Novel variants in SPTAN1 without epilepsy: An expansion of the phenotype.

We describe two unrelated children with de novo variants in the non-erythrocytic alpha-II-spectrin (SPTAN1) gene who have hypoplastic brain structures, intellectual disability, and both fine and gross motor impairments. Using agnostic exome sequencing, we identified a nonsense variant creating a premature stop codon in exon 21 of SPTAN1, and in a second patient we identified an intronic substitution in SPTAN1 prior to exon 50 creating a new donor acceptor site. Neither of these variants has been described previously. Although some of these patients' features are consistent with the known SPTAN1 encephalopathy phenotype, these two children do not have epilepsy, in contrast to reports about nearly every other patient with heterozygous SPTAN1 variants and in all patients with a variant near the C-terminal coding region. Moreover, both children have abnormal thyroid function, which has not been previously reported in association with SPTAN1 variant. We present a detailed discussion of the clinical manifestations of these two unique SPTAN1 variants and provide evidence that both variants result in reduced mRNA expression despite different locations within the gene and clinical phenotypes. These findings expand the motor, cognitive, and behavioral spectrum of the SPTAN1-associated phenotype and invite speculation about underlying pathophysiologies.

Transcriptional deficits in oxidative phosphorylation with statin myopathy.

INTRODUCTION: Hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, or statins, are widely used drugs for hyperlipidemia and are generally well-tolerated, but the can produce skeletal muscle toxicity. The molecular mechanisms driving statin myopathy are unknown. We investigated the effects of statin treatment and eccentric (damaging) exercise on transcriptional patterns between statin myopathy (Sym; N = 9) and statin-tolerant subjects (Asym; N = 6). METHODS: Skeletal muscle biopsies were collected 6 h post-exercise at baseline and after statin treatment. Subjects performed concentric (non-damaging) exercise with one leg and concentric + eccentric exercise with the other leg using a cross-over design between time-points. RESULTS: Sym as compared with Asym demonstrated decreased skeletal muscle gene expression for oxidative phosphorylation-related and mitochondrial ribosomal protein genes before and after statin treatment with eccentric exercise. CONCLUSIONS: These results suggest that pre-existing deficiencies in energy production may contribute to the development of symptoms during statin therapy, especially when muscle is exposed to eccentric exercise.

Expression of two temporally distinct microglia-related gene clusters after spinal cord injury.

The dual role of microglia in cytotoxicity and neuroprotection is believed to depend on the specific, temporal expression of microglial-related genes. To better clarify this issue, we used high-density oligonucleotide microarrays to examine microglial gene expression after spinal cord injury (SCI) in rats. We compared expression changes at the lesion site, as well as in rostral and caudal regions after mild, moderate, or severe SCI. Using microglial-associated anchor genes, we identified two clusters with different temporal profiles. The first, induced by 4 h postinjury to peak between 4 and 24 h, included interleukin-1beta, interleukin-6, osteopontin, and calgranulin, among others. The second was induced 24 h after SCI, and peaked between 72 h and 7 days; it included C1qB, Galectin-3, and p22(phox). These two clusters showed similar expression profiles regardless of injury severity, albeit with slight decreases in expression in mild or severe injury vs. moderate injury. Expression was also decreased rostral and caudal to the lesion site. We validated the expression of selected cluster members at the mRNA and protein levels. In addition, we demonstrated that stimulation of purified microglia in culture induces expression of C1qB, Galectin-3, and p22(phox). Finally, inhibition of p22(phox) activity within microglial cultures significantly suppressed proliferation in response to stimulation, confirming that this gene is involved in microglial activation. Because microglial-related factors have been implicated both in secondary injury and recovery, identification of temporally distinct clusters of genes related to microglial activation may suggest distinct roles for these groups of factors.

Gene expression profiling of experimental traumatic spinal cord injury as a function of distance from impact site and injury severity.

Changes in gene expression contribute to pathophysiological alterations following spinal cord injury (SCI). We examined gene expression over time (4 h, 24 h, 7 days) at the impact site, as well as rostral and caudal regions, following mild, moderate, or severe contusion SCI in rats. High-density oligonucleotide microarrays were used that included approximately 27,000 genes/ESTs (Affymetrix RG-U34; A, B and C arrays), together with multiple analyses (MAS 5.0, dChip). Alterations after mild injury were relatively rapid (4 and 24 h), whereas they were delayed and prolonged after severe injury (24 h and 7 days). The number and magnitude of gene expression changes were greatest at the injury site after moderate injury and increased in rostral and caudal regions as a function of injury severity. Sham surgery resulted in expression changes that were similar to mild injury, suggesting the importance of using time-linked surgical controls as well as naive animals for these kinds of studies. Expression of many genes and ESTs was altered; these were classified functionally based on ontology. Overall representation of these functional classes varied with distance from the site of injury and injury severity, as did the individual genes that contributed to each functional class. Different clustering approaches were used to identify changes in neuronal-specific genes and several transcription factors that have not previously been associated with SCI. This study represents the most comprehensive evaluation of gene expression changes after SCI to date. The results underscore the power of microarray approaches to reveal global genomic responses as well as changes in particular gene clusters and/or families that may be important in the secondary injury cascade.

In vivo and in vitro characterization of novel neuronal plasticity factors identified following spinal cord injury.

Following spinal cord injury, there are numerous changes in gene expression that appear to contribute to either neurodegeneration or reparative processes. We utilized high density oligonucleotide microarrays to examine temporal gene profile changes after spinal cord injury in rats with the goal of identifying novel factors involved in neural plasticity. By comparing mRNA changes that were coordinately regulated over time with genes previously implicated in nerve regeneration or plasticity, we found a gene cluster whose members are involved in cell adhesion processes, synaptic plasticity, and/or cytoskeleton remodeling. This group, which included the small GTPase Rab13 and actin-binding protein Coronin 1b, showed significantly increased mRNA expression from 7-28 days after trauma. Overexpression in vitro using PC-12, neuroblastoma, and DRG neurons demonstrated that these genes enhance neurite outgrowth. Moreover, RNAi gene silencing for Coronin 1b or Rab13 in NGF-treated PC-12 cells markedly reduced neurite outgrowth. Coronin 1b and Rab13 proteins were expressed in cultured DRG neurons at the cortical cytoskeleton, and at growth cones along with the pro-plasticity/regeneration protein GAP-43. Finally, Coronin 1b and Rab13 were induced in the injured spinal cord, where they were also co-expressed with GAP-43 in neurons and axons. Modulation of these proteins may provide novel targets for facilitating restorative processes after spinal cord injury.

Neuronal plasticity after spinal cord injury: identification of a gene cluster driving neurite outgrowth.

Functional recovery after spinal cord injury (SCI) may result in part from axon outgrowth and related plasticity through coordinated changes at the molecular level. We employed microarray analysis to identify a subset of genes the expression patterns of which were temporally coregulated and correlated to functional recovery after SCI. Steady-state mRNA levels of this synchronously regulated gene cluster were depressed in both ventral and dorsal horn neurons within 24 h after injury, followed by strong re-induction during the following 2 wk, which paralleled functional recovery. The identified cluster includes neuritin, attractin, microtubule-associated protein 1a, and myelin oligodendrocyte protein genes. Transcriptional and protein regulation of this novel gene cluster was also evaluated in spinal cord tissue and in single neurons and was shown to play a role in axonal plasticity. Finally, in vitro transfection experiments in primary dorsal root ganglion cells showed that cluster members act synergistically to drive neurite outgrowth.

Effect of heat stress on promoter binding by transcription factors in the cytosol of the archaeon Methanosarcina mazeii.

Regulation of archaeal stress genes is not yet fully understood. This work is part of a research effort aimed at elucidating the molecular mechanisms of transcription initiation and regulation of the stress genes in the hsp70(dnaK) locus of the mesophilic, methanogenic archaeon Methanosarcina mazeii. The locus has the stress genes 5'-grpE-hsp70(dnaK)-hsp40(dnaJ)-3' encoding the chaperone machine components GrpE, Hsp70(DnaK), and Hsp40(DnaJ), respectively, flanked by non-heat shock inducible genes, orf16 and orf11-trkA. Thus, the M. mazeii hsp70(dnaK) locus offers the opportunity for studying heat shock and non-heat shock inducible genes side by side. The objectives of the work reported here were to develop procedures for studying basal transcription factors in the cytosol of M. mazeii and their interaction with these genes' promoters in stressed cells for comparison with unstressed counterparts. The preparation of non-radioactive DNA probes for electrophoretic mobility shift assay (EMSA), and the combination of EMSA with Western blotting for DNA-binding protein identification were standardized for this investigation. DNA probes bearing the genes' promoter regions were used for detecting and identifying DNA-binding proteins in the cytosol of unstressed and heat-shocked cells. Cytosolic TATA-binding protein (TBP) was found to bind the stress-gene promoters in both unstressed and heat-shocked cells but more strongly in the latter. Likewise, in stressed cells TBP-transcription factor B (TFB)(TFIIB) association was increased by comparison with unstressed controls. The level of cytosolic TBP assessed by its DNA-binding activity using EMSA remained unchanged during the various phases of culture growth in the absence of heat stress. The results indicate that heat stress of cells in culture modulates the level and/or the stress-gene promoter-binding activity of the M. mazeii TBP, and enhances TBP-TFB association in the cytosol and DNA binding.