The new neuromuscular disease related with defects in the ASC-1 complex: report of a second case confirms ASCC1 involvement.

Next-generation sequencing technology aided the identification of the underlying genetic cause in a female newborn with a severe neuromuscular disorder. The patient presented generalized hypotonia, congenital bone fractures, lack of spontaneous movements and poor respiratory effort. She died within the first days of life. Karyotyping and screening for several genes related with neuromuscular diseases all tested negative. A male sibling was subsequently born with the same clinical presentation. Whole-exome sequencing was performed with variant filtering assuming a recessive disease model. Analysis focused on genes known to be related firstly with congenital myopathies, extended to muscle diseases and finally to other neuromuscular disorders. No disease-causing variants were identified. A similar disorder was described in patients with recessive variants in two genes: TRIP4 (three families) and ASCC1 (one family), both encoding subunits of the nuclear activating signal cointegrator 1 (ASC-1) complex. Our patient was also found to have a homozygous frameshift variant (c.157dupG, p.Glu53Glyfs*19) in ASCC1 , thereby representing the second known case. This confirms ASCC1 involvement in a severe neuromuscular disease lying within the spinal muscular atrophy or primary muscle disease spectra.

mTOR inhibitors in urinary bladder cancer.

Despite the great scientific advances that have been made in cancer treatment, there is still much to do, particularly with regard to urinary bladder cancer. Some of the drugs used in urinary bladder cancer treatment have been in use for more than 30 years and show reduced effectiveness and high recurrence rates. There have been several attempts to find new and more effective drugs, to be used alone or in combination with the drugs already in use, in order to overcome this situation.The biologically important mammalian target of rapamycin (mTOR) pathway is altered in cancer and mTOR inhibitors have raised many expectations as potentially important anticancer drugs. In this article, the authors will review the mTOR pathway and present their experiences of the use of some mTOR inhibitors, sirolimus, everolimus and temsirolimus, in isolation and in conjunction with non-mTOR inhibitors cisplatin and gemcitabine, on urinary bladder tumour cell lines. The non-muscle-invasive cell line, 5637, is the only one that exhibits a small alteration in the mTOR and AKT phosphorylation after rapalogs exposure. Also, there was a small inhibition of cell proliferation. With gemcitabine plus everolimus or temsirolimus, the results were encouraging as a more effective response was noticed with both combinations, especially in the 5637 and T24 cell lines. Cisplatin associated with everolimus or temsirolimus also gave promising results, as an antiproliferative effect was observed when the drugs were associated, in particular on the 5637 and HT1376 cell lines. Everolimus or temsirolimus in conjunction with gemcitabine or cisplatin could have an important role to play in urinary bladder cancer treatment, depending on the tumour grading.

Challenges with in vitro and in vivo experimental models of urinary bladder cancer for novel drug discovery.

INTRODUCTION: Urinary bladder cancer (UBC) is the second most frequent malignancy of the urinary system and the ninth most common cancer worldwide, affecting individuals over the age of 65. Several investigations have embarked on advancing knowledge of the mechanisms underlying urothelial carcinogenesis, understanding the mechanisms of antineoplastic drugs resistance and discovering new antineoplastic drugs. In vitro and in vivo models are crucial for providing additional insights into the mechanisms of urothelial carcinogenesis. With these models, various molecular pathways involved in urothelial carcinogenesis have been discovered, allowing therapeutic manipulation. AREAS COVERED: This paper provides critical information on existing in vitro and in vivo models to screen the efficacy and toxicity of innovative UBC therapies and point out the challenges for new and improved models. EXPERT OPINION: In our opinion, results obtained with in vitro and in vivo models should be interpreted together, as a set of delicate biological tools that can be used at different stages in the drug discovery process, to address specific questions. With the development of new technologies, new assays and biomarkers are going to play an important role in the study of UBC. The molecular diagnostics and genomic revolution will not only help to develop new drug therapies, but also to achieve tailored therapies.

Quantitative analysis of five sterols in amniotic fluid by GC-MS: application to the diagnosis of cholesterol biosynthesis defects.

Cholesterol and its precursors, namely 7-dehydrocholesterol, desmosterol and lathosterol are important biochemical markers of cholesterol biosynthesis, and their quantification in body fluids is useful for the diagnosis of cholesterol biosynthesis pathway disorders. A rapid and sensitive gas chromatographic-mass spectrometric method was developed and validated for quantitative analysis of five sterols (cholesterol, 7-dehydrocholesterol, desmosterol, lathosterol and sitosterol) in amniotic fluid. The method was linear for all compounds (r(2)>0.99), and intra and inter-assay coefficients of variation were typically below 5%, and inaccuracy was within a +/-12% interval. The method was applied to 330 amniotic fluid samples, grouped by gestational age between 13 and 22 weeks of pregnancy, in order to establish reference intervals for sterols in this specimen. The obtained concentrations (mumol/L) for each sterol was as follows: 22.1758+/-4.2716 at 13 weeks and 78.5082+/-12.9041 at 22 weeks for cholesterol; 0.0039+/-0.0007 at 13 weeks and 0.1150+/-0.0212 at 22 weeks for 7-dehydrocholesterol; 0.1562+/-0.0406 at 13 weeks and 0.7691+/-0.0821 at 22 weeks for desmosterol; 0.0272+/-0.0035 at 13 weeks and 0.8551+/-0.1791 at 22 weeks for lathosterol; and 0.0404+/-0.0039 at 13 weeks and 0.2326+/-0.0386 at 22 weeks for sitosterol. The method was also applied to one pathological sample that showed decreased levels of cholesterol, and higher concentration of 7-dehydrocholesterol, which is consistent with a 7-dehydrocholesterol-reductase deficiency. Our results showed that as long as pregnancy goes on, the concentrations of cholesterol and precursors increase in amniotic fluid, which is related to the increased need for cholesterol by the fetus. The reference range of each sterol in amniotic fluid was calculated at different gestational ages and will be useful for the interpretation and validation of biochemical prenatal diagnosis of inborn errors of sterol biosynthesis.