Exome Sequencing of Index Patients with Retinal Dystrophies as a Tool for Molecular Diagnosis

Background

Retinal dystrophies (RD) are a group of hereditary diseases that lead to debilitating visual impairment and are usually transmitted as a Mendelian trait. Pathogenic mutations can occur in any of the 100 or more disease genes identified so far, making molecular diagnosis a rather laborious process. In this work we explored the use of whole exome sequencing (WES) as a tool for identification of RD mutations, with the aim of assessing its applicability in a diagnostic context.

Methodology/Principal Findings

We ascertained 12 Spanish families with seemingly recessive RD. All of the index patients underwent mutational pre-screening by chip-based sequence hybridization and resulted to be negative for known RD mutations. With the exception of one pedigree, to simulate a standard diagnostic scenario we processed by WES only the DNA from the index patient of each family, followed by in silico data analysis. We successfully identified causative mutations in patients from 10 different families, which were later verified by Sanger sequencing and co-segregation analyses. Specifically, we detected pathogenic DNA variants (∼50% novel mutations) in the genes RP1, USH2A, CNGB3, NMNAT1, CHM, and ABCA4, responsible for retinitis pigmentosa, Usher syndrome, achromatopsia, Leber congenital amaurosis, choroideremia, or recessive Stargardt/cone-rod dystrophy cases.

Conclusions/Significance

Despite the absence of genetic information from other family members that could help excluding nonpathogenic DNA variants, we could detect causative mutations in a variety of genes known to represent a wide spectrum of clinical phenotypes in 83% of the patients analyzed. Considering the constant drop in costs for human exome sequencing and the relative simplicity of the analyses made, this technique could represent a valuable tool for molecular diagnostics or genetic research, even in cases for which no genotypes from family members are available.     

Genetic Instability and Intratumoral Heterogeneity in Neuroblastoma with MYCN Amplification Plus 11q Deletion

Background/Aim

Genetic analysis in neuroblastoma has identified the profound influence of MYCN amplification and 11q deletion in patients’ prognosis. These two features of high-risk neuroblastoma usually occur as mutually exclusive genetic markers, although in rare cases both are present in the same tumor. The purpose of this study was to characterize the genetic profile of these uncommon neuroblastomas harboring both these high-risk features.

Methods

We selected 18 neuroblastomas with MNA plus 11q loss detected by FISH. Chromosomal aberrations were analyzed using Multiplex Ligation-dependent Probe Amplification and Single Nucleotide Polymorphism array techniques.

Results and Conclusion

This group of tumors has approximately the same high frequency of aberrations as found earlier for 11q deleted tumors. In some cases, DNA instability generates genetic heterogeneity, and must be taken into account in routine genetic diagnosis.     

Brain trauma and autophagy: What flies and mice can teach us about conserved responses

Drosophila models have been successfully used to identify many genetic components that affect neurodegenerative disorders. Recently, there has been a growing interest in identifying innate and environmental factors that influence the individual outcomes following traumatic brain injury (TBI). This includes both severe TBI and more subtle, mild TBI (mTBI), which is common in people playing contact sports. Autophagy, as a clearance pathway, exerts protective effects in multiple neurological disease models. In a recent publication, we highlighted the development of a novel repetitive mTBI system using Drosophila, which recapitulates several phenotypes associated with trauma in mammalian models. In particular, flies subjected to mTBI exhibit an acute impairment of the macroautophagy/autophagy pathway that is restored 1 wk following traumatic injury exposure. These phenotypes closely resemble temporary autophagy defects observed in a mouse TBI model. Through these studies, we also identified methods to directly assess autophagic responses in the fly nervous system and laid the groundwork for future studies designed to identify genetic, epigenetic and environmental factors that have an impact on TBI outcomes.     

Predicting microbial traits with phylogenies

Phylogeny reflects genetic and phenotypic traits in Bacteria and Archaea. The phylogenetic conservatism of microbial traits has prompted the application of phylogeny-based algorithms to predict unknown trait values of extant taxa based on the traits of their evolutionary relatives to estimate, for instance, rRNA gene copy numbers, gene contents or tolerance to abiotic conditions. Unlike the ‘macrobial'' world, microbial ecologists face scenarios potentially compromising the accuracy of trait reconstruction methods, as, for example, extremely large phylogenies and limited information on the traits of interest. We review 990 bacterial and archaeal traits from the literature and support that phylogenetic trait conservatism is widespread through the tree of life, while revealing that it is generally weak for ecologically relevant phenotypic traits and high for genetically complex traits. We then perform a simulation exercise to assess the accuracy of phylogeny-based trait predictions in common scenarios faced by microbial ecologists. Our simulations show that ca. 60% of the variation in phylogeny-based trait predictions depends on the magnitude of the trait conservatism, the number of species in the tree, the proportion of species with unknown trait values and the mean distance in the tree to the nearest neighbour with a known trait value. Results are similar for both binary and continuous traits. We discuss these results under the light of the reviewed traits and provide recommendations for the use of phylogeny-based trait predictions for microbial ecologists.Trait-based approaches in community ecology studies are becoming increasingly appealing for microbial ecologists partly because metagenomic sequencing allows surveying molecular functions (Green et al., 2008; Lauro et al., 2009; Burke et al., 2011; Raes et al., 2011; Brown et al., 2014; Fierer et al., 2014). Although genetic data can provide precise information on cellular processes or metabolic pathways, they are generally blind to other ecologically relevant phenotypic traits such as the tolerance to certain abiotic conditions or the specific growth rate (but see Vieira-Silva and Rocha, 2010). Unlike ‘macrobial'' ecologists, who can directly observe phenotypic characters of plants and animals, microbial ecologists usually face situations where most of the phenotypes of their study organisms are unknown. This difficulty relies on the fact that gathering phenotypic (physiological, morphological, biochemical) data requires culturing microbial species. The unbalanced growth of genotypic vs phenotypic information is currently challenging microbial ecologists to work with phylogenetic trees of increasing size (hundreds to thousands of species) in which the percentage of species with unknown traits becomes larger and larger.Recent evidence indicate that phylogeny reflects molecular functions and phenotypes in Bacteria and Archaea (Langille et al., 2013; Martiny et al., 2013). This is due to the phylogenetic conservatism of microbial traits (Martiny et al., 2013), which likely arises from microbial evolution mostly proceeding by vertical gene inheritance rather than horizontal gene transfer (Kurland et al., 2003, see Fraser et al., 2007 for theoretical models on the role of horizontal gene transfer in bacterial speciation). At present, the massive sequencing of microbes in the environment is providing a huge amount of genetic information that is extremely useful to reconstruct the phylogenetic relationships among microbial lineages. This fact has triggered the interest of microbial ecologists to apply the methods developed to predict unobserved trait values of extant taxa based on the traits observed in their evolutionary relatives (Kembel et al., 2012; Langille et al., 2013; Angly et al., 2014, see review in Zaneveld and Thurber, 2014). All these methods are based on the existence of a significant phylogenetic signal or, in other words, in the fact that close relatives have more similar traits than expected by chance. Phylogeny-based trait prediction procedures (PTP hereafter) in microbes have been mainly performed under the phylogenetic generalized least squares framework (Martins and Hansen, 1997; Garland and Ives, 2000). Specifically, the trait value (for continuous traits) or state (for binary traits) of the focal species have been reconstructed through ancestral state reconstructions after rerooting the phylogeny at the most recent common ancestor of the taxon with unobserved trait and the rest of the tree (Kembel et al., 2012). The accuracy of PTP methods has been typically assessed under ‘macrobial'' scenarios containing phylogenies of moderate size, with low-to-medium proportion of species with unknown traits and significant phylogenetic signals. For example, Fagan et al. (2013) predicted population growth rates of mammals in phylogenies of 42–65 species containing 54–64% of unknowns and a significant phylogenetic signal (Blomberg et al., 2003; Blomberg''s K) ranging from 0.68 to 1.42. However, the current microbial scenarios derived from high-throughput sequencing projects face large-sized phylogenies (hundreds to thousands tips) with a high number of species with unknown traits and varying phylogenetic signals jeopardizing the applicability of PTP methods (Zaneveld and Thurber, 2014).The extent to which phylogeny reflects phenotype is strongly dependent on the degree of conservatism with which the focal trait has evolved. For instance, complex traits that involve many genes (for example, photosynthesis or methanogenesis) show higher conservatism than simpler traits, such as the consumption of a specific carbon source (Martiny et al., 2013). Furthermore, certain traits such as those related to genes encoding antibiotic or metal resistance are particularly prone to be horizontally transferred (Bruins et al., 2000), a process that can blur their phylogenetic signal. Therefore, if phylogenetic relatedness is to be used to infer the phenotype, the phylogenetic conservatism of the target trait needs to be quantified in every case.Altogether, the abovementioned observations indicate that the possibility to estimate phenotypes from phylogenies depends on the amount of phylogenetic and phenotypic information available to predict the unobserved trait values. Here we provide a simulation exercise to test the accuracy of the most widely used PTP method in microbial ecology to predict continuous trait values and binary trait states of extant taxa with different amount of phenotypic and phylogenetic information. We simulated several situations faced by microbial ecologists, including phylogenies of different sizes in which a small (P=0.3), medium (P=0.6) or large (P=0.9) proportion of species have unknown trait values. The correlations between the actual and the predicted trait values were obtained for characters evolved under different degree of conservatism. Finally, we put these values in the context of the phylogenetic signals described in the literature for different continuous and binary microbial traits and provide some recommendations for future analyses aimed to predict microbial traits with the help of the phylogenetic information.     

Synthetic Cyclolipopeptides Selective against Microbial,Plant and Animal Cell Targets by Incorporation of D-Amino Acids or Histidine

Cyclolipopeptides derived from the antimicrobial peptide c(Lys-Lys-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Gln) (BPC194) were prepared on solid-phase and screened against four plant pathogens. The incorporation at Lys⁵ of fatty acids of 4 to 9 carbon atoms led to active cyclolipopeptides. The influence on the antimicrobial activity of the Lys residue that is derivatized was also evaluated. In general, acylation of Lys¹, Lys² or Lys⁵ rendered the sequences with the highest activity. Incorporation of a D-amino acid maintained the antimicrobial activity while significantly reduced the hemolysis. Replacement of Phe with a His also yielded cyclolipopeptides with low hemolytic activity. Derivatives exhibiting low phytotoxicity in tobacco leaves were also found. Interestingly, sequences with or without significant activity against phytopathogenic bacteria and fungi, but with differential hemolysis and phytotoxicity were identified. Therefore, this study represents an approach to the development of bioactive peptides with selective activity against microbial, plant and animal cell targets. These selective cyclolipopeptides are candidates useful not only to combat plant pathogens but also to be applied in other fields.     

Natural products from marine invertebrates against <Emphasis Type="Italic">Leishmania</Emphasis> parasites: a comprehensive review

Parasitic infections by Leishmania parasites remains a severe public health problem, especially in developing countries where it is highly endemic. Chemotherapy still remains a first option for the treatment of those diseases, despite the fact that available drugs exhibit a variety of shortcomings. Thus, innovative, less toxic more affordable and effective antileishmanial agents are urgently needed. The marine environment holds an immeasurable bio- and chemical diversity, being a valuable source of natural products with therapeutic potential. As invertebrates comprise about 60 % of all marine organisms, bioprospecting this class of organisms for antileishmanial properties may unravel unique and selective hit molecules. In this context, this review covers results on the literature of marine invertebrate extracts and pure compounds evaluated against Leishmania parasites mainly by in vitro methods. It comprises results obtained from the phyla Porifera, Cnidaria, Bryozoa (Ectoprota), Mollusca, Echinodermata, Annelida, Cetnophora, Platyhelminthes, sub phyla Crustacea (phylum Arthropoda) and Tunicata (phylum Chordata). Moreover, structure–activity relationships and possible mechanisms of action are mentioned, whenever available information is provided. About 70 species of marine invertebrates belonging to seven different phyla are included in this work. Besides a variety of crude extracts, a total of 140 pure compounds was tested against different Leishmania species. Although the research on the antileishmanial potential of marine invertebrates is in its early beginnings, promising results have been achieved that encourage further research. As more extracts and compounds are being screened, the possibility of finding active and selective antileishmanial molecules increases, rising new hope in the search for new treatments against leishmaniases.     

Risk of Advanced Neoplasia in First-Degree Relatives with Colorectal Cancer: A Large Multicenter Cross-Sectional Study

BackgroundFirst-degree relatives (FDR) of patients with colorectal cancer have a higher risk of developing colorectal cancer than the general population. For this reason, screening guidelines recommend colonoscopy every 5 or 10 y, starting at the age of 40, depending on whether colorectal cancer in the index-case is diagnosed at <60 or ≥60 y, respectively. However, studies on the risk of neoplastic lesions are inconclusive. The aim of this study was to determine the risk of advanced neoplasia (three or more non-advanced adenomas, advanced adenoma, or invasive cancer) in FDR of patients with colorectal cancer compared to average-risk individuals (i.e., asymptomatic adults 50 to 69 y of age with no family history of colorectal cancer).ConclusionsIndividuals having two FDR with colorectal cancer showed an increased risk of advanced neoplasia compared to those with average-risk for colorectal cancer. Men had over 2-fold higher risk of advanced neoplasia than women, independent of family history. These data suggest that screening colonoscopy guidelines should be revised in the familial-risk population.