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The objective of the study is to provide age-related normative values for dorsal sural nerve (DSN) and to analyse its application during follow-up of hereditary transthyretin amyloidosis (ATTRv) pre-symptomatic subjects. We consecutively recruited ATTRv pre-symptomatic carriers in which clinical examination, cardiological evaluation, and nerve conduction studies of the sural nerve and DSN were performed. To provide normative data of DSN, neurophysiologic parameters from healthy controls referred to our service were entered into linear regression analyses to check the relative influence of age and height. A correction grid was then derived. We collected 231 healthy subjects: the mean DSN sensory nerve action potential (SNAP) amplitude was 9.99 ± 5.48 µV; the mean conduction velocity was 49.01 ± 5.31 m/s. Significant correlations were found between age and height with DSN SNAP amplitude. Fifteen ATTRv pre-symptomatic carriers were examined. Sural nerve NCS were normal in 12/15 and revealed low/borderline values in three subjects. Considering our correction grid, we found an abnormal DNS amplitude in 9/15 subjects and low/borderline values in 2/15. In ATTRv, early detection of peripheral nerve damage is crucial to start a disease-modifying treatment. DSN may be easily and reliably included in the routine neurophysiological follow-up of ATTRv pre-symptomatic subjects.
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Objective: The aim of our study was to evaluate the long-term efficacy and safety of mexiletine in 112 patients affected by genetically confirmed non-dystrophic myotonias. The study was performed at the Neurophysiologic Division of Fondazione Policlinico Universitario A. Gemelli Istituto di Ricerca e Cura a Carattere Scientifico (IRCCS), Rome and the Children's Hospital Bambino Gesù, Rome. Methods: The treatment was accepted by 59 patients according to clinical severity, individual needs, and concerns about a chronic medication. Forty-three patients were affected by recessive congenita myotonia, 11 by sodium channel myotonia, and five by dominant congenital myotonia. They underwent clinical examination before and after starting therapy, and Electromyography (EMG). A number of recessive myotonia patients underwent a protocol of repetitive nerve stimulations, for detecting and quantifying the transitory weakness, and a modified version of the Timed Up and Go test, to document and quantify the gait impairment. Results: Treatment duration ranged from 1 month to 20 years and the daily dosages in adults ranged between 200 and 600 mg. No patient developed cardiac arrhythmias causing drug discontinuation. Mexiletine was suspended in 13 cases (22%); in three patients, affected by Sodium Channel myotonia, because flecainide showed better efficacy; in one patient because of a gastric cancer antecedent treatment; in four patients because of untreatable dyspepsia; and five patients considered the treatment not necessary. Conclusions: In our experience, mexiletine is very useful and not expensive. We did not observe any hazarding cardiac arrhythmias. Dyspepsia was the most frequent dose-limiting side effect.
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Sodium channel myotonia is a form of muscle channelopathy due to mutations that affect the Nav1.4 channel. We describe seven families with a series of symptoms ranging from asymptomatic to clearly myotonic signs that have in common two novel mutations, p.Ile215Thr and p.Gly241Val, in the first domain of the Nav1.4 channel. The families described have been clinically and genetically evaluated. p.Ile215Thr and p.Gly241Val lie, respectively, on extracellular and intracellular loops of the first domain of the Nav1.4 channel. We assessed that the p.Ile215Thr mutation can be related to a founder effect in people from Southern Italy. Electrophysiological evaluation of the channel function showed that the voltage dependence of the activation for both the mutant channels was significantly shifted toward hyperpolarized potentials (Ile215Thr: -28.6 ± 1.5 mV and Gly241Val: -30.2 ± 1.3 mV vs. WT: -18.5 ± 1.3 mV). The slow inactivation was also significantly affected, whereas fast inactivation showed a different behavior in the two mutants. We characterized two novel mutations of the SCN4A gene expanding the knowledge about genetics of mild forms of myotonia, and we present, to our knowledge, the first homozygous patient with sodium channel myotonia.
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Sodium channel myotonia and paramyotonia congenita are caused by gain-of-function mutations in the skeletal muscle voltage-gated sodium channel hNav1.4. The first-line drug is the sodium channel blocker mexiletine; however, some patients show side effects or limited responses. We previously showed that two hNav1.4 mutations, p.G1306E and p.P1158L, reduce mexiletine potency in vitro, whereas another sodium channel blocker, flecainide, is less sensitive to mutation-induced gating defects. This observation was successfully translated to p.G1306E and p.P1158L carriers. Thus, the aim of this study was to perform a pharmacological characterization of myotonic Nav1.4 mutations clustered near the fast inactivation gate of the channel. We chose seven mutations (p.V1293I, p.N1297S, p.N1297K, p.F1298C, p.G1306E, p.I1310N, and p.T1313M) from the database of Italian and French networks for muscle channelopathies. Recombinant hNav1.4 mutants were expressed in HEK293T cells for functional and pharmacological characterization using the patch-clamp technique. All the studied mutations impair the kinetics and/or voltage dependence of fast inactivation, which is likely the main mechanism responsible for myotonia. The severity of myotonia is well-correlated to the enhancement of window currents generated by the intersection of the activation and fast inactivation voltage dependence. Five of the six mutants displaying a significant positive shift of fast inactivation voltage dependence reduced mexiletine inhibition in an experimental condition mimicking myotonia. In contrast, none of the mutations impairs flecainide block nor does p.T1313M impair propafenone block, indicating that class Ic antiarrhythmics may constitute a valuable alternative. Our study suggests that mutation-driven therapy would be beneficial to myotonic patients, greatly improving their quality of life.
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Transtornos Miotônicos/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Células HEK293 , Humanos , Recém-Nascido , Ativação do Canal Iônico , Masculino , Pessoa de Meia-Idade , Mutação , Transtornos Miotônicos/tratamento farmacológico , Adulto JovemRESUMO
Myotonia congenita (MC) is a skeletal-muscle hyperexcitability disorder caused by loss-of-function mutations in the ClC-1 chloride channel. Mutations are scattered over the entire sequence of the channel protein, with more than 30 mutations located in the poorly characterized cytosolic C-terminal domain. In this study, we characterized, through patch clamp, seven ClC-1 mutations identified in patients affected by MC of various severities and located in the C-terminal region. The p.Val829Met, p.Thr832Ile, p.Val851Met, p.Gly859Val, and p.Leu861Pro mutations reside in the CBS2 domain, while p.Pro883Thr and p.Val947Glu are in the C-terminal peptide. We showed that the functional properties of mutant channels correlated with the clinical phenotypes of affected individuals. In addition, we defined clusters of ClC-1 mutations within CBS2 and C-terminal peptide subdomains that share the same functional defect: mutations between 829 and 835 residues and in residue 883 induced an alteration of voltage dependence, mutations between 851 and 859 residues, and in residue 947 induced a reduction of chloride currents, whereas mutations on 861 residue showed no obvious change in ClC-1 function. This study improves our understanding of the mechanisms underlying MC, sheds light on the role of the C-terminal region in ClC-1 function, and provides information to develop new antimyotonic drugs.
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Canais de Cloreto/genética , Análise Mutacional de DNA , Mutação/genética , Miotonia Congênita/genética , Adolescente , Adulto , Aminoácidos/genética , Feminino , Humanos , Ativação do Canal Iônico/genética , Masculino , Pessoa de Meia-Idade , Miotonia Congênita/tratamento farmacológico , Miotonia Congênita/fisiopatologia , Técnicas de Patch-Clamp , Peptídeos/genética , Domínios Proteicos/genéticaRESUMO
OBJECTIVE: We performed a clinical, functional, and pharmacologic characterization of the novel p.P1158L Nav1.4 mutation identified in a young girl presenting a severe myotonic phenotype. METHODS: Wild-type hNav1.4 channel and P1158L mutant were expressed in tsA201 cells for functional and pharmacologic studies using patch-clamp. RESULTS: The patient shows pronounced myotonia, slowness of movements, and generalized muscle hypertrophy. Because of general discomfort with mexiletine, she was given flecainide with satisfactory response. In vitro, mutant channels show a slower current decay and a rightward shift of the voltage dependence of fast inactivation. The voltage dependence of activation and slow inactivation were not altered. Mutant channels were less sensitive to mexiletine, whereas sensitivity to flecainide was not altered. The reduced inhibition of mutant channels by mexiletine was also observed using clinically relevant drug concentrations in a myotonic-like condition. CONCLUSIONS: Clinical phenotype and functional alterations of P1158L support the diagnosis of myotonia permanens. Impairment of fast inactivation is consistent with the possible role of the channel domain III S4-S5 loop in the inactivation gate docking site. The reduced sensitivity of P1158L to mexiletine may have contributed to the unsatisfactory response of the patient. The success of flecainide therapy underscores the usefulness of in vitro functional studies to help in the choice of the best drug for each individual.
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Mutação , Miotonia Congênita/tratamento farmacológico , Miotonia Congênita/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico , Linhagem Celular , Criança , Diagnóstico Diferencial , Feminino , Flecainida/farmacologia , Flecainida/uso terapêutico , Humanos , Mexiletina/efeitos adversos , Mexiletina/farmacologia , Mexiletina/uso terapêutico , Miotonia Congênita/diagnóstico , Miotonia Congênita/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Testes Farmacogenômicos/métodos , Medicina de Precisão/métodos , Pesquisa Translacional Biomédica , Bloqueadores do Canal de Sódio Disparado por Voltagem/efeitos adversos , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
OBJECTIVE: Although patients with Guillain-Barré syndrome (GBS) are often hospitalized few days after symptoms onset, nerve conduction studies (NCS) abnormalities in early phases of the disease are not well characterized. Our aim was to report early neurophysiological abnormalities from a cohort of GBS patients. METHODS: In this single-center study, we retrospectively reviewed the NCS data of 71 consecutive GBS patients in whom neurophysiology was performed within two weeks after disease onset. We further divided our cohort in three subgroups according to the interval between disease onset and NCS (≤ 4 days; 5-7 days; 8-14 days). RESULTS: A great proportion of patients (37%) with an early NCS (≤ 4 days) showed normal neurophysiological results. The most altered parameters were F waves and their proportion increases in correspondence to number of days after onset. Conduction blocks were observed preferentially in upper limbs, in about a third of cases. CONCLUSION: This study confirms that NCS may be normal in the early phases of GBS syndrome and suggests to perform an extensive neurophysiological evaluation in these patients. SIGNIFICANCE: Our results may help the clinicians in the interpretation of NCS in early-onset GBS.
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Potenciais de Ação/fisiologia , Síndrome de Guillain-Barré/fisiopatologia , Condução Nervosa/fisiologia , Nervos Periféricos/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Masculino , Nervo Mediano/fisiopatologia , Pessoa de Meia-Idade , Nervo Fibular/fisiopatologia , Nervo Radial/fisiopatologia , Estudos Retrospectivos , Nervo Sural/fisiopatologia , Nervo Tibial/fisiopatologia , Nervo Ulnar/fisiopatologia , Adulto JovemRESUMO
Myoclonus consists of sudden, brief, involuntary jerky muscular contractions. Central and peripheral nervous system lesions are involved in the pathogenesis of this movement disorder. Symptomatic or secondary spinal myoclonus is the most common form. A 68-year-old woman was diagnosed with hemiabdominal spinal myoclonus. Occasional and very mild involuntary repetitive movements of the hemiabdomen began immediately after surgery for uterine cancer. After surgery for laparocele, secondary to the uterine cancer surgery, performed under spinal anesthesia, there was severe worsening of movements. Neuroradiological investigations failed to demonstrate spinal injury, while neurophysiological studies showed impairment of the right central somatosensory pathway. Considering the low resolution of magnetic resonance imaging in the evaluation of thoracic level, we suggest an extensive neurophysiological evaluation in patients with spinal myoclonus.
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Músculos Abdominais/fisiopatologia , Mioclonia/diagnóstico , Mioclonia/tratamento farmacológico , Idoso , Anticonvulsivantes/uso terapêutico , Benzotiazóis/uso terapêutico , Clonazepam/uso terapêutico , Diagnóstico Diferencial , Agonistas de Dopamina/uso terapêutico , Quimioterapia Combinada , Eletroencefalografia , Eletromiografia , Feminino , Humanos , Imageamento por Ressonância Magnética , Mioclonia/fisiopatologia , PramipexolRESUMO
OBJECTIVE: We aim to demonstrate the effect of mexiletine on the compound muscle action potential (CMAP) amplitude transitory depression (TD) in a cohort of patients with recessive myotonia congenita. METHODS: We evaluated 21 patients with recessive myotonia congenita referred to our institute from 1990 to 2013 and treated with mexiletine chlorhydrate. All patients underwent prolonged 3 Hz repetitive nerve stimulation (3 Hz-PLRS) before and after the beginning of treatment. RESULTS: We observed in all subjects a reduction of CMAP amplitude TD after the beginning of treatment. The mean value of the TD nadir before starting mexiletine treatment was -62.0% and reduced to -28.8% after the therapy was started (51.6% reduction, p<0.001). CONCLUSIONS: The 3 Hz-PLRS is configured as a neurophysiological test able to indirectly detect and quantify, through the measurement of TD, the clinical phenomenon of the transitory weakness that occurs in myotonic syndromes due to CLCN1 mutations. SIGNIFICANCE: This neurophysiological test might be considered a helpful tool to assess the effect of anti-myotonic drugs, as mexiletine, in recessive myotonia congenita.
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Potencial Evocado Motor/efeitos dos fármacos , Mexiletina/farmacologia , Mexiletina/uso terapêutico , Miotonia Congênita/tratamento farmacológico , Miotonia Congênita/fisiopatologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Adolescente , Adulto , Idoso , Criança , Potencial Evocado Motor/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miotonia Congênita/diagnóstico , Fatores de Tempo , Resultado do Tratamento , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico , Adulto JovemRESUMO
BACKGROUND: Classic 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. PATIENTS AND METHODS: We 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. RESULTS: Typical 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. CONCLUSIONS: About half of the patients with HNPP from our cohort showed an atypical clinical presentation. Neurophysiological examination represents the main tool for a proper diagnosis.
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Deleção Cromossômica , Neuropatia Hereditária Motora e Sensorial , Nervos Periféricos/fisiopatologia , Adolescente , Adulto , Idoso , Cromossomos Humanos Par 17 , Estudos de Coortes , Feminino , Neuropatia Hereditária Motora e Sensorial/genética , Neuropatia Hereditária Motora e Sensorial/patologia , Neuropatia Hereditária Motora e Sensorial/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Condução Nervosa/genética , Condução Nervosa/fisiologia , Síndrome de Smith-Magenis , Adulto JovemRESUMO
OBJECTIVE: Idiopathic brachial plexopathy is a non-progressive disorder characterized by the sudden onset of shoulder pain associated with weakness and sometimes paraesthesia of the arm. Clinical and electrophysiological examinations are the primary diagnostic tools and allow physicians to localize the site of damage. MRI neurography is rarely performed in this setting. METHODS: We herein describe the cases of eight consecutive patients suffering from idiopathic brachial plexopathy. All patients underwent clinical visits, neurophysiological evaluations and MRI neurography. RESULTS: We confirmed the primary role of clinical and neurophysiological evaluations in the diagnosis of idiopathic brachial plexopathy and demonstrate the usefulness of brachial plexus MRI neurography for confirming the presence of inflammatory changes. CONCLUSION: In patients with idiopathic brachial plexopathy, MR neurography is a helpful tool for excluding different aetiologies, such as compression or tumour formation, and/or confirming inflammatory changes.
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Neuropatias do Plexo Braquial/diagnóstico , Imageamento por Ressonância Magnética/métodos , Adulto , Idoso , Neuropatias do Plexo Braquial/tratamento farmacológico , Neuropatias do Plexo Braquial/fisiopatologia , Eletromiografia , Fenômenos Eletrofisiológicos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Condução Nervosa , Esteroides/uso terapêutico , Tomografia Computadorizada por Raios XRESUMO
Practitioners may refer to experienced hand surgeons to differentiate a recurrence in carpal tunnel syndrome (CTS) from a failed carpal tunnel release. The patient may complain about the reappearance of symptoms, whatever is the cause. Nerve conduction studies (NCS) are often required by the practitioner to assist the final diagnosis. We observed abnormal values in NCS in patients who were clinically healed from CTS. We evaluated the changes preoperatively and, then, at 1, 3, 6, 9, and 12 month postoperatively. At the same time, we performed a retrospective study on a group of 37 clinically healed patients. Follow-up ranged from 2 to 20 years. Surgical treatment let the electrophysiological parameters to improve toward physiological values; however, normality is hardly ever reached. This sort of ''electrophysiological scar'' is true for all the parameters measured. In presence of CTS, the latency difference between the radial and median sensory nerve action potentials, recorded following thumb stimulation, produces a double peak shift. The ''double peak shift'' best described this ''electrophysiological scar,'' being a parameter that should measure about zero in the normal population. In conclusion, abnormal postoperative electrophysiological findings cannot substantiate the diagnosis of a poor outcome of a carpal tunnel release nor a recurrence of CTS.
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Síndrome do Túnel Carpal/fisiopatologia , Síndrome do Túnel Carpal/cirurgia , Condução Nervosa/fisiologia , Complicações Pós-Operatórias/fisiopatologia , Potenciais de Ação/fisiologia , Feminino , Humanos , Masculino , Nervo Mediano/fisiopatologia , Nervo Mediano/cirurgia , Exame Neurológico , Estudos Prospectivos , Recidiva , Estudos RetrospectivosRESUMO
OBJECTIVE: We aim to draw clinical-neurophysiological correlations in our cohort of patients affected by IgM-related neuropathy to investigate whether neurophysiological parameters may help differentiate the classical phenotype from atypical forms. METHODS: We retrospectively evaluated patients with IgM-related neuropathy referred to our Institute from 1990 to 2011. All patients underwent extensive laboratory, clinical and neurophysiological evaluation. RESULTS: A classic sensory-ataxic form was observed in 20 of 34 patients, while an atypical phenotype (multiple mononeuropathy, polyneuropathy with predominant motor impairment, painful small-fibre neuropathy) was identified in the remaining 14 cases. Nerve conduction studies revealed in almost all cases a pattern typical of demyelination. A reduced terminal latency index and a prolonged distal motor latency of median nerve, as well as a prolonged distal motor latency and a reduced motor conduction velocity of peroneal nerve when recorded from extensor digitorum brevis, were significantly associated with classic sensory-ataxic phenotype. Conversely, a compound muscle action potential amplitude reduction of peroneal nerve from the tibialis anterior, was mostly associated with atypical forms. CONCLUSIONS: No clear electrophysiological differences between classical forms and atypical cases can be identified in IgM-related neuropathy. Still, we demonstrated that demyelinating abnormalities are more often associated with classical phenotypes, while axonal impairment occurs more often in atypical clinical patterns. SIGNIFICANCE: Performing correlations between clinical and neurophysiological findings in IgM-related neuropathy may help to better understand different disease mechanisms in this heterogeneous form of inflammatory neuropathy.
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Ataxia/etiologia , Doenças Desmielinizantes/etiologia , Imunoglobulina M , Paraproteinemias/complicações , Polineuropatias/diagnóstico , Polineuropatias/etiologia , Idoso , Ataxia/diagnóstico , Doenças Desmielinizantes/diagnóstico , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Nervo Mediano/fisiopatologia , Pessoa de Meia-Idade , Condução Nervosa/fisiologia , Exame Neurológico , Nervo Fibular/fisiopatologia , Fenótipo , Estudos Retrospectivos , Células Receptoras Sensoriais/fisiologiaAssuntos
Eletrodiagnóstico/métodos , Síndrome Miastênica de Lambert-Eaton/diagnóstico , Potenciais de Ação/fisiologia , Adulto , Idoso , Diagnóstico Diferencial , Estimulação Elétrica , Feminino , Humanos , Síndrome Miastênica de Lambert-Eaton/fisiopatologia , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/fisiopatologia , Miastenia Gravis/diagnósticoRESUMO
Familial amyloid polyneuropathy (FAP) is a rare condition caused by mutations of the transthyretin (TTR) gene and it is generally characterized by a length-dependent polyneuropathy affecting prevalently the small fibers. We reviewed clinical, electrophysiological and pathological findings of 15 unrelated patients with genetically confirmed TTR-FAP. All patients presented a progressive sensory-motor polyneuropathy. Pathological findings were negative for amyloid deposits in about half of the cases. Sequence analysis of TTR gene revealed the presence of three different mutations (p.Val30Met, p.Phe64Leu, and p.Ala120Ser). The p.Val30Met was the most frequently identified mutation and it often occurred in apparently sporadic cases. Conversely, the p.Phe64Leu generally presented in a high percentage of familial cases in patients coming from Southern Italy. Clinicians should consider, to avoid misdiagnosis, the screening for TTR mutations in patients presenting with progressive axonal polyneuropathy of undetermined etiology, including apparently sporadic cases with pathological examinations negative for amyloid deposition.
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Neuropatias Amiloides Familiares/diagnóstico , Neuropatias Amiloides Familiares/genética , Mutação/genética , Pré-Albumina/genética , Idoso , Amiloide/metabolismo , Neuropatias Amiloides/diagnóstico , Neuropatias Amiloides/genética , Neuropatias Amiloides/fisiopatologia , Neuropatias Amiloides Familiares/epidemiologia , Neuropatias Amiloides Familiares/fisiopatologia , Feminino , Testes Genéticos , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-IdadeRESUMO
Transitory depression of the compound muscle action potential during repetitive nerve stimulation is a well-documented neurophysiologic finding in recessive myotonia congenita. It represents the neurophysiologic counterpart of the transitory weakness often impairing patients at the beginning of a movement after rest, and it is usually better induced using high-rate nerve stimulations. The authors examined 30 patients with recessive myotonia congenita and carried out a 3 Hz nerve stimulation study to ascertain to what extent this protocol was able to detect the occurrence of transitory depression. Their findings were compared with the results obtained by 12 patients affected by dominant myotonia congenita and 12 patients affected by nondystrophic myotonia due to SCN4A mutations. Molecular genetic analysis of the CLCN1 and SCN4A genes was also performed. The 3 Hz nerve stimulation protocol was well tolerated and showed high sensitivity, resulting positive in 66% of recessive case and good reproducibility, if performed after an adequate period of rest. All dominant cases and all patients affected by myotonia due to SCN4A mutations showed negative results. Molecular studies identified 26 different CLCN1 mutations, 16 of which were novel. Transitory depression confirmed to vary in accordance to CLCN1 mutations. The 3 Hz protocol was well tolerated and showed good sensitivity and reproducibility. Furthermore, this test might be suitable for genotype-phenotype correlation studies.