Linkage of recessive familial amyotrophic lateral sclerosis to chromosome 2q33-q35.

Amyotrophic lateral sclerosis (ALS) usually presents as a sporadic disorder of motor neurons. However, familial forms of ALS have been described--autosomal dominant forms (ALS1, ALS3), clinically indistinguishable from the sporadic form, and autosomal recessive forms with early onset and slower progression of symptoms (ALS2). To localize the gene for one of the autosomal recessive forms of ALS, we applied linkage analysis to a large inbred family from Tunisia. A lod score maximum of Zmax = 8.2 at theta = 0.00 was obtained with marker D2S72 located on chromosome 2q33-q35. The fine mapping of this region suggested that the ALS2 locus lies in the 8 cM segment flanked by D2S155 and D2S115.

A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2.

Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.

Purification and partial characterization of the myelin-associated glycoprotein from adult rat brain.

The myelin-associated glycoprotein was purified from rat central nervous system myelin by selective extraction with lithium diiodosalicylate-phenol followed by gel filtration on a column of Sepharose CL-6B. Amino acid analysis of the purified glycoprotein revealed an excess of acidic over basic amino acids and a relatively high content of nonpolar residues. On the basis of weight, the molecule is about one-third carbohydrate consisting of 5% fucose, 23% mannose, 20% galactose, 34% N-acetylglucosamine, and 18% N-acetylneuraminic acid.

Intramuscular grafts of myoblasts genetically modified to secrete glial cell line-derived neurotrophic factor prevent motoneuron loss and disease progression in a mouse model of familial amyotrophic lateral sclerosis.

Effects of ex vivo GDNF gene delivery on the degeneration of motoneurons were studied in the G1H transgenic mouse model of familial ALS carrying a human superoxide dismutase (SOD1) with a Gly93Ala mutation (Gurney et al., 1994). Retroviral vectors were made to produce human GDNF or E. coli beta-galactosidase (beta-Gal) by transient transfection of the Phoenix cell line and used to infect primary mouse myoblasts. In 6-week-old G1H mice, 50,000 myoblasts per muscle were injected bilaterally into two hindlimb muscles. Untreated G1H and wild-type mice served as additional controls. At 17 weeks of age, 1 week before sacrifice, these muscles were injected with fluorogold (FG) to retrogradely label spinal motoneurons that maintained axonal projections to the muscles. There were significantly more large FG-labeled alpha motoneurons at 18 weeks in GDNF-treated G1H mice than in untreated and beta-Gal-treated G1H mice. A morphometric study of motoneuron size distribution showed that GDNF shifted the size distribution of motoneurons toward larger cells compared with control G1H mice, although the average size and number of large motoneurons in GDNF-treated mice were less than that in wild-type mice. GDNF also prolonged the onset of disease, delayed the deterioration of performance in tests of motor behavior, and slowed muscle atrophy. Quantitative, real-time RT-PCR and PCR showed persistence of transgene mRNA and DNA in muscle for up to 12 weeks postgrafting. These observations demonstrate that ex vivo GDNF gene therapy in a mouse model of FALS promotes the survival of functional motoneurons, suggesting that a similar approach might delay the progression of neurodegeneration in ALS.

Aggregation of mutant Cu/Zn superoxide dismutase proteins in a culture model of ALS.

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene underlie some familial cases of amyotrophic lateral sclerosis (FALS), a neurodegenerative disorder characterized by loss of cortical, brainstem, and spinal motor neurons. To investigate the mechanisms responsible for the toxicity of mutant enzyme, SOD-1 cDNAs bearing mutations found in FALS patients (mSOD) were expressed in cultured spinal motor neurons, dorsal root ganglion (DRG) and hippocampal neurons. Many features of motor neuron disease seen in humans with FALS and in transgenic mouse models were reproduced, including preferential susceptibility of motor neurons to toxicity of mSOD. Abnormal cytoplasmic aggregation of mSOD protein was observed in mSOD-expressing motor neurons, but never in neurons expressing SODwt enzyme, and was followed by evidence of apoptotic cell death. Such aggregates were not observed in nonvulnerable neuronal populations expressing mSOD (DRG or hippocampal neurons). Aggregation of SOD-1 may contribute significantly to the death of motor neurons expressing mutations associated with FALS-1 and the mechanisms leading to aggregation may pertain to the specific vulnerability of motor neurons in this disease.

Polymorphism in the multi-phosphorylation domain of the human neurofilament heavy-subunit-encoding gene.

The C-terminal region of the human neurofilament heavy subunit (NEFH) contains a unique functional domain consisting of 43 repeat motifs of the amino acids (aa) Lys-Ser-Pro (KSP) with either 3- or 5-aa spacers in between. Past studies have demonstrated that the serine in these KSP motifs can be phosphorylated, resulting in heavy phosphorylation of this domain. Recent studies provide strong evidence for a role of neurofilament phosphorylation in the establishment of neurofilament density and axonal caliber. Since it may be hypothesized that mutations in the phosphorylated region are a basis for neuropathological conditions, and since regions of the human genome containing repeat motifs have been demonstrated to be significantly polymorphic, we undertook to identify and characterize polymorphism in this region of the human NEFH gene. We were able to identify an allelic variant of a slightly larger molecular size, containing an additional KSP phosphorylation motif. The variant form of NEFH displays Mendelian inheritance and has a widespread population distribution. In addition, we also identified a point mutation in one individual which would result in a Pro-->Leu substitution in one of the repeat motifs.

Clinical implications of the genetics of ALS and other motor neuron diseases.

Genetic mutations have been identified in the major motor neuron diseases, including ALS, spinal muscular atrophy, bulbospinal muscular atrophy (Kennedy's disease), the hereditary spastic paraplegias, and rarer conditions such as GM2 gangliosidosis (hexosaminidase A deficiency). These include mutations in the SOD1 gene, deletions of the telomeric copy of the SMN gene, expansions of the trinucleotide repeat region in the first exon of the androgen receptor gene, other rare mutations, and diseases where linkage has been established but the gene not identified. Identification of one of these genetic abnormalities will allow specific diagnosis in patients. Because cure is not yet available, presymptomatic testing is seldom indicated; in such cases, careful counseling is appropriate.

Analysis of the KSP repeat of the neurofilament heavy subunit in familiar amyotrophic lateral sclerosis.

We examined the neurofilament heavy subunit (NEFH) as a candidate gene for familial amyotrophic lateral sclerosis. We screened the KSP repeat region of the NEFH gene in 117 unrelated individuals who inherited familial amyotrophic lateral sclerosis as an autosomal trait but who do not have the mutation in the SOD1 locus, and we found no variants in any individual. We conclude that the motor neuron degeneration observed in non-SOD1 familial amyotrophic lateral sclerosis is not due to mutations in the KSP repeat of the NEFH gene.

Definitive molecular diagnosis of facioscapulohumeral dystrophy.

OBJECTIVE: To establish the usefulness of a molecular diagnostic protocol for the autosomal dominant disease facioscapulohumeral dystrophy (FSHD). BACKGROUND: The genetic defect underlying the majority of cases is a deletion on chromosome 4q35 that is not associated with the coding sequence of any known gene. Molecular diagnosis of FSHD involves the visualization of this deletion as a "small" EcoRI restriction fragment. However, molecular diagnostics are complicated because of the homology of the telomeric regions of chromosomes 4q and 10q; the homologous 10q26 EcoRI fragments are also detected, and can fall into the size range considered to be diagnostic for FSHD. It is therefore important to distinguish the 4q35 and 10q26 EcoRI fragments, taking advantage of the presence of additional restriction sites (BlnI) in the alleles of chromosome 10q origin. METHODS: Paired digests of genomic DNA (EcoRI only and EcoRI/BlnI double digest), followed by pulsed field gel electrophoresis (PFGE), were used to establish the molecular diagnosis of FSHD in 82 unrelated index cases (46 familial, 24 proven sporadic with de novo mutations, and 12 with uncertain family history). RESULTS: In all cases fulfilling FSHD diagnostic criteria, a 4q35 EcoRI allele size of < or = 38 kb was present. The smallest 4q35 EcoRI allele in 205 normal control subjects was 41 kb. EcoRI alleles < or = 38 kb of chromosome 10q26 origin were present in 11.2% of this control group. In problematic cases, it was possible to resolve the diagnostic question. CONCLUSIONS: The combination of double digestion with EcoRI and BlnI followed by PFGE is the most reliable molecular protocol for distinguishing patients with FSHD.

Spinobulbar muscular atrophy can mimic ALS: the importance of genetic testing in male patients with atypical ALS.

The clinical presentation of amyotrophic lateral sclerosis (ALS) is variable and overlaps with that of other motor neuron diseases such as spinobulbar muscular atrophy (SBMA; Kennedy disease). With the identification of disease-specific mutations such as the CAG repeat expansion in the androgen receptor in SBMA, an accurate molecular diagnosis can be made in some patients with motor neuron disease. To determine the extent of misdiagnosis of ALS we screened 147 male ALS patients and 100 unrelated male patients from 100 familial ALS (FALS) kindreds for the presence of the SBMA mutation using polymerase chain reaction methods. We show that ALS was clinically misdiagnosed in 2% of sporadic cases and in two of the 100 FALS kindreds. This study underscores the difficulty in distinguishing SBMA from ALS clinically, particularly in patients who lack the classic signs of each disease.

Hereditary spastic paraplegia: advances in genetic research. Hereditary Spastic Paraplegia Working group.

Hereditary spastic paraplegia (HSP) is a diverse group of inherited disorders characterized by progressive lower-extremity spasticity and weakness. Insight into the genetic basis of these disorders is expanding rapidly. Uncomplicated autosomal dominant, autosomal recessive, and X-linked HSP are genetically heterogeneous: different genes cause clinically indistinguishable disorders. A locus for autosomal recessive HSP is on chromosome 8q. Loci for autosomal dominant HSP have been identified on chromosomes 2p, 14q, and 15q. One locus (Xq22) has been identified for X-linked, uncomplicated HSP and shown to be due to a proteolipoprotein gene mutation in one family. The existence of HSP families for whom these loci are excluded indicates the existence of additional, as yet unidentified HSP loci. There is marked clinical similarity among HSP families linked to each of these loci, suggesting that gene products from HSP loci may participate in a common biochemical cascade, which, if disturbed, results in axonal degeneration that is maximal at the ends of the longest CNS axons. Identifying the single gene defects that cause HSPs distal axonopathy may provide insight into factors responsible for development and maintenance of axonal integrity. We review clinical, genetic, and pathologic features of HSP and present differential diagnosis and diagnostic criteria of this important group of disorders. We discuss polymorphic microsatellite markers useful for genetic linkage analysis and genetic counseling in HSP.

The glycinamide ribonucleotide transformylase (GART) gene is not responsible for familial amyotrophic lateral sclerosis.

Though the nature of the underlying metabolic defect which leads to amyotrophic lateral sclerosis (ALS) remains obscure, certain biochemical anomalies have been found, such as, reduced RNA content in ALS motor neurons. Recently, a gene causing the familial form of ALS (FALS) has been assigned to an interval of approximately 10 cM including the locus D21S58 on chromosome 21q22.1. This region includes the GART gene which encodes an enzyme catalyzing three steps in the de novo biosynthesis of purine nucleotides which are precursors for RNA. A defect of this gene might result in reduced RNA production and predispose to premature death of motor neurons. In order to test GART as a candidate we developed two highly informative DNA markers in this region and carried out linkage analyses for FALS. GART is excluded as a candidate for FALS.

Androgen receptor gene polymorphisms in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is more common in men than in women (male to female ratio of approximately 2:1), suggesting a role for a sex-linked factor in the disease. The recent identification of a mutation of the androgen receptor gene in Kennedy's disease or X-linked bulbospinal neuronopathy, a rare form of progressive lower motor neurone degeneration, also associated with clinical signs of androgen insensitivity, raises the possibility that androgen function may be disturbed in other motor neurone disorders, including ALS. The Kennedy's disease mutation consists of an increased size of a highly polymorphic CAG repeat sequence in the first exon of the androgen receptor gene, coding for a polyglutamine tract. We have analysed this CAG repeat sequence in a large number of patients with typical sporadic ALS and in normal controls, in order to test the hypothesis that this polymorphism of the androgen receptor gene may influence susceptibility for ALS. We report that the distribution of alleles relating to the size of the CAG repeat sequence of the androgen receptor gene is similar in ALS and controls, indicating that polymorphisms of the CAG repeat sequence of the androgen receptor gene play a limited role, if any, in susceptibility to ALS.

Absence of mutations in the Mn superoxide dismutase or catalase genes in familial amyotrophic lateral sclerosis.

Familial amyotrophic lateral sclerosis (FALS) is an autosomal dominant, adult onset, neurological disorder caused by the degeneration of motor neurons of the cortex, brainstem and spinal cord. Recently, the defective gene in some FALS families was identified as the Cu/Zn superoxide dismutase (SOD1) gene. However, SOD1 mutations are present in approximately 20% of patients with FALS. We have tested the genes of two more free radical detoxifying enzymes, Mn superoxide dismutase (SOD2) and catalase by single strand conformation analysis (SSCA) for mutations in the remaining FALS cases. No mutations were found in the catalase enzyme in 73 unrelated FALS cases; mutations were not detected in the 66% of the SOD2 gene analyzed. FALS does not appear to be caused by mutations in the SOD2 nor the catalase genes.

Culture models of neurodegenerative disease.

In order to investigate how mutant SOD1 protein or environmental exogenous stressors lead to the death of motor neurons, we have established several in vitro model systems. We describe some features of the various models in order to demonstrate the advantages and shortcomings of each system.

Nitrotyrosination contributes minimally to toxicity of mutant SOD1 associated with ALS.

Enhanced production of nitrotyrosine and subsequent protein nitration has been proposed as the mechanism by which mutant SOD1 causes death of motor neurons in a familial form of amyotrophic lateral sclerosis (FALS-1). We have tested this hypothesis in a primary culture model in which mutant human SOD1 was expressed in motor neurons of dissociated spinal cord cultures. Preventing formation of nitrotyrosine by inhibiting nitric oxide synthase rescued cultured motor neurons from excitotoxic death induced by adding glutamate to the culture medium, but failed to significantly delay death of motor neurons expressing the G93A mutant SOD1. The results do not support generation of nitrotyrosine being the predominant lethal gain of function conferred by mutations in SOD1.

The Cu/Zn superoxide dismutase gene in ALS and parkinsonism-dementia of Guam.

Guam is one of three endemic foci whose indigenous (Chamorro) people have an unusually high incidence of fatal neurodegenerative disorders, amyotrophic lateral sclerosis (ALS) and Parkinsonism-dementia (PD). Recently, mutations in the Cu/Zn superoxide dismutase (SOD-1) gene have been identified in some familial cases of ALS. To investigate if mutations in the SOD-1 gene are also involved in the pathogenesis of ALS and PD of Guam, we analyzed the SOD-1 gene in Chamorros. No mutations were found in Chamorros with ALS or PD, indicating that mutations in the SOD-1 gene do not underlie the high-incidence neurodegenerative disorders of Guam.

Differential expression of neurofilament subunits in the developing corpus callosum.

In the course of development, corticocortical axons seem to first appear in a labile state from which they either mature into a stable state or are eliminated. These state transitions may be related to cytoskeletal modifications. By immunohistochemistry and immunobiochemistry we found that, in the corpus callosum of the cat, the heavy (200 kDa) subunit of neurofilaments (NF) becomes progressively more visible during the first postnatal month. This aspect of cytoskeletal maturation parallels the developmental loss of callosal axons, i.e. probably the stabilization of the axons which are not eliminated. A similar maturation of the heavy subunit was observed in the visual cortical areas 17 and 18. The medium (150 kDa) and to a lesser extent the light (70 kDa) NF subunits are already present a few days after birth.

Cultured glial cells are resistant to the effects of motor neurone disease-associated SOD1 mutations.

Free radical damage has been implicated in the pathophysiology of motor neurone disease (MND); mutations have been identified in the gene encoding Cu/Zn superoxide dismutase (SOD1). There is evidence that glial cell dysfunction may contribute to motor neurone injury, but the exact role of glial cells in MND has yet to be established. The aim of this study was to determine whether expression of mutant SOD1 affects the response of glia to oxidative stress. Stable C6 glioma cells expressing mutant SOD1 and cortical astrocyte cultures from G93A-SOD1 transgenic mice were exposed to: xanthine/xanthine oxidase; hydrogen peroxide; A23187 and 3-morpholinosydonimine. Cell viability was measured using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Neither C6 glioma cells nor cortical astrocytes expressing mutant SOD1 were more susceptible to any of the free radical generating systems compared to control cells. These results suggest that astrocytes are resistant to the toxic effects of mutant SOD1 widely reported for neuronal cells.

Cells from individuals with SOD-1 associated familial amyotrophic lateral sclerosis do not have an increased susceptibility to radiation-induced free radical production or DNA damage.

Oxidative stress may play a role in the pathogenesis of familial amyotrophic lateral sclerosis (FALS). Superoxide dismutases (SODs) are enzymes that can influence free radical processes in irradiated cells and there is some evidence that manipulation of SODs can affect survival of cells after radiation treatments. SOD-1 associated FALS mutants may have an altered radiation response due to an enhanced generation of hydroxyl radicals or a compromised ability to neutralize free radicals. We have investigated the ability of the lymphoblastoid cell lines from FALS patients with SOD-1 gene mutations, patients with sporadic ALS and controls to handle oxidative stress induced by ionising radiation by measuring levels of intracellular reactive oxygen species and production of DNA double-strand breaks. Levels of reactive oxygen species, expressed as the slope of the relative fluorescence of a radical-reactive fluorochrome, in the cells from familial ALS patients with SOD-1 gene mutations (2.14+/-1.06 Gy(-1)) and patients with sporadic ALS (1.38+/-0.21 Gy(-1)) were not significantly different from the controls (1.54+/-0.39 Gy(-1)). No significant difference was observed in the production of DNA double-strand breaks between three groups. The ability of lymphoblastoid cells from FALS patients with SOD-1 gene mutations to scavenge radiation-induced free radicals is not compromised nor is their ability to protect DNA damage induced by ionising radiation.