Search

Contains fulltext : 229735.pdf (Publisher’s version ) (Closed access) BACKGROUND AND PURPOSE: The aim was to define the radiological picture of facioscapulohumeral muscular dystrophy 2 (FSHD2) in comparison with FSHD1 and to explore correlations between imaging and clinical/molecular data. METHODS: Upper girdle and/or lower limb muscle magnetic resonance imaging scans of 34 molecularly confirmed FSHD2 patients from nine European neuromuscular centres were analysed. T1-weighted and short-tau inversion recovery (STIR) sequences were used to evaluate the global pattern and to assess the extent of fatty replacement and muscle oedema. RESULTS: The most frequently affected muscles were obliquus and transversus abdominis, semimembranosus, soleus and gluteus minimus in the lower limbs; trapezius, serratus anterior, latissimus dorsi and pectoralis major in the upper girdle. Iliopsoas, popliteus, obturator internus and tibialis posterior in the lower limbs and subscapularis, spinati, sternocleidomastoid and levator scapulae in the upper girdle were the most spared. Asymmetry and STIR hyperintensities were consistent features. The pattern of muscle involvement was similar to that of FSHD1, and the combined involvement of trapezius, abdominal and hamstring muscles, together with complete sparing of iliopsoas and subscapularis, was detected in 91% of patients. Peculiar differences were identified in a rostro-caudal gradient, a predominant involvement of lower limb muscles compared to the upper girdle, and in the higher percentage of STIR hyperintensities in FSHD2. CONCLUSION: This multicentre study defines the pattern of muscle involvement in FSHD2, providing useful information for diagnostics and clinical trial design. Both similarities and differences between FSHD1 and FSHD2 were detected, which is also relevant to better understand the pathogenic mechanisms underlying the FSHD-related disease spectrum.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is characterized by extreme variability in symptoms, with females being less severely affected than males and presenting a higher proportion of asymptomatic carriers. The sex-related factors involved in the disease are not known. Here, we have utilized myoblasts isolated from FSHD patients (FSHD myoblasts) to investigate the effect of estrogens on muscle properties. Our results demonstrated that estrogens counteract the differentiation impairment of FSHD myoblasts without affecting cell proliferation or survival. Estrogen effects are mediated by estrogen receptor beta (ERbeta), which reduces chromatin occupancy and transcriptional activity of double homeobox 4 (DUX4), a protein whose aberrant expression has been implicated in FSHD pathogenesis. During myoblast differentiation, we observed that the levels and activity of DUX4 increased progressively and were associated with its enhanced recruitment in the nucleus. ERbeta interfered with this recruitment by relocalizing DUX4 in the cytoplasm. This work identifies estrogens as a potential disease modifier that underlie sex-related differences in FSHD by protecting against myoblast differentiation impairments in this disease.

Background Facioscapulohumeral muscular dystrophy is associated with an epigenetic defect on 4q subtelomere. This dystrophy is caused by contraction of the D4Z4 macrosatellite array on chromosome 4qter in FSHD1, or by functional impairment of SMCHD1, a chromatin modifier binding to D4Z4, in FSHD2. Both genetic defects lead to D4Z4 DNA hypomethylation associated with inappropriate expression in skeletal muscle of the D4Z4-encoded DUX4 transcription factor in the presence of a polymorphic polyadenylation signal (PAS) distal to the last D4Z4 unit (4qA). Currently, hypomethylation is not diagnostic per se because of the interference of non-pathogenic arrays and because it does not interrogate the presence of DUX4-PAS. Importantly, the methylation status of the DUX4-PAS critical region has not been thoroughly investigated. Methods We investigated, by bisulfite sequencing, the DNA methylation levels of the region distal to the D4Z4 array critical to FSHD development in PAS-positive alleles. Results Comparison of FSHD1, FSHD2 and control subjects showed highly significant differences of methylation levels in all CpGs tested. Noteworthy, one of these CpGs (CpG6) is able to discriminate the affected individuals with a sensitivity of 0.95 in a fully characterized samples, supporting the potential usefulness of this assay for FSHD diagnosis. Moreover, our study evidenced a relationship between PAS-specific methylation and severity of the disease. Conclusions These data point to CpGs distal to the D4Z4 array as a critical region that summarizes multiple factors affecting epigenetics of FSHD. Additionally, methylation analysis of this region allows the establishment of a rapid and sensitive tool for FSHD diagnosis

ARCHAEA-ExPRESs is an mRNA modification technology that makes use of components derived from the Archaeon Methanocaldococcus jannaschii, namely the tRNA splicing endonuclease (MJ-EndA) and its natural substrate, the bulge-helix-bulge (BHB) structure (1). These components can perform both cis- and trans-splicing in cellular and animal models and may provide a convenient way to modulate gene expression using components independent of cellular regulatory networks. To use MJ-EndA in stable expression mammalian systems, we developed variants characterized by high efficiency and sustainable in vivo activity. The MJ-EndA variants were created by the introduction of proper localization signals followed by mutagenesis and direct selection in mammalian cells. Of note, enzyme selection used an in vivo selection method based on puromycin resistance conferred to cells by BHB-mediated intron splicing from an out-of-frame puromycin N-acetyl transferase (PAC) gene. This approach yielded several endonuclease variants, the best of which showed 40-fold higher activity compared to the parental enzyme and stable processing of 30% of the target mRNA. Notably, these variants showed complete compatibility with long-term expression in mammalian cells, suggesting that they may be usefully applied in functional genomics and genetically modified animal models.

Using precursor tRNA molecules to study RNA-protein interactions, we have identified an RNA motif recognized by eukaryotic RNase P (EC 3.1.26.5). Analysis of circularly permuted precursors indicates that interruptions in the sugar-phosphate backbone are not tolerated in the acceptor stem, in the T stem-loop, or between residues A-9 and G-10. Prokaryotic RNase P will function with a minihelix consisting of the acceptor stem connected directly to the T stem-loop. Eukaryotic RNase P cannot use such a minimal substrate unless a linker sequence is added in the gap where the D stem and anticodon stem-loop were deleted.

Utilizing a procedure for the purification of RNase P from Xenopus laevis germinal vesicle (GV) extracts, according to which the contamination by a large, cytoplasmic, cylindrical structure (1) is avoided, we demonstrate that the X.laevis enzyme, like the HeLa RNase P, is precipitated by anti-Th antibodies and an RNA molecule (XL RNA), 320 nucleotides long, copurifies with the activity. The sequence of XL RNA is 60% homologous to HeLa H1 RNA, therefore the two molecules seem related.

Investigation of the mechanism of cleavage site selection by Xenopus RNAase P reveals that the acceptor stem, a 7 bp helix common to all tRNA precursors, is required for cleavage. We propose that Xenopus RNAase P recognizes conserved features of the mature tRNA and that the cleavage site is selected by measuring the length of the acceptor stem. In support of this, we demonstrate that insertion of 2 bp in the acceptor stem of yeast pre-tRNA(3Leu) relocates the cleavage site 2 bases 3' to the original one. In addition, insertion of 1 bp in the acceptor stem of the end-matured yeast pre-tRNA(Phe) generates an RNAase P cleavage site: the enzyme produces a mature tRNA with the characteristic 7 bp stem and releases one 5' flanking nucleotide. Since it has previously been shown that cleavage sites of the splicing endonuclease are determined by the length of the anticodon stem, RNAase P and the splicing endonuclease apparently use different stems to determine their cutting sites.