Targeted Next-Generation Sequencing in a Large Cohort of Genetically Undiagnosed Patients with Neuromuscular Disorders in Spain

This article belongs to the Special Issue Genetic Advances in Neuromuscular Disorders: From Gene Identification to Gene Therapy.The term neuromuscular disorder (NMD) includes many genetic and acquired diseases and differential diagnosis can be challenging. Next-generation sequencing (NGS) is especially useful in this setting given the large number of possible candidate genes, the clinical, pathological, and genetic heterogeneity, the absence of an established genotype-phenotype correlation, and the exceptionally large size of some causative genes such as TTN, NEB and RYR1. We evaluated the diagnostic value of a custom targeted next-generation sequencing gene panel to study the mutational spectrum of a subset of NMD patients in Spain. In an NMD cohort of 207 patients with congenital myopathies, distal myopathies, congenital and adult-onset muscular dystrophies, and congenital myasthenic syndromes, we detected causative mutations in 102 patients (49.3%), involving 42 NMD-related genes. The most common causative genes, TTN and RYR1, accounted for almost 30% of cases. Thirty-two of the 207 patients (15.4%) carried variants of uncertain significance or had an unidentified second mutation to explain the genetic cause of the disease. In the remaining 73 patients (35.3%), no candidate variant was identified. In combination with patients’ clinical and myopathological data, the custom gene panel designed in our lab proved to be a powerful tool to diagnose patients with myopathies, muscular dystrophies and congenital myasthenic syndromes. Targeted NGS approaches enable a rapid and cost-effective analysis of NMD- related genes, offering reliable results in a short time and relegating invasive techniques to a second tier.This study was granted by FIS PI15/01898, funded by ISCIII and FEDER, ‘Una manera de hacer Europa’ and by Fundación Mutua Madrileña in the “Convocatoria de ayudas a la Investigación en Salud 2015”. It was also funded by an ACCI grant from CIBERER. Daniel Natera-de Benito is the recipient of a grant from the Instituto de Salud Carlos III (Contrato Rio Hortega, CM17/00044)

Sequential targeted exome sequencing of 1001 patients affected by unexplained limb-girdle weakness

Several hundred genetic muscle diseases have been described, all of which are rare. Their clinical and genetic heterogeneity means that a genetic diagnosis is challenging. We established an international consortium, MYO-SEQ, to aid the work-ups of muscle disease patients and to better understand disease etiology. Exome sequencing was applied to 1001 undiagnosed patients recruited from more than 40 neuromuscular disease referral centers; standardized phenotypic information was collected for each patient. Exomes were examined for variants in 429 genes associated with muscle conditions. We identified suspected pathogenic variants in 52% of patients across 87 genes. We detected 401 novel variants, 116 of which were recurrent. Variants in CAPN3, DYSF, ANO5, DMD, RYR1, TTN, COL6A2, and SGCA collectively accounted for over half of the solved cases; while variants in newer disease genes, such as BVES and POGLUT1, were also found. The remaining well-characterized unsolved patients (48%) need further investigation. Using our unique infrastructure, we developed a pathway to expedite muscle disease diagnoses. Our data suggest that exome sequencing should be used for pathogenic variant detection in patients with suspected genetic muscle diseases, focusing first on the most common disease genes described here, and subsequently in rarer and newly characterized disease genes

Preliminary safety and efficacy of first-line pertuzumab combined with trastuzumab and taxane therapy for HER2-positive locally recurrent or metastatic breast cancer (PERUSE).

BACKGROUND: Pertuzumab combined with trastuzumab and docetaxel is the standard first-line therapy for HER2-positive metastatic breast cancer, based on results from the phase III CLEOPATRA trial. PERUSE was designed to assess the safety and efficacy of investigator-selected taxane with pertuzumab and trastuzumab in this setting. PATIENTS AND METHODS: In the ongoing multicentre single-arm phase IIIb PERUSE study, patients with inoperable HER2-positive advanced breast cancer (locally recurrent/metastatic) (LR/MBC) and no prior systemic therapy for LR/MBC (except endocrine therapy) received docetaxel, paclitaxel or nab-paclitaxel with trastuzumab [8\u2009mg/kg loading dose, then 6\u2009mg/kg every 3\u2009weeks (q3w)] and pertuzumab (840\u2009mg loading dose, then 420\u2009mg q3w) until disease progression or unacceptable toxicity. The primary end point was safety. Secondary end points included overall response rate (ORR) and progression-free survival (PFS). RESULTS: Overall, 1436 patients received at least one treatment dose (initially docetaxel in 775 patients, paclitaxel in 589, nab-paclitaxel in 65; 7 discontinued before starting taxane). Median age was 54\u2009years; 29% had received prior trastuzumab. Median treatment duration was 16\u2009months for pertuzumab and trastuzumab and 4\u2009months for taxane. Compared with docetaxel-containing therapy, paclitaxel-containing therapy was associated with more neuropathy (all-grade peripheral neuropathy 31% versus 16%) but less febrile neutropenia (1% versus 11%) and mucositis (14% versus 25%). At this preliminary analysis (52 months' median follow-up), median PFS was 20.6 [95% confidence interval (CI) 18.9-22.7] months overall (19.6, 23.0 and 18.1\u2009months with docetaxel, paclitaxel and nab-paclitaxel, respectively). ORR was 80% (95% CI 78%-82%) overall (docetaxel 79%, paclitaxel 83%, nab-paclitaxel 77%). CONCLUSIONS: Preliminary findings from PERUSE suggest that the safety and efficacy of first-line pertuzumab, trastuzumab and taxane for HER2-positive LR/MBC are consistent with results from CLEOPATRA. Paclitaxel appears to be a valid alternative taxane backbone to docetaxel, offering similar PFS and ORR with a predictable safety profile. CLINICALTRIALS.GOV: NCT01572038

Final results from the PERUSE study of first-line pertuzumab plus trastuzumab plus a taxane for HER2-positive locally recurrent or metastatic breast cancer, with a multivariable approach to guide prognostication

Background: The phase III CLinical Evaluation Of Pertuzumab And TRAstuzumab (CLEOPATRA) trial established the combination of pertuzumab, trastuzumab and docetaxel as standard first-line therapy for human epidermal growth factor receptor 2 (HER2)-positive locally recurrent/metastatic breast cancer (LR/mBC). The multicentre single-arm PERtUzumab global SafEty (PERUSE) study assessed the safety and efficacy of pertuzumab and trastuzumab combined with investigator-selected taxane in this setting. Patients and methods: Eligible patients with inoperable HER2-positive LR/mBC and no prior systemic therapy for LR/mBC (except endocrine therapy) received docetaxel, paclitaxel or nab-paclitaxel with trastuzumab and pertuzumab until disease progression or unacceptable toxicity. The primary endpoint was safety. Secondary endpoints included progression-free survival (PFS) and overall survival (OS). Prespecified subgroup analyses included subgroups according to taxane, hormone receptor (HR) status and prior trastuzumab. Exploratory univariable analyses identified potential prognostic factors; those that remained significant in multivariable analysis were used to analyse PFS and OS in subgroups with all, some or none of these factors. Results: Of 1436 treated patients, 588 (41%) initially received paclitaxel and 918 (64%) had HR-positive disease. The most common grade 653 adverse events were neutropenia (10%, mainly with docetaxel) and diarrhoea (8%). At the final analysis (median follow-up: 5.7 years), median PFS was 20.7 [95% confidence interval (CI) 18.9-23.1] months overall and was similar irrespective of HR status or taxane. Median OS was 65.3 (95% CI 60.9-70.9) months overall. OS was similar regardless of taxane backbone but was more favourable in patients with HR-positive than HR-negative LR/mBC. In exploratory analyses, trastuzumab-pretreated patients with visceral disease had the shortest median PFS (13.1 months) and OS (46.3 months). Conclusions: Mature results from PERUSE show a safety and efficacy profile consistent with results from CLEOPATRA and median OS exceeding 5 years. Results suggest that paclitaxel is a valid alternative to docetaxel as backbone chemotherapy. Exploratory analyses suggest risk factors that could guide future trial design

REDUCED TITANIUM-DIOXIDE AS SUPPORT FOR ZIEGLER-NATTA CATALYSTS

Titanium tetrachloride heterogenized on reduced TiO2 has been studied as a catalyst for ethylene polymerization. The catalyst has good storage stability and exhibits good activity for ethylene polymerization. The polymer chains grow linearly during ca. 1 h, giving an average molecular weight of up to 2.5 X 10(6) which indicates that practically no beta-elimination occurs. The activity of the catalyst at 50-degrees-C, based on Ti(III), is 7.6 X 10(6) PE/mol Ti h bar and based on the quantity of polyethylene formed it is 1.25 X 10(6) g PE/mol Ti h bar. The molecular weight of the polymer can be controlled with the addition of hydrogen; under 0.5 bar hydrogen, polyethylene with a molecular weight of 411,000 and a relatively low polydispersity index of 2.2 is obtained. The catalyst shows good thermal stability; the Arrhenius activation energy is 31.8 kJ/mol for the polymerization. The catalyst is also active for propylene polymerization, giving 3 X 10(6) g PP/mol Ti h bar with the high isotacticity of 93%. (C) 1994 John Wiley & Sons, Inc.32592993

Interaction Between Saccharomyces Cerevisiae And Chrysotile

The interaction between Saccharomyces cerevisiae and chrysotile fibers was studied by scanning electron microscopy. The yeast cells adhere preferentially to the fibrils. In the extreme case, all the adhered fibrils were broken, resulting in a complete coverage of the surface. The chrysotile covered cells showed less buds, but retained metabolic capacities, and were fully active in fermentation experiments after one year. The interaction degree was depending on contact time and adhesion medium. The longer the contact period, the stronger the interaction between the cells and the fibers. Cells adhered in water show poor entrapment after short contact time, but were highly entrapped after longer periods and did not show any agglomerates. Cells adhered in the presence of nutrients showed a lower entrapment and a higher degree of cellular growth.23035Bandypadhyay, K.K., Ghose, T.K., Studies on immobilized Saccharomyces cerevisiae. III Physiology of growth and metabolism on various supports (1982) Biotechnol. Bioeng., 24, pp. 805-815Broaddus, V.C., Asbestos, the mesothelial cell and malignancy: A mater of life or death (1997) Am. J. Respir. Cell Mol. Biol., 17, pp. 657-659Ciesarová, Z., Smogrovicová, D., Dömény, Z., Enhancement of yeast ethanol tolerance by calcium and magnesium (1996) Folia Microbiol., 41, pp. 485-488Cullen, M.R., Chrysotile asbestos: Enough is enough (1998) The Lancet, 351, pp. 1377-1378Fukumori, N., Aoki, N., Sasaki, M., Biological effects of ingested asbestos fibers (VII). Finestructural findings of intravenously injected fibers (1995) Ann. Rep. Tokyo Metr. Res. Lab. P.H., 46, pp. 265-270Fukumori, N., Aoki, N., Sasaki, M., Electron microscopic observations of asbestos fibers in the rat lung after intratracheal instillation (1996) Ann. Rep. Tokyo Metr. Res. Lab. P.H., 47, pp. 303-308Fukumori, N., Aoki, N., Sasaki, M., Ultrastructural changes of macrophages on phagocytosis of asbestos fibers (1997) Ann. Rep. Tokyo Metr. Res. Lab. P.H., 48, pp. 312-317Joekes, I., Moran, P.J.S., Rodrigues, J.A.R., Wendhausen, R., Tonella, E., Cassiola, F., Characterization of Saccharomyces cerevisiae immobilized onto chrysotile for ethanol production (1998) J. Chem Technol. Biotechnol., 73, pp. 54-58Kida, K., Morimura, S., Sonoda, Y., Yanoh, T., The importance of the surface charge on support media for microbial adhesion (1992) J. Ferm. Bioeng., 73, pp. 323-325Kogan, F.M., Nikitina, O.V., Solubility of chrysotile asbestos and basalt fibers in relation to their fibrogenic and carcinogenic action (1994) Environ. Health Perspect., 102, pp. 205-206Langer, A.M., Nolan, R.P., Chrysotile biopersistence in the lungs of persons in the general population and exposed workers (1994) Environ. Health Perspect., 102, pp. 235-239Mozes, N., Marchal, F., Hermesse, M.P., Van Haecht, J.L., Reuliaux, L., Leonard, A.J., Rouxhet, P.G., Immobilization of microorganisms by adhesion: Interplay of electrostatic and nonelectrostatic interactions (1987) Biotechnol. Bioeng., 30, pp. 439-450Nolan, R.P., Langer, A.M., Addison, J., Lung content analysis of cases occupationally exposed to chrysotile asbestos (1994) Environ. Health Perspect., 102, pp. 245-250Norton, S., D'Amore, T., Physiological effects of yeast cell immobilization: Applications for brewing (1994) Enzyme Microb. Technol., 16, pp. 365-375Parizotto, O., Comerlato, M.H., Pedroso, P.R., Moran, P.J.S., Carvalho, M., Joekes, I., (1989) Preparation of chrysotile with high specific surface area. Pat. BR. 8903849Walker, G.M., Maynard, A., Accumulation of magnesium ions during fermentative metabolism in Saccharomyces cerevisiae (1997) J. Industrial Microbiol. Biotechnol., 18, pp. 1-3Wendhausen, R., Moran, P.J.S., Joekes, I., Rodrigues, J.A.R., Continuous process for large-scale preparation of chiral alcohols with baker's yeast immobilized on chrysotile fibers (1998) J. Mol. Cat. B: Enzymatic, 5, pp. 57-6

Impulsivity and compulsive buying: examining the moderating role of socioeconomic status

People nowadays are more impulsive especially the recent generation when it comes to making choices. This impulsivity is partly fed by today\u27s consumer culture, where we want more things in greater quantity. This could create a phenomenon called impulsive buying where a person has an almost irrepressible urge to go buy and shop for things whether or not they actually need it. And so the researchers wanted to investigate if impulsivity and compulsive buying have a relationship, and if a person\u27s socioeconomic status would have a moderating effect on that relationship. The researchers will gather their data by way of a survey. Their participants being employees of De La Salle University (DLSU) and companies in Metro Manila. This is because the researchers believe the employees of these institutions will offer a diverse range of different socioeconomic status

<i>GlycoMine^struct</i>:A new bioinformatics tool for highly accurate mapping of the human N-linked and O-linked glycoproteomes by incorporating structural features

Glycosylation plays an important role in cell-cell adhesion, ligand-binding and subcellular recognition. Current approaches for predicting protein glycosylation are primarily based on sequence-derived features, while little work has been done to systematically assess the importance of structural features to glycosylation prediction. Here, we propose a novel bioinformatics method called GlycoMinestruct(http://glycomine.erc.monash.edu/Lab/GlycoMine_Struct/) for improved prediction of human N- and O-linked glycosylation sites by combining sequence and structural features in an integrated computational framework with a two-step feature-selection strategy. Experiments indicated that GlycoMinestruct outperformed NGlycPred, the only predictor that incorporated both sequence and structure features, achieving AUC values of 0.941 and 0.922 for N- and O-linked glycosylation, respectively, on an independent test dataset. We applied GlycoMinestruct to screen the human structural proteome and obtained high-confidence predictions for N- and O-linked glycosylation sites. GlycoMinestruct can be used as a powerful tool to expedite the discovery of glycosylation events and substrates to facilitate hypothesis-driven experimental studies

SecretEPDB: a comprehensive web-based resource for secreted effector proteins of the bacterial types III, IV and VI secretion systems

Bacteria translocate effector molecules to host cells through highly evolved secretion systems. By definition, the function of these effector proteins is to manipulate host cell biology and the sequence, structural and functional annotations of these effector proteins will provide a better understanding of how bacterial secretion systems promote bacterial survival and virulence. Here we developed a knowledgebase, termed SecretEPDB (Bacterial Secreted Effector Protein DataBase), for effector proteins of type III secretion system (T3SS), type IV secretion system (T4SS) and type VI secretion system (T6SS). SecretEPDB provides enriched annotations of the aforementioned three classes of effector proteins by manually extracting and integrating structural and functional information from currently available databases and the literature. The database is conservative and strictly curated to ensure that every effector protein entry is supported by experimental evidence that demonstrates it is secreted by a T3SS, T4SS or T6SS. The annotations of effector proteins documented in SecretEPDB are provided in terms of protein characteristics, protein function, protein secondary structure, Pfam domains, metabolic pathway and evolutionary details. It is our hope that this integrated knowledgebase will serve as a useful resource for biological investigation and the generation of new hypotheses for research efforts aimed at bacterial secretion systems