The genetic cause of some familial nonsyndromic renal cell carcinomas (RCC) defined by at least two affected first-degree relatives is unknown. By combining whole-exome sequencing and tumor profiling in a family prone to cases of RCC, we identified a germline BAP1 mutation c.277A>G (p.Thr93Ala) as the probable genetic basis of RCC predisposition. This mutation segregated with all four RCC-affected relatives. Furthermore, BAP1 was found to be inactivated in RCC-affected individuals from this family. No BAP1 mutations were identified in 32 familial cases presenting with only RCC. We then screened for germline BAP1 deleterious mutations in familial aggregations of cancers within the spectrum of the recently described BAP1-associated tumor predisposition syndrome, including uveal melanoma, malignant pleural mesothelioma, and cutaneous melanoma. Among the 11 families that included individuals identified as carrying germline deleterious BAP1 mutations, 6 families presented with 9 RCC-affected individuals, demonstrating a significantly increased risk for RCC. This strongly argues that RCC belongs to the BAP1 syndrome and that BAP1 is a RCC-predisposition gene.

and †Research Unit on Cellular Biology (URBC), University of Namur (FUNDP), rue de Bruxelles 61, B-5000 Namur, BelgiumBackground information. Aging of human skeletal muscle results in a decline in muscle mass and force, and excessive turnover of muscle fibres, such as in muscular dystrophies, further increases this decline. Although it has been shown in rodents, by cross-age transplantation of whole muscles, that the environment plays an important role in this process, the implication of proliferating aging of the muscle progenitors has been poorly investigated, particularly in humans, since the regulation of cell proliferation differs between rodents and humans. The myogenic differentiation of human myoblasts is regulated by the muscle-specific regulatory factors. Cross-talk between the muscle-specific regulatory factors and the cell cycle regulators is essential for differentiation. The aim of the present study was to determine the effects of replicative senescence on the myogenic programme of human myoblasts.Results. We showed that senescent myoblasts, which could not re-enter the cell cycle, are still able to differentiate and form multinucleated myotubes. However, these myotubes are significantly smaller. The expression of musclespecific regulatory factors and cell cycle regulators was analysed in proliferating myoblasts and compared with senescent cells. We have observed a delay and a decrease in the muscle-specific regulatory factors and the cyclin-dependent kinase inhibitor p57 during the early step of differentiation in senescent myoblasts, as well as an increase in the fibroblastic markers.Conclusions. Our results demonstrate that replicative senescence alters the expression of the factors triggering muscle differentiation in human myoblasts and could play a role in the regenerative defects observed in muscular diseases and during normal skeletal-muscle aging.

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A CTG repeat amplification is responsible for the dominantly inherited neuromuscular disorder, myotonic dystrophy type 1 (DM1), which is characterized by progressive muscle wasting and weakness. The expanded (CTG)n tract not only alters the myogenic differentiation of the DM1 muscle precursor cells but also reduces their proliferative capacity. In this report, we show that these muscle precursor cells containing large CTG expansion sequences have not exhausted their proliferative capacity, but have entered into premature senescence. We demonstrate that an abnormal accumulation of p16 is responsible for this defect because the abolition of p16 activity overcomes early growth arrest and restores an extended proliferative capacity. Our results suggest that the accelerated telomere shortening measured in DM1 cells does not contribute to the aberrant induction of p16. We propose that a cellular stress related to the amplified CTG repeat promotes premature senescence mediated by a p16-dependent pathway in DM1 muscle precursor cells. This mechanism is responsible for the reduced proliferative capacity of the DM1 muscle precursor cells and could participate in both the impaired regeneration and atrophy observed in the DM1 muscles containing large CTG

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The regenerative capacity of skeletal muscle will depend on the number of available satellite cells and their proliferative capacity. We have measured both parameters in ageing, and have shown that although the proliferative capacity of satellite cells is decreasing during muscle growth, it then stabilizes in the adult, whereas the number of satellite cells decreases during ageing. We have also developed a model to evaluate the regenerative capacity of human satellite cells by implantation into regenerating muscles of immunodeficient mice. Using telomere measurements, we have shown that the proliferative capacity of satellite cells is dramatically decreased in muscle dystrophies, thus hampering the possibilities of autologous cell therapy. Immortalization by telomerase was unsuccessful, and we currently investigate the factors involved in cell cycle exits in human myoblasts. We have also observed that insulin-like growth factor-1 (IGF-1), a factor known to provoke hypertrophy, does not increase the proliferative potential of satellite cells, which suggests that hypertrophy is provoked by increasing the number of satellite cells engaged in differentiation, thus possibly decreasing the compartment of reserve cells. We conclude that autologous cell therapy can be applied to specific targets when there is a source of satellite cells which is not yet exhausted. This is the case of Oculo-Pharyngeal Muscular Dystrophy (OPMD), a late onset muscular dystrophy, and we participate to a clinical trial using autologous satellite cells isolated from muscles spared by the disease.

Insulin-like growth factor-1 (IGF-1) has been shown to induce skeletal muscle hypertrophy, to prevent the loss of muscle mass with ageing and to improve the muscle phenotype of dystrophic mice. We previously developed a model of IGF-1-induced hypertrophy of human myotubes, in which hypertrophy was not only characterized by an increase in myotube size and myosin content but also by an increased recruitment of reserve cells for fusion. Here, we describe a new mechanism of IGF-1-induced hypertrophy by demonstrating that IGF-1 signals exclusively to myotubes but not to reserve cells, leading, under the control of the transcription factor NFATc2, to the secretion of IL-13 that will secondly recruit reserve cells for differentiation and fusion. In addition, we show that IGF-1 also signals to myotubes to stimulate protein metabolism via Akt by (1) activating the mTOR-p70S6K-S6 pathway and inhibiting GSK-3␤, both involved in the control of protein translation, and (2) inhibiting the Foxo1-atrogin-1 protein degradation pathway. Journal of Cell Science 671 IL-13-mediated reserve cell recruitment (Abbott et al., 1998;Horsley et al., 2001), the data in the literature concerning the role of calcineurin in skeletal muscle hypertrophy are again often contradictory since some studies in rodent models show that IGF-1-induced hypertrophy can be suppressed using the calcineurin inhibitors cyclosporine A or FK506 Semsarian et al., 1999b), whereas other groups see no effect of these inhibitors on hypertrophy and no increase in calcineurin activity in the presence of IGF-1 (Bodine et al., 2001b;Rommel et al., 2001).The ability of IGF-1 to act as an anabolic factor on skeletal muscle and to counterbalance the signalling pathways of muscle atrophy has led to the proposition that IGF-1 could be used as a therapeutic agent to combat muscle atrophy related to age (sarcopenia) or to various diseases. However all data available until now describing the mechanisms of IGF-1-induced hypertrophy have been obtained in rodent models, and very little is known about the effects and the signalling pathways of IGF-1 in human skeletal muscle. It is becoming increasingly evident that the results obtained in rodent models cannot always be directly transposed to man. For example, whereas a twofold increase was observed in the lifespan of myoblasts from transgenic mice overexpressing IGF-1 in muscle (Chakravarthy et al., 2000), we recently showed in human myoblasts that IGF-1 has no effect on the proliferative lifespan, suggesting a different mechanism of regulation in these two species (Jacquemin et al., 2004).We previously developed an in vitro model of human myotube hypertrophy induced by IGF-1 where cultures were exposed to IGF-1 only 3 days after the induction of differentiation, a time when most of the myoblasts have already fused into myotubes and no more proliferation is observed. This model allows us to distinguish between the different effects of IGF-1 on proliferation, differentiation and hypertrophy (Jacquemin et al., 2004). In these condition...

In myotonic dystrophy, muscleblind-like protein 1 (MBNL1) protein binds specifically to expanded CUG or CCUG repeats, which accumulate as discrete nuclear foci, and this is thought to prevent its function in the regulation of alternative splicing of pre-mRNAs. There is strong evidence for the role of the MBNL1 gene in disease pathology, but the roles of two related genes, MBNL2 and MBNL3, are less clear. Using new monoclonal antibodies specific for each of the three gene products , we found that MBNL2 decreased during human fetal development and myoblast culture , while MBNL1 was unchanged. In Duchenne muscular dystrophy muscle , MBNL2 was elevated in immature , regenerating fibres compared with mature fibres , supporting some developmental role for MBNL2. MBNL3 was found only in C2C12 mouse myoblasts. Both MBNL1 and MBNL2 were partially sequestered by nuclear foci of expanded repeats in adult muscle and cultured cells from myotonic dystrophy patients. In adult muscle nucleoplasm , both proteins were reduced in myotonic dystrophy type 1 compared with an age-matched control. In normal human myoblast cultures , MBNL1 and MBNL2 always co-distributed but their distribution could change rapidly from nucleoplasmic to cytoplasmic. Myotonic dystrophy type 1 (DM1) is a progressive multisystemic disorder showing considerable clinical variation between individuals. DM1 is characterized by skeletal muscle weakness, wasting and pain, as well as myotonia.1 Other symptoms may include cardiac arrhythmias, cataracts, insulin resistance, hypogonadism, neurological problems and premature male balding.1-4 The genetic mutation responsible for DM1 has been identified as the expansion of a CTG repeat in exon 15 in the 3Ј-untranslated region of the DM protein kinase (DMPK) gene on chromosome 19q13.3. [5][6][7] The largest germline expansions occur during maternal transmission but the length of repeats may also increase somatically in affected individuals. 8 The size of the CTG expansion is related to the disease severity. More than 50 CTG repeats cause mild to classical adult-onset DM and 700 to greater than 3000 repeats often result in the severe congenital form of the disease. However, repeat size in muscle and other tissues can be much higher than in lymphocytes.9 A second form of DM (DM2) is due to a CCTG repeat in intron 1 of the ZNF9 gene on chromosome 3q21.3. 10Clinical features of DM1 and DM2 are similar but not identical. DM2 patients may show proximal rather than distal muscle involvement, and the severe congenital form occurs in DM1 only. The number of repeats in DM2 may be 10-fold greater than in DM1.