Final Report Summary - GEVAD (Rare and Common Genetic Variants analysis in ASDs and ADHD) The main aim of GEVAD the identification of rare and common genetic variants that increase the susceptibility to autism. The proposal consisted of three research plans that were developed as follows: 1) GEVAD-1: miRNA genes in autism spectrum disorder: a case-control association studyMicroRNAs (miRNAs) are a class of non-coding RNA molecules of 19 to 23 bp that work as post-transcriptional regulators. They are involved in a wide range of biological processes including cell cycle control, cell growth, differentiation, apoptosis and neuronal differentiation. Common and rare variants in miRNAs genes are likely to have a significant role in phenotypic variation, including disease susceptibility. In this study we aimed to test the possible contribution to autism spectrum disorder (ASD) of common variants in a selection of miRNAs genes through a case-control association study design in four European populations. We genotyped 350 tagSNPs in the discovery sample (DS), capturing the allelic variability of a selection of 163 miRNAs genes. The discovery sample (DS) for the case-control association study consisted of 636 autistic patients (311 Spanish, 247 Dutch and 78 German) and 673 gender-matched controls (322 Spanish, 269 Dutch and 82 German). A replication study (RS) was conducted in an independent European sample of 449 cases (232 Italian, 124 German and 93 Spanish) and 415 gender-matched controls (175 Italian, 131 German and 109 Spanish). The most significant associations in the DS were obtained for the following miRNAs: miR-133b/miR-206 cluster (rs16882131, P = 0.00037; rs17578851, P = 0.00872); miR-17/miR-18a/miR-19a/miR-20a/miR-19b-1/miR92a-1 cluster (rs6492538, P = 0.00199); mir-106b/mir-93/mir-25 cluster (rs4729575, P = 0.00645) and miR-219-1 (rs107822, P = 0.00881). The RS was performed on 28 SNPs that included all the polymorphisms of those genes showing association in the DS sample. None of these SNPs achieved a significant association after Bonferroni correction in the replica study. However, the pooled (DS and RS) showed significant associations after applying permutation tests for markers rs16882131 (P= 0.00157 permP = 0.042) and rs6492538 (P = 0.00074 permP = 0.0185). A Mantel-Haenszel meta-analysis for rs16882131C showed an OR of 1.23 (95% CI= 1.08 to 1.41) whereas for rs6492538C an OR = 1.27 (95 % CI = 1.1 to 1.48) was obtained. Our data suggest that common allelic variants in two miRNA clusters on chromosome 6 (miR-133b and miR-206) and 13 (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR92a-1) may be involved in autism susceptibility.Both clusters include miRNAs that regulate potential candidate genes for autism: miR-206 and mir-133b have been described to regulate the MET proto-oncogene, previously associated with autism. Also, mir-133b has been implicated in the maturation of midbrain dopaminergic neurons through the down-regulation of the transcription factor PITX3. MiR-19a regulates PTEN, reported to be involved in autism.We also investigated the presence of novel rare variants in pre-miRNA genes (70-80 nt) using exome sequencing data in 41 patients (21 of them from multiplex families). The rare variants study implicated several miRNA genes, although their function is unknown in most cases. A conserved variant in the mature miRNA-541 is predicted to diminish miRNA stability. This gene is a brain-specific miRNA and has been involved in neurite outgrowth. Functional studies are needed to establish whether these rare miRNA variants have a role in the disease aetiology. 2) GEVAD-2: Copy number variants analysis in autism The sample of 21 ASD individuals from multiplex families included in the exome sequencing project was processed for genomewide inspection of copy number variants using the CytoScan HD array (Affymetrix, Santa Clara, CA, USA). This study was carried out to rule out families with reported fully penetrant structural variants before the exome sequencing. This analysis, followed by experimental validation, revealed six inherited CNVs present in five sib-pairs that involved the following genes: COL4A3-MFF, FHIT, MRPL36-NDUFS6, CTNND2, GRM1 and ASAH1. Structural variants spanning the genes FHIT and CTNND2 had previously been described in autism. 3) GEVAD-3: Exome sequencing of ten ASD multiplex familiesRecently, whole exome sequencing (WES) efforts have focused mainly on rare de novo variants in singleton families. Although these studies have provided pioneering insights, de novo variants probably explain only a small proportion of the autism risk variance. In the present study we performed exome sequencing of ten autism multiplex families with the aim of investigating the role of heterozygous rare variants that are co-inherited in affected sibs. The pool of variants selected in our study is enriched with genes involved in neuronal functions or previously reported in psychiatric disorders. Our data suggest that rare truncating variants have a predominant role in the etiology of autism. We investigated the possible correlation between the severity of the different type of rare variants and intellectual disability, by using the non-verbal intellectual quotient (NVIQ). The results showed that only truncating variants contribute significantly to NVIQ (P = 0.007). Furthermore, truncating variants correlated with lower NVIQ scores (r = -0.517 P = 0.016). We subsequently investigated whether truncating mutations might play a major role in autism etiology by comparing the number of those that were transmitted to affected sibs with those that were not transmitted. We found a significant difference, considering the total number of rare variants, between disrupting mutations that were co-inherited by the affected probands (16 nonsense SNVs and 20 frame-shift indels) and those not transmitted (9 nonsense SNVs and 9 frameshift indels) (Fisher exact test, P = 0.015). The list of 220 genes harbouring rare predicted pathogenic mutations and genes found in the CNV study (GEVAD-2) were considered together and analysed by ingenuity pathway analysis (IPA) to identify canonical networks that may suggest interactions of potential ASD candidate genes. The best scoring network was obtained after considering only direct interactions (score = 89). It is noteworthy that this network included genes previously involved in autism or other psychiatric disorders such as SCN1A, PAX3, KDM5C, TSC1, NF1, CYFIP1, KCNQ3 and LAMA5. The YWHAZ gene, found disrupted in one sib-pair, was the most interconnected node in this network and represents the most intriguing candidate gene of our study: it encodes a post-synaptic protein, interacts with known autism genes (TSC1, TSC2, DISC1, UBE3A, CYFIP1, among others) and belongs to a gene family whose members have been associated with psychiatric or developmental disorders.We have also exome-sequenced 20 singleton autism families, and the results of this project are currently being analysed.Potential impact and use:Although many studies have provided convincing evidence for multiple interacting genetic factors as the main causative determinants of autism, these are still poorly known. Recent GWAS and exome sequencing studies, including our work, have identified a number of new genetic variants and genes involved in the disease, but more studies are needed to delineate the complete genetic landscape of the disorder. Also, the relative contribution of these variants / genes to the phenotype has to be properly weighted. Until this occurs, a useful genetic diagnosis and the development of new therapeutic interventions will have to wait.