Complete sequencing of the SMN2 gene in SMA patients detects SMN gene deletion junctions and variants in SMN2 that modify the SMA phenotype.

Spinal muscular atrophy (SMA) is a progressive motor neuron disease caused by loss or mutation of the survival motor neuron 1 (SMN1) gene and retention of SMN2. We performed targeted capture and sequencing of the SMN2, CFTR, and PLS3 genes in 217 SMA patients. We identified a 6.3 kilobase deletion that occurred in both SMN1 and SMN2 (SMN1/2) and removed exons 7 and 8. The deletion junction was flanked by a 21 bp repeat that occurred 15 times in the SMN1/2 gene. We screened for its presence in 466 individuals with the known SMN1 and SMN2 copy numbers. In individuals with 1 SMN1 and 0 SMN2 copies, the deletion occurred in 63% of cases. We modeled the deletion junction frequency and determined that the deletion occurred in both SMN1 and SMN2. We have identified the first deletion junction where the deletion removes exons 7 and 8 of SMN1/2. As it occurred in SMN1, it is a pathogenic mutation. We called variants in the PLS3 and SMN2 genes, and tested for association with mild or severe exception patients. The variants A-44G, A-549G, and C-1897T in intron 6 of SMN2 were significantly associated with mild exception patients, but no PLS3 variants correlated with severity. The variants occurred in 14 out of 58 of our mild exception patients, indicating that mild exception patients with an intact SMN2 gene and without modifying variants occur. This sample set can be used in the association analysis of candidate genes outside of SMN2 that modify the SMA phenotype.

Serum creatinine is a biomarker of progressive denervation in spinal muscular atrophy.

OBJECTIVE: Identifying simple biomarkers that can predict or track disease progression in patients with spinal muscular atrophy (SMA) remains an unmet clinical need. To test the hypothesis that serum creatinine (Crn) could be a prognostic biomarker for monitoring progression of denervation in patients with SMA, we determined whether serum Crn concentration correlates with disease severity in patients with SMA. METHODS: We examined a cohort of 238 patients with SMA with 1,130 Crn observations between 2000 and 2016. Analyses were corrected for age, and 156 patients with SMA had dual-energy x-ray absorptiometry data available for correction for lean mass. We investigated the relationship between Crn and SMA type, survival motor neuron 2 (SMN2) copies, and Hammersmith Functional Motor Scale (HFMS) score as primary outcomes. In addition, we tested for associations between Crn and maximum ulnar compound muscle action potential amplitude (CMAP) and motor unit number estimation (MUNE). RESULTS: Patients with SMA type 3 had 2.2-fold (95% confidence interval [CI] 1.93-2.49; p < 0.0001) higher Crn levels compared to those with SMA type 1 and 1.7-fold (95% CI 1.52-1.82; p < 0.0001) higher Crn levels compared to patients with SMA type 2. Patients with SMA type 2 had 1.4-fold (95% CI 1.31-1.58; p < 0.0001) higher Crn levels than patients with SMA type 1. Patients with SMA with 4 SMN2 copies had 1.8-fold (95% CI 1.57-2.11; p < 0.0001) higher Crn levels compared to patients with SMA with 2 SMN2 copies and 1.4-fold (95% CI 1.24-1.58; p < 0.0001) higher Crn levels compared to patients with SMA with 3 SMN2 copies. Patients with SMA with 3 SMN2 copies had 1.4-fold (95% CI 1.21-1.56; p < 0.0001) higher Crn levels than patients with SMA with 2 SMN2 copies. Mixed-effect model revealed significant differences in Crn levels among walkers, sitters, and nonsitters (p < 0.0001) and positive associations between Crn and maximum CMAP (p < 0.0001) and between Crn and MUNE (p < 0.0001). After correction for lean mass, there were still significant associations between Crn and SMA type, SMN2 copies, HFMS, CMAP, and MUNE. CONCLUSIONS: These findings indicate that decreased Crn levels reflect disease severity, suggesting that Crn is a candidate biomarker for SMA progression. We conclude that Crn measurements should be included in the routine analysis of all patients with SMA. In future studies, it will be important to determine whether Crn levels respond to molecular and gene therapies.

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.

Impaired kidney structure and function in spinal muscular atrophy.

OBJECTIVE: To determine changes in serum profiles and kidney tissues from patients with spinal muscular atrophy (SMA) type 1 compared with age- and sex-matched controls. METHODS: In this cohort study, we investigated renal structure and function in infants and children with SMA type 1 in comparison with age- and sex-matched controls. RESULTS: Patients with SMA had alterations in serum creatinine, cystatin C, sodium, glucose, and calcium concentrations, granular casts and crystals in urine, and nephrocalcinosis and fibrosis. Nephrotoxicity and polycystic kidney disease PCR arrays revealed multiple differentially expressed genes, and immunoblot analysis showed decreased calcium-sensing receptors and calbindin and increased insulin-like growth factor-binding proteins in kidneys from patients with SMA. CONCLUSIONS: These findings demonstrate that patients with SMA type 1, in the absence of disease-modifying therapies, frequently manifest impaired renal function as a primary or secondary consequence of their disease. This study provides new insights into systemic contributions to SMA disease pathogenesis and the need to identify coadjuvant therapies.

Transcriptomic Analysis of Octanoic Acid Response in Drosophila sechellia Using RNA-Sequencing.

The dietary specialist fruit fly Drosophila sechellia has evolved to specialize on the toxic fruit of its host plant Morinda citrifolia Toxicity of Morinda fruit is primarily due to high levels of octanoic acid (OA). Using RNA interference (RNAi), prior work found that knockdown of Osiris family genes Osiris 6 (Osi6), Osi7, and Osi8 led to increased susceptibility to OA in adult D. melanogaster flies, likely representing genes underlying a Quantitative Trait Locus (QTL) for OA resistance in D. sechellia While genes in this major effect locus are beginning to be revealed, prior work has shown at least five regions of the genome contribute to OA resistance. Here, we identify new candidate OA resistance genes by performing differential gene expression analysis using RNA-sequencing (RNA-seq) on control and OA-exposed D. sechellia flies. We found 104 significantly differentially expressed genes with annotated orthologs in D. melanogaster, including six Osiris gene family members, consistent with previous functional studies and gene expression analyses. Gene ontology (GO) term enrichment showed significant enrichment for cuticle development in upregulated genes and significant enrichment of immune and defense responses in downregulated genes, suggesting important aspects of the physiology of D. sechellia that may play a role in OA resistance. In addition, we identified five candidate OA resistance genes that potentially underlie QTL peaks outside of the major effect region, representing promising new candidate genes for future functional studies.