Consensus from the Brazilian Academy of Neurology for the diagnosis, genetic counseling, and use of disease-modifying therapies in 5q spinal muscular atrophy / Consenso da Academia Brasileira de Neurologia para diagnóstico, aconselhamento genético e uso de terapias modificadoras na atrofia muscular espinhal 5q

Abstract Spinal muscular atrophy linked to chromosome 5 (SMA-5q) is an autosomal recessive genetic disease caused by mutations in the SMN1. SMA-5q is characterized by progressive degeneration of the spinal cord and bulbar motor neurons, causing severe motor and respiratory impairment with reduced survival, especially in its more severe clinical forms. In recent years, highly effective disease-modifying therapies have emerged, either acting by regulating the splicing of exon 7 of the SMN2 gene or adding a copy of the SMN1 gene through gene therapy, providing a drastic change in the natural history of the disease. In this way, developing therapeutic guides and expert consensus becomes essential to direct the use of these therapies in clinical practice. This consensus, prepared by Brazilian experts, aimed to review the main available disease-modifying therapies, critically analyze the results of clinical studies, and provide recommendations for their use in clinical practice for patients with SMA-5q. This consensus also addresses aspects related to diagnosis, genetic counseling, and follow-up of patients under drug treatment. Thus, this consensus provides valuable information regarding the current management of SMA-5q, helping therapeutic decisions in clinical practice and promoting additional gains in outcomes.

Resumo Atrofia muscular espinhal ligada ao cromossomo 5 (AME-5q) é uma doença genética de herança autossômica recessiva causada por mutações no gene SMN1. A AME-5q cursa com degeneração progressiva dos motoneurônios medulares e bulbares, acarretando grave comprometimento motor e respiratório com redução da sobrevida, especialmente nas suas formas clínicas mais graves. Nos últimos anos, terapias modificadoras da doença altamente eficazes, ou que atuam regulando o splicing do exon 7 do gene SMN2 ou adicionando uma cópia do gene SMN1 via terapia gênica, têm surgido, proporcionando uma mudança drástica na história natural da doença. Dessa forma, o desenvolvimento de guias terapêuticos e de consensos de especialistas torna-se importante no sentido de direcionar o uso dessas terapias na prática clínica. Este consenso, preparado por especialistas brasileiros, teve como objetivos revisar as principais terapias modificadoras de doença disponíveis, analisar criticamente os resultados dos estudos clínicos dessas terapias e prover recomendações para seu uso na prática clínica para pacientes com AME-5q. Aspectos relativos ao diagnóstico, aconselhamento genético e seguimento dos pacientes em uso das terapias também são abordados nesse consenso. Assim, esse consenso promove valiosas informações a respeito do manejo atual da AME-5q auxiliando decisões terapêuticas na prática clínica e promovendo ganhos adicionais nos desfechos finais.

Gene therapy in neuromuscular disorders / Terapia gênica nas doenças neuromusculares

Abstract Monogenic neuromuscular disorders are potentially treatable through gene therapy. Using viral vectors, a therapeutic transgene aims to restore normal levels of a protein not produced by the defective gene, or to silence a gene whose expression leads to toxic effects. Spinal Muscular Atrophy (SMA) is a good example of a monogenic disease that currently has an AAV9-based vector gene therapy as a therapeutic option. In this review, we intend to discuss the viral vectors and their mechanisms of action, in addition to reviewing the clinical trials that supported the approval of gene therapy (AVXS-101) for SMA as well as neuromuscular diseases that are potentially treatable with gene replacement therapy.

Resumo Doenças neuromusculares monogênicas são potencialmente tratáveis através de terapia gênica. Utilizando-se de vetores virais, um transgene terapêutico objetiva repor os níveis normais de uma proteina não produzida pelo gene defeituoso ou silenciar um gene cuja expressão leva a efeitos tóxicos. A Atrofia Muscular Espinhal (AME) é um bom exemplo de doença monogenica que atualmente tem uma terapia gênica com vetor viral AAV9 como opção terapêutica. Nesta revisão, pretendemos discutir os vetores virais e macanismos de ação utilizados, além de revisar os ensaios clínicos que embasaram a aprovação da terapia gênica (AVXS-101) para AME, assim como doenças neuromusculares potencialmente tratáveis com terapia de reposição gênica.

Managing intrathecal administration of nusinersen in adolescents and adults with 5q-spinal muscular atrophy and previous spinal surgery / Manejo da administração intratecal de nusinersena em adolescentes e adultos com atrofia muscular espinhal 5q e cirurgia prévia de coluna

ABSTRACT Background: Spinal muscular atrophy (SMA) is a neurodegenerative disease of lower motor neurons associated with frequent occurrence of spinal deformity. Nusinersen is an antisense oligonucleotide that increases SMN protein level and is administrated by frequent intrathecal lumbar injections. Thus, spinal deformities and previous spinal surgery are important challenges for drug delivery in SMA. Objective: To report imaging methods used for Nusinersen injection in SMA patients. Methods: Nusinersen injection procedures in SMA types 2 and 3 patients who had previous spinal surgery were analyzed retrospectively to describe the imaging and puncture procedures, as well as the occurrence of complications. Results: Nine SMA patients (14 to 50 years old) underwent 57 lumbar punctures for nusinersen injection. Six patients had no interlaminar space available; in five of them, a transforaminal approach was used, and another one underwent a surgery to open a posterior bone window for the injections. Transforaminal puncture was performed using CT scan in three cases and fluoroscopy in the other two, with a similar success rate. One patient in the transforaminal group had post-procedure radiculitis, and another one had vagal reaction (hypotension). In three cases, with preserved interlaminar space, injections were performed by posterior interlaminar puncture, and only one adverse event was reported (post-puncture headache). Conclusion: In SMA patients with previous spinal surgery, the use of imaging-guided intervention is necessary for administering intrathecal nusinersen. Transforaminal technique is indicated in patients for whom the interlaminar space is not available, and injections should always be guided by either CT or fluoroscopy.

RESUMO Introdução: A atrofia muscular espinal (AME) é uma desordem neurodegenerativa dos motoneurônios inferiores frequentemente associada à ocorrência de deformidade da coluna vertebral. Nusinersena é um oligonucleotídeo antisense que aumenta os níveis da proteína SMN, sendo administrado através de injeções lombares intratecais frequentes. Assim, deformidades da coluna vertebral e abordagem cirúrgica prévia são desafios importantes para a administração de medicamentos na AME. Objetivo: descrever os métodos de imagens utilizados para administração do Nusinersena nos pacientes com AME. Métodos: Os procedimentos de administração de nusinersena em pacientes com AME dos tipos 2 e 3 submetidos à cirurgia prévia da coluna foram analisados retrospectivamente para descrever os métodos de imagem e punção, e a ocorrência de complicações. Resultados: Nove pacientes com AME (14 a 50 anos) foram submetidos a 57 punções lombares para administração de nusinersena. Seis pacientes tinham enxerto ósseo ou nenhum espaço interlaminar disponível; em cinco deles foi utilizada uma abordagem transforaminal, e outra paciente foi submetida à abertura cirúrgica de janela óssea para as injeções. A punção transforaminal foi realizada usando tomografia computadorizada (TC) em três casos e fluoroscopia nos outros dois, com taxa de sucesso semelhante. Um paciente no grupo de abordagem transforaminal apresentou radiculite pós-procedimento e outro apresentou reação vagal (hipotensão). Em três casos, com espaço interlaminar preservado, foram realizadas técnica de punção interlaminar posterior e apenas um evento adverso foi relatado (cefaleia pós-punção). Conclusão: Em pacientes com AME e cirurgia prévia, o uso de intervenção guiada por imagem é necessário para a administração de nusinersena. A técnica transforaminal é indicada nos casos onde o espaço interlaminar não está disponível, devendo ser guiada por TC ou técnicas de imagem fluoroscópica.

SMN1 gene deletion analysis using mid-trimester amniotic fluid cells by real-time PCR / 中华检验医学杂志

Objectives To investigate the prenatal diagnosis method of spinal muscular atrophy with amniotic fluid sample.Methods Totally 1 064 amniotic fluid samples from mid-trimester pregnant women were enrolled during January 2015 and January 2016 in 4 hospitals.Genetic analysis was performed for detecting potential contamination of maternal tissue by a genetic technique based on short tandem repeat ( STR) markers.Deletion of SMN1 gene was detected in 1 062 uncontaminated amniotic fluid samples by real-time PCR and multiplex ligation-dependent probe amplification ( MLPA) respectively.Results Two contaminated amniotic fluid samples were detected within 1 064 mid-trimester pregnant women by STR genotyping.The other 1 062 uncontaminated amniotic fluid samples were tested by real-time PCR.There were 37 samples with heterozygous deletion of Exon 7 of SMN1 gene ( 3.67%) , 34 samples with heterozygous deletion of Exon 8 of SMN1 gene (3.2%) and two samples with homozygous deletion of Exon 7 and Exon8 of SMN1 gene ( 0.19%) respectively , while other samples observed with no deletion of Exon 7 and Exon8 in SMN1 gene.Totally 41 samples with heterozygous or homozygous deletion of SMN 1 gene and 55 samples with undetected deletion of SMN 1 gene were confirmed by MLPA and the results showed 100%consistence with that of real-time PCR.Conclusions Both real-time PCR and MLPA are suitable for detecting the deletion of SMN 1 gene with amniotic fluid sample . Real-time PCR exhibits less sample requirement and time compared with MLPA .

Genetic diagnosis of 154 patients with suspected spinal muscular atrophy and its clinical value / 中华检验医学杂志

Objective To explore the clinical value of genetic diagnosis of SMA,the homozygous deletion of survival motor neuron 1 (SMN1) gene in suspected spinal muscular atrophy (SMA) patients were analyzed in this study.Methods A total of 154 patients suspected with SMA and 20 healthy volunteers were recruited from January 2007 to December 2014 in the Genetic Diagnosis Center of the First People's Hospital of Yunnan Province and the Department of Neurology of the Fourth Affiliated Hospital of Kunming Medical University.Potential deletions in exons 7 and 8 of SMN1 gene were screened by use of polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method in both 154 patients suspected with SMA and 20 healthy volunteers.The frequencies of the deletions in exons 7 and 8 of SMN1 were calculated and statistical analysis of different deletion types among 3 SMA groups was performed with SPSS 13.0 software package.Results Among 154 suspected SMA patients,101 cases with homozygous deletions of exon 7 of SMN1 gene were detected,which accounted 65.6％ (101/154) of the suspected SMA patients.Among the 101 SMA patients,97.0％ (98/101) of the patients with both homozygous deletions of exons 7 and 8 for SMN1 gene and 3.0％ (3/101) of the patients with homozygous deletions of only exon 7 for SMN1 gene were detected.The patient with only deletion of exon 8 for SMN1 gene was notdetected.Four cases with negative results were subjected to be followed-up,but they were characteristic of SMA symptom by clinical re-visit.Thus,total 105 patients were confirmed with SMA,among them,68 were type Ⅰ SMA,27 were type Ⅱ SMA,and 10 were type Ⅲl SMA,which accounted for 64.8％ (68/105),25.7％ (27/105) and 9.5％ (10/105) of the SMA patients,respectively.Type Ⅳ SMA was not observed in these patients.No deletion was detected among 20 healthy volunteers.Conclusions PCR-RFLP assay is a noninvasive,simple,high sensitive and specific method for SMA diagnosis,which can be considered as the first-line genetic test for the suspected SMA patients.It will help to improve the accuracy of clinical diagnosis and the detection rate by strengthening the clinical diagnostic criteria and re-evaluating the suspected patients after negative genetic diagnosis.

Identification of two survival motor neuron gene 1 gene mutations and evaluation of their effects on full-length survival motor neuron gene 1 transcripts / 中华神经科杂志

Objective To perform mutation analysis of survival motor neuron gene 1 (SMN1 in two spinal muscular atrophy (SMA) patients and their parents to evaluate the effects of the two SMN1 gene mutations on the transcript levels of the gene and preliminarily predict their effects on the structure and function of SMN protein.Methods Mutation analysis of SMN1 gene was carried out by multiplex ligationdependent probe amplification,reverse transcript-polymerase chain reaction (RT-PCR) and cloning sequencing.Transmission of the mutations was confirmed by the mutation analysis in patients' parents.The full-length SMN1 (SMN1-fl) transcript levels of the patients carrying these subtle mutations were detected using quantitative RT-PCR.Results The two patients were diagnosed as SMA Ⅱ and SMA Ⅲ.They carried p.Val19GlyfsX21 and p.Ala2Gly SMN1 mutations in SMN1 gene,respectively.Both of the two mutations were originated from their fathers.Compared with the healthy individuals (23.5 ± 4.9),the two patients had a significant reduction in the level of SMN1-fl transcripts (t =3.322,P =0.011 (p.Ala2Gly) ;t =6.964,P =0.000 (p.Val19GlyfsX21)).However,compared with the healthy carriers (14.1 ±4.5),the patient with p.Ala2Gly mutation had no significant reduction in the level of SMN1-fl transcripts (13.9 ±3.6,t =0.058,P =0.955) ; however,the patient with p.Val19GlyfsX21 mutation had a significant reduction (4.9± 2.4,t =3.725,P =0.004).Conclusions Two SMN1 gene mutations are identified in our study.The mutation p.Val19GlyfsX21 is a novel mutation and p.Ala2Gly is firstly reported in Chinese SMA patients.p.Val19GlyfsX21 may possibly lead to decreased SMN1-fl mRNA by nonsense-mediated messenger RNA decay,however,p.Ala2Gly has no obvious effects on the amount of the SMN1-fl transcripts,indicating that its deleterious effect may be occurring at SMN protein level or the function of SMN protein.

Subtle mutation analysis of survival motor neuron gene in families with spinal muscular atrophy / 中华检验医学杂志

ObjectiveTo establish a analytical system for the survival motor neuron (SMN) subtle mutation,and evaluate its application in two families with spinal muscular atrophy (SMA).MethodsSMN genes in seven family members from two SMA families were analyzed at both transcript level and genomic level,by the use of the conventional PCR-RFLP,allele-specific PCR,multiplex ligation-dependent probe amplification (MLPA) and T subcloning and sequencing of SMNI gene.ResultsIn family A,the patient had a single SMN1 copy who was carrying nonsense mutation L228X,which was also found in his father.In family B,as the patient's sample was unavailable,the father was indeed a carrier with one normal SMN1 allele and the other SMN1 allele carrying a frameshift mutation 22_23insA.The remaining family members were SMA carriers with one SMN1 copy.ConclusionThis analytical system for SMN subtle mutation offers viable molecular basis for genetic counseling and prenatal diagnosis in SMA families.