1 gene were ampli®ed from genomic DNA by polymerase chain reaction (PCR), using previously described primers [6]. An analysis with single-strand conformation polymorphism of the PCR products (PCR-SSCP) was performed spanning all the ®ve exons of the SOD-1 gene as previously described [6]. In order to verify the putative presence of mutations in the SOD-1 gene of ALS patients, sequencing of the sense and antisense strands of the PCR products was performed on an automated DNA sequencer (ALF DNA Sequencer, Pharmacia LKB, NY, USA). Cu/Zn SOD activity of erythrocyte lysate from heparinized whole blood from the patients (n ˆ 3) and non-affected family members of FALS (n ˆ 9) and age-matched control subjects (n ˆ 10) was measured by the method of Nebot et al. [11] in triplicate. Brie¯y, the assay was based on the SOD-mediated increase in the rate of autoxidation of 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo(c)¯uorene (BXT-01050) in alkaline solution at 378C. One SOD activity unit (U-525) has been de®ned as the activity that doubles the autoxidation background. SSCP analysis was performed in order to individuate band shifts indicating the presence of SOD1 gene mutations. No abnormally migrated band was observed in the PCR products of exon 1 to 5 from all members of the two analyzed ALS families compared to control subjects. The absence of mutations was con®rmed in the patients by SOD1 gene sequence analysis. Cu/Zn SOD1 erythrocyte activity was not statistically different (P ˆ 0:875) in the patients from two FALS families as compared to the non-affected FALS individuals and unrelated normal control subjects (Table 1). These data showed no evident relationship between Cu/Zn SOD1 activity alterations and the age at onset or the progression of the disease in the families. This study shows no mutation in SOD1 exon 1 to 5 and no activity alteration in the two analyzed ALS families. The genetic studies have shown ALS families to be a heterogeneous group, with predominant autosomal dominant pattern of inheritance with varying degrees of penetrance [18]. Published data reported heterogeneity among FALS patients about clinical picture, genetic abnormalities and biochemical alterations. The SOD1 mutations account for only about

20% of all cases of FALS [1]. To date genetic abnormalities have not been identi®ed in the remaining ALS patients with a con®rmed familial history. Human SOD1 catalyzes the dismutation of the toxic superoxide anion radical O22 to O2 and H2O2. Free radicals seem to cause neuronal injury in several neurological disorders including Parkinson's disease and ischaemic brain injury. It is still unknown why the damage is selectively at the level of motor neurons, given that SOD1 gene is broadly expressed. To date studies on continental Europe families showed no evidence of a signi®cant SOD1 gene involvement in the transmission of the disease [2,17]. We have studied two unrelated Italian families with FALS characterized by different clinical phenotype. No mutation was observed in exon 1 to 5 in living patients of the analyzed ALS families by SOD1 gene sequence analysis. The activity of the Cu/Zn SOD enzyme was not different in patients and subjects at risk compared to health controls included in the study, con®rming the data that the decrease/increase of enzyme activity is detectable only in the presence of some mutations. These molecular ®ndings from a small sample of Italian ALS families con®rm published data showing an apparent heterogeneous distribution of genetic abnormalities in ALS patients among different ethnic groups [9]. However, the number of Italian ALS families tested so far is clearly not large enough to conclude signi®cantly that in continental Europe families SOD1 gene is involved in the transmission of the disease less than in different populations. If the rate of SOD1 gene mutations in continental Europe studies will remain low in the next investigations too, the occurrence of ancestral SOD1 gene mutations in British, American and Japanese families can be hypothesized. This molecular abnormality may play a different role in each ethnic group according to its speci®c genetic background. An extension of the genetic approach to sporadic ALS patients is needed to con®rm this observation and to understand the pathogenic mechanism of the disease. It has been speculated about involvement of an alternative genetic factor to explain the clinical variability among SOD1 FALS but also what causes the disease in FALS without SOD1 gene mutation. It could explain the remarkable clinic heterogeneity in the Italian ALS families investigated in this study. Whether these two different disease expressions are due to different aetiologies

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or represent a diverse outcome of the same process is unknown. Given these ®ndings, we can conclude that probably in the continental Europe ethnic group SOD1 mutated FALS patients incidence is less than in other investigated populations [1,4,12]. Finally, a large scale screening of SOD1 and eventually other putative genes mutations is needed to better characterize the aetiology of FALS cases in Europe. This work was supported by Telethon Italia Fondazione ONLUS (grant no. C.27). The authors also thank Dr Silvia Piacentini (Department of Neurological and Psychiatric Sciences, University of Florence, Florence, Italy) for her helpful comments in the preparation of the manuscript. [1] Aoki, M., Abe, K., Houi, K., Okasawara, M., Matsubara, Y., Kobayashi, T., Mochio, S., Narisawa, K. and Itojama, Y., Variance of age at onset in a Japanese family with amyotrophic lateral sclerosis associated with a novel Cu/Zn superoxide dismutase mutation, Ann. Neurol., 37 (1995) 676±679. [2] Bachus, R., Claus, A., Megow, D., Brockmoller, J., Porstmann, T., Gericke, C.A., Riepe, M., Kuther, G., Zierz, S. and Ludolph, A.C., Cu,Zn SOD in German families with ALS, J. Neurol. Sci., 129 (1995) 93±95. [3] Ceroni, M., Malaspina, A., Poloni, T.E., Alimonti, D., Rognoni, F., Habgood, J., Imbesi, F., Antonelli, P., Alfonsi, E., Curti, D. and deBelleroche, J., Clustering of ALS patients in central Italy due to the occurrence of the L84F SOD1 gene mutation, Neurology, 53 (1999) 1064±1071. [4] Cudkovitz, M.E., McKenna-Yasek, R.N. and Sapp, B.S., Epidemiology of mutations in Superoxide Dismutase in Amyotrophic Lateral Sclerosis, Ann. Neurol., 41 (1997) 210±221. [5] Deng, H-X., Tainer, J.A., Mitsumoto, H., Ohnishi, A., He, X., Hung, W.Y., Zhao, Y., Juneja, T., Hentati, A. and Siddique, T., Two novel SOD1 mutations in patients with familial amyotrophic lateral sclerosis, Hum. Mol. Genet., 4 (1995) 1113±1116. [6] Jackson, M., Al-Chalabi, A., Enayat, Z.E., Chioza, B., Leigh, P.N. and Morrison, K.E., Copper/Zinc superoxide dismutase 1 and sporadic amyotrophic lateral sclerosis: analysis of 155 cases and identi®cation of a novel insertion mutation, Ann. Neurol., 42 (1997) 803±807. [7] Kurland, L.T. and Mulder, D.W., Epidemiologic investigations of amyotrophic lateral sclerosis. Familial aggregation indicative of dominant inheritance, Neurology, 5 (1955) 182±196. [8] Li, T.-M., Alberman, E. and Swash, M., Comparison of sporadic and familial disease among 580 cases of motor neuron disease, J. Neurol. Neurosurg. Psychiatry, 51 (1988) 778±784. [9] Malaspina, A., Zaman, R., Mazzini, L., Camana, C., Poloni, E., Curti, D. and Ceroni, M., Heterogeneous distribution of

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