Estudo de 43 pacientes com atrofia muscular espinhal e seu diagnóstico molecular

Abstract

Resumo: A atrofia muscular espinhal e uma doenca degenerativa das celulas do como anterior da medula, com quatro tipos descritos (I, II, IIIa/b, IV). Somente os tipos I, II, Illa e Illb apresentam comprometimento do cromossomo 5ql3, em que dois genes sao os causadores da doenca: o primeiro codifica a proteina de sobrevivencia do neuronio motor (SMN) e o segundo codifica a proteina inibidora da apoptose neuronal (NAIP). Ha delecao dos exons 7 e 8 em mais de 90% dos casos, ambos envolvidos na codificacao do SMN. A delecao do exon 5, envolvido na codificacao do NAIP, ocorre em 45% a 70% dos pacientes com atrofia muscular espinhal tipo I, e em menos de 20% dos casos tipo II e III. Com o proposito de desenvolver e introduzir o diagnostico molecular nas investigacoes de rotina da atrofia muscular espinhal, selecionaram-se 43 pacientes com diagnostico ja estabelecido de atrofia muscular espinhal, baseando-se no quadro clinico, dosagens enzimaticas, resultados de biopsia muscular e eletromiografia. Pesquisou-se a delecao dos exons envolvidos no DNA extraido de biopsias musculares atraves da analise por reacao em cadeia da polimerase. Relacionaram-se os resultados com os respectivos pacientes, por meio de dados de anamnese, exame fisico, achados de biopsia muscular e eletroneuromiografia. Nove pacientes foram classificados como tendo atrofia muscular espinhal tipo I; quatorze pacientes, como atrofia muscular espinhal tipo II, e 20, como atrofia muscular espinhal tipo III (12 Illa e 8 Illb). Nos pacientes com atrofia muscular espinhal tipo I, houve delecao do NAIP em 5 dos 9 casos (55%); naqueles com o tipo II, houve delecao em 3 dos 14 casos (21,4%). Todos os casos considerados como tipo I e II demonstraram delecao simultanea dos exons 7 e 8. No tipo III, o exon 5 esteve deletado em 5 dos 20 casos (25%), mas com delecao do exon 7 em 18 dos 20 pacientes (90%) e do exon 8 em 19 deles (95%). A delecao do NAIP teve relacao estatisticamente significante com os casos de fraqueza muscular da porcao distai dos membros superiores (p < 0,05) e com o achado de nucleos internos na biopsia muscular (p < 0,05). A delecao do exon 8 foi estatisticamente significante na presenca de fibras angulares atroficas (p < 0,05) e de fibras redondas atroficas dispersas (p < 0,05) na biopsia muscular. A biopsia muscular foi condizente com desinervacao em 80% dos casos, e a eletromiografia em 88,8%, enquanto que a delecao concomitante dos exons 7 e 8 esteve presente em 93% dos casos nos tres tipos de atrofia muscular espinhal. Ha estreita relacao entre o diagnostico clinico e o diagnostico molecular, corroborando os indices de delecao descritos na literatura. A analise pela reacao em cadeia da polimerase, portanto, serve como exame diagnostico tao seguro e preciso quanto a biopsia muscular e a eletromiografia. alem de menos invasiva.

Abstract: Spinal muscle atrophy results from degeneration of motor neurons in the anterior horn of the spinal cord. There are four clinical types (I, II, IIIa/b, IV), but only three forms (I, II, Ilia and Illb) were found to be linked to the 5ql3 region o f chromosome 5, where 2 genes were postulated: one was designated as the Survival Motor Neuron (SMN) gene, and the other as the Neuronal Apoptosis Inhibitory Protein (NAIP) gene. Deletion of exons 7 and 8 occurs in over 90% of cases, both related to SMN codification. Deletion of exon 5 (related to NAIP gene) occurs in 45% to 70% of type I spinal muscle atrophy patients, and in less than 20% o f type II and III patients. The purpose o f this study is to develop and to establish molecular research in routine investigation of spinal muscle atrophy. Forty-three patients were included in this study, all o f them with previous diagnosis o f spinal muscle atrophy, according to their clinical features, muscular enzymes levels, electromyography and anatomopathologic results. The DNA was isolated from muscle biopsies. Polimerase chain reaction (PCR) amplification of SMN exons 7 and 8, and NAIP exon 5 was carried out and to clinical history, physical exam, electromyography and anatomopathologic results. A population o f 9 type I, fourteen patients type II and 20 patients type III (12 Ilia and 8 Illb) was enrolled into this study. Type I spinal muscle atrophy patients presented deletion of exon 5 (NAIP gene) in 5 cases (55%), and type II patients presented this deletion in 3 cases (21,4%). Type I and type II patients showed concomitant deletion of exons 7 and 8 in 100% o f cases. In type III patients, exon 5 was deleted in 5 cases (25%), with deletion of exon 7 in 18 patients (90%) and deletion o f exon 8 in 19 (95%) of them. Distal impairment of upper limbs was significantly correlated to the cases of NAIP deletion (p < 0,05), as to the presence of internal nucleus in muscle biopsy {p < 0,05). Deletion o f exon 8 was significantly correlated when associated to atrophic angulated fibers (p < 0,05) as well to the presence o f atrophic dispersed rounded fibers (p < 0,05) in muscle biopsy. Muscle biopsy presented with denervation in 80% of cases, and electromyography with the same pattern in 88,8%. Concomitant deletion of exons 7 and 8 occurred in 93% o f cases, in all three types of spinal muscle atrophy. Precise identification of deletion by PCR in spinal muscle atrophy patients provides an important diagnostic achievement, which may be considered as good as the muscle biopsy and electromyography, but less invasive.