Effect of exercise on telomere length and telomere proteins expression in mdx mice.

In Duchenne muscular dystrophy (DMD), telomere shortening has been postulated to contribute to the failure of regenerative activity promoting the premature senescence of satellite cells. The aim of the present study was to investigate the telomere length and the expression of telomeric repeat-binding factor-1 (TRF1), poly (ADP-ribose) polymerase-1 (PARP1) and mouse telomerase reverse transcriptase (MTERT) in gastrocnemius, tibialis anterior and diaphragm muscles of the murine model of DMD, the mdx mouse and whether a chronic protocol of forced exercise impacts on them. Our results confirmed a telomere shortening in mdx muscles, more evident in the diaphragm, in which exercise induced a greater shortening than in wild-type mice. Moreover, we showed for the first time in mdx an increased TRF1 and PARP1 expression and an augmented activity of MTERT, further enhanced by exercise. These results reinforce the hypothesis that a deregulation of mechanisms involved in telomere length occurs and may pave the way for the test of compounds targeting proteins modulating telomere maintenance as a novel strategy to treat dystrophinopathies.

Deletion of REXO1L1 locus in a patient with malabsorption syndrome, growth retardation, and dysmorphic features: a novel recognizable microdeletion syndrome?

BACKGROUND: Copy number variations (CNVs) can contribute to genetic variation among individuals and/or have a significant influence in causing diseases. Many studies consider new CNVs' effects on protein family evolution giving rise to gene duplicates or losses. "Unsuccessful" duplicates that remain in the genome as pseudogenes often exhibit functional roles. So, changes in gene and pseudogene number may contribute to development or act as susceptibility alleles of diseases. CASE PRESENTATION: We report a de novo heterozygous 271 Kb microdeletion at 8q21.2 region which includes the family of REXO1L genes and pseudogenes in a young man affected by global development delay, progeroid signs, and gastrointestinal anomalies. Molecular and cellular analysis showed that the REXO1L1 gene hemizygosity in a patient's fibroblasts induces genetic instability and increased apoptosis after treatment with different DNA damage-induced agents. CONCLUSIONS: The present results support the hypothesis that low copy gene number within REXO1L1 cluster could play a significant role in this complex clinical and cellular phenotype.

A Phase 1/2 Study of Flavocoxid, an Oral NF-κB Inhibitor, in Duchenne Muscular Dystrophy.

Flavocoxid is a blended extract containing baicalin and catechin with potent antioxidant and anti-inflammatory properties due to the inhibition of the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) enzymes, nuclear factor-κB (NF-κB), tumor necrosis factor (TNF)-alpha, and the mitogen-activated protein kinases (MAPKs) pathways. This phase 1/2 study was designed to assess the safety and tolerability of flavocoxid in patients with Duchenne muscular dystrophy (DMD). Thirty-four patients were recruited: 17 were treated with flavocoxid at an oral dose of 250 or 500 mg, according to body weight, for one year; 17 did not receive flavocoxid and served as controls. The treatment was well tolerated and nobody dropped out. Flavocoxid induced a significant reduction in serum interleukin (IL)-1 beta and TNF-alpha only in the group of DMD boys on add-on therapy (flavocoxid added to steroids for at least six months). The decrease in IL-1 beta was higher in younger boys. The serum H2O2 concentrations significantly decreased in patients treated with flavocoxid alone with a secondary reduction of serum glutathione peroxidase (GPx) levels, especially in younger boys. The exploratory outcome measures failed to show significant effects but there was a trend showing that the younger boys who received treatment were faster at performing the Gowers' maneuver, while the older boys who received treatment were faster at doing the 10-m walk test (10MWT). Therefore, a double-blind, placebo-controlled study for at least two/three years is warranted to verify flavocoxid as a steroid substitute or as add-on therapy to steroids.

Modulation of neuronal nitric oxide synthase and apoptosis by the isoflavone genistein in Mdx mice.

Dystrophin lack in DMD causes neuronal nitric oxide synthase (nNOS) membrane delocalization which in turn promotes functional muscle ischemia, and exacerbates muscle injury. Apoptosis and the exhaustion of muscle regenerative capacity are implicated in Duchenne muscular dystrophy (DMD) pathogenesis and therefore are relevant therapeutic targets. Genistein has been reported to have pro-proliferative effects, promoting G1/S cell phase transition through the induction of cyclin D1, and anti-apoptotic properties. We previously showed that genistein could reduce muscle necrosis and enhance regeneration with an augmented number of myogenin-positive satellite cells and myonuclei, ameliorating muscle function in mdx mice. In this study we evaluated the underlying mechanisms of genistein effect on muscle specimens of mdx and wild type mice treated for five weeks with genistein (2 mg/kg/i.p. daily) or vehicle. Western blot analysis show that genistein increased cyclin D1 and nNOS expression; and showed an antiapoptotic effect, modulating the expression of BAX and Bcl-2. Our results suggest that this isoflavone might enhance the regenerative spurt in mdx mice muscle restoring nNOS, promoting G1/S phase transition in muscle cell, and inhibiting apoptosis. Further studies with longer time treatment or using different experimental approaches are needed to better investigate the underlying mechanisms of such results.

Clinical, Molecular, and Functional Characterization of CLCN1 Mutations in Three Families with Recessive Myotonia Congenita.

Myotonia congenita (MC) is an inherited muscle disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. Both recessive (Becker's disease) or dominant (Thomsen's disease) MC are caused by mutations in the CLCN1 gene encoding the voltage-dependent chloride ClC-1 channel, which is quite exclusively expressed in skeletal muscle. More than 200 CLCN1 mutations have been associated with MC. We provide herein a detailed clinical, molecular, and functional evaluation of four patients with recessive MC belonging to three different families. Four CLCN1 variants were identified, three of which have never been characterized. The c.244A>G (p.T82A) and c.1357C>T (p.R453W) variants were each associated in compound heterozygosity with c.568GG>TC (p.G190S), for which pathogenicity is already known. The new c.809G>T (p.G270V) variant was found in the homozygous state. Patch-clamp studies of ClC-1 mutants expressed in tsA201 cells confirmed the pathogenicity of p.G270V, which greatly shifts the voltage dependence of channel activation toward positive potentials. Conversely, the mechanisms by which p.T82A and p.R453W cause the disease remained elusive, as the mutated channels behave similarly to WT. The results also suggest that p.G190S does not exert dominant-negative effects on other mutated ClC-1 subunits. Moreover, we performed a RT-PCR quantification of selected ion channels transcripts in muscle biopsies of two patients. The results suggest gene expression alteration of sodium and potassium channel subunits in myotonic muscles; if confirmed, such analysis may pave the way toward a better understanding of disease phenotype and a possible identification of new therapeutic options.

Dysregulation of the Fas/FasL system in mononuclear cells recovered from peritoneal fluid of women with endometriosis.

In endometriosis, regurgitating endometrial cells fail to undergo apoptosis and implant themselves outside the uterus, particularly in the peritoneum. We studied Fas and FasL behaviour by evaluating the percentages of mFas and mFasL-bearing mononuclear cells from peritoneal fluid, the level of Fas and FasL gene expression at both mRNA and protein levels in the same cells, and the sFas and sFasL values in peritoneal fluid of 80 endometriotic women, at four stages of disease severity. We found no variation in percentage of mFas-bearing mononuclear cells; high and unchanging levels of Fas mRNA and protein, and high and invariable sFas values. Overproduction of sFas antagonises mFas function and plays a role as a decoy in the peritoneal fluid. The mFasL-bearing mononuclear cells and protein levels decreased from the minimal to the severe stage of disease. In contrast to FasL protein, FasL mRNA was overexpressed throughout the course of the disease. sFasL values were high and increased as the disease worsened. Our results showed a non-linear ratio between FasL mRNA and FasL protein levels. Abnormally elevated FasL mRNA may be due to dysregulation in several mechanisms controlling mRNA turnover. The high level of sFasL would be expected to down-regulate FasL activity and compete with the membrane form for mFas binding. As a consequence, mFas-bearing mononuclear cells may be unable to kill and in turn, may themselves become targets for killing by FasL-expressing endometriotic cells.

Skeletal phenotype of mandibuloacral dysplasia associated with mutations in ZMPSTE24.

Mandibuloacral dysplasia (MAD) is a rare recessively inherited premature aging disease characterized by skeletal and metabolic anomalies. It is part of the spectrum of diseases called laminopathies and results from mutations in genes regulating the synthesis of the nuclear laminar protein, lamin A. Homozygous or compound heterozygous mutations in the LMNA gene, which encodes both the precursor protein prelamin A and lamin C, are the commonest cause of MAD type A. In a few cases of MAD type B, mutations have been identified in the ZMPSTE24 gene encoding a zinc metalloproteinase important in the post-translational modification of lamin A. Here we describe a new case of MAD resulting from compound heterozygote mutations in ZMPSTE24 (p.N256S/p.Y70fs). The patient had typical skeletal changes of MAD, but in addition a number of unusual skeletal features including neonatal tooth eruption, amorphous calcific deposits, submetaphyseal erosions, vertebral beaking, severe cortical osteoporosis and delayed fracture healing. Treatment with conventional doses of pamidronate improved estimated volumetric bone density in the spine but did not arrest cortical bone loss. We reviewed the literature on cases of MAD associated with proven LMNA and ZMPSTE24 mutations and found that the unusual features described above were all substantially more prevalent in patients with mutations in ZMPSTE24 than in those with LMNA mutations. We conclude that MAD associated with ZMPSTE24 mutations has a more severe phenotype than that associated with LMNA mutations--probably reflecting the greater retention of unprocessed farnesylated prelamin A in the nucleus, which is toxic to cells.