Intragenic and structural variation in the SMN locus and clinical variability in spinal muscular atrophy.

Clinical severity and treatment response vary significantly between patients with spinal muscular atrophy. The approval of therapies and the emergence of neonatal screening programmes urgently require a more detailed understanding of the genetic variants that underlie this clinical heterogeneity. We systematically investigated genetic variation other than SMN2 copy number in the SMN locus. Data were collected through our single-centre, population-based study on spinal muscular atrophy in the Netherlands, including 286 children and adults with spinal muscular atrophy Types 1-4, including 56 patients from 25 families with multiple siblings with spinal muscular atrophy. We combined multiplex ligation-dependent probe amplification, Sanger sequencing, multiplexed targeted resequencing and digital droplet polymerase chain reaction to determine sequence and expression variation in the SMN locus. SMN1, SMN2 and NAIP gene copy number were determined by multiplex ligation-dependent probe amplification. SMN2 gene variant analysis was performed using Sanger sequencing and RNA expression analysis of SMN by droplet digital polymerase chain reaction. We identified SMN1-SMN2 hybrid genes in 10% of spinal muscular atrophy patients, including partial gene deletions, duplications or conversions within SMN1 and SMN2 genes. This indicates that SMN2 copies can vary structurally between patients, implicating an important novel level of genetic variability in spinal muscular atrophy. Sequence analysis revealed six exonic and four intronic SMN2 variants, which were associated with disease severity in individual cases. There are no indications that NAIP1 gene copy number or sequence variants add value in addition to SMN2 copies in predicting the clinical phenotype in individual patients with spinal muscular atrophy. Importantly, 95% of spinal muscular atrophy siblings in our study had equal SMN2 copy numbers and structural changes (e.g. hybrid genes), but 60% presented with a different spinal muscular atrophy type, indicating the likely presence of further inter- and intragenic variabilities inside as well as outside the SMN locus. SMN2 gene copies can be structurally different, resulting in inter- and intra-individual differences in the composition of SMN1 and SMN2 gene copies. This adds another layer of complexity to the genetics that underlie spinal muscular atrophy and should be considered in current genetic diagnosis and counselling practices.

Analysis of FUS, PFN2, TDP-43, and PLS3 as potential disease severity modifiers in spinal muscular atrophy.

OBJECTIVE: To investigate mutations in genes that are potential modifiers of spinal muscular atrophy (SMA) severity. METHODS: We performed a hypothesis-based search into the presence of variants in fused in sarcoma (FUS), transactive response DNA-binding protein 43 (TDP-43), plastin 3 (PLS3), and profilin 2 (PFN2) in a cohort of 153 patients with SMA types 1-4, including 19 families. Variants were detected with targeted next-generation sequencing and confirmed with Sanger sequencing. Functional effects of the identified variants were analyzed in silico and for PLS3, by analyzing expression levels in peripheral blood. RESULTS: We identified 2 exonic variants in FUS exons 5 and 6 (p.R216C and p.S135N) in 2 unrelated patients, but clinical effects were not evident. We identified 8 intronic variants in PLS3 in 33 patients. Five PLS3 variants (c.1511+82T>C; c.748+130 G>A; c.367+182C>T; c.891-25T>C (rs145269469); c.1355+17A>G (rs150802596)) potentially alter exonic splice silencer or exonic splice enhancer sites. The variant c.367+182C>T, but not RNA expression levels, corresponded with a more severe phenotype in 1 family. However, this variant or level of PLS3 expression did not consistently correspond with a milder or more severe phenotype in other families or the overall cohort. We found 3 heterozygous, intronic variants in PFN2 and TDP-43 with no correlation with clinical phenotype or effects on splicing. CONCLUSIONS: PLS3 and FUS sequence variants do not modify SMA severity at the population level. Specific variants in individual patients or families do not consistently correlate with disease severity.

A Comparative Study of SMN Protein and mRNA in Blood and Fibroblasts in Patients with Spinal Muscular Atrophy and Healthy Controls.

BACKGROUND: Clinical trials to test safety and efficacy of drugs for patients with spinal muscular atrophy (SMA) are currently underway. Biomarkers that document treatment-induced effects are needed because disease progression in childhood forms of SMA is slow and clinical outcome measures may lack sensitivity to detect meaningful changes in motor function in the period of 1-2 years of follow-up during randomized clinical trials. OBJECTIVE: To determine and compare SMN protein and mRNA levels in two cell types (i.e. PBMCs and skin-derived fibroblasts) from patients with SMA types 1-4 and healthy controls in relation to clinical characteristics and SMN2 copy numbers. MATERIALS AND METHODS: We determined SMN1, SMN2-full length (SMN2-FL), SMN2-delta7 (SMN2-Δ7), GAPDH and 18S mRNA levels and SMN protein levels in blood and fibroblasts from a total of 150 patients with SMA and 293 healthy controls using qPCR and ELISA. We analyzed the association with clinical characteristics including disease severity and duration, and SMN2 copy number. RESULTS: SMN protein levels in PBMCs and fibroblasts were higher in controls than in patients with SMA (p<0.01). Stratification for SMA type did not show differences in SMN protein (p>0.1) or mRNA levels (p>0.05) in either cell type. SMN2 copy number was associated with SMN protein levels in fibroblasts (p = 0.01), but not in PBMCs (p = 0.06). Protein levels in PBMCs declined with age in patients (p<0.01) and controls (p<0.01)(power 1-beta = 0.7). Ratios of SMN2-Δ7/SMN2-FL showed a broad range, primarily explained by the variation in SMN2-Δ7 levels, even in patients with a comparable SMN2 copy number. Levels of SMN2 mRNA did not correlate with SMN2 copy number, SMA type or age in blood (p = 0.7) or fibroblasts (p = 0.09). Paired analysis between blood and fibroblasts did not show a correlation between the two different tissues with respect to the SMN protein or mRNA levels. CONCLUSIONS: SMN protein levels differ considerably between tissues and activity is age dependent in patients and controls. SMN protein levels in fibroblasts correlate with SMN2 copy number and have potential as a biomarker for disease severity.

Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways.

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins. Interactomes of WT and mutant ALS proteins were very similar except for OPTN and UBQLN2, in which mutations caused loss or gain of protein interactions. Several of the identified interactomes showed a high degree of overlap: shared binding partners of ATXN2, FUS and TDP-43 had roles in RNA metabolism; OPTN- and UBQLN2-interacting proteins were related to protein degradation and protein transport, and C9orf72 interactors function in mitochondria. To confirm that this overlap is important for ALS pathogenesis, we studied fragile X mental retardation protein (FMRP), one of the common interactors of ATXN2, FUS and TDP-43, in more detail in in vitro and in vivo model systems for FUS ALS. FMRP localized to mutant FUS-containing aggregates in spinal motor neurons and bound endogenous FUS in a direct and RNA-sensitive manner. Furthermore, defects in synaptic FMRP mRNA target expression, neuromuscular junction integrity, and motor behavior caused by mutant FUS in zebrafish embryos, could be rescued by exogenous FMRP expression. Together, these results show that interactomics analysis can provide crucial insight into ALS disease mechanisms and they link FMRP to motor neuron dysfunction caused by FUS mutations.

Quantification of SMN protein in leucocytes from spinal muscular atrophy patients: effects of treatment with valproic acid.

BACKGROUND: Spinal muscular atrophy (SMA) is caused by the homozygous deletion of the survival motor neuron (SMN)1 gene. The nearly identical SMN2 gene produces small amounts of full-length mRNA and functional SMN protein, due to a point mutation in a critical splicing site. Increasing SMN protein production by histone deacetylase inhibiting drugs such as valproic acid (VPA) is an experimental treatment strategy for SMA. OBJECTIVE: To investigate whether an SMN-specific ELISA could detect changes in SMN protein expression in peripheral blood mononuclear cells (PBMCs) after treatment with VPA. METHODS: The authors developed a sensitive SMN-specific ELISA. Six patients with SMA types 2 and 3 participated in the study. Recombinant SMN calibration curves were used to calculate SMN protein levels in PBMCs before and after 4 months of VPA treatment. RESULTS: The SMN ELISA was able to detect small differences in SMN protein concentrations, and differences in SMN protein levels in Epstein-Barr virus immortalised lymphocyte cell lines from SMA type 1 and 2 patients, carriers and healthy individuals (p<0.05). The mean SMN protein level in PBMCs from SMA patients was 22% (SD 15%) of the value in a healthy control. VPA treatment resulted in significantly increased SMN protein levels in five out of six SMA patients compared with baseline values (p<0.05), but did not restore SMN levels to normal values. CONCLUSIONS: SMN protein quantification by this SMN ELISA is a useful additional tool for evaluating the effects of experimental treatment in SMA.

Down-regulation of GRK2 after oxygen and glucose deprivation in rat hippocampal slices: role of the PI3-kinase pathway.

G protein-coupled receptor kinase 2 (GRK2) modulates G protein-coupled receptor desensitization and signaling. We previously described down-regulation of GRK2 expression in vivo in rat neonatal brain following hypoxia-ischemia. In this study, we investigated the molecular mechanisms involved in GRK2 down-regulation, using organotypic cultures of neonatal rat hippocampal slices exposed to oxygen and glucose deprivation (OGD). We observed a 40% decrease in GRK2 expression 4 h post-OGD. No changes in GRK2 protein occurred after exposure of hippocampal slices to glucose deprivation only. No significant alterations in GRK2 mRNA expression were detected, suggesting a post-transcriptional effect of OGD on GRK2 expression. Blockade of the proteasome pathway by MG132 prevented OGD-induced decrease of GRK2. It has been shown that extracellular signal-regulated kinase-dependent phosphorylation of GRK2 at Ser670 triggers its turnover via the proteasome pathway. However, despite a significant increase of pSer670-GRK2 after OGD, inhibition of the extracellular signal-regulated kinase pathway by PD98059 did neither prevent the hypoxia-ischemia-induced increase in pSer670-GRK2 nor the down-regulation of GRK2 protein. Interestingly, inhibition of phosphoinositide-3-kinase with wortmannin inhibits both OGD-induced phosphorylation of GRK2 on Ser670 and the GRK2 decrease. In conclusion, OGD-induced phosphoinositide-3-kinase-dependent phosphorylation of GRK2 on Ser670 is a novel mechanism leading to down-regulation of GRK2 protein via a proteasome-dependent pathway.

A phosphatidylinositol transfer protein alpha-dependent survival factor protects cultured primary neurons against serum deprivation-induced cell death.

Selective neuronal loss is a prominent feature in both acute and chronic neurological disorders. Recently, a link between neurodegeneration and a deficiency in the lipid transport protein phosphatidylinositol transfer protein alpha (PI-TPalpha) has been demonstrated. In this context it may be of importance that fibroblasts overexpressing PI-TPalpha are known to produce and secrete bioactive survival factors that protect fibroblasts against UV-induced apoptosis. In the present study it was investigated whether the conditioned medium of cells overexpressing PI-TPalpha (CMalpha) has neuroprotective effects on primary neurons in culture. We show that CMalpha is capable of protecting primary, spinal cord-derived motor neurons from serum deprivation-induced cell death. Since the conditioned medium of wild-type cells was much less effective, we infer that the neuroprotective effect of CMalpha is linked (in part) to the PI-TPalpha-dependent production of arachidonic acid metabolites. The neuroprotective activity of CMalpha is partly inhibited by suramin, a broad-spectrum antagonist of G-protein coupled receptors. Western blot analysis shows that brain cortex and spinal cord express relatively high levels of PI-TPalpha, suggesting that the survival factor may be produced in neuronal tissue. We propose that the bioactive survival factor is implicated in neuronal survival. If so, PI-TPalpha could be a promising target to be evaluated in studies on the prevention and treatment of neurological disorders.