New sample of drosophilids from the Font Groga site, Barcelona (Spain)

A new sample of drosophilids was obtained from Font Groga (Barcelona) on 9th October 2013. Flies were netted over 12 baits containing fermenting bananas placed along a trail from 4 to 7 pm. The number of flies classified according to species and sex is presented in Table 1. The most abundant species is D. subobscura (62.60%). This is expected because the sample was obtained during its autumn peak of expansion. Also interesting is to find again D. suzukii, and in a percentage similar (9.20%) to that obtained in 2012 sample. This species invaded recently many European regions and seems it is well established. We have finally estimated the species diversity using H" (Shannon diversity index) and J (Shannon uniformity index). The values obtained were 0.990 and 0.615, respectively. They are similar to those estimates obtained in the same site by Calabria in autumn 2007 and higher than those of Canals et al. in late autumn 2012

Annual climatic effects on the autumnal Drosophilids fauna composition at the Font Groga site (Tibidabo, Barcelona)

The abundance of different drosophilid species was studied in a series of six consecutive years during autumn at the Font Groga site (Tibidabo, Barcelona). Furthermore, the relation between these abundances and the following climatological variables (Tmean: mean temperature, Tmax: maximum temperature, Tmin: minimum temperature, Hm: mean humidity and Rf: rainfall) were studied. The most common species observed at this location were D. subobscura, D. melanogaster/D.simulans and the invasive species D. suzukii. Other species were trapped depending on the year (D. immigrans, D. buzzatti, D. cameraria, D. phalerata and D. hydei), and the values of several diversity indexes were computed for each annual collections. In general, in those years where D. melanogaster/D. simulans flies were abundant, D. subobscura and D. suzukii were in low frequencies. From the analyses of the climatic variables and the three most abundant species, it seemed that D. subobscura would need environmental conditions characterized by a low Tmin, not a very high Tmax (but with a large difference between both), a certain degree of humidity and scarce rainfall. D. suzukii presented a similar pattern, but not so accused, whereas D. melanogaster / D.simulans group would need high Tmin, low Tmax (with a small difference between Tmin and Tmax values) and low levels of humidity and rainfall. Finally, in these autumnal samples our results would indicate a certain association between the abundances of D. subobscura and D. suzukii, although their breeding sites are clearly different

Exploring allele specific methylation in drug dependence susceptibility

Drug dependence is a neuropsychiatric condition that involves genetic, epigenetic and environmental factors. Allele-specific methylation (ASM) is a common and stable epigenetic mechanism that involves genetic variants correlating with differential levels of methylation at CpG sites. We selected 182 single-nucleotide polymorphisms (SNPs) described to influence cis ASM in human brain regions to evaluate their possible contribution to drug dependence susceptibility. We performed a case-control association study in a discovery sample of 578 drug-dependent patients (including 428 cocaine-dependent subjects) and 656 controls from Spain, and then, we followed-up the significant associations in an independent sample of 1,119 cases (including 589 cocaine-dependent subjects) and 1,092 controls. In the discovery sample, we identified five nominal associations, one of them replicated in the follow-up sample (rs6020251). The pooled analysis revealed an association between drug dependence and rs6020251 but also rs11585570, both overcoming the Bonferroni correction for multiple testing. We performed the same analysis considering only cocaine-dependent patients and obtained similar results. The rs6020251 variant correlates with differential methylation levels of cg17974185 and lies in the first intron of the CTNNBL1 gene, in a genomic region with multiple histone marks related to enhancer and promoter regions in brain. Rs11585570 is an eQTL in brain and blood for the SCP2 and ECHDC2 genes and correlates with differential methylation of cg27535305 and cg13461509, located in the promoter regions of both genes. To conclude, using an approach that combines genetic and epigenetic data, we highlighted the CTNNBL1, SCP2 and ECHDC2 genes as potential contributors to drug dependence susceptibility

Association of the PLCB1 gene with drug dependence

Genetic factors involved in the susceptibility to drug addiction still remain largely unknown. MiRNAs seem to play key roles in the drug-induced plasticity of the brain that likely drives the emergence of addiction. In this work we explored the role of miRNAs in drug addiction. With this aim, we selected 62 SNPs located in the 3'UTR of target genes that are predicted to alter the binding of miRNA molecules and performed a case-control association study in a Spanish sample of 735 cases (mainly cocaine-dependent subjects with multiple drug dependencies) and 739 controls. We found an association between rs1047383 in the PLCB1 gene and drug dependence that was replicated in an independent sample (663 cases and 667 controls). Then we selected 9 miRNAs predicted to bind the rs1047383 region, but none of them showed any effect on PLCB1 expression. We also assessed two miRNAs binding a region that contains a SNP in linkage disequilibrium with rs1047383, but although one of them, hsa-miR-582, was found to downregulate PLCB1, no differences were observed between alleles. Finally, we explored the possibility that PLCB1 expression is altered by cocaine and we observed a significant upregulation of the gene in the nucleus accumbens of cocaine abusers and in human dopaminergic-like neurons after cocaine treatment. Our results, together with previous studies, suggest that PLCB1 participates in the susceptibility to drug dependence

Involvement of the 14-3-3 gene family in autism spectrum disorder and schizophrenia: Genetics, transcriptomics and functional analyses

The 14-3-3 protein family are molecular chaperones involved in several biological functions and neurological diseases. We previously pinpointed YWHAZ (encoding 14-3-3ζ) as a candidate gene for autism spectrum disorder (ASD) through a whole-exome sequencing study, which identified a frameshift variant within the gene (c.659-660insT, p.L220Ffs*18). Here, we explored the contribution of the seven human 14-3-3 family members in ASD and other psychiatric disorders by investigating the: (i) functional impact of the 14-3-3ζ mutation p.L220Ffs*18 by assessing solubility, target binding and dimerization; (ii) contribution of common risk variants in 14-3-3 genes to ASD and additional psychiatric disorders; (iii) burden of rare variants in ASD and schizophrenia; and iv) 14-3-3 gene expression using ASD and schizophrenia transcriptomic data. We found that the mutant 14-3-3ζ protein had decreased solubility and lost its ability to form heterodimers and bind to its target tyrosine hydroxylase. Gene-based analyses using publicly available datasets revealed that common variants in YWHAE contribute to schizophrenia (p = 6.6 × 10-7), whereas ultra-rare variants were found enriched in ASD across the 14-3-3 genes (p = 0.017) and in schizophrenia for YWHAZ (meta-p = 0.017). Furthermore, expression of 14-3-3 genes was altered in post-mortem brains of ASD and schizophrenia patients. Our study supports a role for the 14-3-3 family in ASD and schizophrenia

Genètica i epigenètica de les addiccions i trastorns comòrbids

[cat] Aquesta Tesi té com a objectiu principal contribuir a l’estudi i a la identificació de factors genètics i epigenètics de susceptibilitat a les addiccions i a trastorns comòrbids, tant en humans com en models animals. Els objectius concrets d’aquest treball són els següents: CAPÍTOL 1. Estudi de factors genètics de risc a l’addicció a drogues identificats prèviament. 1.1. Realització d’un estudi de rèplica d’associacions a la dependència de drogues, i de cocaïna en particular, prèviament identificades en estudis d’associació genètica a escala genòmica. 1.2. Avaluació de l’efecte d’NFAT5, un gen candidat a l'addicció a cocaïna descrit prèviament pel grup, sobre la síntesi de la dopamina a través de la regulació de l’expressió del gen DDC en dues línies cel·lulars neurals dopaminèrgiques. CAPÍTOL 2. Identificació de factors epigenètics de susceptibilitat a l’addicció a les drogues i al menjar. 2.1. Estudi de la contribució a la dependència de drogues de variants genètiques amb un efecte sobre la metilació específica d’al·lel (ASM). 2.2. Identificació de variants genètiques de susceptibilitat a la dependència de drogues que poden alterar el lloc d’unió de miRNAs, i posterior avaluació funcional de les variants identificades. 2.3. Exploració dels perfils transcriptòmics (mRNAs i miRNAs) i metilòmics en un model de ratolí d’addicció al menjar. CAPÍTOL 3. Identificació de factors epigenètics de susceptibilitat a trastorns comòrbids a l’addicció a les drogues. 3.1. Exploració de la contribució de variants genètiques amb un efecte en l’ASM al trastorn per dèficit d'atenció amb hiperactivitat (TDAH). 3.2. Anàlisi de la funció de C2orf82 al cervell i de la seva contribució al TDAH: determinació de l’expressió del gen en diferents àrees cerebrals de ratolí i caracterització fenotípica d’un model murí genoanul·lat. 3.3. Avaluació de l’efecte pleotròpic de variants genètiques que presenten ASM en diferents trastorns psiquiàtrics.[eng] Addiction is a complex neuropsychiatric disorder characterized by compulsive and uncontrolled use of an addictive stimulus such as drugs of abuse or food. Its etiology involves genetic and environmental factors, with epigenetics as the interplay between them. Although family and twin studies have estimated a high contribution of genetics to the disease, the underlying mechanisms remain largely unknown. In addition, multiple studies have shown that addiction is highly comorbid with other psychiatric disorders, which can be explained, at least in part, by shared genetics. The work presented in this Doctoral Thesis has contributed to the study and identification of genetic and epigenetic factors underlying drug and food addiction, as well as comorbid disorders. First, we have replicated a variant previously identified in a genome wide association study for drug dependence and we have discarded that NFAT5, a candidate gene for cocaine dependence, modulates dopamine synthesis by regulating DDC expression in dopaminergic neural cell lines. Subsequently, we focused on the identification of genes and epigenetic risk factors for drug and food addiction. We identified three variants associated with drug addiction that alter the methylation of CpG sites or the binding of miRNAs, pinpointing CTNNBL1, SCP2, ECHDC2 and PLCB1 as candidate genes for the disorder. Furthermore, we have identified several mRNAs and miRNAs that are differentially expressed in two brain areas of mice with extreme behaviours towards food addiction-like phenotype (vulnerable and resilient), some of these changes explained by promoter methylation alterations. Then, we have identified multiple variants affecting the methylation of CpG sites associated with ADHD. Some of these variants have an impact on the expression of three genes, ARTN, PIDD1 and C2orf82, pointing them as candidates to contribute to the disorder. For the C2orf82 gene, we have explored its expression in the mouse brain, obtaining the highest expression in the nucleus accumbens, and we have also phenotyped a knockout mouse model, observing hypolocomotion, impulsivity and lower cocaine motivation. Finally, we have identified multiple variants that affect brain methylation and expression of eight genes with pleiotropic effects on several psychiatric disorders

Identification of genetic variants influencing methylation in brain with pleiotropic effects on psychiatric disorders.

Psychiatric disorders affect 29% of the global population at least once in the lifespan, and genetic studies have proved a shared genetic basis among them, although the underlying molecular mechanisms remain largely unknown. DNA methylation plays an important role in complex disorders and, remarkably, enrichment of common genetic variants influencing allele-specific methylation (ASM) has been reported among variants associated with specific psychiatric disorders. In the present study we assessed the contribution of ASM to a set of eight psychiatric disorders by combining genetic, epigenetic and expression data. We interrogated a list of 3896 ASM tagSNPs in the brain in the summary statistics of a cross-disorder GWAS meta-analysis of eight psychiatric disorders from the Psychiatric Genomics Consortium, including more than 162,000 cases and 276,000 controls. We identified 80 SNPs with pleiotropic effects on psychiatric disorders that show an opposite directional effect on methylation and gene expression. These SNPs converge on eight candidate genes: ZSCAN29, ZSCAN31, BTN3A2, DDAH2, HAPLN4, ARTN, FAM109B and NAGA. ZSCAN29 shows the broadest pleiotropic effects, showing associations with five out of eight psychiatric disorders considered, followed by ZSCAN31 and BTN3A2, associated with three disorders. All these genes overlap with CNVs related to cognitive phenotypes and psychiatric traits, they are expressed in the brain, and seven of them have previously been associated with specific psychiatric disorders, supporting our results. To sum up, our integrative functional genomics analysis identified eight psychiatric disease risk genes that impact a broad list of disorders and highlight an etiologic role of SNPs that influence DNA methylation and gene expression in the brain

Exploring genetic variation that influences brain methylation in attention-deficit/hyperactivity disorder

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder caused by an interplay of genetic and environmental factors. Epigenetics is crucial to lasting changes in gene expression in the brain. Recent studies suggest a role for DNA methylation in ADHD. We explored the contribution to ADHD of allele-specific methylation (ASM), an epigenetic mechanism that involves SNPs correlating with differential levels of DNA methylation at CpG sites. We selected 3896 tagSNPs reported to influence methylation in human brain regions and performed a case-control association study using the summary statistics from the largest GWAS meta-analysis of ADHD, comprising 20,183 cases and 35,191 controls. We observed that genetic risk variants for ADHD are enriched in ASM SNPs and identified associations with eight tagSNPs that were significant at a 5% false discovery rate (FDR). These SNPs correlated with methylation of CpG sites lying in the promoter regions of six genes. Since methylation may affect gene expression, we inspected these ASM SNPs together with 52 ASM SNPs in high LD with them for eQTLs in brain tissues and observed that the expression of three of those genes was affected by them. ADHD risk alleles correlated with increased expression (and decreased methylation) of ARTN and PIDD1 and with a decreased expression (and increased methylation) of C2orf82. Furthermore, these three genes were predicted to have altered expression in ADHD, and genetic variants in C2orf82 correlated with brain volumes. In summary, we followed a systematic approach to identify risk variants for ADHD that correlated with differential cis-methylation, identifying three novel genes contributing to the disorder

MicroRNA signatures associated with vulnerability to food addiction in mice and humans

Food addiction is characterized by a loss of behavioral control over food intake and is associated with obesity and other eating disorders. The mechanisms underlying this behavioral disorder are largely unknown. We aimed to investigate the changes in miRNA expression promoted by food addiction in animals and humans and their involvement in the mechanisms underlying the behavioral hallmarks of this disorder. We found sharp similitudes between miRNA signatures in the medial prefrontal cortex (mPFC) of our animal cohort and circulating miRNA levels in our human cohort, which allowed us to identify several miRNAs of potential interest in the development of this disorder. Tough decoy (TuD) inhibition of miRNA-29c-3p in the mouse mPFC promoted persistence of the response and enhanced vulnerability to developing food addiction, whereas miRNA-665-3p inhibition promoted compulsion-like behavior and also enhanced food addiction vulnerability. In contrast, we found that miRNA-137-3p inhibition in the mPFC did not lead to the development of food addiction. Therefore, miRNA-29c-3p and miRNA-665-3p could be acting as protective factors with regard to food addiction. We believe the elucidation of these epigenetic mechanisms will lead to advances toward identifying innovative biomarkers and possible future interventions for food addiction and related disorders based on the strategies now available to modify miRNA activity and expression