ELSEVIER
Neuroscience Letters 205 (1996) 79-82
NHRDSCIENCE lflTiilS
A novel two-base mutation in the Cu/Zn superoxide dismutase gene associated with familial amyotrophic lateral sclerosis in Japan Mitsuya Morita ",c, Masashi Aoki", Koji Abe a,*, Takafumi Hasegawa a, Ryo Sakuma., Yoshiaki O n o d e r a a, N o b u m i c h i Ichikawa b, M a s a t o y o Nishizawa c, Yasuto I t o y a m a a aDepartment of Neurology, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-77, Japan bDepartment of Neurology, Sendai Red Cross Hospital, Sendai, Japan CDepartment of Neurology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Tochigi 329-04. Japan
Received 4 December 1995; revised version received 16 January 1996; accepted 16 January 1996
Abstract
We have identified a novel two-base mutation in exon 1 of the Cu/Zn superoxide dismutase (SOD1) gene (TGC to "ITI'), which resulted in Cys 6 to Phe substitution in a Japanese family with amyotrophic lateral sclerosis (ALS). This is the first case of familial ALSassociated two-base change of the SOD1 gene. Similar to several mutations in exon 1 of the SOD1 gene such as Ala4 to Val, Ala4 to Thr and Va114 to Met, affected members of the present family showed a rapid progression of motor dysfunction. Although Ala4, Cys 6 and Val7 reside in the middle of the first fl-strand of the SOD 1, a family with a mutation of Val7 to Glu associates with slow progression of the disease. These findings suggest that clinical courses are variable with each mutation, even in the same exon. Keywords: Amyotrophic lateral sclerosis; Superoxide dismutase; Mutation; Gene
A m y o t r o p h i c lateral sclerosis (ALS), a degenerative disorder of cortical and spinal motor neurons, causes a progressive paralysis that results in fatality usually within 5 years of the onset. Familial ALS (about 5 - 1 0 % of ALS) is usually expressed as an autosomal dominant trait and clinically indistinguishable from sporadic cases. Although the pathophysiologic process of ALS remains unknown, recent studies showed that some pedigrees with familial A L S carried mutations in the Cu/Zn superoxide dismutase (SOD 1) gene. Rosen et al. and Deng et al. discovered 14 different mutations within exons 1, 2, 4 and 5 of the SOD1 gene and reported that the mutation Ala 4 to Val in exon 1 was most common and it was characterized by rapidly progressive clinical course [4,13]. In Japanese families with ALS, Nakano et al. and Hirano et al. reported point mutations of Ala 4 to Thr [9] and Val 7 to Glu [7] in exon 1, respectively. Here we report a novel two-base mutation resulting in an aminoacid substitution of Cys 6 to Phe in exon 1 of the SOD 1 gene in a Japanese family with ALS. * Corresponding author. Tel.: +81 22 2739294; fax: +81 22 2725818.
W e have studied one Japanese family. The proband noticed fasciculations in the right upper and lower extremities at age 59. Muscle weakness and atrophy in her left lower extremity rapidly developed. Deep tendon reflexes were decreased in left extremities, while those were increased in right extremities. Within a half year of the onset, she became unable to raise arms and legs and had to use a wheel chair, and bulbar symptom began to appear. Jaw jerk was present, but was not increased. Other neurological symptoms and signs such as sensory, cerebellar, extrapyramidal and autonomic functions were normal. Serum creatine kinase was slightly increased as 389 IU/1. Muscle biopsy specimen of left biceps brachii showed typical neurogenic change. Electromyographic analysis showed a typical neurogenic pattern. The diagnosis of ALS was thus confirmed by these clinical, electromyographic and pathological findings. Her father noticed weakness of lower extremities at age 4 l, and died of respiratory failure about 1 year after the onset of disease. He was suspected to be affected with A L S because of neurological symptoms and signs. Other members of this family did not suffer from ALS.
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved Pll: S0304- 3940(96) 12378-6
M. Morita et al. / Neuroscience Letters 205 (1996) 79-82
80
Mutant +ys Ala
Normal
Ala
Lys
Val
( ~
Val
Leu
Lys
Val ( ~
Val
Leu
Lys
:t: A .% • *..- :
~"
~ L
/~
Jl
& I
'|•
• I AI I
!
]/1
!~,
l
i
1 li'
I
I
I
I
I
?ti ~
'
',
J
I
•
1
FI
',11,,11 i
Ilil
"I l
!,,
1 1
•
i
!i ii
I J,
.......... I
II! ',/I
I!I,,rl !
"
Ik, I I-Y \iV k'-, ,jv. iIII
,11~
i
"" :! ; ::
j I A I ', A
I
ii ' VI
;i] I / /,v
~=:'-~: ............................... ~---"~-" I I I I I I I I I I I I I I
....
' " " " '
"~'-~. . . . .
''
' "
~-:"~-",:...'~,..~
' "
-
' ";
-~'l[
'1
Fig. 1. Sequence analysis of two-base mutation in the Cu/Zn SOD gene. Left panel shows normal sequence and right panel shows a GC-to-TT transition (arrowhead) that results in the substitution of Cys6 by Phe. The enzyme activity of SODI was measured in the proband and in age- and sex-matched normal Japanese controls (mean age + SD, 59.4 -+ 2.2; n = 5) by the nitrite method [3,10]. In brief, heparinized blood was centrifuged and the plasma was discarded. After being washed with saline, the cell pellet was diluted with water to lyse the erythrocytes. Ethanol and chloroform were then added to remove the hemoglobin, and the water-ethanol layer was separated to measure SOD1 activity. SOD1 activity in erythrocytes was significantly lower in the patient (1.36 cytochrome c unit/rag red cell protein) as compared to the normal controls (mean _+ SD, 5.37 _+ 1.12). SOD1 activity in this patient was 25.3% of that in controls. DNA was extracted from the peripheral blood leukocytes of the proband as well as 41 control Japanese subjects after obtaining informed consents. An analysis with single-strand conformational polymorphism of the products of polymerase chain reaction (PCR-SSCP) was performed covering the five exons of the SOD1 gene as previously described [11] with a fluorescein-isothiocyanate (FITC)-labeled primer on an automated D N A sequencer (A.L.F. DNA Sequencer, Pharmacia LKB, NJ, USA). DNA sequence of exon 1 of the SOD1 gene was analyzed on this patient based on its abnormal pattern of
SSCP. Exon 1 of the SOD1 gene was amplified by PCR as previously described [3]. The PCR products were subcloned into a Bluescript SK + plasmid vector (Stratagene, La Jolla, CA) and the sequence was determined using the automated sequence analyzer. By the SSCP analysis, an abnormally migrating band was observed in the PCR product of exon 1 from this patient. PCR products of exons 2, 3, 4, 5 did not show abnormal patterns. Forty-one control subjects were also screened by the same method, but showed no abnormal band patterns. Nine plasmid clones of PCR products from exon 1 were sequenced, and in three of them a novel twobase mutation was found in the SODI gene (TGC to ~), which resulted in Cys 6 to Phe substitution (Fig. 1). No mutations were found in the other clones, so sequence analysis revealed a heterozygosity in this patient. Cysteine is translated from codon TGC or TGT. Therefore, the C to T change in Cys 6 might be a polymorphism. The Cys 6 is well conserved among species in human, cow, pig, mouse, Xenopus, swordfish and Drosophila [12]. Although segregation of the mutation could not be confirmed by the family members because of the death of her father, the Cys ° to Phe substitution is most likely the cause of ALS of this family. Since the initial discovery of the relations between mutations in the SOD1 gene and
M. Morita et al. / Neuro~cience Letters 205 (1996) 79-82
ALS in some families, the number of reported mutations has grown to more than 30 [1]. Although all mutations that were previously reported were point mutations, this is the first case of familial ALS-associated two-base change of the SOD1 gene. In terms of mutations in exon 1, substitutions of Ala 4 to Val or Thr, and Val 7 to Glu were previously reported near the region of Cys 6 to Phe substitution of the SODI gene. These three aminoacids locate in the middle of the first fl-strand, and are strongly conserved among species [12]. Rosen et al. reported that a mutation of Ala n to Val was associated with severe clinical features and rapid progression [12]. Nakano et al. reported a family with a mutation of Ala 4 to Thr showing rapid progression and lower penetrance [9]. However, Hirano et al. reported a family with a mutation of Val 7 to Glu with slow progression of the disease [7]. Substitutions of Va114 to Met and Glu 21 to Lys in exon 1 were also reported [5,8]. Deng et al. speculated that mutant at Va114 was related to defective packing and stability of the SOD1 enzyme, and therefore this mutant was associated with more serious clinical features of ALS [5]. Jones et al. found a mutation of Glu 21 to Lys in a sporadic ALS case, but a clinical course was not fully described [8]. In the present family with C y s 6 to Phe substitution, affected members also showed a rapid progression of motor dysfunction. Thus, the clinical courses are quite different among the families with the different mutations within the same region of exon 1. Aoki et al. reported a mutation His 46 to Arg in exon 2 of the SODI gene in two Japanese families with very slow progression of ALS [3]. In these families clinical features were almost identical. In contrast, they also reported the family with a mutation Leu 84 to Val, in which the progression of the disease was very rapid, but the age at onset varied with sex or generation [2]. These findings suggest that clinical courses are very variable with mutations among exons, even within the same exon. Furthermore, a clinical variance in the same pedigree suggests that other factors such as sexual or environmental factors could also affect age at onset and clinical courses of the disease. Although some reports suggested that SODI mutations showed a reduction of enzyme activity, the exact relationship between the enzyme activity and the mechanism leading to motor neuron degeneration is still obscure. A recent report showed that an overexpression of human mutated SOD I gene caused ALS-like symptoms and motor neuron degeneration in transgenic mice [6]. Moreover, Andersen et al. reported a family in which symptoms were observed only in patients with homozygous mutations, and they showed no reduction in erythrocyte SOD1 activity [1 ]. These findings suggest that motor neuron degeneration is caused by the gain of deleterious interaction of mutated SODI rather than a simple loss of the activity. It is suggested that the mutations of SOD1 gene may alter the three-dimensional structure of SOD1, that disturb both the subunit folding and the dimer formation
81
[4,7], and that these changes might contribute to gain an adverse property of the enzyme. We thank Drs. Y. Aoki, T. Kameya and K. Fuchigami for their excellent technical assistance. This work was partly supported by Grant-in-Aid for Scientific Research on Priority Areas (Kanazawa I) 06272204, and Grant-inAid for Scientific Research (C) 06807055 from the Ministry of Education, Science and Culture of Japan, and by a grant (Hirai S and Yanagisawa N) from the Ministry of Health and Welfare of Japan. [1] Andersen, P.M., Nilsson, P., Ala-Hurula, V., Keranen, M.-L., Tarvainen, 1., Haltia, T., Nilsson, L., Binzer, M., Forsgren, L. and Marklund, S.L., Amyotrophic lateral sclerosis associated with homozygosity for an Asp90Ala mutation in CuZn-superoxide dismutase, Nature Genet., I 0 (1995) 61 ~ 6 . [2] Aoki, M., Abe, K., Houi, K., Ogasawara, M., Matsubara, Y., Kobayashi, T., Mochio, S., Narisawa, K. and lloyama, 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. [3] Aoki, M., Ogasawara, M., Matsubara, Y., Narisawa, K., Nakamura, S., Itoyama, Y. and Abe, K., Familial amyotrophic lateral sclerosis (ALS) in Japan associated with H46R mutation in Cu/Zn superoxide dismutase gene: a possible new subtype of familial ALS, J. Neurol. Sci., 126 (1994) 77-83. [4] Deng, H.X., Hentati, A., Tainer, J.A., Iqbal, Z., Cayabyab, A., Hung, W.Y., Getzoff, E.D., Hu, P., Herzfeldt, B., Roos, R.P., Warner, C., Deng, G., Soriano, E., Smyth, C., Parge, H.E., Ahmed, A., Roses, AD., Hallewell, R.A.. Pericak-Vance, M.A. and Siddique, T., Amyotrophie lateral sclerosis and structural defects in Cu,Zn superoxide dismutase, Science, 261 (1993) 1047-1051. [5] Deng, H.X., Tainer, J.A., Mitsumoto, H., Ohnishi, A., He, X.X., Hung, W.Y., Zhao, Y.L., Juneja, T., Hentati, A. and Siddique, T., Two novel SODI mutations in patients with familial amyotrophic lateral sclerosis, Hum. Mol. Genet., 4 (1995) I t 13--1 116. [6] Gurney, M.E., Pu, H., Chiu, A.Y., l)al, C.M., Polchow, C.Y, Alexander, D.D., Caliendo, J., Hentati. A., Kwon, Y.W,, Deng, H.X., Chen, W., Zhai, P., Sufit, R.L. and Siddique, T , Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation, Science, 264 (1994) 1772-175'5. [7] Hirano, M , Fujii, J., Nagai, Y., Sonobe, M.. Okamoto, K., Araki, H., Taniguchi, N. and Ueno, S., A new variant Cu/Zn superoxide dismutase (Val7--~ Glu) deduced from lymphocyte mRNA sequences from Japanese patients with familial amyotrophic lateral sclerosis, Biochem. Biophys. Res. Commun., 2()4 (1994) 572 577. [8] Jones, C.T., Swingler, R.J., Simpson, S.A. and Brock, I).J.H.. Superoxide dismutase mutations in an unselected cohort of Scottish amyotrophic lateral sclerosis patients. J. Med. Genet., 32 (1995) 290-292. [9] Nakano, R., Sato, S., lnuzuka, T., Sakimura, K., Mishina, M., Takahashi, H., Ikuta, F., Honma, Y., Fujii, J., Taniguchi, N. and Tsuji, S., A novel mutation in Cu/Zn superoxide dismutase gene in Japanese familial amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun., 200 (1994) 695-703. [10] Oyanagui, Y., Reevaluation of assay methods and establishment of kit for superoxide dismutase activity, Anal. Biochem., 142 (1984) 290-296. [11] Pramatarova, A., Figlewicz, D.A., Krizus, A., Han, F.Y., Ceballos, P.I., Nicole, A., Dib, M., Meininger, V., Brown, R.H. and Rouleau, G.A., Identification of new mutations in the Cu/Zn superoxide dismutase gene of patients with familial amyotrophic lateral sclerosis, Am. J. Hum. Genet., 56 (1995) 592-596.
82
M. Morita et al. / Neuroscience Letters 205 (1996) 79-82
[12] Rosen, D.R., Bowling, A.C., Patterson, D., Usdin, T.B., Sapp, P., Mezey, E., McKenna, Y.D., O'Regan, J., Rahmani, Z., Ferrante, R.J., Brownstein, M.J., Kowall, N.W., Beal, M.F., Horvitz, H.R. and Brown, Jr., R.H., A frequent ala 4 to val superoxide dismutase-I mutation is associated with a rapidly progressive familial amyotrophic lateral sclerosis, Hum. Mol. Genet., 3 (1994) 981987. [13] Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O'Regan, J.P., Deng,
H.X., Rahmani, Z., Krizus, A., McKenna-Yasek, D., Cayabyab, A., Gaston, S.M., Berger, R., Tanzi, R.E., Halperin, J.J., Herzfeldt, B., Van den Vergh, R., Hung, W.Y., Bird, T., Deng, G., Mulder, D.W., Smyth, C., Laing, N.G., Soriano, E., PericakVance, M.A., Haines, J., Rouleau, G.A., Gusella, J.S., Horvitz. H.R. and Brown, Jr., R.H., Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis, Nature, 362 (1993) 59~2.