Duchenne muscular Dystrophy (DMD) is an inherited disease caused by mutations in the dystrophin gene that disrupt the open reading frame, while in frame mutations result in Becker muscular dystrophy ...(BMD). Ullrich congenital muscular dystrophy (UCMD) is due to mutations affecting collagen VI genes. Specific muscle miRNAs (dystromirs) are potential non-invasive biomarkers for monitoring the outcome of therapeutic interventions and disease progression. We quantified miR-1, miR-133a,b, miR-206 and miR-31 in serum from patients with DMD, BMD, UCMD and healthy controls. MiR-1, miR-133a,b and miR-206 were upregulated in DMD, but unchanged in UCMD compared to controls. Milder DMD patients had higher levels of dystromirs than more severely affected patients. Patients with low forced vital capacity (FVC) values, indicating respiratory muscle weakness, had low levels of serum miR-1 and miR-133b. There was no significant difference in the level of the dystromirs in BMD compared to controls. We also assessed the effect of dystrophin restoration on the expression of the five dystromirs in serum of DMD patients treated systemically for 12 weeks with antisense oligomer eteplirsen that induces skipping of exon 51 in the dystrophin gene. The dystromirs were also analysed in muscle biopsies of DMD patients included in a single dose intramuscular eteplirsen clinical trial. Our analysis detected a trend towards normalization of these miRNA between the pre- and post-treatment samples of the systemic trial, which however failed to reach statistical significance. This could possibly be due to the small number of patients and the short duration of these clinical trials. Although longer term studies are needed to clarify the relationship between dystrophin restoration following therapeutic intervention and the level of circulating miRNAs, our results indicate that miR-1 and miR-133 can be considered as exploratory biomarkers for monitoring the progression of muscle weakness and indirectly the remaining muscle mass in DMD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Nav1.7 sodium channel plays an important role in pain and is also upregulated in prostate cancer. To investigate the mechanisms regulating physiological and pathophysiological Nav1.7 expression ...we identified the core promoter of this gene (
SCN9A) in the human genome.
In silico genomic analysis revealed a putative
SCN9A 5′ non-coding exon ∼
64,000 nucleotides from the translation start site, expression of which commenced at three very closely-positioned transcription initiation sites (TISs), as determined by 5′ RACE experiments. The genomic region around these TISs possesses numerous core elements of a TATA-less promoter within a well-defined CpG island. Importantly, it acted as a promoter when inserted upstream of luciferase in a fusion construct. Moreover, the activity of the promoter-luciferase construct ostensibly paralleled endogenous Nav1.7 mRNA levels
in vitro, with both increased in a quantitatively and qualitatively similar manner by numerous factors (including NGF, phorbol esters, retinoic acid, and Brn-3a transcription factor over-expression).
OBJECTIVE:To expand the clinical phenotype of autosomal dominant congenital spinal muscular atrophy with lower extremity predominance (SMA-LED) due to mutations in the dynein, cytoplasmic 1, heavy ...chain 1 (DYNC1H1) gene.
METHODS:Patients with a phenotype suggestive of a motor, non–length-dependent neuronopathy predominantly affecting the lower limbs were identified at participating neuromuscular centers and referred for targeted sequencing of DYNC1H1.
RESULTS:We report a cohort of 30 cases of SMA-LED from 16 families, carrying mutations in the tail and motor domains of DYNC1H1, including 10 novel mutations. These patients are characterized by congenital or childhood-onset lower limb wasting and weakness frequently associated with cognitive impairment. The clinical severity is variable, ranging from generalized arthrogryposis and inability to ambulate to exclusive and mild lower limb weakness. In many individuals with cognitive impairment (9/30 had cognitive impairment) who underwent brain MRI, there was an underlying structural malformation resulting in polymicrogyric appearance. The lower limb muscle MRI shows a distinctive pattern suggestive of denervation characterized by sparing and relative hypertrophy of the adductor longus and semitendinosus muscles at the thigh level, and diffuse involvement with relative sparing of the anterior-medial muscles at the calf level. Proximal muscle histopathology did not always show classic neurogenic features.
CONCLUSION:Our report expands the clinical spectrum of DYNC1H1-related SMA-LED to include generalized arthrogryposis. In addition, we report that the neurogenic peripheral pathology and the CNS neuronal migration defects are often associated, reinforcing the importance of DYNC1H1 in both central and peripheral neuronal functions.
Cranial growth and development is a complex process which affects the closely related traits of head circumference (HC) and intracranial volume (ICV). The underlying genetic influences shaping these ...traits during the transition from childhood to adulthood are little understood, but might include both age-specific genetic factors and low-frequency genetic variation. Here, we model the developmental genetic architecture of HC, showing this is genetically stable and correlated with genetic determinants of ICV. Investigating up to 46,000 children and adults of European descent, we identify association with final HC and/or final ICV + HC at 9 novel common and low-frequency loci, illustrating that genetic variation from a wide allele frequency spectrum contributes to cranial growth. The largest effects are reported for low-frequency variants within TP53, with 0.5 cm wider heads in increaser-allele carriers versus non-carriers during mid-childhood, suggesting a previously unrecognized role of TP53 transcripts in human cranial development.
We have previously shown that the Brn-3b transcription factor is subjected to post-transcriptional gene regulation by specific microRNAs (mir-23 and mir-214) in the ND7 and SHSY-5Y neuronal cell ...lines (Calissano et al., 2007). As Brn-3b plays an essential role in the survival of retinal ganglion cells in the rat (Erkman et al., 1996; Gan et al., 1996; Gan et al., 1999; Erkman et al., 2000), we wanted to investigate whether mir-23 and mir-214 are expressed and target Brn-3b mRNA in a retinal ganglion cell line (RGC-5) thus potentially killing the cells expressing it.
Here we show that, possibly due to its pro-survival role, Brn-3b is protected from degradation by microRNAs in RGC-5 cells in contrast to its fate in other cell types. This seems to be accomplished by i) the lack of expression of one of the two microRNAs targeting its 3′UTR and by ii) the requirement of at least two distinct microRNAs to mediate its down-regulation in retinal ganglion cells.
We speculate that this mechanism could have a widespread role in the regulation of mRNAs encoding for essential proteins.
Purpose
Duchenne muscular dystrophy (DMD) is a severe and fatal neuromuscular disease. With the current developments on novel therapeutic strategies for DMD, the need to carefully monitor disease ...progression or regression upon treatment using molecular markers has become urgent.
Experimental design
2D LC protein fractionation was performed on patient serum samples, followed by LC‐MS/MS‐based identifications with label‐free quantifications.
Results
Protein signatures were compared between patients and healthy (child and adult) controls and between ambulant and nonambulant patients. Various myofibrillar proteins demonstrated differences between DMD patients and controls, likely due to leakiness and breakdown of muscle fibers. Previously reported biomarkers, such as muscle‐derived titin, myosin, and carbonic anhydrase I (CA1), were verified. MS‐based results were compared with ELISA for vitamin D binding protein (GC), fibulin‐1 (FBLN1), gelsolin (GSN), and carbonic anhydrase 1 (CA1).
Conclusions and clinical relevance
The combined results of MS‐ and ELISA‐based quantifications indicated more studies are needed to validate this serum protein signature for DMD patients. With these data promising candidate biomarkers have been identified for a rare genetic disease using serum proteome analysis.
Abstract The POU family transcription factor Brn-3a is required for the differentiation and survival of sensory neurones, and is phosphorylated in neuroblastoma cells following treatment with ...all-trans retinoic acid (RA). Mutation of serines-121 and -122 of Brn-3a to alanine blocks its phosphorylation and impairs RA-mediated neurite outgrowth. Here we show that this deficit in differentiation is mimicked by a single mutation at serine-122, and demonstrate a similar requirement for a second residue, threonine-39. Like Brn-3a, the neuropeptide Galanin has been implicated in the development of sensory neurones. We show that Brn-3a over-expression acts synergistically with RA treatment to up-regulate Galanin promoter activity; that the activity of the N-terminal transcriptional activation domain of Brn-3a is increased following RA treatment; and that both these effects require threonine-39 and serine-122. In addition, we demonstrate that the RA-mediated activation of Galanin promoter activity and Brn-3a N-terminal transcriptional activity are both blocked by pan-MEK inhibitors, and show that the expression of a constitutively-active mutant of MEK1, but not MEK5, is sufficient to increase Brn-3a activity. These results reveal an important role for the ERK1/2 pathway in Brn-3a regulation during RA-mediated neuronal differentiation and define the neuropeptide Galanin as a novel target of this transcription factor.
The post-transcriptional control of mRNA levels is a very powerful mechanism which allows cells to quickly change the amount of specific proteins. In this study, we wanted to analyze whether the ...Brn-3b transcription factor, essential for the proper development of mouse retinal ganglion cells, is subjected to such post-transcriptional regulation. In particular, due to its conservation amongst different species, we wanted to study the role of its 3′ untranslated region (3′UTR).
We show that the 3′UTR of the Brn-3b mRNA does indeed contain regulatory sequences that mediate mRNA degradation upon serum starvation-induced differentiation of ND7 neuroblastoma cells. The specific region mediating this effect has been characterized and two different microRNAs that potentially regulate the stability of Brn-3b have been identified. Moreover we show that Dicer, one of the key enzymes in the production of microRNAs, is strongly up-regulated in ND7 cells subjected to differentiation.
Brn-3a is a transcription factor expressed in a subset of neurons of the peripheral nervous system. Its role encompasses the activation of genes involved in neuronal differentiation and survival. ...While a lot of data have been produced on Brn-3a target promoters, very little is known about the upstream regulatory signals that mediate its activation in response to differentiation. In this work, we describe for the first time that Brn-3a is phosphorylated in IMR-32 neuroblastoma cells in response to differentiation induced by retinoic acid treatment and that its post-translational modification is potentially mediated by the activation of the MAPK/ERK pathway. Furthermore, we show that the mutation of a putative phosphorylated amino acid strongly reduces the ability of Brn-3a to mediate the differentiation of IMR-32 cells.
PDF
