Angela Romano

Contribution of major amyotrophic lateral sclerosis genes to the etiology of sporadic disease

Objectives: To quantify the overall contribution of mutations in the currently known amyotrophic lateral sclerosis (ALS) genes in a large cohort of sporadic patients and to make genotype–phenotype correlations. Methods: Screening for SOD1, TARDBP, FUS, ANG, ATXN2, OPTN, and C9ORF72 was carried out in 480 consecutive patients with sporadic ALS (SALS) and in 48 familial ALS (FALS) index patients admitted to a single Italian referral center. Results: Mutations were detected in 53 patients, with a cumulative frequency of 11. Seven of them were novel. The highest frequencies of positive cases were obtained in TARDBP (2.7%), C9ORF72 (2.5%), and SOD1 (2.1%). The overall group of mutated patients was indistinguishable from that without mutations as no significant differences were observed with regard to age and site of onset, frequency of clinical phenotypes, and survival. However, by separately evaluating genotype–phenotype correlation in single genes, clinical differences were observed among different genes. Duration of disease was significantly shorter in patients harboring the C9ORF72 expansion and longer in the SOD1 group. A high frequency of predominant upper motor neuron phenotype was observed among patients with TARDBP mutations. Two patients, 1 with C9ORF72 and 1 with SOD1 mutation, had concurrent ANG mutations. Mutations were detected in 43.7% of patients with FALS. Conclusions: A considerable proportion of patients with SALS harbored mutations in major ALS genes. This result has relevant implications in clinical practice, namely in genetic counseling. The detection of double mutations in 2 patients raises the hypothesis that multiple mutations model may explain genetic architecture of SALS. Neurology® 2012;79:66–72

Mutations in the 3′ untranslated region of FUS causing FUS overexpression are associated with amyotrophic lateral sclerosis

Mutations in the gene encoding fused-in-sarcoma (FUS) have been identified in a subset of patients with sporadic and familial amyotrophic lateral sclerosis (ALS). Variants in the 3 untranslated region (3UTR) of FUS have also been reported in ALS patients, but their pathogenic role has not been assessed. We sequenced the whole 3UTR of FUS in 420 ALS patients who were negative for mutations in the currently known ALS genes and in 480 ethnically matched controls. We detected four 3UTR variants (c.*48 G>A, c.*59 G>A, c.*108 C>T and c.*110 G>A) in four sporadic and in one familial ALS patients compared with none in controls (P = 0.02).We investigated whether these variants impaired FUS expression in primary fibroblast cultures from three patients harbouring the c.*59 G>A, c.*108 C>T and c.*110 G>A variants, respectively. The pattern of FUS expression was also investigated in fibroblasts from one ALS patient with FUS R521C mutation, in two ALS patients without mutations in the known ALS genes and in four control individuals. By immunostaining and immunoblotting, large amounts of FUS were observed in both the cytoplasm and nuclei of mutant 3UTR FUS fibroblasts. In FUS R521C mutant fibroblasts, we observed a slight increase of FUS in the cytoplasm associated with a remarkable loss of detection in nuclei. Our findings show that mutations in 3UTR of FUS are overrepresented in ALS patients and result into translation de-regulation of FUS. Overexpression and mislocalization of wild-type FUS likely contribute to ALS pathogenesis in these cases.

Classification of familial amyotrophic lateral sclerosis by family history: effects on frequency of genes mutation

Objective To classify familial amyotrophic lateral sclerosis (FALS) on the base of family history, and to determine whether frequency of mutations in major amyotrophic lateral sclerosis (ALS) genes varies in different FALS categories. Methods Included in the study are 53 FALS families. Patients were classified as definite, probable and possible FALS, according to recently proposed criteria. Seven ALS-associated genes, including SOD1, TARDBP, FUS, ANG, ATXN2, OPTN and C9ORF72, were analysed. Results Thirteen patients (24.5%) were included in the definite group. The great majority of our FALS cases (40/53, 75.5%) were families with only two affected relatives; of these, 31 (58.5%) were included in the probable, and 9 (17%) in the possible FALS categories. The percentage of mutations was 61.5% in definite, 41.9% in probable and 11.1% in possible FALS. With respect to probable FALS, if cases with parent-to-child transmission of the disease were considered separately, the mutational load increased to 61.5%, as observed in definite FALS. Conclusions Our findings provide evidence that frequency of mutations in currently known ALS genes varies widely among different FALS categories. Families with only two affected relatives have heterogeneous genetic components, the chance to detect mutations being higher in cases with parent-to-child transmission.

Primary fibroblasts cultures reveal TDP-43 abnormalities in amyotrophic lateral sclerosis patients with and without SOD1 mutations

TAR DNA-binding protein 43 (TDP-43) is a major component of the pathologic inclusions observed in thexa0motor neurons of amyotrophic lateral sclerosis (ALS) patients. We examined TDP-43 expression in primary fibroblasts cultures from 22 ALS patients, including cases with SOD1 (nxa0= 4), TARDBP (nxa0= 4), FUS (nxa0= 2), and C9ORF72 (nxa0= 3) mutations and 9 patients without genetic defect. By using a phosphorylation-independent antibody, 15 patients showed notable alterations of TDP-43 level in the nuclear or cytoplasmic compartments. In particular, a marked accumulation of TDP-43 was observed in the cytoplasm of all cases with C9ORF72 and TARDBP mutations, 1 patient with FUS mutation and 3 patients without genetic defect. Patients with SOD1 mutations revealed a significant reduction of TDP-43 in the nuclei without cytoplasmic mislocalization. These changes were associated with the presence of truncated and phosphorylated TDP-43 species. Our results show that fibroblasts recapitulate some of hallmark TDP-43 abnormalities observed in neuronal cells. The reduction of full-length TDP-43 level in mutant SOD1 cells indicates that at least some SOD1 mutations alter TDP-43 metabolism.

Clinical, neurophysiological and pathological findings of HNPP patients with 17p12 deletion: a single-centre experience

BACKGROUNDnClassic clinical manifestations of HNPP are characterized by recurrent painless mononeuropathies, but a minority of patients present with an atypical clinical pattern, including CMT-like neuropathy, acute or chronic inflammatory demyelinating neuropathy-like polyneuropathy, and carpal tunnel syndrome. Electrophysiological examination plays a central role in the diagnosis of HNPP, disclosing a non-uniform conduction slowing, more pronounced at entrapment sites.nnnPATIENTS AND METHODSnWe report clinical, electrophysiological and pathological findings from 73 patients with HNPP, coming from 53 unrelated families, followed at our Institute of Neurology over a 20-year period.nnnRESULTSnTypical presentation with recurrent multiple mononeuropathies was observed in 28/64 (44%) patients. In the remaining 36/64 (56%), we observed an atypical clinical presentation, characterized by generalized weakness and cramps, chronic ulnar neuropathy, carpal tunnel syndrome, chronic sensory polyneuropathy, Guillain-Barrè-like presentation, and CMT-like presentation. Nine patients were asymptomatic for neuropathic symptoms. Nerve conduction studies showed in all cases a sensori-motor demyelinating polyneuropathy with conduction abnormalities preferentially localized at common entrapment sites. When performed, sural nerve biopsy disclosed the focal thickening of the myelin sheath in all patients.nnnCONCLUSIONSnAbout half of the patients with HNPP from our cohort showed an atypical clinical presentation. Neurophysiological examination represents the main tool for a proper diagnosis.

Ultrasound evaluation in transthyretin-related amyloid neuropathy

Introduction: Familial amyloid polyneuropathy is a rare condition caused by mutations of the transthyretin gene (TTR). We assessed the pattern of nerve ultrasound (US) abnormalities in patients with TTR‐related neuropathy. Methods: Seven patients with TTR‐related neuropathy (TTR‐N) and 5 asymptomatic TTR‐mutation carriers (TTR‐C) underwent neurological examination, nerve conduction studies, and US evaluation. Results: Multifocal US abnormalities were identified in 6 of 7 TTR‐N patients. A single patient with only a mild sensory polyneuropathy had normal nerves on US evaluation. In the TTR‐C, we only detected an enlarged ulnar nerve at the elbow. Interestingly, disease severity correlated with number of nerves affected on US evaluation. Conclusions: No specific pattern of US abnormalities was identified in this cohort. However, in TTR‐related amyloid neuropathy, US may be a helpful tool in monitoring disease progression, and/or clinical response to pharmacological treatment. Muscle Nerve 50: 372–376, 2014

Sural nerve pathology in ALS patients: a single-centre experience

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease of upper and lower motor neurons. Sensory involvement is thought not to be a feature of ALS. We reviewed 17 cases of sural nerve biopsies performed in a large cohort of ALS patients referred to our centre over a 23-year period. More than two-third of biopsies revealed a variable degree of axonal loss. In one case, pathological findings suggested the concomitant presence of an inherited neuropathy, subsequently confirmed by genetic evaluation. In another case, pathological and neurographic data were similar to those of an inflammatory demyelinating neuropathy, but the clinical course corroborated the diagnosis of ALS. Our data confirm that sensory nerve involvement may be found in ALS patients. This finding should prompt physicians to carefully investigate a possible alternative diagnosis, but does not exclude the possibility that the patient may have ALS.

AL amyloid neuropathy mimicking a chronic inflammatory demyelinating polyneuropathy

09 December 2011 A 73-year-old man was admitted to our Department with a 12-month history of progressive weakness and sensory disturbances in all four limbs. He was the product of non-consanguineous healthy parents and there were no neuropsychiatric or other significant medical illnesses in his family history. Neurological examination revealed diffuse weakness of proximal and distal muscles of the upper and lower limbs, graded 4 on the Medical Research Council scale, paraesthesias and hypoesthesias with stocking-and-glove distribution and the absence of tendon reflexes. Extensive laboratory studies proved normal, with no signs of ongoing infection, malignancy, malabsorption, systemic autoimmune or metabolic disorders. Serum immunofixation electrophoresis revealed the presence of a small IgG λ monoclonal component. Nerve conduction studies revealed reduced amplitude of sensory nerve action potentials (SNAPs) and of compound muscle action potentials (CMAPs) in all tested nerves with low-borderline nerve conduction velocities (NCV) and prolonged distal motor latencies (DML) and mean F waves (Table I). Electromyography showed in all four limbs a neurogenic pattern: denervation (fibrillation potentials and positive sharp waves) and rapidly firing motor unit potentials. Considering clinical and neurophysiological findings, a chronic inflammatory demyelinating polyneuropathy (CIDP) was suspected and, after obtaining the patient’s informed consent, right sural nerve biopsy was performed to confirm the diagnosis. HE staining documented a severe loss of fibers (Figure 1A). Around a single epineural artery, the presence of eosinophilic proteinaceous material suggested amyloid deposition (Figure 1B), which was confirmed by Congo red staining (Figure 1C); this also revealed minute amyloid deposits around epineural arterioles (Figure 1D), in the endoneurim (Figure 1E) and around endoneural capillaries (Figure 1F). Direct immunofluorescence on cryostat sections using anti-λ light-chain sera documented an AL amyloid neuropathy (Figure 1G–1I). Urine immunofixation demonstrated a λ-type Bence-Jones protein; bone marrow needle aspiration and biopsy showed a high percentage of plasma cells (around 20%); serum-free λ light chains were increased (195.50 mg/l; normal value 5.71– 26.30) with a reduced k/λ ratio (0.05; normal value 0.26– 1.65). Cardiac imaging with echocardiography and magnetic resonance revealed severe left ventricular hypertrophy with gross diastolic dysfunction, suggestive of an infiltrative cardiac disease. NT-proBNP (brain natriuretic peptide) was elevated (47069 pg/ml; normal value <150). Direct sequencing of transthyretin (TTR) exons 1–4 and related exon-intron boundaries, performed after informed consent, was normal and excluded familial amyloid polyneuropathy. A final diagnosis of AL amyloidosis was formulated. Unfortunately, 3 weeks later, the patient suffered sudden death, before starting chemotherapy. LETTER TO THE EDITOR

Charcot-Marie-Tooth type 2 and distal hereditary motor neuropathy: Clinical, neurophysiological and genetic findings from a single-centre experience

OBJECTIVESnCMT is a group of heterogeneous motor and sensory neuropathies divided into demyelinating (CMT1) and axonal forms (CMT2). Distal Hereditary Motor Neuropathy (dHMN) is a motor neuropathy/neuronopathy which resembles CMT. Final genetic diagnosis is poor in CMT2 and in dHMN when compared with CMT1. Our aim is to report clinical, neurophysiological and genetic findings in a cohort of patients with axonal inherited neuropathies.nnnPATIENTS AND METHODSnWe report clinical, neurophysiological and genetic findings from 45 patients with CMT2 or dHMN, coming from 39 unrelated families, observed in our Institute of Neurology over a 20-year period.nnnRESULTSnClinical and electrophysiological examinations showed that 38 patients had CMT2 and 7 patients presented dHMN. Extensive genetic evaluation showed 6 mutations in MFN2, 4 mutations in HSPB1, 2 mutations in BSCL2, 3 mutations in GJB1, 1 mutation in MPZ.nnnCONCLUSIONnSince next-generation sequencing will not be easily accessible, epidemiological data and clinical phenotyping remain the best strategy for clinicians to reach a correct genetic diagnosis in CMT2 and dHMN patients.

A novel compound heterozygous ALS2 mutation in two Italian siblings with juvenile amyotrophic lateral sclerosis

ISSN 2167-8421 print/ISSN 2167-9223 online