This Article  • Abstract  • PDF  • Reply to article  • Send to a friend  • Save in My Folder  • Save to citation manager  • Permissions  Citing Articles  • Citation map  • Citing articles on HighWire  • Citing articles on Web of Science (7)  • Contact me when this article is cited  Related Content  • Related article  • Similar articles in this journal  Topic Collections  • Amyotrophic Lateral Sclerosis  • Neurogenetics  • Neuromuscular diseases  • Genetic Counseling/ Testing/ Therapy  • Genetic Disorders  • Alert me on articles by topic  Social Bookmarking   What's this?

Yasushi Takehisa, MD; Hiroshi Ujike, MD, PhD; Hideki Ishizu, MD, PhD; Seishi Terada, MD; Takashi Haraguchi, MD; Yuji Tanaka, MD, PhD; Tetsuya Nishinaka, MD, PhD; Keigo Nobukuni, MD; Yuetsu Ihara, MD, PhD; Reiko Namba, MD; Takeshi Yasuda, MD, PhD; Masahiro Nishibori, MD, PhD; Toshiyuki Hayabara, MD, PhD; Shigetoshi Kuroda, MD, PhD

Arch Neurol. 2001;58:736-740.

ABSTRACT
Background  Mutations in the SOD1 gene are responsible for approximately 25% of all familial amyotrophic lateral sclerosis (ALS) cases. However, the correlation between the clinical and pathological features and the various SOD1 gene mutations has not been well characterized.

Objectives  To screen the SOD1 gene in search of potential mutations and to obtain clinical and pathological data for 2 Japanese families with ALS.

Design  Clinical histories and neurological findings, gross and microscopic pathological features, and DNA analysis of the SOD1 gene.

Results  The 2 families with ALS showed a novel missense mutation in the SOD1 gene, which was heterozygous for point mutation TTG to TCG, causing substitution of leucine for serine at codon 126 (Leu126Ser) in exon 5. Clinically, patients showed slower disease progression and lack of upper motor neuron signs. Neuropathologically, the autopsied patient showed the form of familial ALS with posterior column involvement, and the pontocerebellar tract and the dentate nuclei of the cerebellum were also involved. Furthermore, abundant Lewy body–like hyaline inclusions were observed in the affected motor and nonmotor neurons.

Conclusions  Familial ALS with a novel Leu126Ser mutation in the SOD1 gene showed mild clinical features and lack of upper motor neuron signs. We believe that Leu126Ser might be associated with the clinical features and that the mutation site in the SOD1 gene and disease duration might be associated with the formation of Lewy body–like hyaline inclusions.

INTRODUCTION

Jump to Section  • Top  • Introduction  • Patients and methods  • Results  • Comment  • Author information  • References

IN APPROXIMATELY 5% to 10% of individuals, amyotrophic lateral sclerosis (ALS) is inherited as an autosomal dominant trait.¹ Approximately 25% of familial ALS (FALS) cases are associated with mutations in the copper/zinc superoxide dismutase (SOD1) gene^{2, 3} encoded on chromosome arm 21q22.1.⁴ More than 60 kinds of mutations in the SOD1 gene have been identified,^{5, 6} mostly single base pair (bp) substitutions in an exon or at a splice junction, except for 3 cases with 2- or 4-bp deletions.^{7, 8, 9}

The correlation between the clinical and pathological features and the various SOD1 gene mutations has not been well characterized. Patients with FALS and the Ala4Val mutation show defined clinical features and rapid progression, and they usually die within 1 year of disease onset.^{5, 9} However, other mutations in the SOD1 gene produce a wide spectrum of clinical features, including age of onset, disease duration, and severity and distribution of clinical symptoms.⁵ We describe 4 patients with ALS in 2 families with a novel mutation at codon 126 of the SOD1 gene and show their characteristic clinical and pathological features.

PATIENTS AND METHODS

Jump to Section  • Top  • Introduction  • Patients and methods  • Results  • Comment  • Author information  • References

Pedigrees of family A and B are shown in Figure 1A-B.

View larger version (46K): [in this window] [in a new window] Figure 1. A, Pedigree of family A (patients 1 and 2). B, Pedigree of family B (patients 3 and 4). Black symbols indicate affected individuals; diagonal lines across symbols, dead individuals; and arrows, probands. C, Direct sequencing of exon 5 in the SOD1 gene from the 5' end. A heterozygous single base pair (bp) substitution of TTG to TCG (encoding leucine to serine) was detected at codon 126. The result was confirmed by sequencing from the 3' end of exon 5. D, AvaI digestion of polymerase chain reaction products of SOD1 exon 5 from patients with familial amyotrophic lateral sclerosis and controls. Controls (lane 4) show only 1 fragment of amplified polymerase chain reaction product of 205 bp, whereas patients with familial amyotrophic lateral sclerosis (lane 1, patient 1; lane 2, patient 2; and lane 3, patient 3) showed 1 fragment (205 bp) of amplified polymerase chain reaction product and 2 cleavages (138 and 67 bp) by AvaI.

FAMILY A

Patient 1

A 52-year-old man first noticed weakness of the left lower limb that extended to the other limbs during the next 6 months. At age 54 years he developed dysphagia and dysarthria. Neurological examination revealed muscular weakness, atrophy and fasciculation of all limbs, and decreased deep tendon reflex. The Babinski sign was absent. Cognitive function, the cranial nerves, sensation, and the autonomic system were intact. Electromyography revealed active denervation discharge in muscles of all limbs. At age 58 years he was given respirator support because of dyspnea; he died 4 days later. The pyramid sign was not observed until his death. Disease duration was 6 years. An autopsy was performed.

Patient 2

A 28-year-old man (the nephew of patient 1) first noticed right lower limb weakness that progressed to the other limbs during the next year. Neurological examination revealed weakness, atrophy, and fasciculation of all limbs and decreased deep tendon reflex. The Babinski sign was absent. Electromyography showed a neurogenic pattern in all muscles examined. Muscle biopsy of the quadriceps femoralis pathologically revealed neurogenic change. Patient 2 died at age 36 years, with a disease duration of 8 years.

FAMILY B

Patient 3

A 74-year-old man who experienced right hemiparesis by cerebral infarction at age 71 years first noticed lower limb weakness that progressed to the upper limbs and dysarthria and dysphagia during the next year. Deep tendon reflex was decreased and the Babinski sign was absent. Patient 3 died of pneumonia at age 76 years, and disease duration was expected to be 20 months if we assume the age when he noticed lower limb weakness to be the onset of disease.

Patient 4

A 54-year-old woman (the sister of patient 3) first noticed lower limb weakness that progressed to the upper limbs during the next 2 years. On neurological examination, the muscles of all limbs showed marked weakness and atrophy with fasciculation and decreased deep tendon reflex. The Babinski sign was absent. Patient 4 died at age 68 years.

MOLECULAR GENETIC STUDIES

Genomic DNA was extracted from heparin-anticoagulated peripheral blood samples from patients 1 and 3 and from paraffin-embedded tissue from the quadriceps femoralis biopsy sample from patient 2. The region that encoded the 5 exons of the SOD1 gene was amplified by polymerase chain reaction, which was performed with pairs of primers⁶ described previously. Polymerase chain reaction products were sequenced by the protocol of the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Applied Biosystems, Santa Clara, Calif).

NEUROPATHOLOGICAL STUDIES

The brain and spinal cord of patient 1 were fixed in 10% buffered formalin. Multiple tissue blocks were embedded in paraffin; sectioned at 7-µm thickness; and stained with hematoxylin-eosin, Klüver-Barrera, and Holzer stains. For immunohistochemical analysis, polyclonal primary antibodies were used against ubiquitin (Dako, Glostrup, Denmark), -synuclein (Dako), neurofilament (Dako), and rabbit SOD1 (provided by M. Nishibori, MD, PhD, Department of Pharmacology, Okayama University Medical School, Okayama, Japan). Immunostaining was performed using the avidin-biotin peroxidase complex method with a Vectastain ABC Elite kit (Vector, Burlingame, Calif).

RESULTS

Jump to Section  • Top  • Introduction  • Patients and methods  • Results  • Comment  • Author information  • References

MOLECULAR GENETICS

The probands had a heterozygous point mutation in exon 5 of the SOD1 gene (codon 126 TTG to TCG) that resulted in an amino acid substitution from leucine to serine (Leu126Ser) (Figure 1C). No mutations were detected in the other exons. The mutation was confirmed by sequencing the complementary strand. Because this mutation created an AvaI restriction site, it was analyzed using polymerase chain reaction–restriction fragment length polymorphism (Figure 1D). AvaI digestion in exon 5 of the SOD1 gene was found in all 3 patients examined but not in 108 controls (mean age, 68.7 years).

NEUROPATHOLOGICAL FINDINGS

Degeneration of the cerebral cortices, basal ganglia, thalamus, and midbrain was unremarkable. There were no abnormalities in the striatum, globus pallidus, substantia nigra, thalamus, hypothalamus, and red nuclei. There was mild loss of neurons with gliosis in the pontine, hypoglossal nerve, and vestibular nuclei. The tracts of the pontine transverse fibers showed severe degeneration accompanied by fibrillary gliosis. In the cerebellum, the dentate nucleus showed grumose degeneration, and patchy loss of Purkinje cells was also observed. Histological examination showed diffuse cell loss in the anterior horn, middle root zone of the posterior column, spinocerebellar tracts, and Clark column nuclei, with preservation of Onuf nuclei (Figure 2). Demyelination of the corticospinal tract was observed. Bunina bodies were not found. We found Lewy body–like hyaline inclusions (LBHIs) in neurites and neurons in the anterior horn, hypoglossal nerve nucleus, dentate nucleus of the cerebellum, and pontine transverse fibers. The LBHIs were labeled by the antibody against SOD1, ubiquitin, and neurofilament but not labeled by the antibody against -synuclein (Figure 3).

View larger version (141K): [in this window] [in a new window] Figure 2. Sections through the cervical cord (A), thoracic cord (B), and lumbar cord (C) of patient 1 showing severe degeneration of the corticospinal tract, the spinocerebellar tracts, and the middle root zones of the posterior column (Klüver-Barrera stain, original magnification x80).

View larger version (57K): [in this window] [in a new window] Figure 3. Lewy body–like hyaline inclusions in anterior horn cells of patient 1 in family A with an SOD1 mutation (Leu126Ser). Optical microscopy shows the Lewy body–like hyaline inclusions, composed of the eosinophilic core and the pale halo in hematoxylin-eosin–stained sections, and immunoreactive for SOD1 and ubiquitin but not for -synuclein. A, Hematoxylin-eosin, original magnification x300; B-D, immunoperoxidase stains with rabbit anti-SOD1, anti–-synuclein, and anti-ubiquitin antibody, original magnification x300.

COMMENT

Jump to Section  • Top  • Introduction  • Patients and methods  • Results  • Comment  • Author information  • References

We described 4 patients with FALS in 2 familes with a novel Leu126Ser mutation in exon 5 of the SOD1 gene. We could not examine the SOD1 genes of other nonaffected members of the family because they declined gene analysis. Therefore, it is likely that Leu126Ser can be a rare polymorphism. However, other mutations at codon 126 have been found to be associated with FALS, and 108 control subjects did not have such mutant alleles. This result strongly indicates that Leu126Ser must be responsible for the pathogenesis of FALS in these patients.

Clinically, the patients' ages of onset were disparate; however, they showed similar mild progression of illness and long disease duration of 6, 8, and 14 years (excluding patient 3) compared with the duration in patients with FALS and the SOD1 mutation (mean duration, 3.9 years).⁶ Disease onset in patient 3 is late (age 74 years); however, he experienced cerebral infarction at age 72 years. We expect that he might have noticed disease onset lately because of aging and his complication of cerebral infarction; therefore, it is difficult to compare simply patient 3 with the other patients with Leu126Ser. Although the mother of patient 2 must be carrying the mutant gene, she is presently healthy at age 65 years and has declined gene analysis. Moreover, none of the patients had clinical features of upper motor neuron involvement in life, but the autopsy case showed corticospinal tract involvement. The lack of upper motor neuron features in life probably reflects the severe early lower motor neuron degeneration and late upper motor neuron changes.

Familial ALS is neuropathologically classified into 2 forms. The classical form is similar to sporadic ALS, in which degeneration is restricted to motor neurons only. The other form has posterior column involvement and is characterized by degeneration of middle root zones of the posterior column and spinocerebellar tracts, in addition to the lesion of the motor neuron system.^{10, 11} Contrary to the mild clinical features, the neuropathological findings in patient 1 showed the form of FALS with posterior column involvement, and the degeneration extended to the pontocerebellar tract and the dentate nuclei of the cerebellum, which are usually spared in FALS. The reason for the discrepancy between the clinical and neuropathological findings in patient 1 is not clear, but it is possible that the mutation site of the SOD1 gene and disease duration might be significant. Different types of mutations at codon 126 have been described in 4 patients (Table 1).^{2, 7, 8, 9} Three patients had 2-bp deletions and the other had Leu126stop. Although few clinical data are provided, at least 2 of these patients showed rapid progression compared with the patients with Leu126Ser in this study, and their duration was less than 2 years. The difference of progression might be because the 2-bp deletion in codon 126 generates a frameshift that introduces a premature stop codon. The patient with the 2-bp deletion in the report by Kato et al⁸ showed longer duration (11 years) than the other 2 patients; however, he was given respirator support within 1 year of disease onset and it is impossible to compare this patient with the other 2.

View this table: [in this window] [in a new window] Table 1. Comparison of Clinical Findings in Patients With FALS and a Mutation in Codon 126 of the SOD1 Gene*

Approximately 20 autopsied FALS cases with SOD1 mutation, including patient 1 in this study, are currently available for analysis of neurological findings. Nine kinds of SOD1 mutations were found in autopsied cases¹⁰: Ala4Thr,¹² Ala4Val,^{5, 13} His46Arg,¹⁴ His48Glu,⁶ Glu100Gly,¹¹ Ilu113Thr,^{15, 16, 17} Val118Leu,¹⁸ a 2-bp deletion in codon 126, and Leu126Ser (patient 1 in this study) (Table 2). Among them, LBHIs are observed only in patients with Ala4Thr, Ala4Val, His46Arg, a 2-bp deletion in codon 126,¹⁰ and Leu126Ser. The major components of the LBHIs are 15- to 25-nm granule-coated fibril and granular materials, which are positive for SOD1 antibody by immunoelectron microscope^{8, 9, 19, 20}; therefore, LBHIs include SOD1 as core protein. In patients with FALS and posterior column degeneration, LBHIs are characteristically found in the lower motor neuron and are rarely seen beyond the motor neuron system.²⁰ In our patient 1, LBHIs are observed in the affected nonmotor neurons. In patients with FALS, LBHIs are observed not only in long survivors with His46Arg, a 2-bp deletion in codon 126,⁸ and Leu126Ser (present study), but also in short survivors with Ala4Thr and Ala4Val. In contrast, astrocytic hyaline inclusions are observed only in long-surviving patients with FALS who have 2-bp deletions in codon 126⁸ or Leu126Ser. Astrocytic hyaline inclusions are not found in short-surviving patients with FALS who have 2-bp deletions in codon 126.^{7, 9} The essential common constituents between LBHIs and astrocytic hyaline inclusions are immunoelectoron microscopically SOD1-positive granular-coated fibril.^{9, 13, 19} These common properties of the 2 types of inclusions might represent a reflection of the same disease process. Because the published literature concerning FALS autopsy cases with mutation of the SOD1 gene is remarkably limited in number, we cannot conclude but assume that the mutation site of the SOD1 gene and disease duration might be related to the formation of LBHIs. Further pathological and molecular analyses of a large number of patients with FALS are necessary to confirm this assumption.

View this table: [in this window] [in a new window] Table 2. Comparison of Clinical Findings and Neuropathological Findings in Patients With FALS and a Mutation in the SOD1 Gene*

AUTHOR INFORMATION

Jump to Section  • Top  • Introduction  • Patients and methods  • Results  • Comment  • Author information  • References

Accepted for publication November 21, 2000.

This work was supported by a research grant from Zikei Hospital, Okayama, Japan.

We thank M. Kobashi and M. Onbe for their skillful technical assistance.

From the Departments of Neuropsychiatry (Drs Takehisa, Ujike, Ishizu, Terada, Haraguchi, Tanaka, Nishinaka, and Kuroda) and Pharmacology (Dr Nishibori), Okayama University Medical School; Department of Neurology, Clinical Research Institute National Sanatorium Minamiokayama Hospital (Drs Nobukuni, Ihara, Namba, and Hayabara); and the Department of Neurology, Kawasaki Medical School Hospital (Dr Yasuda), Okayama, Japan.

Corresponding author and reprints: Yasushi Takehisa, Department of Neuropsychiatry, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.

REFERENCES

Jump to Section  • Top  • Introduction  • Patients and methods  • Results  • Comment  • Author information  • References

1. de Belleroche J, Orrell RW, Virgo L. Amyotrophic lateral sclerosis. J Neuropathol Exp Neurol. 1996;55:747-757. ISI | PUBMED 2. Deng HX, Hentati A, Tainer JA, et al. Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxide dismutase. Science. 1993;261:1047-1051. FREE FULL TEXT 3. Rosen DR, Siddique T, Patterson D, et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993;362:59-62. FULL TEXT | PUBMED 4. Siddique T, Figlewicz DA, Pericak-Vance MA, et al. Linkage of a gene causing familial amyotrophic lateral sclerosis to chromosome 21 and evidence of genetic-locus heterogeneity. N Engl J Med. 1991;324:1381-1384. ABSTRACT 5. Cudkowicz ME, McKenna-Yasek D, Sapp PE, et al. Epidemiology of mutations in superoxide dismutase in amyotrophic lateral sclerosis. Ann Neurol. 1997;41:210-221. FULL TEXT | ISI | PUBMED 6. Shaw CE, Enayat ZE, Chioza BA, et al. Mutations in all five exons of SOD1 may cause ALS. Ann Neurol. 1998;43:390-394. FULL TEXT | ISI | PUBMED 7. Kadekawa J, Fujimura H, Ogawa Y, et al. Clinicopathological study of a patient with familial amyotrophic lateral sclerosis associated with a two base pair deletion in the copper/zinc superoxide dismutase SOD1 gene. Acta Neuropathol. 1997;94:617-622. FULL TEXT | PUBMED 8. Kato S, Shimoda M, Watanabe Y, Nakashima K, Takahashi K, Ohama E. Familial amyotrophic lateral sclerosis with a two base pair deletion in superoxide dismutase 1 gene. J Neuropathol Exp Neurol. 1996;55:1089-1101. ISI | PUBMED 9. Takahashi K, Nakamura H, Okada E. Hereditary amyotrophic lateral sclerosis. Arch Neurol. 1972;27:292-299. FREE FULL TEXT 10. Hirano A. Neuropathology of familial amyotrophic lateral sclerosis patients with superoxide dismutase 1 gene mutation. Neuropathology. 1998;18:363-369. 11. Ince PG, Tomkins J, Slade JY, Thatcher NM, Shaw PJ. Amyotrophic lateral sclerosis associated with genetic abnormalities in the gene encoding Cu/Zn superoxide dismutase. J Neuropathol Exp Neurol. 1998;57:895-904. ISI | PUBMED 12. Takahashi H, Makifuchi T, Nakano R, et al. Familial amyotrophic lateral sclerosis with a mutation in the Cu/Zn superoxide dismutase gene. Acta Neuropathol (Berl). 1994;88:185-188. PUBMED 13. Shibata N, Hirano A, Kobayashi M, et al. Intense superoxide dismutase-1 immunoreactivity in intracytoplasmic hyaline inclusions of familial amyotrophic lateral sclerosis with posterior column involvement. J Neuropathol Exp Neurol. 1996;55:481-490. ISI | PUBMED 14. Saida K, Ooi N, Nabeshima K, Asada Y, Kumamoto K, Matsukura S. An autopsy case of familial amyotrophic lateral screlosis with an His46Arg substitution in exon 2 of Cu/Zn superoxide dismutase gene [Japanese abstract]. Clin Neurol. 1999;39:287. 15. Rouleau GA, Clark AW, Rooke K, et al. SOD1 mutation is associated with accumulation of neurofilaments in amyotrophic lateral sclerosis. Ann Neurol. 1996;39:128-131. FULL TEXT | ISI | PUBMED 16. Kokubo Y, Kuzuhara S, Narita Y, et al. Accumulation of neurofilaments and SOD1-immunoreactive products in a patient with familial amyotrophic lateral sclerosis with I113T SOD1 mutation. Arch Neurol. 1999;56:1506-1508. FREE FULL TEXT 17. Orrell RW, King AW, Hilton DA, Campbell MJ, Lane RJ, de Belleroche JS. Familial amyotrophic lateral sclerosis with a point mutation of SOD-1. J Neurol Neurosurg Psychiatry. 1995;59:266-270. FREE FULL TEXT 18. Shimizu T, Kawata A, Kato S, et al. Autonomic failure in ALS with a novel SOD1 gene mutation. Neurology. 2000;54:1534-1537. FREE FULL TEXT 19. Kato S, Saito M, Hirano A, Ohama E. Recent advances in research on neuropathological aspects of familial amyotrophic lateral sclerosis with superoxide dismutase 1 gene mutations. Histol Histopathol. 1999;14:973-989. ISI | PUBMED 20. Kato S, Hayashi H, Nakashima K, et al. Pathological characterization of astrocytic hyaline inclusions in familial amyotrophic lateral sclerosis. Am J Pathol. 1997;151:611-620. ABSTRACT

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter     What's this?

RELATED ARTICLE

Archives of Neurology Reader's Choice: Continuing Medical Education
Arch Neurol. 2001;58(5):840-841.
FULL TEXT  

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES

Immature Copper-Zinc Superoxide Dismutase and Familial Amyotrophic Lateral Sclerosis
Seetharaman et al.
Exp. Biol. Med. 2009;234:1140-1154.
ABSTRACT | FULL TEXT