Clinical Phenotype of Pediatric and Adult Patients With Spinal Muscular Atrophy With Four SMN2 Copies: Are They Really All Stable?

OBJECTIVE: The aim of this study was to provide an overview of the clinical phenotypes associated with 4 SMN2 copies. METHODS: Clinical phenotypes were analyzed in all the patients with 4 SMN2 copies as part of a nationwide effort including all the Italian pediatric and adult reference centers for spinal muscular atrophy (SMA). RESULTS: The cohort includes 169 patients (102 men and 67 women) with confirmed 4 SMN2 copies (mean age at last follow-up = 36.9 ± 19 years). Six of the 169 patients were presymptomatic, 8 were classified as type II, 145 as type III (38 type IIIA and 107 type IIIB), and 8 as type IV. The remaining 2 patients were asymptomatic adults identified because of a familial case. The cross-sectional functional data showed a reduction of scores with increasing age. Over 35% of the type III and 25% of the type IV lost ambulation (mean age = 26.8 years ± 16.3 SD). The risk of loss of ambulation was significantly associated with SMA type (p < 0.0001), with patients with IIIB and IV less likely to lose ambulation compared to type IIIA. There was an overall gender effect with a smaller number of women and a lower risk for women to lose ambulation. This was significant in the adult (p = 0.009) but not in the pediatric cohort (p = 0.43). INTERPRETATION: Our results expand the existing literature on natural history of 4 SMN2 copies confirming the variability of phenotypes in untreated patients, ranging from type II to type IV and an overall reduction of functional scores with increasing age. ANN NEUROL 2023;94:1126-1135.

Experience of a 2-year spinal muscular atrophy NBS pilot study in Italy: towards specific guidelines and standard operating procedures for the molecular diagnosis.

BACKGROUND: Spinal muscular atrophy (SMA) is due to the homozygous absence of SMN1 in around 97% of patients, independent of the severity (classically ranked into types I-III). The high genetic homogeneity, coupled with the excellent results of presymptomatic treatments of patients with each of the three disease-modifying therapies available, makes SMA one of the golden candidates to genetic newborn screening (NBS) (SMA-NBS). The implementation of SMA in NBS national programmes occurring in some countries is an arising new issue that the scientific community has to address. We report here the results of the first Italian SMA-NBS project and provide some proposals for updating the current molecular diagnostic scenario. METHODS: The screening test was performed by an in-house-developed qPCR assay, amplifying SMN1 and SMN2. Molecular prognosis was assessed on fresh blood samples. RESULTS: We found 15 patients/90885 newborns (incidence 1:6059) having the following SMN2 genotypes: 1 (one patient), 2 (eight patients), 2+c.859G>C variant (one patient), 3 (three patients), 4 (one patient) or 6 copies (one patient). Six patients (40%) showed signs suggestive of SMA at birth. We also discuss some unusual cases we found. CONCLUSION: The molecular diagnosis of SMA needs to adapt to the new era of the disease with specific guidelines and standard operating procedures. In detail, SMA diagnosis should be felt as a true medical urgency due to therapeutic implications; SMN2 copy assessment needs to be standardised; commercially available tests need to be improved for higher SMN2 copies determination; and the SMN2 splicing-modifier variants should be routinely tested in SMA-NBS.

Longitudinal evaluation of SMN levels as biomarker for spinal muscular atrophy: results of a phase IIb double-blind study of salbutamol.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder, due to the loss of function of the survival motor neuron (SMN1) gene. The first treatment for the condition, recently approved, is based on the reduction of exon 7 skipping in mRNAs produced by a highly homologous gene (SMN2). The primary objective of the present study was to evaluate the applicability of the dosage of SMN gene produts in blood, as biomarker for SMA, and the safety of oral salbutamol, a beta2-adrenergic agonist modulating SMN2 levels. METHODS: We have performed a 1-year multicentre, double-blind, placebo-controlled study with salbutamol in 45 adult patients with SMA. Patients assumed 4 mg of salbutamol or placebo/three times a day. Molecular tests were SMN2 copy number, SMN transcript and protein levels. We have also explored the clinical effect, by the outcome measures available at the time of study design. RESULTS: Thirty-six patients completed the study. Salbutamol was safe and well tolerated. We observed a significant and progressive increase in SMN2 full-length levels in peripheral blood of the salbutamol-treated patients (p<0.00001). The exploratory analysis of motor function showed an improvement in most patients. CONCLUSIONS: Our data demonstrate safety and molecular efficacy of salbutamol. We provide the first longitudinal evaluation of SMN levels (both transcripts and protein) in placebo and in response to a compound modulating the gene expression: SMN transcript dosage in peripheral blood is reliable and may be used as pharmacodynamic marker in clinical trials with systemic compounds modifying SMN2levels. TRIAL REGISTRATION NUMBER: EudraCT no. 2007-001088-32.

Spinal muscular atrophy associated with progressive myoclonic epilepsy: A rare condition caused by mutations in ASAH1.

OBJECTIVE: To present the clinical features and the results of laboratory investigations in three patients with spinal muscular atrophy associated with progressive myoclonic epilepsy (SMA-PME), a rare condition caused by mutations in the N-acylsphingosine amidohydrosilase 1 (ASAH1) gene. METHODS: The patients were submitted to clinical evaluation, neurophysiologic investigations (that included wakefulness and sleep electroencephalography [EEG], video-polygraphic recording with jerk-locked back-averaging, multimodal evoked potentials, and electromyography), brain magnetic resonance imaging (MRI), biochemical screening, muscle and skin biopsies, and molecular genetic analysis. RESULTS: The main clinical features were onset in childhood with proximal muscular weakness, generalized epilepsy with absences and myoclonic seizures, cognitive impairment of variable degree; the course was progressive with muscle wasting and uncontrolled epileptic seizures. In one patient, earlier onset before the age of 2 years was associated with a more complex clinical picture, with abnormal eye movements, progressive cognitive impairment, and a more rapid and severe course. EEG/polygraphic data were consistent with PME, demonstrating generalized spike-and-wave discharges, evidence of positive and negative myoclonia, and prominent photosensitivity. In one patient, transcranial magnetic stimulation showed a hyperexcitable motor cortex, whereas somatosensory evoked potentials were unaffected. Possible involvement of the central acoustic and visual pathways was suggested by abnormal auditory and visual evoked potentials. Muscle biopsies showed typical signs of neurogenic damage. Molecular genetic analysis showed mutations of the ASAH1 gene. SIGNIFICANCE: Our data indicate that SMA-PME associated with ASAH1 mutations is a genetically distinct condition with specific clinical and neurophysiologic features. Further studies are warranted to explore the role of the ASAH1 gene in muscle and brain function.

Characterization of the Sarcidano Horse Coat Color Genes.

The goal of this study was to contribute to the general knowledge of the Sarcidano Horse, both by the identification of the genetic basis of the coat color and by updating the exact locations of the genotyping sites, based on the current EquCab3.0 genome assembly version. One-hundred Sarcidano Horses, living in semi-feral condition, have been captured to perform health and biometric checks. From that total number, 70 individual samples of whole blood were used for DNA extraction, aimed to characterize the genetic basis of the coat color. By genotyping and sequencing analyses of the MC1R Exon 1 and ASIP Exon 3, a real image of the coat color distribution in the studied population has been obtained. Chestnut and Black resulted in the most representative coat colors both from a phenotypic and genotypic point of view, that is suggestive of no human domestication or crossbreeding with domestic breed. Due to its ancient origin and genetic isolation, an active regional plan for the conservation of this breed would be desirable, focused on maintenance of resident genotypes and genetic resources. Collection and management of DNA, sperm, embryos, with the involvement of research centers and Universities, could be a valid enhancing strategy.

Salbutamol increases survival motor neuron (SMN) transcript levels in leucocytes of spinal muscular atrophy (SMA) patients: relevance for clinical trial design.

BACKGROUND: Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by mutations of the SMN1 gene. Based on severity, three forms of SMA are recognised (types I-III). All patients usually have 2-4 copies of a highly homologous gene (SMN2) which produces insufficient levels of functional survival motor neuron (SMN) protein. Recently, evidence has been provided that SMN2 expression can be enhanced in vitro by salbutamol, a ß2-adrenergic agonist. This compound has also been shown to improve motor function of SMA patients in two different pilot trials. AIM: To evaluate the in vivo molecular efficacy of salbutamol in SMA patients. METHODS: Twelve type II-III patients took salbutamol orally for 6 months. SMN2 full length transcript levels were determined in peripheral blood leucocytes by absolute real-time PCR, at baseline and after 3 and 6 months of treatment. RESULTS: A significant and constant increase in SMN2 full length transcript levels was detected; the response was directly proportional to SMN2 gene copy number. CONCLUSIONS: The data strongly support salbutamol as a candidate for treating SMA, and suggest that SMN2 copy number may predict the molecular response to treatment and may be a useful randomisation parameter in a double blind placebo controlled clinical trial design.

SMA-miRs (miR-181a-5p, -324-5p, and -451a) are overexpressed in spinal muscular atrophy skeletal muscle and serum samples.

Background: Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by the degeneration of the second motor neuron. The phenotype ranges from very severe to very mild forms. All patients have the homozygous loss of the SMN1 gene and a variable number of SMN2 (generally 2-4 copies), inversely related to the severity. The amazing results of the available treatments have made compelling the need of prognostic biomarkers to predict the progression trajectories of patients. Besides the SMN2 products, few other biomarkers have been evaluated so far, including some miRs. Methods: We performed whole miRNome analysis of muscle samples of patients and controls (14 biopsies and 9 cultures). The levels of muscle differentially expressed miRs were evaluated in serum samples (51 patients and 37 controls) and integrated with SMN2 copies, SMN2 full-length transcript levels in blood and age (SMA-score). Results: Over 100 miRs were differentially expressed in SMA muscle; 3 of them (hsa-miR-181a-5p, -324-5p, -451a; SMA-miRs) were significantly upregulated in the serum of patients. The severity predicted by the SMA-score was related to that of the clinical classification at a correlation coefficient of 0.87 (p<10-5). Conclusions: miRNome analyses suggest the primary involvement of skeletal muscle in SMA pathogenesis. The SMA-miRs are likely actively released in the blood flow; their function and target cells require to be elucidated. The accuracy of the SMA-score needs to be verified in replicative studies: if confirmed, its use could be crucial for the routine prognostic assessment, also in presymptomatic patients. Funding: Telethon Italia (grant #GGP12116).

Clinical phenotypes and trajectories of disease progression in type 1 spinal muscular atrophy.

The advent of clinical trials has highlighted the need for natural history studies reporting disease progression in type 1 spinal muscular atrophy. The aim of this study was to assess functional changes using the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) scale in a cohort of type 1 infants. Nutritional and respiratory longitudinal data were also recorded. Patients were classified according to the severity of the phenotype and age of onset. SMN2 copies were also assessed. Twenty patients were included, eight with early onset most severe phenotype, eight with the more typical type 1 phenotype and 4, who achieved some head control, with a milder phenotype. Both baseline values and trajectories of progression were different in the three subgroups (p = 0.0001). Infants with the most severe phenotype had the lowest scores (below 20) on their first assessment and had the most rapid decline. Those with the typical phenotype had scores generally between 20 and 40 and also had a fast decline. The infants with the milder phenotype had the highest scores, generally above 35, and a much slower deterioration. Infants with three SMN2 copies had an overall milder phenotype and milder progression while two SMN2 copies were found in all three subgroups.

Longitudinal assessments in discordant twins with SMA.

We report longitudinal clinical and neurophysiological assessments in twins affected by spinal muscular atrophy (SMA) with discordant phenotypes. The boy had the homozygous deletion of SMN1, a typical type 1 SMA course, and died at the age of eight months. His twin sister, asymptomatic at the time of the diagnosis in her brother, had the same genetic defect but she developed clinical and electrophysiological signs of type 2 SMA. The reduction of tendon reflexes was the first clinical sign at the age of 4 months, followed within few weeks, by a mild decrement in the amplitude of the compound motor action potentials. After the age of 9 months, she showed a sudden clinical and electrophysiological deterioration. Among molecular tests, we determined SMN2 copy number, SMN2 and Plastin 3 transcript levels in peripheral blood, and observed no relevant differences between twins.

Clinical and molecular cross-sectional study of a cohort of adult type III spinal muscular atrophy patients: clues from a biomarker study.

Proximal spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations of the SMN1 gene. Based on severity, three forms of SMA are recognized (types I-III). All patients usually have 2-4 copies of a highly homologous gene (SMN2), which produces insufficient levels of functional survival motor neuron (SMN) protein due to the alternative splicing of exon 7. The availability of potential candidates to the treatment of SMA has raised a number of issues, including the availability of biomarkers. This study was aimed at evaluating whether the quantification of SMN2 products in peripheral blood is a suitable biomarker for SMA. Forty-five adult type III patients were evaluated by Manual Muscle Testing, North Star Ambulatory Assessment scale, 6-min walk test, myometry, forced vital capacity, and dual X-ray absorptiometry. Molecular assessments included SMN2 copy number, levels of full-length SMN2 (SMN2-fl) transcripts and those lacking exon 7 and SMN protein. Clinical outcome measures strongly correlated to each other. Lean body mass correlated inversely with years from diagnosis and with several aspects of motor performance. SMN2 copy number and SMN protein levels were not associated with motor performance or transcript levels. SMN2-fl levels correlated with motor performance in ambulant patients. Our results indicate that SMN2-fl levels correlate with motor performance only in patients preserving higher levels of motor function, whereas motor performance was strongly influenced by disease duration and lean body mass. If not taken into account, the confounding effect of disease duration may impair the identification of potential SMA biomarkers.

De novo mutations in ATP1A3 cause alternating hemiplegia of childhood.

Alternating hemiplegia of childhood (AHC) is a rare, severe neurodevelopmental syndrome characterized by recurrent hemiplegic episodes and distinct neurological manifestations. AHC is usually a sporadic disorder and has unknown etiology. We used exome sequencing of seven patients with AHC and their unaffected parents to identify de novo nonsynonymous mutations in ATP1A3 in all seven individuals. In a subsequent sequence analysis of ATP1A3 in 98 other patients with AHC, we found that ATP1A3 mutations were likely to be responsible for at least 74% of the cases; we also identified one inherited mutation in a case of familial AHC. Notably, most AHC cases are caused by one of seven recurrent ATP1A3 mutations, one of which was observed in 36 patients. Unlike ATP1A3 mutations that cause rapid-onset dystonia-parkinsonism, AHC-causing mutations in this gene caused consistent reductions in ATPase activity without affecting the level of protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in ATP1A3.

SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous mutations of the SMN1 gene. Three forms of SMA are recognized (type I-III) on the basis of clinical severity. All patients have at least one or more (usually 2-4) copies of a highly homologous gene (SMN2), which produces insufficient levels of functional SMN protein, because of alternative splicing of exon 7. Recently, evidence has been provided that SMN2 expression can be enhanced by pharmacological treatment. However, no reliable biomarkers are available to test the molecular efficacy of the treatments. At present, the only potential biomarker is the dosage of SMN products in peripheral blood. However, the demonstration that SMN full-length (SMN-fl) transcript levels are reduced in leukocytes of patients compared with controls remains elusive (except for type I). We have developed a novel assay based on absolute real-time PCR, which allows the quantification of SMN1-fl/SMN2-fl transcripts. For the first time, we have shown that SMN-fl levels are reduced in leukocytes of type II-III patients compared with controls. We also found that transcript levels are related to clinical severity as in type III patients SMN2-fl levels are significantly higher compared with type II and directly correlated with functional ability in type II patients and with age of onset in type III patients. Moreover, in haploidentical siblings with discordant phenotype, the less severely affected individuals showed significantly higher transcript levels. Our study shows that SMN2-fl dosage in leukocytes can be considered a reliable biomarker and can provide the rationale for SMN dosage in clinical trials.