Cardiac function evaluation in children with spinal muscular atrophy: A case-control study.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by degeneration of lower motor neurons, resulting in progressive muscle weakness and atrophy. However, little is known regarding the cardiac function of children with SMA. METHODS: We recruited SMA patients younger than 18 years of age from January 1, 2022, to April 1, 2022, in the First Affiliated Hospital of Sun Yat-sen University. All patients underwent a comprehensive cardiac evaluation before treatment, including history taking, physical examination, blood tests of cardiac biomarkers, assessment of echocardiography and electrocardiogram. Age/gender-matched healthy volunteers were recruited as controls. RESULTS: A total of 36 SMA patients (26 with SMA type 2 and 10 with SMA type 3) and 40 controls were enrolled in the study. No patient was clinically diagnosed with heart failure. Blood tests showed elevated values of creatine kinase isoenzyme M and isoenzyme B (CK-MB) mass and high-sensitivity cardiac troponin T (hs-cTnT) in spinal muscular atrophy (SMA) patients. Regarding echocardiographic parameters, SMA children were detected with lower global left and right ventricular longitudinal strain, abnormal diastolic filling velocities of trans-mitral and trans-tricuspid flow. The results revealed no clinical heart dysfunction in SMA patients, but subclinical ventricular dysfunction was seen in SMA children including the diastolic function and myocardial performance. Some patients presented with elevated heart rate and abnormal echogenicity of aortic valve or wall. Among these SMA patients, seven patients (19.4%) had scoliosis. The Cobb's angles showed a significant negative correlation with LVEDd/BSA, but no correlation with other parameters, suggesting that mild scoliosis did not lead to significant cardiac dysfunction. CONCLUSIONS: Our findings warrant increased attention to the cardiac status and highlight the need to investigate cardiac interventions in SMA children.

Response of plasma microRNAs to nusinersen treatment in patients with SMA.

OBJECTIVE: Spinal muscular atrophy (SMA) is a common genetic cause of infant mortality. Nusinersen treatment ameliorates the clinical outcome of SMA, however, some patients respond well, while others have limited response. We investigated microRNAs in blood samples from SMA patients and their response to nusinersen treatment evaluating the potential of circulating microRNAs as biomarkers for SMA. METHODS: In a discovery cohort study, microRNA next-generation sequencing was performed in blood samples from SMA patients (SMA type 2, n = 10; SMA type 3, n = 10) and controls (n = 7). The dysregulated microRNAs were further analysed in the therapeutic response cohort comprised of SMA type 1 patients (n = 22) who had received nusinersen treatment, at three time points along the treatment course (baseline, 2 and 6 months of treatment). The levels of the studied microRNAs were correlated to the SMA clinical outcome measures. RESULTS: In the discovery cohort, 69 microRNAs were dysregulated between SMA patients and controls. In the therapeutic response cohort, the baseline plasma levels of miR-107, miR-142-5p, miR-335-5p, miR-423-3p, miR-660-5p, miR-378a-3p and miR-23a-3p were associated with the 2 and 6 months response to nusinersen treatment. Furthermore, the levels of miR-107, miR-142-5p, miR-335-5p, miR-423-3p, miR-660-5p and miR-378-3p at 2 months of treatment were associated with the response after 6 months of nusinersen treatment. INTERPRETATION: Blood microRNAs could be used as biomarkers to indicate SMA patients' response to nusinersen and to monitor the efficacy of the therapeutic intervention. In addition, some of these microRNAs provide insight into processes involved in SMA that could be exploited as novel therapeutic targets.

Phosphoethanolamine Elevation in Plasma of Spinal Muscular Atrophy Type 1 Patients.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by degeneration or loss of lower motor neurons. The survival of motor neuron (SMN) 1 gene, which produces the SMN protein, has been identified as a responsible gene for the disease. SMN is ubiquitously expressed in any tissue and may play an important role on the metabolism in the human body. However, no appropriate biomarkers reflecting the alteration in the metabolism in SMA have been identified. METHODS: Low-molecular-weight metabolites were extracted from plasma of 20 human infants (9 SMA type 1 patients and 11 controls) and 9 infant mice (5 SMA-model mice, 4 control mice), and derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide. Finally, the derivatized products were applied to Gas Chromatography/Mass Spectrometry apparatus. To confirm the metabolite abnormality in SMA type 1 patients, we performed SMN-silencing experiment using a hepatocyte-derived cell line (HepG2). RESULTS: We performed a comprehensive metabolomics analysis of plasma from the patients with SMA type 1 and controls, and found that phosphoethanolamine (PEA) was significantly higher in the patients than in the controls. HepG2 experiment also showed that SMN-silencing increased PEA levels. However, comprehensive metabolomics analysis of plasma from SMA-model mice and control mice showed different profile compared to human plasma; there was no increase of PEA even in the SMA-model mice plasma. CONCLUSION: Our data suggested that PEA was one of the possible biomarkers of human SMA reflecting metabolic abnormalities due to the SMN protein deficiency.

Whole-blood dysregulation of actin-cytoskeleton pathway in adult spinal muscular atrophy patients.

OBJECTIVE: Recent advances in therapeutics have improved prognosis for severely affected spinal muscular atrophy (SMA) type 1 and 2 patients, while the best method of treatment for SMA type 3 patients with later onset of disease is unknown. To better characterize the SMA type 3 population and provide potential therapeutic targets, we aimed to understand gene expression differences in whole blood of SMA type 3 patients (n = 31) and age- and gender-matched controls (n = 34). METHODS: We performed the first large-scale whole blood transcriptomic screen with L1000, a rapid, high-throughput gene expression profiling technology that uses 978 landmark genes to capture a representation of the transcriptome and predict expression of 9196 additional genes. RESULTS: The primary downregulated KEGG pathway in adult SMA type 3 patients was "Regulation of Actin Cytoskeleton," and downregulated expression of key genes in this pathway, including ROCK1, RHOA, and ACTB, was confirmed in the same whole blood samples using RT-qPCR. SMA type 3 patient-derived fibroblasts had lower expression of these genes compared to control fibroblasts from unaffected first-degree relatives. Overexpression of SMN levels using an AAV vector in fibroblasts did not normalize ROCK1, RHOA, and ACTB mRNA expression, indicating the involvement of additional genes in cytoskeleton dynamic regulation. INTERPRETATION: Our findings from whole blood and patient-derived fibroblasts suggest SMA type 3 patients have decreased expression of actin cytoskeleton regulators. These observations provide new insights and potential therapeutic targets for SMA patients with longstanding denervation and secondary musculoskeletal pathophysiology.

Neurofilament light chain in serum of adolescent and adult SMA patients under treatment with nusinersen.

OBJECTIVE: To determine the diagnostic and monitoring value of serum neurofilament light chain (NfL) in spinal muscular atrophy (SMA). METHODS: We measured serum NfL in 46 SMA patients at baseline and over 14 months of treatment with the antisense-oligonucleotide (ASO) nusinersen using the ultrasensitive single molecule array (Simoa) technology. Serum NfL levels of SMA patients were compared to controls and related to cerebrospinal fluid (CSF) NfL, blood-CSF barrier function quantified by the albumin blood/CSF ratio (Qalb) and motor scores (Hammersmith Functional Motor Scale Expanded, HFMSE; Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised, ALSFRS-R). RESULTS: Serum NfL levels of SMA patients were in the range of controls (p = 0.316) and did not correlate with CSF NfL (ρ = 0.302, p = 0.142) or Qalb (ρ = - 0.160, p = 0.293). During therapy, serum NfL levels were relatively stable with notable concentration changes in single SMA patients, however, within the control range. Higher NfL levels were associated with worse motor performance in SMA (baseline: HFMSE ρ = - 0.330, p = 0.025, ALSFRS-R ρ = - 0.403, p = 0.005; after 10 months: HFMSE ρ = - 0.525, p = 0.008, ALSFRS-R ρ = - 0.537, p = 0.007), but changes in motor scores did not correlate with changes in serum NfL. CONCLUSION: Diagnostic and monitoring performance of serum NfL measurement seems to differ between SMA subtypes. Unlike to SMA type 1, in adolescent and adult SMA type 2 and 3 patients, neurodegeneration is not reflected by increased NfL levels and short-term therapeutic effects cannot be observed. Long-term follow-up has to be performed to see if even low levels of NfL might be good prognostic markers.

Detection of early nocturnal hypoventilation in neuromuscular disorders.

Objective Nocturnal hypoventilation (NH) is a complication of respiratory involvement in neuromuscular disorders (NMD) that can evolve into symptomatic daytime hypercapnia if not treated proactively with non-invasive ventilation. This study aimed to assess whether NH can be detected in the absence of other signs of nocturnal altered gas exchange. Methods We performed nocturnal transcutaneous coupled (tc) pCO2/SpO2 monitoring in 46 consecutive cases of paediatric-onset NMD with a restrictive respiratory defect (forced vital capacity < 60%). Nocturnal hypoventilation was defined as tcPCO2 > 50 mmHg for > 25% of recorded time, and hypoxemia as tcSpO2 < 88% for > 5 minutes. Daytime symptoms and bicarbonate were recorded after overnight monitoring. Results Twenty-nine of 46 consecutive patients showed NH. Twenty-three patients did not have nocturnal hypoxemia and 18 were clinically asymptomatic. In 20 patients, PaCO2 in daytime blood samples was normal. Finally, 13/29 patients with NH had isolated nocturnal hypercapnia without nocturnal hypoxia, clinical NH symptoms, or daytime hypercapnia. Conclusions Paediatric patients with NMD can develop NH in the absence of clinical symptoms or significant nocturnal desaturation. Therefore, monitoring of NH should be included among nocturnal respiratory assessments of these patients as an additional tool to determine when to commence non-invasive ventilation.

Natural history of infantile-onset spinal muscular atrophy.

OBJECTIVE: Infantile-onset spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality, typically resulting in death preceding age 2. Clinical trials in this population require an understanding of disease progression and identification of meaningful biomarkers to hasten therapeutic development and predict outcomes. METHODS: A longitudinal, multicenter, prospective natural history study enrolled 26 SMA infants and 27 control infants aged <6 months. Recruitment occurred at 14 centers over 21 months within the NINDS-sponsored NeuroNEXT (National Network for Excellence in Neuroscience Clinical Trials) Network. Infant motor function scales (Test of Infant Motor Performance Screening Items [TIMPSI], The Children's Hospital of Philadelphia Infant Test for Neuromuscular Disorders, and Alberta Infant Motor Score) and putative physiological and molecular biomarkers were assessed preceding age 6 months and at 6, 9, 12, 18, and 24 months with progression, correlations between motor function and biomarkers, and hazard ratios analyzed. RESULTS: Motor function scores (MFS) and compound muscle action potential (CMAP) decreased rapidly in SMA infants, whereas MFS in all healthy infants rapidly increased. Correlations were identified between TIMPSI and CMAP in SMA infants. TIMPSI at first study visit was associated with risk of combined endpoint of death or permanent invasive ventilation in SMA infants. Post-hoc analysis of survival to combined endpoint in SMA infants with 2 copies of SMN2 indicated a median age of 8 months at death (95% confidence interval, 6, 17). INTERPRETATION: These data of SMA and control outcome measures delineates meaningful change in clinical trials in infantile-onset SMA. The power and utility of NeuroNEXT to provide "real-world," prospective natural history data sets to accelerate public and private drug development programs for rare disease is demonstrated. Ann Neurol 2017;82:883-891.

Serum cytokine and chemokine profiles in patients with juvenile muscular atrophy of distal upper extremity (Hirayama disease).

Juvenile muscular atrophy of the distal upper extremity (Hirayama disease) is characterized by adolescent-onset muscular weakness of the distal upper limb. Several studies showed the contribution of atopic disposition and hyperIgEaemia to the disease process, but it has not been well clarified. To identify cytokine and chemokine profiles in Hirayama disease, serum samples were analyzed using multiplex magnetic bead-based assay. Eotaxin, MCP-1 and RANTES levels were significantly higher in Hirayama disease (N=11) than in normal controls (N=12). These chemokines are associated with inflammatory cell recruitment. Allergic inflammation may involve in the pathogenesis of Hirayama disease.

Responses to Fasting and Glucose Loading in a Cohort of Well Children with Spinal Muscular Atrophy Type II.

OBJECTIVE: To examine the impact of fasting and glucose tolerance on selected metabolic variables in children with spinal muscular atrophy (SMA) type II in a well state, secondary to reports of glucose regulation abnormalities in SMA. STUDY DESIGN: In this prospective pilot study, 6 children aged 7-11 years with SMA type II participated in an oral glucose tolerance test and a supervised medical fast during 2 overnight visits at the University of Utah. At baseline, a dual-energy x-ray absorptiometry scan was performed to determine body composition. Laboratory test results were obtained at baseline and in response to the respective interventions. Data analysis was descriptive. Prefasting and postfasting data were evaluated using the Wilcoxon signed-rank test. RESULTS: Based on the dual-energy x-ray absorptiometry scan, all 6 children were variably obese at baseline. All 6 exhibited hyperinsulinemia, and 3 of 6 met formal American Diabetes Association criteria for impaired glucose tolerance. According to homeostatic insulin resistance calculations, 5 of the 6 participants were insulin-resistant. All 6 participants tolerated a monitored fast for 20 hours without hypoglycemia (blood glucose <54 mg/dL). Free fatty acid levels increased significantly from prefasting to postfasting, whereas levels of several plasma amino acids decreased significantly during fasting. CONCLUSION: Children with SMA type II defined as obese using objective variables are at increased risk for impaired glucose tolerance regardless of whether or not they visually appear obese. Further studies are needed to determine the prevalence of impaired glucose tolerance and tolerance for fasting within the broader heterogeneous SMA population and to develop appropriate guidelines for intervention.

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.

The effect of long term ventilatory support on hemodynamics in children with spinal muscle atrophy (SMA) type II.

BACKGROUND: Bi-level Positive Airway Pressure (Bi-PAP) treatment improves breathing efficiency and ventilation in children with SMA type II, but the effects of positive airway pressure swings on heart rate (HR) and blood pressure are not known. Here we studied children with SMA to determine whether Bi-PAP administered during sleep is associated with changes in hemodynamics. METHODS: Ten children aged 8-12 years on long term Bi-PAP therapy were evaluated during a routine overnight sleep study. We recorded HR, ECG, thoraco-abdominal movements and blood gases. Blood pressure was estimated indirectly from pulse transit time (PTT) and the efficiency ("work") of breathing from the phase angle between chest and abdominal movements. We compared periods of unsupported (spontaneous) and supported (i.e., on Bi-PAP) breathing during a split-night study. We also compared periods when Bi-PAP was judged optimal with periods that were sub-optimal due to mask leakage. RESULTS: HR and PTT during unsupported breathing and on optimal Bi-PAP were comparable (p=0.85 and 0.79, respectively), as were blood gases (SaO2, TcO2, TcCO2p=0.79, 0.88, 0.79, respectively). Breathing efficiency improved as expected when Bi-PAP was optimal (decrease in phase angle from 42 degrees to 22 degrees ). Sub-optimal Bi-PAP due to air leaking from the mask was associated with marked increases in breath-to-breath variability of HR, PTT and phase angle. CONCLUSIONS: Bi-PAP therapy does not appear to adversely influence hemodynamics in children with SMA if pressures are optimized and the mask is correctly applied and sealed.

A feasibility study for the newborn screening of spinal muscular atrophy.

PURPOSE: The natural history of spinal muscular atrophy suggests that for maximum effect, therapeutics will need to be administered in the earliest phases of the disease. This will require the adoption of techniques for the genetic analysis of affected individuals at the newborn stage. Our objective was to examine the feasibility surrounding the newborn screening for spinal muscular atrophy. METHODS: We investigated the application of real-time polymerase chain reaction technology for newborn screening. A multiplex assay was designed to identify homozygous deletions in SMN1 exon 7 and validated using 266 samples with defined SMN1 and SMN2 copy numbers. Sensitivity and specificity were then evaluated as part of a newborn screening strategy using DNA from 153 blood spots. RESULTS: Real-time technology validation demonstrated correct exclusion of all normal and carrier samples, and identified the homozygous SMN1 exon 7 deletions in all 32 affected samples. In the series of blood spots, all 59 affected samples were correctly identified yielding an analytic sensitivity of 100%; 56 normal and 39 carrier samples were correctly excluded yielding an analytic specificity of 100% for this blood spot series. CONCLUSION: We demonstrate that effective molecular technology exists and that ethics may soon warrant the newborn screening of spinal muscular atrophy.

A novel cell immunoassay to measure survival of motor neurons protein in blood cells.

BACKGROUND: The motor neuron degenerative disease spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality and is caused by mutations in the survival of motor neurons (SMN) gene that reduce the expression levels of the SMN protein. A major goal of current therapeutic approaches is to increase SMN levels in SMA patients. The purpose of this study was to develop a reliable assay to measure SMN protein levels from peripheral blood samples. METHODS: We developed a novel cell immunoassay to quantitatively measure SMN levels from peripheral blood mononuclear cells (PBMCs) using a single anti-SMN antibody. RESULTS: SMN levels determined by the cell immunoassay are comparable to levels determined by Western blot, but in contrast, the immunoassay does not involve cell lysis, requires a small amount of patient material, and can be done on a large number of samples simultaneously. SMN levels from PBMCs are not influenced by cell type heterogeneity. CONCLUSION: SMN levels measured from total PBMCs provide an important snapshot of SMN protein expression, which should be a useful aid in SMA diagnosis, and a surrogate marker of efficacy of treatment in SMA clinical trials.

Autonomic dysfunction in cases of spinal muscular atrophy type 1 with long survival.

In Japan, quite a few patients with spinal muscular atrophy type 1 (SMA type 1) survive with mechanical ventilation. Since a patient with SMA type 1 and continuous artificial ventilation exhibited excessive perspiration and tachycardia, we examined the autonomic functions in three cases of SMA type 1, undergoing mechanical ventilation. Two cases exhibited the common sympathetic-vagal imbalance on R-R interval analysis involving 24-h Holter ECG recordings in addition to an abnormality in finger cold-induced vasodilatation. Furthermore, one case showed blood pressure and heart rate fluctuation with the paroxysmal elevation, and a high plasma concentration of norepinephrine during tachycardia. These findings suggest that autonomic dysfunction should be examined in SMA type 1 patients with long survival, although the pathogenesis remains to be clarified.

HyperIgEaemia in patients with juvenile muscular atrophy of the distal upper extremity (Hirayama disease).

BACKGROUND: Juvenile muscular atrophy of the distal upper extremity (Hirayama disease) is characterised by anterior horn cell loss in the lower cervical cord, presumably caused by anterior displacement of the dural sac during neck flexion. A recent report suggests that atopy and IgE may contribute to anterior horn damage. OBJECTIVE: To investigate whether IgE is a contributing factor in Hirayama disease. METHODS: Serum total IgE and allergen specific IgE were examined in 20 consecutive patients, and their correlations with clinical profiles investigated. RESULTS: Past or present history of allergy/atopy was found in only four patients (20%), but serum IgE was raised in 14 (70%). Patients with hyperIgEaemia had more severe clinical disabilities than those without (p = 0.01). In patients whose history of Hirayama disease was less than five years, serum total IgE was higher than in those with the disease for five years or more (p = 0.05). CONCLUSIONS: The results suggest that hyperIgEaemia is often associated with Hirayama disease and can facilitate its pathophysiology, particularly in the early phases of the disease. HyperIgEaemia does not appear to involve the anterior horn cells primarily.

Distal spinal muscular atrophy of upper limb (Hirayama disease) associated with high serum lead levels.

Hirayama disease causes unilateral or asymmetrical bilateral distal weakness and atrophy of upper limbs. We report a 6 1/2-year-old female with Hirayama disease and associated high serum lead levels. This report highlights the occurrence of this condition in younger children and the need to further study the role of lead in its pathophysiology.