RESUMO
High-throughput sequencing (HTS) technologies have contributed to expand current knowledge of the biology of complex diseases, including nonalcoholic fatty liver disease (NAFLD). Genome-wide association studies, whole exome sequencing, and sequencing of entire genes are used to identify variants and/or mutations that predispose to the disease pathogenesis. Here, we present a tutorial that may guide readers to manage high volume of genetics data in the context of Next-Generation Sequencing (NGS) studies.
Assuntos
Exoma , Hepatopatia Gordurosa não Alcoólica , Biologia Computacional , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hepatopatia Gordurosa não Alcoólica/genética , SoftwareRESUMO
Genome-wide association studies of complex diseases, including nonalcoholic fatty liver disease (NAFLD), have demonstrated that a large number of variants are implicated in the susceptibility of multiple traits - a phenomenon known as pleiotropy that is increasingly being explored through phenome-wide association studies. We focused on the analysis of pleiotropy within variants associated with hematologic traits and NAFLD. We used information retrieved from large public National Health and Nutrition Examination Surveys, Genome-wide association studies, and phenome-wide association studies based on the general population and explored whether variants associated with NAFLD also present associations with blood cell-related traits. Next, we applied systems biology approaches to assess the potential biological connection/s between genes that predispose affected individuals to NAFLD and nonalcoholic steatohepatitis, and genes that modulate hematological-related traits-specifically platelet count. We reasoned that this analysis would allow the identification of potential molecular mediators that link NAFLD with platelets. Genes associated with platelet count are most highly expressed in the liver, followed by the pancreas, heart, and muscle. Conversely, genes associated with NAFLD presented high expression levels in the brain, lung, spleen, and colon. Functional mapping, gene prioritization, and functional analysis of the most significant loci (P < 1 × 10-8) revealed that loci involved in the genetic modulation of platelet count presented significant enrichment in metabolic and energy balance pathways. In conclusion, variants in genes influencing NAFLD exhibit pleiotropic associations with hematologic traits, particularly platelet count. Likewise, significant enrichment of related genes with variants influencing platelet traits was noted in metabolic-related pathways. Hence, this approach yields novel mechanistic insights into NAFLD pathogenesis.
Assuntos
Sistema Hematopoético , Hepatopatia Gordurosa não Alcoólica , Estudo de Associação Genômica Ampla , Humanos , Hepatopatia Gordurosa não Alcoólica/genética , FenótipoRESUMO
The human transcriptome comprises a myriad of non protein-coding RNA species, including long noncoding RNAs (lncRNAs), which have a remarkable role in transcriptional and epigenetic regulation. We hypothesized that variants in lncRNAs influence the susceptibility to nonalcoholic fatty liver disease (NAFLD). Using next generation sequencing, we performed a survey of genetic variation associated with randomly selected lncRNA-genomic regions located within both experimentally validated and computationally predicted regulatory elements. We used a two-stage (exploratory, n = 96 and replication, n = 390) case-control approach that included well-characterized patients with NAFLD diagnosed by liver biopsy. We sequenced > 263 megabase pairs at quality score > Q17, in a total of 2,027,565 reads, including 170 lncRNA-genomic regions. In the sequencing analysis and the validated dataset, we found that the rs2829145 A/G located in a lncRNA (lnc-JAM2-6) was associated with NAFLD and the disease severity. Prediction of regulatory elements in lnc-JAM2-6 showed potential sequence-specific binding motifs of oncogenes MAFK and JUND, and the transcription factor CEBPB that is involved in inflammatory response. The A-allele was significantly associated with NAFLD as disease trait (p = 0.0081) and the disease severity (NASH-nonalcoholic steatohepatitis vs controls: OR 2.36 [95% CI: 1.54-3.62], p = 0.000078). The A-allele carriers also have significantly higher body mass index and glucose-related traits compared with homozygous GG. Hence, our results suggest that variation in lncRNAs contributes to NAFLD severity, while pointing toward the complexity of the genetic component of NAFLD, which involves still unexplored regulatory regions of the genome.
Assuntos
Hepatopatia Gordurosa não Alcoólica/genética , RNA Longo não Codificante/genética , Epigênese Genética , Feminino , Expressão Gênica , Predisposição Genética para Doença , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
It is well known that animals exposed to stressful stimuli during their early life develop different neurological disorders when they become adults. In this study, we evaluated the effect of acute cold stress on gamma-aminobutyric acid (GABA) and L-Serine (L-Ser) transporters in vitro, using the uptake of [(3)H]-GABA and [(3)H]L-Ser by synaptosomes-enriched fractions isolated from rat cerebral cortex during postnatal development. GABA and L-Ser uptake studies in vitro will be used in this investigation as a colateral evidence of changes in the expression of transporters of GABA and L-Ser. We observed that the maximum velocity (V (max)) in L-Ser and GABA uptake after stress session increased in all stages studied. In contrast, K (m) values of L-Ser uptake enhancent in almost age calculated, excluding at PD21 after cold stress during development, at the same time as K (m) (uptake affinity) values of GABA increased in just about age considered but not at PD5 compared with the control group. Finally we investigated the mechanism by which cells regulate the substrate affinity of L-Ser and GABA transporters. We demonstrated a significantly increase in total PKC activity to PD5 from PD21. Pretreatment with PKC inhibitor: staurosporine (SP) led to a restoration of control uptake in several postnatal-days suggesting a relationship between amino acids system and PKC activation. These findings suggest that a single exposure to postnatal cold stress at different periods after birth modifies both GABA and L-Ser transporters and the related increase in total PKC activity could be intracellular events that participate in neuronal plasticity by early life stress, which could be relevant to function of transporters in the adult rat brain.