Iis--integrated Interactome System: A Web-based Platform For The Annotation, Analysis And Visualization Of Protein-metabolite-gene-drug Interactions By Integrating A Variety Of Data Sources And Tools.

High-throughput screening of physical, genetic and chemical-genetic interactions brings important perspectives in the Systems Biology field, as the analysis of these interactions provides new insights into protein/gene function, cellular metabolic variations and the validation of therapeutic targets and drug design. However, such analysis depends on a pipeline connecting different tools that can automatically integrate data from diverse sources and result in a more comprehensive dataset that can be properly interpreted. We describe here the Integrated Interactome System (IIS), an integrative platform with a web-based interface for the annotation, analysis and visualization of the interaction profiles of proteins/genes, metabolites and drugs of interest. IIS works in four connected modules: (i) Submission module, which receives raw data derived from Sanger sequencing (e.g. two-hybrid system); (ii) Search module, which enables the user to search for the processed reads to be assembled into contigs/singlets, or for lists of proteins/genes, metabolites and drugs of interest, and add them to the project; (iii) Annotation module, which assigns annotations from several databases for the contigs/singlets or lists of proteins/genes, generating tables with automatic annotation that can be manually curated; and (iv) Interactome module, which maps the contigs/singlets or the uploaded lists to entries in our integrated database, building networks that gather novel identified interactions, protein and metabolite expression/concentration levels, subcellular localization and computed topological metrics, GO biological processes and KEGG pathways enrichment. This module generates a XGMML file that can be imported into Cytoscape or be visualized directly on the web. We have developed IIS by the integration of diverse databases following the need of appropriate tools for a systematic analysis of physical, genetic and chemical-genetic interactions. IIS was validated with yeast two-hybrid, proteomics and metabolomics datasets, but it is also extendable to other datasets. IIS is freely available online at: http://www.lge.ibi.unicamp.br/lnbio/IIS/.

A Draft of the Human Septin Interactome

Background: Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins. Methodology/Principal Findings: Here, we performed yeast two-hybrid screens with human septins 1-10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments. Conclusions/Significance: If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the ""group rule"", i.e. members of the same group (e. g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.

Interactome mapping of the phosphatidylinositol 3-kinase-mammalian target of rapamycin pathway identifies deformed epidermal autoregulatory factor-1 as a new glycogen synthase kinase-3 interactor.

The phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) pathway plays pivotal roles in cell survival, growth, and proliferation downstream of growth factors. Its perturbations are associated with cancer progression, type 2 diabetes, and neurological disorders. To better understand the mechanisms of action and regulation of this pathway, we initiated a large scale yeast two-hybrid screen for 33 components of the PI3K-mTOR pathway. Identification of 67 new interactions was followed by validation by co-affinity purification and exhaustive literature curation of existing information. We provide a nearly complete, functionally annotated interactome of 802 interactions for the PI3K-mTOR pathway. Our screen revealed a predominant place for glycogen synthase kinase-3 (GSK3) A and B and the AMP-activated protein kinase. In particular, we identified the deformed epidermal autoregulatory factor-1 (DEAF1) transcription factor as an interactor and in vitro substrate of GSK3A and GSK3B. Moreover, GSK3 inhibitors increased DEAF1 transcriptional activity on the 5-HT1A serotonin receptor promoter. We propose that DEAF1 may represent a therapeutic target of lithium and other GSK3 inhibitors used in bipolar disease and depression.

Interactome analysis reveals that FAM161A, deficient in recessive retinitis pigmentosa, is a component of the Golgi-centrosomal network.

Defects in FAM161A, a protein of unknown function localized at the cilium of retinal photoreceptor cells, cause retinitis pigmentosa, a form of hereditary blindness. By using different fragments of this protein as baits to screen cDNA libraries of human and bovine retinas, we defined a yeast two-hybrid-based FAM161A interactome, identifying 53 bona fide partners. In addition to statistically significant enrichment in ciliary proteins, as expected, this interactome revealed a substantial bias towards proteins from the Golgi apparatus, the centrosome and the microtubule network. Validation of interaction with key partners by co-immunoprecipitation and proximity ligation assay confirmed that FAM161A is a member of the recently recognized Golgi-centrosomal interactome, a network of proteins interconnecting Golgi maintenance, intracellular transport and centrosome organization. Notable FAM161A interactors included AKAP9, FIP3, GOLGA3, KIFC3, KLC2, PDE4DIP, NIN and TRIP11. Furthermore, analysis of FAM161A localization during the cell cycle revealed that this protein followed the centrosome during all stages of mitosis, likely reflecting a specific compartmentalization related to its role at the ciliary basal body during the G0 phase. Altogether, these findings suggest that FAM161A's activities are probably not limited to ciliary tasks but also extend to more general cellular functions, highlighting possible novel mechanisms for the molecular pathology of retinal disease.

Computational analysis of the LRRK2 interactome

LRRK2 was identified in 2004 as the causative protein product of the Parkinson’s disease locus designated PARK8. In the decade since then, genetic studies have revealed at least 6 dominant mutations in LRRK2 linked to Parkinson’s disease, alongside one associated with cancer. It is now well established that coding changes in LRRK2 are one of the most common causes of Parkinson’s. Genome-wide association studies (GWAs) have, more recently, reported single nucleotide polymorphisms (SNPs) around the LRRK2 locus to be associated with risk of developing sporadic Parkinson’s disease and inflammatory bowel disorder. The functional research that has followed these genetic breakthroughs has generated an extensive literature regarding LRRK2 pathophysiology; however, there is still no consensus as to the biological function of LRRK2. To provide insight into the aspects of cell biology that are consistently related to LRRK2 activity, we analysed the plethora of candidate LRRK2 interactors available through the BioGRID and IntAct data repositories. We then performed GO terms enrichment for the LRRK2 interactome. We found that, in two different enrichment portals, the LRRK2 interactome was associated with terms referring to transport, cellular organization, vesicles and the cytoskeleton. We also verified that 21 of the LRRK2 interactors are genetically linked to risk for Parkin- son’s disease or inflammatory bowel disorder. The implications of these findings are discussed, with particular regard to potential novel areas of investigation.

Reconstruction and analysis of the NF-kB pathway interactome: a systems biology approach

Background. One of the phenomena observed in human aging is the progressive increase of a systemic inflammatory state, a condition referred to as “inflammaging”, negatively correlated with longevity. A prominent mediator of inflammation is the transcription factor NF-kB, that acts as key transcriptional regulator of many genes coding for pro-inflammatory cytokines. Many different signaling pathways activated by very diverse stimuli converge on NF-kB, resulting in a regulatory network characterized by high complexity. NF-kB signaling has been proposed to be responsible of inflammaging. Scope of this analysis is to provide a wider, systemic picture of such intricate signaling and interaction network: the NF-kB pathway interactome. Methods. The study has been carried out following a workflow for gathering information from literature as well as from several pathway and protein interactions databases, and for integrating and analyzing existing data and the relative reconstructed representations by using the available computational tools. Strong manual intervention has been necessarily used to integrate data from multiple sources into mathematically analyzable networks. The reconstruction of the NF-kB interactome pursued with this approach provides a starting point for a general view of the architecture and for a deeper analysis and understanding of this complex regulatory system. Results. A “core” and a “wider” NF-kB pathway interactome, consisting of 140 and 3146 proteins respectively, were reconstructed and analyzed through a mathematical, graph-theoretical approach. Among other interesting features, the topological characterization of the interactomes shows that a relevant number of interacting proteins are in turn products of genes that are controlled and regulated in their expression exactly by NF-kB transcription factors. These “feedback loops”, not always well-known, deserve deeper investigation since they may have a role in tuning the response and the output consequent to NF-kB pathway initiation, in regulating the intensity of the response, or its homeostasis and balance in order to make the functioning of such critical system more robust and reliable. This integrated view allows to shed light on the functional structure and on some of the crucial nodes of thet NF-kB transcription factors interactome. Conclusion. Framing structure and dynamics of the NF-kB interactome into a wider, systemic picture would be a significant step toward a better understanding of how NF-kB globally regulates diverse gene programs and phenotypes. This study represents a step towards a more complete and integrated view of the NF-kB signaling system.

Identification of SNPs in the cystic fibrosis interactome influencing pulmonary progression in cystic fibrosis

There is growing evidence that the great phenotypic variability in patients with cystic fibrosis (CF) not only depends on the genotype, but apart from a combination of environmental and stochastic factors predominantly also on modifier gene effects. It has been proposed that genes interacting with CF transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) are potential modifiers. Therefore, we assessed the impact of single-nucleotide polymorphisms (SNPs) of several of these interacters on CF disease outcome. SNPs that potentially alter gene function were genotyped in 95 well-characterized p.Phe508del homozygous CF patients. Linear mixed-effect model analysis was used to assess the relationship between sequence variants and the repeated measurements of lung function parameters. In total, we genotyped 72 SNPs in 10 genes. Twenty-five SNPs were used for statistical analysis, where we found strong associations for one SNP in PPP2R4 with the lung clearance index (P ≤ 0.01), the specific effective airway resistance (P ≤ 0.005) and the forced expiratory volume in 1 s (P ≤ 0.005). In addition, we identified one SNP in SNAP23 to be significantly associated with three lung function parameters as well as one SNP in PPP2R1A and three in KRT19 to show a significant influence on one lung function parameter each. Our findings indicate that direct interacters with CFTR, such as SNAP23, PPP2R4 and PPP2R1A, may modify the residual function of p.Phe508del-CFTR while variants in KRT19 may modulate the amount of p.Phe508del-CFTR at the apical membrane and consequently modify CF disease.

Proteomic characterization of the FUS/TLS interactome

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed RNA-binding protein, that has been discovered as fused to transcription factors in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis [1]. To date, FUS has been implicated in a variety of cellular processes such as gene expression control, transcriptional regulation, pre-mRNA splicing and miRNA processing [2]. In addition, some evidences link FUS to genome stability control and DNA damage response. In fact, mice lacking FUS are hypersensitive to ionizing radiation and show high levels of chromosome instability and in response to double-strand breaks, FUS gets phosphorylated by the protein kinase ATM [3, 4, 5]. Moreover, upon DNA damage stress, FUS mediates Ebp1 (ErbB3 receptor-binding protein) SUMOylation, a post-translational modification that is required for its onco-suppressive activity, by acting as SUMO E3 ligase [6]. The study aims to investigate the role of FUS in DNA damage response and SUMOylation, two cellular pathways tightly interconnected to each other. Moreover, we will exploit biochemical and mass spectrometry-based approaches in order to identify other potential substrates of the E3 SUMO ligase activity of FUS. Preliminary results of mass spectrometric identification of FUS interacting proteins, in HEK293 and SHSY5Y cells, highlighted the interaction of FUS with several proteins involved in DNA damage response and many of those have been described already as target of SUMOylation, such as XRCC5, DDX5, PARP1, Nucleophosmin, and others. These evidences strengthen the hypothesis that FUS might represent a link between these pathways, even thou its exact role still needs to be clearly addressed. [1] Vance C. et al. (2009) Science 323(5918): p. 1208-11 [2] Fiesel FC., Kahle PJ. (2011) FEBS J. 278(19): p. 3550-68 [3] Kuroda M. et al. (2000) Embo J. 19(3): p. 453-62 [4] Hicks GG. et al. (2000) Nat Genet. 24(2):p. 175-9 [5] Gardiner M. et al. (2008) Biochem J. 415(2): p. 297-307 [6] Oh SM. et al. (2010) Oncogene 29(7): p. 1017-30

A comprehensive two-hybrid analysis to explore the yeast protein interactome

Protein–protein interactions play crucial roles in the execution of various biological functions. Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions. We previously developed a system to examine two-hybrid interactions in all possible combinations between the ≈6,000 proteins of the budding yeast Saccharomyces cerevisiae. Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins. Unexpectedly, these data do not largely overlap with those obtained by the other project [Uetz, P., et al. (2000) Nature (London) 403, 623–627] and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast. Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins. Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks. They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport. Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.

Uncovering changes in the redox protein interactome of PTEN

Phosphatase and tensin homolog (PTEN) is a redox-sensitive, dual-specificity protein phosphatase involved in regulating a number of cellular processes including metabolism, apoptosis, cell proliferation and survival. It acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of a redox regulation of PTEN downstream signaling has been reported, the effect of cellular oxidative stress or direct PTEN oxidation on the PTEN interactome is still poorly defined. To investigate this, PTEN-GST fusion protein was prepared in its reduced form and an H2O2-oxidized form that was reversible by DTT treatment, and these were immobilized on a glutathione-sepharose-based support. The immobilized protein was incubated with cell lysate to capture interacting proteins. Captured proteins were eluted from the beads, analyzed by LC-MSMS and comparatively quantified using label-free methods. After subtraction of interactors that were also present in the resin and GST controls, 97 individual protein interactors were identified, including several that are novel. Fourteen interactors that varied significantly with the redox status of PTEN were identified, including thioredoxin and peroxiredoxin-1. Except for one interactor, their binding was higher for oxidized PTEN. Using western blotting, altered binding to PTEN was confirmed for 3 selected interactors (Prdx1, Trx, and Anxa2) and DDB1 was validated as a novel interactor with unaltered binding. Our results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome which is important for the cellular function of PTEN. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.

A proteo-liposome system for the analysis of the intracellular interactome of membrane proteins using amyloid precursor protein as a model

Transmembrane proteins play crucial roles in many important physiological processes. The intracellular domain of membrane proteins is key for their function by interacting with a wide variety of cytosolic proteins. It is therefore important to examine this interaction. A recently developed method to study these interactions, based on the use of liposomes as a model membrane, involves the covalent coupling of the cytoplasmic domains of membrane proteins to the liposome membrane. This allows for the analysis of interaction partners requiring both protein and membrane lipid binding. This thesis further establishes the liposome recruitment system and utilises it to examine the intracellular interactome of the amyloid precursor protein (APP), most well-known for its proteolytic cleavage that results in the production and accumulation of amyloid beta fragments, the main constituent of amyloid plaques in Alzheimer’s disease pathology. Despite this, the physiological function of APP remains largely unclear. Through the use of the proteo-liposome recruitment system two novel interactions of APP’s intracellular domain (AICD) are examined with a view to gaining a greater insight into APP’s physiological function. One of these novel interactions is between AICD and the mTOR complex, a serine/threonine protein kinase that integrates signals from nutrients and growth factors. The kinase domain of mTOR directly binds to AICD and the N-terminal amino acids of AICD are crucial for this interaction. The second novel interaction is between AICD and the endosomal PIKfyve complex, a lipid kinase involved in the production of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol-3-phosphate, which has a role in controlling ensdosome dynamics. The scaffold protein Vac14 of the PIKfyve complex binds directly to AICD and the C-terminus of AICD is important for its interaction with the PIKfyve complex. Using a recently developed intracellular PI(3,5)P2 probe it is shown that APP controls the formation of PI(3,5)P2 positive vesicular structures and that the PIKfyve complex is involved in the trafficking and degradation of APP. Both of these novel APP interactors have important implications of both APP function and Alzheimer’s disease. The proteo-liposome recruitment method is further validated through its use to examine the recruitment and assembly of the AP-2/clathrin coat from purified components to two membrane proteins containing different sorting motifs. Taken together this thesis highlights the proteo-liposome recruitment system as a valuable tool for the study of membrane proteins intracellular interactome. It allows for the mimicking of the protein in its native configuration therefore identifying weaker interactions that are not detected by more conventional methods and also detecting interactions that are mediated by membrane phospholipids.

LAP1 interactome and its functional features throughout spermatogenesis

The lamina-associated polypeptide 1 (LAP1) is a type II transmembrane protein of the inner nuclear membrane encoded by the human gene TOR1AIP1. LAP1 is involved in maintaining the nuclear envelope structure and appears be involved in the positioning of lamins and chromatin. In the nuclear envelope, LAP1 is suggested to exist as a complex with A-type and B-type lamins, torsins and emerin. The presence of such complexes suggests that LAP1 may cooperate functionally with these proteins in tissues where they play a critical role. Therefore, the identification of LAP1 binding partners and the signalling pathways where LAP1 participates, is crucial for a better understanding of LAP1 functions. The work described in this thesis addresses novel human LAP1 associated proteins found through bioinformatic tools. Public databases allowed for the discovery of the LAP1 interactome, which was manually curated, identifying several functionally relevant proteins. Subsequently, the integration of multiple bioinformatic tools established novel functions to LAP1 such as DNA damage response and telomere association. In conjunction, bioinformatic results also reinforced the association of LAP1 with mitosis, and the already identified role of LAP1 in nuclear morphology. Interestingly, this association of LAP1 with the regulation of the nuclear envelope structure and mitosis progression, shares functional elements with spermatogenesis. Therefore, this work additionally described the localization of LAP1 and some of its interactors throughout the spermatogenic cycle, in mouse and human testis. The results established that the activity of LAP1 during the mouse spermatogenic cycle is most evident from stage VIII until the end of spermiogenesis, which is characteristic of manchette development. Concomitantly, some LAP1 interactors studied in this work share a similar localization, namely, PP1γ2, Lamin B1 and Lamin A/C. The results obtained from the study of LAP1 throughout different periods of the male reproductive system attributed potential new biological functions to LAP1. Thereby, this work can be the foundation of future studies regarding LAP1 and the regulation of multiple cellular processes and disease conditions.

Contribution to drug discovery and development for tauopathies using yeast as a model

This work aimed to contribute to drug discovery and development (DDD) for tauopathies, while expanding our knowledge on this group of neurodegenerative disorders, including Alzheimer’s disease (AD). Using yeast, a recognized model for neurodegeneration studies, useful models were produced for the study of tau interaction with beta-amyloid (Aβ), both AD hallmark proteins. The characterization of these models suggests that these proteins co-localize and that Aβ1-42, which is toxic to yeast, is involved in tau40 phosphorylation (Ser396/404) via the GSK-3β yeast orthologue, whereas tau seems to facilitate Aβ1-42 oligomerization. The mapping of tau’s interactome in yeast, achieved with a tau toxicity enhancer screen using the yeast deletion collection, provided a novel framework, composed of 31 genes, to identify new mechanisms associated with tau pathology, as well as to identify new drug targets or biomarkers. This genomic screen also allowed to select the yeast strain mir1Δ-tau40 for development of a new GPSD2TM drug discovery screening system. A library of unique 138 marine bacteria extracts, obtained from the Mid-Atlantic Ridge hydrothermal vents, was screened with mir1Δ-tau40. Three extracts were identified as suppressors of tau toxicity and constitute good starting points for DDD programs. mir1Δ strain was sensitive to tau toxicity, relating tau pathology with mitochondrial function. SLC25A3, the human homologue of MIR1, codes for the mitochondrial phosphate carrier protein (PiC). Resorting to iRNA, SLC25A3 expression was silenced in human neuroglioma cells, as a first step towards the engineering of a neural model for replicating the results obtained in yeast. This model is essential to understand the mechanisms of tau toxicity at the mitochondrial level and to validate PiC as a relevant drug target. The set of DDD tools here presented will foster the development of innovative and efficacious therapies, urgently needed to cope with tau-related disorders of high human and social-economic impact.

Mutations in FGF17, IL17RD, DUSP6, SPRY4, and FLRT3 Are Identified in Individuals with Congenital Hypogonadotropic Hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) and its anosmia-associated form (Kallmann syndrome [KS]) are genetically heterogeneous. Among the >15 genes implicated in these conditions, mutations in FGF8 and FGFR1 account for ∼12% of cases; notably, KAL1 and HS6ST1 are also involved in FGFR1 signaling and can be mutated in CHH. We therefore hypothesized that mutations in genes encoding a broader range of modulators of the FGFR1 pathway might contribute to the genetics of CHH as causal or modifier mutations. Thus, we aimed to (1) investigate whether CHH individuals harbor mutations in members of the so-called "FGF8 synexpression" group and (2) validate the ability of a bioinformatics algorithm on the basis of protein-protein interactome data (interactome-based affiliation scoring [IBAS]) to identify high-quality candidate genes. On the basis of sequence homology, expression, and structural and functional data, seven genes were selected and sequenced in 386 unrelated CHH individuals and 155 controls. Except for FGF18 and SPRY2, all other genes were found to be mutated in CHH individuals: FGF17 (n = 3 individuals), IL17RD (n = 8), DUSP6 (n = 5), SPRY4 (n = 14), and FLRT3 (n = 3). Independently, IBAS predicted FGF17 and IL17RD as the two top candidates in the entire proteome on the basis of a statistical test of their protein-protein interaction patterns to proteins known to be altered in CHH. Most of the FGF17 and IL17RD mutations altered protein function in vitro. IL17RD mutations were found only in KS individuals and were strongly linked to hearing loss (6/8 individuals). Mutations in genes encoding components of the FGF pathway are associated with complex modes of CHH inheritance and act primarily as contributors to an oligogenic genetic architecture underlying CHH.

Bioinformatics-driven identification and examination of candidate genes for non-alcoholic fatty liver disease.

ObjectiveCandidate genes for non-alcoholic fatty liver disease (NAFLD) identified by a bioinformatics approach were examined for variant associations to quantitative traits of NAFLD-related phenotypes.Research Design and MethodsBy integrating public database text mining, trans-organism protein-protein interaction transferal, and information on liver protein expression a protein-protein interaction network was constructed and from this a smaller isolated interactome was identified. Five genes from this interactome were selected for genetic analysis. Twenty-one tag single-nucleotide polymorphisms (SNPs) which captured all common variation in these genes were genotyped in 10,196 Danes, and analyzed for association with NAFLD-related quantitative traits, type 2 diabetes (T2D), central obesity, and WHO-defined metabolic syndrome (MetS).Results273 genes were included in the protein-protein interaction analysis and EHHADH, ECHS1, HADHA, HADHB, and ACADL were selected for further examination. A total of 10 nominal statistical significant associations (P<0.05) to quantitative metabolic traits were identified. Also, the case-control study showed associations between variation in the five genes and T2D, central obesity, and MetS, respectively. Bonferroni adjustments for multiple testing negated all associations.ConclusionsUsing a bioinformatics approach we identified five candidate genes for NAFLD. However, we failed to provide evidence of associations with major effects between SNPs in these five genes and NAFLD-related quantitative traits, T2D, central obesity, and MetS.