Autism Linked to Increased Oncogene Mutations but Decreased Cancer Rate

Autism spectrum disorder (ASD) is one phenotypic aspect of many monogenic, hereditary cancer syndromes. Pleiotropic effects of cancer genes on the autism phenotype could lead to repurposing of oncology medications to treat this increasingly prevalent neurodevelopmental condition for which there is currently no treatment. To explore this hypothesis we sought to discover whether autistic patients more often have rare coding, single-nucleotide variants within tumor suppressor and oncogenes and whether autistic patients are more often diagnosed with neoplasms. Exome-sequencing data from the ARRA Autism Sequencing Collaboration was compared to that of a control cohort from the Exome Variant Server database revealing that rare, coding variants within oncogenes were enriched for in the ARRA ASD cohort (p<1.0x10^-8). In contrast, variants were not significantly enriched in tumor suppressor genes. Phenotypically, children and adults with ASD exhibited a protective effect against cancer, with a frequency of 1.3% vs. 3.9% (p<0.001), but the protective effect decreased with age. The odds ratio of neoplasm for those with ASD relative to controls was 0.06 (95% CI: 0.02, 0.19; p<0.0001) in the 0 to 14 age group; 0.35 (95% CI: 0.14, 0.87; p = 0.024) in the 15 to 29 age group; 0.41 (95% CI: 0.15, 1.17; p = 0.095) in the 30 to 54 age group; and 0.49 (95% CI: 0.14, 1.74; p = 0.267) in those 55 and older. Both males and females demonstrated the protective effect. These findings suggest that defects in cellular proliferation, and potentially senescence, might influence both autism and neoplasm, and already approved drugs targeting oncogenic pathways might also have therapeutic value for treating autism.     

Genome-wide copy number variation analysis of a Branchio-oto-renal syndrome cohort identifies a recombination hotspot and implicates new candidate genes

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial arch anomalies, hearing loss and renal dysmorphology. Although haploinsufficiency of EYA1 and SIX1 are known to cause BOR, copy number variation analysis has only been performed on a limited number of BOR patients. In this study, we used high-resolution array-based comparative genomic hybridization on 32 BOR probands negative for coding-sequence and splice-site mutations in known BOR-causing genes to identify potential disease-causing genomic rearrangements. Of the >1,000 rare and novel copy number variants we identified, four were heterozygous deletions of EYA1 and several downstream genes that had nearly identical breakpoints associated with retroviral sequence blocks, suggesting that non-allelic homologous recombination seeded by this recombination hotspot is important in the pathogenesis of BOR. A different heterozygous deletion removing the last exon of EYA1 was identified in an additional proband. Thus, in total five probands (14 %) had deletions of all or part of EYA1. Using a novel disease-gene prioritization strategy that includes network analysis of genes associated with other deletions suggests that SHARPIN (Sipl1), FGF3 and the HOXA gene cluster may contribute to the pathogenesis of BOR.     

Effective mosquito and arbovirus surveillance using metabarcoding

Effective vector and arbovirus surveillance requires timely and accurate screening techniques that can be easily upscaled. Next‐generation sequencing (NGS) is a high‐throughput technology that has the potential to modernize vector surveillance. When combined with DNA barcoding, it is termed ‘metabarcoding.’ The aim of our study was to establish a metabarcoding protocol to characterize pools of mosquitoes and screen them for virus. Pools contained 100 morphologically identified individuals, including one Ross River virus (RRV) infected mosquito, with three species present at different proportions: 1, 5, 94%. Nucleic acid extracted from both crude homogenate and supernatant was used to amplify a 269‐bp section of the mitochondrial cytochrome c oxidase subunit I (COI) locus. Additionally, a 67‐bp region of the RRV E2 gene was amplified from synthesized cDNA to screen for RRV. Amplicon sequencing was performed using an Illumina MiSeq, and bioinformatic analysis was performed using a DNA barcode database of Victorian mosquitoes. Metabarcoding successfully detected all mosquito species and RRV in every positive sample tested. The limits of species detection were also examined by screening a pool of 1000 individuals, successfully identifying the species and RRV from a single mosquito. The primers used for amplification, number of PCR cycles and total number of individuals present all have effects on the quantification of species in mixed bulk samples. Based on the results, a number of recommendations for future metabarcoding studies are presented. Overall, metabarcoding shows great promise for providing a new alternative approach to screening large insect surveillance trap catches.     

Seizures Are Regulated by Ubiquitin-specific Peptidase 9 X-linked (USP9X), a De-Ubiquitinase

Epilepsy is a common disabling disease with complex, multifactorial genetic and environmental etiology. The small fraction of epilepsies subject to Mendelian inheritance offers key insight into epilepsy disease mechanisms; and pathologies brought on by mutations in a single gene can point the way to generalizable therapeutic strategies. Mutations in the PRICKLE genes can cause seizures in humans, zebrafish, mice, and flies, suggesting the seizure-suppression pathway is evolutionarily conserved. This pathway has never been targeted for novel anti-seizure treatments. Here, the mammalian PRICKLE-interactome was defined, identifying prickle-interacting proteins that localize to synapses and a novel interacting partner, USP9X, a substrate-specific de-ubiquitinase. PRICKLE and USP9X interact through their carboxy-termini; and USP9X de-ubiquitinates PRICKLE, protecting it from proteasomal degradation. In forebrain neurons of mice, USP9X deficiency reduced levels of Prickle2 protein. Genetic analysis suggests the same pathway regulates Prickle-mediated seizures. The seizure phenotype was suppressed in prickle mutant flies by the small-molecule USP9X inhibitor, Degrasyn/WP1130, or by reducing the dose of fat facets a USP9X orthologue. USP9X mutations were identified by resequencing a cohort of patients with epileptic encephalopathy, one patient harbored a de novo missense mutation and another a novel coding mutation. Both USP9X variants were outside the PRICKLE-interacting domain. These findings demonstrate that USP9X inhibition can suppress prickle-mediated seizure activity, and that USP9X variants may predispose to seizures. These studies point to a new target for anti-seizure therapy and illustrate the translational power of studying diseases in species across the evolutionary spectrum.     

Simulating an invasion: unsealed water storage (rainwater tanks) and urban block design facilitate the spread of the dengue fever mosquito,Aedes aegypti,in Brisbane,Australia

Aedes aegypti (Linnaeus) was once highly prevalent across eastern Australia, resulting in epidemics of dengue fever. Drought conditions have led to a rapid rise in semi-permanent, urban water storage containers called rainwater tanks known to be critical larval habitat for the species. The presence of these larval habitats has increased the risk of establishment of highly urbanised, invasive mosquito vectors such as Ae. aegypti. Here we use a spatially explicit network model to examine the role that unsealed rainwater tanks may play in population connectivity of an Ae. aegypti invasion in suburbs of Brisbane, a major Australian city. We characterise movement between rainwater tanks as a diffusion-like process, limited by a maximum distance of movement, average life expectancy, and a probability that Ae. aegypti will cross wide open spaces such as roads. The simulation model was run against a number of scenarios that examined population spread through the rainwater tank network based on non-compliance rates of tanks (unsealed or sealed) and road grids. We show that Ae. aegypti tank infestation and population spread was greatest in areas of high tank density and road lengths were shortest e.g. cul-de-sacs. Rainwater tank non-compliance rates of over 30% show increased connectivity when compared to less than 10%, suggesting rainwater tanks non-compliance should be maintained under this level to minimize the spread of an invading Ae. aegypti population. These results presented as risk maps of Ae. aegypti spread across Brisbane, can assist health and government authorities on where to optimally target rainwater tank surveillance and educational activities.

     

Evaluation of entomopathogenic fungi as potential biological control agents of the dengue mosquito,Aedes aegypti (Diptera: Culicidae)

Dengue is a global health concern. Growing insecticide resistance in the primary mosquito vector, Aedes aegypti, limits the effectiveness of vector control, so alternative tools are urgently needed. One approach is the use of biopesticides comprising entomopathogenic fungi, e.g., Beauveria bassiana and Metarhizium anisopliae. These fungi may decrease disease transmission by reducing mosquito vector longevity and also occur worldwide, although many isolates have not been tested for virulence against mosquitoes. Ninety-three isolates of entomopathogenic fungi representing six species (B. bassiana, M. anisopliae, Isaria fumosorosea, I. farinosa, I. flavovirescens, and Lecanicillium spp.) were screened as potential biological control agents of Aedes aegypti. A hierarchical, multi-criteria experimental design was undertaken to find suitable isolates. Initial screening was performed via in vitro assays measuring radial growth and spore persistence, eliminating isolates with poor growth or viability on nutrient-rich substrate. Subsequent measurements of spore persistence revealed that only nine of 30 strains tested had half-lives exceeding 3 weeks. Ten isolates were chosen for in vivo bioassays against adult Ae. aegypti. From these assays, two Australian isolates of B. bassiana, FI-277 and FI-278, appeared to be most promising. Both isolates were shown to be virulent against Ae. aegypti at 20, 26, and 32°C. Spreading spores manually onto substrate was found to be more efficacious than spraying. Ae. aegypti infected by manually-spread spores on cotton substrate were found to have an LT₅₀ of 3.7±0.3 days. These characteristics suggest that FI-277 has promise as a dengue mosquito biocontrol agent, either alone or combined with conventional chemical insecticides.     

RABL6A,a Novel RAB-Like Protein,Controls Centrosome Amplification and Chromosome Instability in Primary Fibroblasts

RABL6A (RAB-like 6 isoform A) is a novel protein that was originally identified based on its association with the Alternative Reading Frame (ARF) tumor suppressor. ARF acts through multiple p53-dependent and p53-independent pathways to prevent cancer. How RABL6A functions, to what extent it depends on ARF and p53 activity, and its importance in normal cell biology are entirely unknown. We examined the biological consequences of RABL6A silencing in primary mouse embryo fibroblasts (MEFs) that express or lack ARF, p53 or both proteins. We found that RABL6A depletion caused centrosome amplification, aneuploidy and multinucleation in MEFs regardless of ARF and p53 status. The centrosome amplification in RABL6A depleted p53−/− MEFs resulted from centrosome reduplication via Cdk2-mediated hyperphosphorylation of nucleophosmin (NPM) at threonine-199. Thus, RABL6A prevents centrosome amplification through an ARF/p53-independent mechanism that restricts NPM-T199 phosphorylation. These findings demonstrate an essential role for RABL6A in centrosome regulation and maintenance of chromosome stability in non-transformed cells, key processes that ensure genomic integrity and prevent tumorigenesis.     

PRICKLE1 Interaction with SYNAPSIN I Reveals a Role in Autism Spectrum Disorders

The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in Prickle1^+/− mice and Drosophila, yeast, and neuronal cell lines. We show that mice with Prickle1 mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.     

Avian defensive behavior and blood-feeding success of the West Nile vector mosquito, Culex pipiens

Avian defensive behavior against host-seeking arthropods influencestransmission of vector-borne pathogens by affecting mosquitobiting rate, either by preventing vector–host contactor by increasing the rate of multiple host feeding. We exposedhouse sparrows (Passer domesticus L.) and chickens (Gallus gallusdomesticus L.) to Culex pipiens pipiens L. overnight in a largeobservation cage and measured avian defensive behavior ratesand mosquito blood-feeding success. Both bird species exhibiteda range of defensive behaviors, 90% of which were foot stomps,head movements, and wing shakes. Total behavior rates increasedproportionately with mosquito density in both species, increasedafter the first hour of mosquito exposure, and decreased asindividual birds were exposed to mosquitoes multiple times.Mosquito blood-feeding success on house sparrows was high overall(82 ± 5%) and independent of behavior rates. Blood-feedingsuccess on chicks was lower (58 ± 5%) and negativelycorrelated with defensive behavior rate after the first hourof mosquito exposure. Results revealed a higher percentage ofpartial blood meals on chicks (18 ± 3% of all blood mealson chicks) than on house sparrows (4.9 ± 3%). Birds ofboth species ate an average of 9.4 ± 1.2% of mosquitoes,and this percentage was positively correlated with defensivebehavior. High mosquito feeding success on house sparrows supportsits role as a potential amplifying host of West Nile virus.