DARTpaths, an in silico platform to investigate molecular mechanisms of compounds.

SUMMARY: Xpaths is a collection of algorithms that allow for the prediction of compound-induced molecular mechanisms of action by integrating phenotypic endpoints of different species; and proposes follow-up tests for model organisms to validate these pathway predictions. The Xpaths algorithms are applied to predict developmental and reproductive toxicity (DART) and implemented into an in silico platform, called DARTpaths. AVAILABILITY AND IMPLEMENTATION: All code is available on GitHub https://github.com/Xpaths/dartpaths-app under Apache license 2.0, detailed overview with demo is available at https://www.vivaltes.com/dartpaths/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

From man to fly - convergent evidence links FBXO25 to ADHD and comorbid psychiatric phenotypes.

BACKGROUND: Mental disorders, including Attention-Deficit/Hyperactivity Disorder (ADHD), have a complex etiology, and identification of underlying genetic risk factors is challenging. This study used a multistep approach to identify and validate a novel risk gene for ADHD and psychiatric comorbidity. METHODS: In a single family, severely affected by ADHD and cooccurring disorders, we applied single nucleotide polymorphism (SNP)-array analysis to detect copy-number variations (CNVs) linked to disease. Genes present in the identified CNV were subsequently tested for their association with ADHD in the largest data set currently available (n = 55,374); this gene-set and gene-based association analyses were based on common genetic variants. Significant findings were taken forward for functional validation using Drosophila melanogaster as biological model system, altering gene expression using the GAL4-UAS system and a pan-neuronal driver, and subsequently characterizing locomotor activity and sleep as functional readouts. RESULTS: We identified a copy number gain in 8p23.3, which segregated with psychiatric phenotypes in the family and was confirmed by quantitative RT-PCR. Common genetic variants in this locus were associated with ADHD, especially those in FBXO25 and TDRP. Overexpression of the FBXO25 orthologue in two Drosophila models consistently led to increased locomotor activity and reduced sleep compared with the genetic background control. CONCLUSIONS: We combine ADHD risk gene identification in an individual family with genetic association testing in a large case-control data set and functional validation in a model system, together providing an important illustration of an integrative approach suggesting that FBXO25 contributes to key features of ADHD and comorbid neuropsychiatric disorders.

Homozygous and heterozygous disruptions of ANK3: at the crossroads of neurodevelopmental and psychiatric disorders.

AnkyrinG, encoded by the ANK3 gene, is involved in neuronal development and signaling. It has previously been implicated in bipolar disorder and schizophrenia by association studies. Most recently, de novo missense mutations in this gene were identified in autistic patients. However, the causative nature of these mutations remained controversial. Here, we report inactivating mutations in the Ankyrin 3 (ANK3) gene in patients with severe cognitive deficits. In a patient with a borderline intelligence, severe attention deficit hyperactivity disorder (ADHD), autism and sleeping problems, all isoforms of the ANK3 gene, were disrupted by a balanced translocation. Furthermore, in a consanguineous family with moderate intellectual disability (ID), an ADHD-like phenotype and behavioral problems, we identified a homozygous truncating frameshift mutation in the longest isoform of the same gene, which represents the first reported familial mutation in the ANK3 gene. The causality of ANK3 mutations in the two families and the role of the gene in cognitive function were supported by memory defects in a Drosophila knockdown model. Thus we demonstrated that ANK3 plays a role in intellectual functioning. In addition, our findings support the suggested association of ANK3 with various neuropsychiatric disorders and illustrate the genetic and molecular relation between a wide range of neurodevelopmental disorders.

Converging evidence does not support GIT1 as an ADHD risk gene.

Attention-Deficit/Hyperactivity Disorder (ADHD) is a common neuropsychiatric disorder with a complex genetic background. The G protein-coupled receptor kinase interacting ArfGAP 1 (GIT1) gene was previously associated with ADHD. We aimed at replicating the association of GIT1 with ADHD and investigated its role in cognitive and brain phenotypes. Gene-wide and single variant association analyses for GIT1 were performed for three cohorts: (1) the ADHD meta-analysis data set of the Psychiatric Genomics Consortium (PGC, N = 19,210), (2) the Dutch cohort of the International Multicentre persistent ADHD CollaboraTion (IMpACT-NL, N = 225), and (3) the Brain Imaging Genetics cohort (BIG, N = 1,300). Furthermore, functionality of the rs550818 variant as an expression quantitative trait locus (eQTL) for GIT1 was assessed in human blood samples. By using Drosophila melanogaster as a biological model system, we manipulated Git expression according to the outcome of the expression result and studied the effect of Git knockdown on neuronal morphology and locomotor activity. Association of rs550818 with ADHD was not confirmed, nor did a combination of variants in GIT1 show association with ADHD or any related measures in either of the investigated cohorts. However, the rs550818 risk-genotype did reduce GIT1 expression level. Git knockdown in Drosophila caused abnormal synapse and dendrite morphology, but did not affect locomotor activity. In summary, we could not confirm GIT1 as an ADHD candidate gene, while rs550818 was found to be an eQTL for GIT1. Despite GIT1's regulation of neuronal morphology, alterations in gene expression do not appear to have ADHD-related behavioral consequences. © 2015 Wiley Periodicals, Inc.

Altered GPM6A/M6 dosage impairs cognition and causes phenotypes responsive to cholesterol in human and Drosophila.

Glycoprotein M6A (GPM6A) is a neuronal transmembrane protein of the PLP/DM20 (proteolipid protein) family that associates with cholesterol-rich lipid rafts and promotes filopodia formation. We identified a de novo duplication of the GPM6A gene in a patient with learning disability and behavioral anomalies. Expression analysis in blood lymphocytes showed increased GPM6A levels. An increase of patient-derived lymphoblastoid cells carrying membrane protrusions supports a functional effect of this duplication. To study the consequences of GPM6A dosage alterations in an intact nervous system, we employed Drosophila melanogaster as a model organism. We found that knockdown of Drosophila M6, the sole member of the PLP family in flies, in the wing, and whole organism causes malformation and lethality, respectively. These phenotypes as well as the protrusions of patient-derived lymphoblastoid cells with increased GPM6A levels can be alleviated by cholesterol supplementation. Notably, overexpression as well as loss of M6 in neurons specifically compromises long-term memory in the courtship conditioning paradigm. Our findings thus indicate a critical role of correct GPM6A/M6 levels for cognitive function and support a role of the GPM6A duplication for the patient's phenotype. Together with other recent findings, this study highlights compromised cholesterol homeostasis as a recurrent feature in cognitive phenotypes.

APC16 is a conserved subunit of the anaphase-promoting complex/cyclosome.

Error-free chromosome segregation depends on timely activation of the multi-subunit E3 ubiquitin ligase APC/C. Activation of the APC/C initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for destruction. The APC/C is the principle target of the mitotic checkpoint, which prevents segregation while chromosomes are unattached to spindle microtubules. We now report the identification and characterization of APC16, a conserved subunit of the APC/C. APC16 was found in association with tandem-affinity-purified mitotic checkpoint complex protein complexes. APC16 is a bona fide subunit of human APC/C: it is present in APC/C complexes throughout the cell cycle, the phenotype of APC16-depleted cells copies depletion of other APC/C subunits, and APC16 is important for APC/C activity towards mitotic substrates. APC16 sequence homologues can be identified in metazoans, but not fungi, by four conserved primary sequence stretches. We provide evidence that the C. elegans gene K10D2.4 and the D. rerio gene zgc:110659 are functional equivalents of human APC16. Our findings show that APC/C is composed of previously undescribed subunits, and raise the question of why metazoan APC/C is molecularly different from unicellular APC/C.

Investigating cytosolic 5'-nucleotidase II family genes as candidates for neuropsychiatric disorders in Drosophila (114/150 chr).

Cytosolic 5'-nucleotidases II (cNT5-II) are an evolutionary conserved family of 5'-nucleotidases that catalyze the intracellular hydrolysis of nucleotides. In humans, the family is encoded by five genes, namely NT5C2, NT5DC1, NT5DC2, NT5DC3, and NT5DC4. While very little is known about the role of these genes in the nervous system, several of them have been associated with neuropsychiatric disorders. Here, we tested whether manipulating neuronal expression of cNT5-II orthologues affects neuropsychiatric disorders-related phenotypes in the model organism Drosophila melanogaster. We investigated the brain expression of Drosophila orthologues of cNT5-II family (dNT5A-CG2277, dNT5B-CG32549, and dNT5C-CG1814) using quantitative real-time polymerase chain reaction (qRT-PCR). Using the UAS/Gal4 system, we also manipulated the expression of these genes specifically in neurons. The knockdown was subjected to neuropsychiatric disorder-relevant behavioral assays, namely light-off jump reflex habituation and locomotor activity, and sleep was measured. In addition, neuromuscular junction synaptic morphology was assessed. We found that dNT5A, dNT5B, and dNT5C were all expressed in the brain. dNT5C was particularly enriched in the brain, especially at pharate and adult stages. Pan-neuronal knockdown of dNT5A and dNT5C showed impaired habituation learning. Knockdown of each of the genes also consistently led to mildly reduced activity and/or increased sleep. None of the knockdown models displayed significant alterations in synaptic morphology. In conclusion, in addition to genetic associations with psychiatric disorders in humans, altered expression of cNT5-II genes in the Drosophila nervous system plays a role in disease-relevant behaviors.

Serum Metabolomic Analysis of Coronary Heart Disease Patients with Stable Angina Pectoris Subtyped by Traditional Chinese Medicine Diagnostics Reveals Biomarkers Relevant to Personalized Treatments.

To improve the treatment of patients with coronary heart disease (CHD), personalized treatments based on potential biomarkers could make a difference. To investigate if such potential biomarkers could be found for CHD inhomogeneous, we combined traditional Chinese medicine based diagnosis with untargeted and targeted metabolomics analyses. Shi and Xu patient subtype groups of CHD with angina pectoris were identified. Different metabolites including lipids, fatty acids and amino acids were further analyzed with targeted metabolomics and mapped to disease-related pathways. The long-chain unsaturated lipids ceramides metabolism, bile acid metabolism were differentially affected in the Xu subtype groups. While, Shi-subtype patients seemed to show inflammation, anomalous levels of bioactive phospholipids and antioxidant molecules. Furthermore, variations in the endothelial damage response and energy metabolism found based on ELISA analysis are the key divergence points between different CHD subtypes. The results showed Xu subtype patients might benefit from long-chain unsaturated lipids ceramides as therapeutic targets. Shi subtype patients might benefit more from levels of polyunsaturated fatty acid consumption and treatments that help in restoring energy balance. Metabolic differences can be essential for treatment protocols. Thus, patient group specific differences can serve as important information to refine current treatment approaches in a personalized manner.

Towards a reporting guideline for developmental and reproductive toxicology testing in C. elegans and other nematodes.

Implementation of reliable methodologies allowing Reduction, Refinement, and Replacement (3Rs) of animal testing is a process that takes several decades and is still not complete. Reliable methods are essential for regulatory hazard assessment of chemicals where differences in test protocol can influence the test outcomes and thus affect the confidence in the predictive value of the organisms used as an alternative for mammals. Although test guidelines are common for mammalian studies, they are scarce for non-vertebrate organisms that would allow for the 3Rs of animal testing. Here, we present a set of 30 reporting criteria as the basis for such a guideline for Developmental and Reproductive Toxicology (DART) testing in the nematode Caenorhabditis elegans. Small organisms like C. elegans are upcoming in new approach methodologies for hazard assessment; thus, reliable and robust test protocols are urgently needed. A literature assessment of the fulfilment of the reporting criteria demonstrates that although studies describe methodological details, essential information such as compound purity and lot/batch number or type of container is often not reported. The formulated set of reporting criteria for C. elegans testing can be used by (i) researchers to describe essential experimental details (ii) data scientists that aggregate information to assess data quality and include data in aggregated databases (iii) regulators to assess study data for inclusion in regulatory hazard assessment of chemicals.

From Rare Copy Number Variants to Biological Processes in ADHD.

OBJECTIVE: Attention deficit hyperactivity disorder (ADHD) is a highly heritable psychiatric disorder. The objective of this study was to define ADHD-associated candidate genes and their associated molecular modules and biological themes, based on the analysis of rare genetic variants. METHODS: The authors combined data from 11 published copy number variation studies in 6,176 individuals with ADHD and 25,026 control subjects and prioritized genes by applying an integrative strategy based on criteria including recurrence in individuals with ADHD, absence in control subjects, complete coverage in copy number gains, and presence in the minimal region common to overlapping copy number variants (CNVs), as well as on protein-protein interactions and information from cross-species genotype-phenotype annotation. RESULTS: The authors localized 2,241 eligible genes in the 1,532 reported CNVs, of which they classified 432 as high-priority ADHD candidate genes. The high-priority ADHD candidate genes were significantly coexpressed in the brain. A network of 66 genes was supported by ADHD-relevant phenotypes in the cross-species database. Four significantly interconnected protein modules were found among the high-priority ADHD genes. A total of 26 genes were observed across all applied bioinformatic methods. Lookup in the latest genome-wide association study for ADHD showed that among those 26 genes, POLR3C and RBFOX1 were also supported by common genetic variants. CONCLUSIONS: Integration of a stringent filtering procedure in CNV studies with suitable bioinformatics approaches can identify ADHD candidate genes at increased levels of credibility. The authors' analytic pipeline provides additional insight into the molecular mechanisms underlying ADHD and allows prioritization of genes for functional validation in validated model organisms.

Contribution of Intellectual Disability-Related Genes to ADHD Risk and to Locomotor Activity in Drosophila.

OBJECTIVE: Attention deficit hyperactivity disorder (ADHD) is a common, highly heritable neuropsychiatric disorder. ADHD often co-occurs with intellectual disability, and shared overlapping genetics have been suggested. The aim of this study was to identify novel ADHD genes by investigating whether genes carrying rare mutations linked to intellectual disability contribute to ADHD risk through common genetic variants. Validation and characterization of candidates were performed using Drosophila melanogaster. METHODS: Common genetic variants in a diagnostic gene panel of 396 autosomal intellectual disability genes were tested for association with ADHD risk through gene set and gene-wide analyses, using ADHD meta-analytic data from the Psychiatric Genomics Consortium for discovery (N=19,210) and ADHD data from the Lundbeck Foundation Initiative for Integrative Psychiatric Research for replication (N=37,076). The significant genes were functionally validated and characterized in Drosophila by assessing locomotor activity and sleep upon knockdown of those genes in brain circuits. RESULTS: The intellectual disability gene set was significantly associated with ADHD risk in the discovery and replication data sets. The three genes most consistently associated were MEF2C, ST3GAL3, and TRAPPC9. Performing functional characterization of the two evolutionarily conserved genes in Drosophila melanogaster, the authors found that their knockdown in dopaminergic (dMEF2) and circadian neurons (dTRAPPC9) resulted in increased locomotor activity and reduced sleep, concordant with the human phenotype. CONCLUSIONS: This study reveals that a large set of intellectual disability-related genes contribute to ADHD risk through effects of common alleles. Utilizing this continuity, the authors identified TRAPPC9, MEF2C, and ST3GAL3 as novel ADHD candidate genes. Characterization in Drosophila suggests that TRAPPC9 and MEF2C contribute to ADHD-related behavior through distinct neural substrates.

Integration of expression profiles and genetic mapping data to identify candidate genes in intracranial aneurysm.

Intracranial aneurysm (IA) is a complex genetic disease for which, to date, 10 loci have been identified by linkage. Identification of the risk-conferring genes in the loci has proven difficult, since the regions often contain several hundreds of genes. An approach to prioritize positional candidate genes for further studies is to use gene expression data from diseased and nondiseased tissue. Genes that are not expressed, either in diseased or nondiseased tissue, are ranked as unlikely to contribute to the disease. We demonstrate an approach for integrating expression and genetic mapping data to identify likely pathways involved in the pathogenesis of a disease. We used expression profiles for IAs and nonaneurysmal intracranial arteries (IVs) together with the 10 reported linkage intervals for IA. Expressed genes were analyzed for membership in Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways. The 10 IA loci harbor 1,858 candidate genes, of which 1,561 (84%) were represented on the microarrays. We identified 810 positional candidate genes for IA that were expressed in IVs or IAs. Pathway information was available for 294 of these genes and involved 32 KEGG biological function pathways represented on at least 2 loci. A likelihood-based score was calculated to rank pathways for involvement in the pathogenesis of IA. Adherens junction, MAPK, and Notch signaling pathways ranked high. Integration of gene expression profiles with genetic mapping data for IA provides an approach to identify candidate genes that are more likely to function in the pathology of IA.

A homozygous FITM2 mutation causes a deafness-dystonia syndrome with motor regression and signs of ichthyosis and sensory neuropathy.

A consanguineous family from Pakistan was ascertained to have a novel deafness-dystonia syndrome with motor regression, ichthyosis-like features and signs of sensory neuropathy. By applying a combined strategy of linkage analysis and whole-exome sequencing in the presented family, a homozygous nonsense mutation, c.4G>T (p.Glu2*), in FITM2 was identified. FITM2 and its paralog FITM1 constitute an evolutionary conserved protein family involved in partitioning of triglycerides into cellular lipid droplets. Despite the role of FITM2 in neutral lipid storage and metabolism, no indications for lipodystrophy were observed in the affected individuals. In order to obtain independent evidence for the involvement of FITM2 in the human pathology, downregulation of the single Fitm ortholog, CG10671, in Drosophila melanogaster was pursued using RNA interference. Characteristics of the syndrome, including progressive locomotor impairment, hearing loss and disturbed sensory functions, were recapitulated in Drosophila, which supports the causative nature of the FITM2 mutation. Mutation-based genetic counseling can now be provided to the family and insight is obtained into the potential impact of genetic variation in FITM2.

A combined binary interaction and phenotypic map of C. elegans cell polarity proteins.

The establishment of cell polarity is an essential process for the development of multicellular organisms and the functioning of cells and tissues. Here, we combine large-scale protein interaction mapping with systematic phenotypic profiling to study the network of physical interactions that underlies polarity establishment and maintenance in the nematode Caenorhabditis elegans. Using a fragment-based yeast two-hybrid strategy, we identified 439 interactions between 296 proteins, as well as the protein regions that mediate these interactions. Phenotypic profiling of the network resulted in the identification of 100 physically interacting protein pairs for which RNAi-mediated depletion caused a defect in the same polarity-related process. We demonstrate the predictive capabilities of the network by showing that the physical interaction between the RhoGAP PAC-1 and PAR-6 is required for radial polarization of the C. elegans embryo. Our network represents a valuable resource of candidate interactions that can be used to further our insight into cell polarization.

Summaries of plenary, symposia, and oral sessions at the XXII World Congress of Psychiatric Genetics, Copenhagen, Denmark, 12-16 October 2014.

The XXII World Congress of Psychiatric Genetics, sponsored by the International Society of Psychiatric Genetics, took place in Copenhagen, Denmark, on 12-16 October 2014. A total of 883 participants gathered to discuss the latest findings in the field. The following report was written by student and postdoctoral attendees. Each was assigned one or more sessions as a rapporteur. This manuscript represents topics covered in most, but not all of the oral presentations during the conference, and contains some of the major notable new findings reported.

Familial intracranial aneurysms: an analysis of 346 multiplex Finnish families.

BACKGROUND AND PURPOSE: Genetic risk factors are considered important in the development, growth, and rupture of intracranial aneurysms; however, few have been identified. We analyzed intracranial aneurysm families with at least 2 affected persons and determined relationships between affected persons and assessed the inheritance patterns of aneurysms. METHODS: Families with > or =2 members with verified diagnoses of intracranial aneurysms were recruited from Kuopio and Helsinki, Finland. Families with a diagnosis of other heritable disorders that have associated intracranial aneurysms, such as autosomal dominant polycystic kidney disease, were excluded. RESULTS: We identified 346 Finnish multiplex families with 160 (46.2%) male and 186 (53.8%) female index cases. There were a total of 937 aneurysm cases, with an average of 2.7 cases per family. The majority of the families had only 2 affected relatives (n=206; 59.5%), although there were families with up to 6 (n=10), 7 (n=1), 8 (n=1), or 10 (n=2) affected persons. The affected relatives of the index cases included 108 sisters, 116 brothers, 105 parents, 30 children, 15 grandparents, 102 aunts or uncles, and 64 cousins. Of the 937 affected persons, 569 (60.7%) were alive and available for genetic analysis. Inheritance patterns consistent with autosomal recessiveness were observed in 198 (57.2%), autosomal dominance in 126 (36.4%), and autosomal dominance with incomplete penetrance in 19 (5.5%) of the families. CONCLUSIONS: The collection is the most extensive published to date and extends previous observations of familial aggregation that are consistent with a major gene effect.

Drosophila models of early onset cognitive disorders and their clinical applications.

The number of genes known to cause human monogenic diseases is increasing rapidly. For the extremely large, genetically and phenotypically heterogeneous group of intellectual disability (ID) disorders, more than 600 causative genes have been identified to date. However, knowledge about the molecular mechanisms and networks disrupted by these genetic aberrations is lagging behind. The fruit fly Drosophila has emerged as a powerful model organism to close this knowledge gap. This review summarizes recent achievements that have been made in this model and envisions its future contribution to our understanding of ID genetics and neuropathology. The available resources and efficiency of Drosophila place it in a position to tackle the main challenges in the field: mapping functional modules of ID genes to provide conceptually novel insights into the genetic control of cognition, tailored functional studies to improve 'next-generation' diagnostics, and identification of reversible ID phenotypes and medication. Drosophila's behavioral repertoire and powerful genetics also open up perspectives for modeling genetically complex forms of ID and neuropsychiatric disorders, which overlap in their genetic etiologies. In conclusion, Drosophila provides many opportunities to advance future medical genomics of early onset cognitive disorders.

Neuroinformatic analyses of common and distinct genetic components associated with major neuropsychiatric disorders.

Major neuropsychiatric disorders are highly heritable, with mounting evidence suggesting that these disorders share overlapping sets of molecular and cellular underpinnings. In the current article we systematically test the degree of genetic commonality across six major neuropsychiatric disorders-attention deficit hyperactivity disorder (ADHD), anxiety disorders (Anx), autistic spectrum disorders (ASD), bipolar disorder (BD), major depressive disorder (MDD), and schizophrenia (SCZ). We curated a well-vetted list of genes based on large-scale human genetic studies based on the NHGRI catalog of published genome-wide association studies (GWAS). A total of 180 genes were accepted into the analysis on the basis of low but liberal GWAS p-values (<10(-5)). 22% of genes overlapped two or more disorders. The most widely shared subset of genes-common to five of six disorders-included ANK3, AS3MT, CACNA1C, CACNB2, CNNM2, CSMD1, DPCR1, ITIH3, NT5C2, PPP1R11, SYNE1, TCF4, TENM4, TRIM26, and ZNRD1. Using a suite of neuroinformatic resources, we showed that many of the shared genes are implicated in the postsynaptic density (PSD), expressed in immune tissues and co-expressed in developing human brain. Using a translational cross-species approach, we detected two distinct genetic components that were both shared by each of the six disorders; the 1st component is involved in CNS development, neural projections and synaptic transmission, while the 2nd is implicated in various cytoplasmic organelles and cellular processes. Combined, these genetic components account for 20-30% of the genetic load. The remaining risk is conferred by distinct, disorder-specific variants. Our systematic comparative analysis of shared and unique genetic factors highlights key gene sets and molecular processes that may ultimately translate into improved diagnosis and treatment of these debilitating disorders.

The EBAX-type Cullin-RING E3 ligase and Hsp90 guard the protein quality of the SAX-3/Robo receptor in developing neurons.

Although protein quality control (PQC) is generally perceived as important for the development of the nervous system, the specific mechanisms of neuronal PQC have remained poorly understood. Here, we report that C. elegans Elongin BC-binding axon regulator (EBAX-1), a conserved BC-box protein, regulates axon guidance through PQC of the SAX-3/Robo receptor. EBAX-1 buffers guidance errors against temperature variations. As a substrate-recognition subunit in the Elongin BC-containing Cullin-RING ubiquitin ligase (CRL), EBAX-1 also binds to DAF-21, a cytosolic Hsp90 chaperone. The EBAX-type CRL and DAF-21 collaboratively regulate SAX-3-mediated axon pathfinding. Biochemical and imaging assays indicate that EBAX-1 specifically recognizes misfolded SAX-3 and promotes its degradation in vitro and in vivo. Importantly, vertebrate EBAX also shows substrate preference toward aberrant Robo3 implicated in horizontal gaze palsy with progressive scoliosis (HGPPS). Together, our findings demonstrate a triage PQC mechanism mediated by the EBAX-type CRL and DAF-21/Hsp90 that maintains the accuracy of neuronal wiring.