Familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a disorder characterized clinically by progressive muscular weakness, wasting, and spasticity due to the degeneration of upper and lower motor neurons. The etiology of sporadic ALS is unknown, but in 5 to 10% of patients the condition is familial (FALS) with a predominantly autosomal dominant pattern of inheritance. Approximately 20% of FALS pedigrees have a mutation in the Cu/Zn superoxide dismutase (SOD1) gene.^1,2 At least 46 different SOD1 mutations are known to exist,³ but the precise mechanism by which mutant SOD1 expression mediates motor neuron injury has yet to be elucidated. Many authors report pathologic differences between familial and sporadic ALS, with prominent involvement of the posterior columns, spinocerebellar tracts, and Clarke's column in FALS.^4-6 Prospective clinicopathologic studies blind to the family history are required to resolve this issue. The finding, in apparently sporadic ALS, of mutations in the neurofilamentous heavy chain (NFH) and SOD1 genes raises questions about the distinction between familial and sporadic disease.^7,8 By studying the detailed molecular pathology of ALS patients with known genetic mutations, we may gain new insight into the pathogenesis of both the familial and sporadic forms of ALS.

Here we report the clinical, genetic, and neuropathologic findings in a patient with a rapidly progressive form of FALS associated with a point mutation in exon 2 (codon 48, CAT > CAG; histidine > glutamine) of the SOD1 gene.

Patient report. The patient, a 54-year-old woman, presented with a 7-month history of progressive limb weakness. She described pain in the lower back and right hand, followed by progressive weakness of the right arm such that there was no movement in the limb after 2 months. Weakness of the left shoulder and both hips developed more slowly over the next 6 months and were accompanied by orthopnea and dysphagia. She had a past history of rheumatic fever and Sydenham's chorea in childhood, two episodes of deep vein thrombosis in the legs, hypertension with pregnancy, and one spontaneous abortion. A strong family history of ALS was obtained (figure 1). The proband (III-6) died at 55 years and her mother (II-9) died at 56 years of definite ALS within 12 months of symptomatic onset. The maternal grandmother (I-2) died of a rapidly progressive "crippling" illness in her late 30s. Two first cousins from separate families died, one (III-2) was 58 years old at the time of death with definite ALS and the other (III-1), was 38 years old at the time of death of a rapidly progressive muscle disease with ventilatory failure (diagnosed as ALS).

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Figure 1. The familial ALS kindred is depicted. Shading indicates an affected individual (half shading = probably affected), circles represent females, squares represent males, the diagonal line across the symbol indicates that the individual is deceased, the arrow indicates the proband.

Examination of our patient at the time of presentation revealed widespread fasciculation, muscle wasting and weakness in the limbs and trunk, but she was still able to walk unaided. There was a brisk jaw jerk, decreased gag reflex on the right, and asymmetry of palatal movement but no fasciculation or wasting of the tongue. The right arm was hypotonic and wasted, but the tendon reflexes in the left arm were brisk. In the legs there was spasticity, exaggerated knee jerks, and an extensor plantar response on the left. There was no sensory deficit or ataxia. In the absence of lower motor neuron signs in the tongue we classified her as having "probable ALS" by the El Escorial criteria.⁹ Her vital capacity was markedly diminished at 0.85 L, with decreased diaphragmatic movement on fluoroscopy. Her hematologic parameters, erythrocyte sedimentation rate (ESR), creatine kinase, electrolytes, glucose, calcium, thyroid function, liver function, vitamin B12, folate, serum electrophoresis, and immunoglobulin levels were normal. Nerve conduction studies demonstrated normal sensory and motor action potential amplitude and conduction velocity with no evidence of multifocal conduction block. Although no motor activity could be detected in the right arm, EMG demonstrated denervation in two other limbs. She died of respiratory failure 9 nine months after the onset of her illness. An autopsy was performed.

Methods. Mutation detection. DNA was extracted from the patient's blood, and four exons (1, 2, 4, and 5) of the SOD1 gene were amplified using published primer sequences.^1,2 Polymerase chain reaction (PCR) fragments were analyzed by single-strand conformational polymorphism (SSCP). If an abnormal migration pattern was found, PCR fragments were subcloned using the pGEM-T vector system (Promega, Southampton, UK), and recombinant colonies were identified. Normal and mutated clones were identified by SSCP analysis of PCR-generated fragments, and the purified plasmid DNA was sequenced by a T7 sequencing kit (Pharmacia Biotech, Milton Keynes, UK). At least two mutated and two normal clones were sequenced, both forward and reverse strand sequencing reactions were carried out.

Specimen processing for light and electron microscopy. The patient's brain and spinal cord were formalin fixed using a standardized protocol. Sporadic ALS, Alzheimer's disease, and normal control brains were used as controls. Blocks of tissue were taken from throughout the brain and spinal cord. Paraffin-embedded sections were cut at 7 µm and stained with standard histologic stains including hematoxylin-eosin, Luxol fast blue/cresyl violet (LFB), and modified Bielschowsky. For electron microscopy the anterior horn of the cervical spinal cord was dissected out, postfixed in 1% osmium tetraoxide, and processed to epoxy resin-Agar 100 (Agar Scientific, Essex, UK). Sections were cut at 80-nm intervals and stained with Reynold's lead citrate and uranyl actetate. For immunohistochemical analysis the following primary antibodies were used: antiubiquitin (polyclonal; Dako, High Wycombe, UK), antineurofilaments (including RT97, a monoclonal antibody[mAb] directed against phosphorylated epitopes on neurofilament heavy (NFH) and medium (NFM) chain and mAb 147 directed against phosphorylated epitopes on NFM, both from Prof. B. Anderton); SMI 31 and SMI 32 directed against phosphorylated epitopes on NFH (both monoclonal; Affiniti, Cambridge, UK), anti-Tau 8073 (polyclonal; from Dr. J-M Gallo), anti-Cu/Zn SOD(SOD1) and Mn SOD (SOD2) (both polyclonal; The Binding Site, Birmingham, UK). Detection was by biotinylated F(Ab)2-secondary antibodies (Dako) and horseradish peroxidase avidin-biotin complex, and visualized with diaminobenzidine. Negative controls were examined using secondary antibodies and detection, in the absence of primary antibody.

Results. Mutation analysis. A novel point mutation(T to G) was identified in exon 2 of the SOD1 gene that resulted in an amino acid substitution of histidine to glycine in the copper binding domain of the SOD1 molecule (figure 2). A brief report of this mutation and others has previously been published.¹⁰

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Figure 2. Direct sequencing of the subcloned normal and mutant Cu/Zn superoxide dismutase gene, showing a single base pair mutation of T > G in one of the chromosomes of the patient, causing an amino acid change of histidine 48 > glycine in the copper-binding domain.

Macroscopic findings. The brain and spinal cord appeared normal aside from atrophy of the anterior spinal roots.

Light microscopy. Histopathologic examination of the cerebrum revealed extensive corpora amylacea in the subpial area and some fibrous thickening of blood vessel walls with mild perivascular lymphocytic cuffing throughout the brain, brainstem, and cerebellum. Mild myelin loss, indicating central axonal degeneration, was present in the lateral corticospinal and spinocerebellar pathways of the cervical cord with minimal involvement of the dorsal columns (figure 3A). There was striking neuronal loss in the anterior horns and many surviving motor neurons appeared either chromatolytic and swollen or atrophic (figure 3B).

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Figure 3. Light microscopic findings in the spinal cord (A, B, F-H), motor cortex (C), and brainstem (D, E). Myelin staining of the cervical cord demonstrates mild pallor of the lateral corticospinal and anterior spinocerebellar tracts with relative sparing of the dorsal columns (A, Luxol fast blue and modified Bielschowsky stain). (B) At low magnification motor neurons in the anterior horn of the cervical cord are severely depleted and the remaining cells appear either atrophic or swollen. (C) Large pyramidal neurons in the precentral gyrus immunolabeled with antibodies to phosphorylated neurofilaments. Hypoglossal nucleus motor neurons immunolabeled for ubiquitin with skeinlike (D), Lewy body-like, and proximal axonal inclusions (E). Anterior horn cells in the cervical cord contain hyaline (F), granular inclusions (G; immunolabeled for ubiquitin), and a single vacuole (H). (H&E, original magnification ×800 to 1,000 before 66% reduction.)

In the cortex abnormalities were confined to the primary motor area. The Betz cells were not severely depleted and there was no obvious loss of layer V pyramidal neurons, although there were occasional chromatolytic Betz cells that reacted intensely with mAbs 147 and RT97 directed against phosphorylated neurofilaments (figure 3C). In some Betz cells and large pyramidal neurons there were granular ubiquitinated inclusions and occasional ubiquitinated Lewy bodylike inclusions. There was no abnormal staining with antibodies directed against SOD1, SOD2, or tau.

In the brainstem the pyramids showed mild pallor with LFB. There was a moderate loss of motor neurons in the hypoglossal nucleus with mild astrocytosis and occasional chromatolytic neurons. Skein-like and Lewy body-like ubiquitinated inclusions were present in many motor neurons, often in the presence of proximal axonal spheroids (figure 3, D and E). A small proportion of ubiquitinnegative hyaline inclusions also stained positively for phosphorylated neurofilaments.

In the cervical cord many motor neurons contained hyaline inclusions that failed to stain for phosphorylated neurofilaments (figure 3F). Other motor neurons showed intense perikaryal neurofilamentous labeling, and granular ubiquitinated inclusions were detected(figure 3G). A single, large vacuole was seen in an anterior horn cell (figure 3H). In the lumbar cord there were many more surviving neurons. Some were atrophic whereas others were swollen, chromatolytic, and contained hyaline inclusions with staining properties identical to those seen in the cervical cord. No Hirano or Bunina body inclusions were demonstrated. No abnormal staining was demonstrated for SOD1, SOD2, or tau.

Electron microscopy. Ultrastructural studies of the cervical cord revealed a large number of swollen neurons with accumulations of lipofuscin and 10- to 20-nm neurofilaments (figure 4).

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Figure 4. Electron microscopic findings of anterior horn cells in the cervical cord. Abundant lipofuscin granules are present in the perikaryal cytoplasm and a large filamentous inclusion abuts the nucleus (bar = 10 µm). The filaments are approximately 10 to 20 nm in diameter, a characteristic of neurofilaments (bar = 1 µm). Uranyl lead stain used.

Discussion. The clinical and neuropathologic findings in this patient are entirely consistent with the diagnosis of definite ALS according to the El Escorial criteria.⁹ We demonstrated all of the cardinal cellular and molecular abnormalities that typify sporadic ALS including corticospinal tract degeneration, motor neuron loss, astrogliosis, chromatolysis, axonal spheroids, lipofuscin pigment, hyaline inclusions, neurofilamentous accumulations, and ubiquitinated inclusions in the perikarya of motor neurons.^11-13 Lewy body-like inclusions (which are seen in 15% of sporadic ALS patients¹⁴) are also present. Bunina and Hirano bodies were absent but have been described previously in familial and sporadic patients.¹⁵

The presence of ubiquitinated inclusions in cortical pyramidal neurons and an isolated vacuole are somewhat atypical but both have been described in sporadic ALS.¹⁶ Interestingly transgenic animals overexpressing other SOD1 mutations also display motor neuron vacuolation.¹⁷ The striking chromatolytic changes and filamentous inclusions in the cervical and lumbar motor neurons of our patient are also seen in rapidly progressive sporadic ALS,¹⁸ suggesting that these features may reflect the rate of disease progression rather than a specific effect of mutant SOD1. That many of these filamentous accumulations were not labeled by antibodies directed against phosphorylated neurofilamentous epitopes suggests that altered phosphorylation does not always accompany perikaryal accumulation of neurofilaments, and thus may not be a prerequisite for altered neurofilament function in this disorder.¹⁹ Our results confirm that neurofilament and ubiquitin accumulation is a common end point of the biochemical disturbances triggered by either SOD1 mutation or unknown factors in sporadic ALS.

The three published pathologic reports^20-22 of patients with known SOD1 mutations vary considerably in their findings. Orrell et al.²⁰ described a patient with an isoleucine 113 > threonine (I1 13T) mutation, with an atypical clinical course of 20 years duration and atypical neuropathology. Neurons of the globus pallidus, substantia nigra, locus coeruleus, and inferior olivary nuclei displayed neurofibrillary tangles, some of which labeled with antibodies to neurofilament and tau but were ubiquitin negative. Rouleau et al.²¹ reported a patient with an identical I1 13T mutation and a more typical clinical course who demonstrated extensive neurofilamentous accumulation in motor neurons. These findings mirror the cellular pathology of our patient, but we have reported more detailed molecular studies of ubiquitin or SOD immunoreactivity. Takahashi et al.²² described a patient with an alanine 4 > threonine mutation who had an 18-month disease duration with prominent sensory symptoms, and posterior column and spinocerebellar involvement pathologically. They reported ubiquitin immunoreactive Lewy bodylike inclusions in the motor neurons of the spinal cord and brainstem similar to our patient. A report²³ on two patients with a mutation at the same codon, but different amino acid substitution, (alanine 4 > valine) was limited to describing SOD1 antibody immunohistochemistry and suggested a reduction of SOD1 labeling in affected neurons. Our patient certainly showed no accumulation of tau, SOD1, or SOD2, suggesting that toxic deposition of mutant SOD1 is not responsible for initiating the disease.

The principle biochemical action of SOD1 is to convert potentially toxic superoxide radicals into hydrogen peroxide, but the current weight of experimental evidence suggests a toxic gain of function for mutant SOD1 rather than inadequate oxygen radical scavenging.²⁴ One hypothesis is that mutations in the molecule might affect its tightly folded structure and so decrease the ability of SOD1 to bind copper ions and shield other proteins from its potent toxic effects. The importance of the copper binding domain is emphasized by its strict conservation over evolutionary development.²⁵ The rapid disease progression seen in affected members of our pedigree was associated with a histidine > glutamine mutation at the codon 48 copper binding site. Although this might suggest a role for abnormal copper binding in the etiology of ALS, patients with a neighboring mutation in the copper binding site at codon 46, histidine> arginine, had a mean disease duration of 17.3 years.²⁶ Given the heterogeneity of clinical characteristics seen in some pedigrees,^19,27 additional genetic or environmental factors must influence disease expression.

The neuropathologic features described in this report occur in both familial and sporadic ALS patients, and do not support claims that they are different pathologic entities. Mutations in the SOD1 gene cause neurofilamentous, hyaline and ubiquitinated inclusions in upper and lower motor neurons that are not due to toxic SOD1 accumulation.

Acknowledgments

We thank Drs. P.L. Lantos, J. Cavanagh, and A. Al-Chalabi for their helpful comments and criticism.