RESUMEN
BACKGROUND: The involvement of microRNAs (miRNAs) in neuronal differentiation and synaptic plasticity suggests a role for miRNAs in psychiatric disorders; association analyses and functional approaches were used to evaluate the implication of miRNAs in the susceptibility for panic disorder. METHODS: Case-control studies for 712 single-nucleotide polymorphisms (SNPs) tagging 325 human miRNA regions were performed in 203 Spanish patients with panic disorder and 341 control subjects. A sample of 321 anxiety patients and 642 control subjects from Finland and 102 panic disorder patients and 829 control subjects from Estonia was used as a replica. Reporter-gene assays and miRNA overexpression experiments in neuroblastoma cells were used to functionally evaluate the spectrum of genes regulated by the associated miRNAs. RESULTS: Two SNPs associated with panic disorder: rs6502892 tagging miR-22 (p < .0002), and rs11763020 tagging miR-339 (p < .00008). Other SNPs tagging miR-138-2, miR-488, miR-491, and miR-148a regions associated with different panic disorder phenotypes. Replication in the north-European sample supported several of these associations, although they did not pass correction for multiple testing. Functional studies revealed that miR-138-2, miR-148a, and miR-488 repress (30%-60%) several candidate genes for panic disorder--GABRA6, CCKBR and POMC, respectively--and that miR-22 regulates four other candidate genes: BDNF, HTR2C, MAOA, and RGS2. Transcriptome analysis of neuroblastoma cells transfected with miR-22 and miR-488 showed altered expression of a subset of predicted target genes for these miRNAs and of genes that might be affecting physiological pathways related to anxiety. CONCLUSIONS: This work represents the first report of a possible implication of miRNAs in the etiology of panic disorder.
Asunto(s)
Ansiedad/genética , Predisposición Genética a la Enfermedad , MicroARNs/genética , Trastorno de Pánico/genética , Polimorfismo de Nucleótido Simple/genética , Adulto , Estudios de Casos y Controles , Línea Celular Tumoral , Comparación Transcultural , Estonia , Femenino , Finlandia , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Estudio de Asociación del Genoma Completo/métodos , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Monoaminooxidasa/genética , Monoaminooxidasa/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/patología , Trastorno de Pánico/etnología , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , Receptores de Colecistoquinina/genética , Receptores de Colecistoquinina/metabolismo , Receptores de GABA-A/metabolismo , Receptores de Serotonina 5-HT2/genética , Receptores de Serotonina 5-HT2/metabolismo , España , Transfección , Adulto JovenRESUMEN
BACKGROUND: Neurotrophins and their receptors are key molecules in the regulation of neuronal differentiation and survival. They mediate the survival of neurons during development and adulthood and are implicated in synaptic plasticity. The human neurotrophin-3 receptor gene NTRK3 yields two major isoforms, a full-length kinase-active form and a truncated non-catalytic form, which activates a specific pathway affecting membrane remodeling and cytoskeletal reorganization. The two variants present non-overlapping 3'UTRs, indicating that they might be differentially regulated at the post-transcriptional level. Here, we provide evidence that the two isoforms of NTRK3 are targeted by different sets of microRNAs, small non-coding RNAs that play an important regulatory role in the nervous system. RESULTS: We identify one microRNA (miR-151-3p) that represses the full-length isoform of NTRK3 and four microRNAs (miR-128, miR-485-3p, miR-765 and miR-768-5p) that repress the truncated isoform. In particular, we show that the overexpression of miR-128 - a brain enriched miRNA - causes morphological changes in SH-SY5Y neuroblastoma cells similar to those observed using an siRNA specifically directed against truncated NTRK3, as well as a significant increase in cell number. Accordingly, transcriptome analysis of cells transfected with miR-128 revealed an alteration of the expression of genes implicated in cytoskeletal organization as well as genes involved in apoptosis, cell survival and proliferation, including the anti-apoptotic factor BCL2. CONCLUSIONS: Our results show that the regulation of NTRK3 by microRNAs is isoform-specific and suggest that neurotrophin-mediated processes are strongly linked to microRNA-dependent mechanisms. In addition, these findings open new perspectives for the study of the physiological role of miR-128 and its possible involvement in cell death/survival processes.
Asunto(s)
MicroARNs/metabolismo , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptor trkC/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Biología Computacional , Perfilación de la Expresión Génica , Humanos , MicroARNs/genética , Factores de Crecimiento Nervioso/metabolismo , Neuroblastoma , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/metabolismo , Receptor trkC/antagonistas & inhibidores , Regulación hacia ArribaRESUMEN
MicroRNAs (miRNA) are recognized posttranscriptional gene repressors involved in the control of almost every biological process. Allelic variants in these regions may be an important source of phenotypic diversity and contribute to disease susceptibility. We analyzed the genomic organization of 325 human miRNAs (release 7.1, miRBase) to construct a panel of 768 single-nucleotide polymorphisms (SNPs) covering approximately 1 Mb of genomic DNA, including 131 isolated miRNAs (40%) and 194 miRNAs arranged in 48 miRNA clusters, as well as their 5-kb flanking regions. Of these miRNAs, 37% were inside known protein-coding genes, which were significantly associated with biological functions regarding neurological, psychological or nutritional disorders. SNP coverage analysis revealed a lower SNP density in miRNAs compared with the average of the genome, with only 24 SNPs located in the 325 miRNAs studied. Further genotyping of 340 unrelated Spanish individuals showed that more than half of the SNPs in miRNAs were either rare or monomorphic, in agreement with the reported selective constraint on human miRNAs. A comparison of the minor allele frequencies between Spanish and HapMap population samples confirmed the applicability of this SNP panel to the study of complex disorders among the Spanish population, and revealed two miRNA regions, hsa-mir-26a-2 in the CTDSP2 gene and hsa-mir-128-1 in the R3HDM1 gene, showing geographical allelic frequency variation among the four HapMap populations, probably because of differences in natural selection. The designed miRNA SNP panel could help to identify still hidden links between miRNAs and human disease.
Asunto(s)
Enfermedades Genéticas Congénitas/genética , Estudio de Asociación del Genoma Completo/métodos , MicroARNs/genética , Polimorfismo de Nucleótido Simple , Mapeo Cromosómico , ADN/genética , ADN/aislamiento & purificación , Frecuencia de los Genes , Variación Genética , Genoma Humano , Genotipo , Geografía , Humanos , Linfocitos/fisiología , Polimorfismo de Nucleótido Simple/genética , Valores de Referencia , EspañaRESUMEN
Genetic and functional data indicate that variation in the expression of the neurotrophin-3 receptor gene (NTRK3) may have an impact on neuronal plasticity, suggesting a role for NTRK3 in the pathophysiology of anxiety disorders. MicroRNA (miRNA) posttranscriptional gene regulators act by base-pairing to specific sequence sites, usually at the 3'UTR of the target mRNA. Variants at these sites might result in gene expression changes contributing to disease susceptibility. We investigated genetic variation in two different isoforms of NTRK3 as candidate susceptibility factors for anxiety by resequencing their 3'UTRs in patients with panic disorder (PD), obsessive-compulsive disorder (OCD), and in controls. We have found the C allele of rs28521337, located in a functional target site for miR-485-3p in the truncated isoform of NTRK3, to be significantly associated with the hoarding phenotype of OCD. We have also identified two new rare variants in the 3'UTR of NTRK3, ss102661458 and ss102661460, each present only in one chromosome of a patient with PD. The ss102661458 variant is located in a functional target site for miR-765, and the ss102661460 in functional target sites for two miRNAs, miR-509 and miR-128, the latter being a brain-enriched miRNA involved in neuronal differentiation and synaptic processing. Interestingly, these two variants significantly alter the miRNA-mediated regulation of NTRK3, resulting in recovery of gene expression. These data implicate miRNAs as key posttranscriptional regulators of NTRK3 and provide a framework for allele-specific miRNA regulation of NTRK3 in anxiety disorders.