Integrated systems approach identifies genetic nodes and networks in late-onset Alzheimer's disease.

The genetics of complex disease produce alterations in the molecular interactions of cellular pathways whose collective effect may become clear through the organized structure of molecular networks. To characterize molecular systems associated with late-onset Alzheimer's disease (LOAD), we constructed gene-regulatory networks in 1,647 postmortem brain tissues from LOAD patients and nondemented subjects, and we demonstrate that LOAD reconfigures specific portions of the molecular interaction structure. Through an integrative network-based approach, we rank-ordered these network structures for relevance to LOAD pathology, highlighting an immune- and microglia-specific module that is dominated by genes involved in pathogen phagocytosis, contains TYROBP as a key regulator, and is upregulated in LOAD. Mouse microglia cells overexpressing intact or truncated TYROBP revealed expression changes that significantly overlapped the human brain TYROBP network. Thus the causal network structure is a useful predictor of response to gene perturbations and presents a framework to test models of disease mechanisms underlying LOAD.

Biopsy and blood-based molecular biomarker of inflammation in IBD.

OBJECTIVE: IBD therapies and treatments are evolving to deeper levels of remission. Molecular measures of disease may augment current endpoints including the potential for less invasive assessments. DESIGN: Transcriptome analysis on 712 endoscopically defined inflamed (Inf) and 1778 non-inflamed (Non-Inf) intestinal biopsies (n=498 Crohn's disease, n=421 UC and 243 controls) in the Mount Sinai Crohn's and Colitis Registry were used to identify genes differentially expressed between Inf and Non-Inf biopsies and to generate a molecular inflammation score (bMIS) via gene set variance analysis. A circulating MIS (cirMIS) score, reflecting intestinal molecular inflammation, was generated using blood transcriptome data. bMIS/cirMIS was validated as indicators of intestinal inflammation in four independent IBD cohorts. RESULTS: bMIS/cirMIS was strongly associated with clinical, endoscopic and histological disease activity indices. Patients with the same histologic score of inflammation had variable bMIS scores, indicating that bMIS describes a deeper range of inflammation. In available clinical trial data sets, both scores were responsive to IBD treatment. Despite similar baseline endoscopic and histologic activity, UC patients with lower baseline bMIS levels were more likely treatment responders compared with those with higher levels. Finally, among patients with UC in endoscopic and histologic remission, those with lower bMIS levels were less likely to have a disease flare over time. CONCLUSION: Transcriptionally based scores provide an alternative objective and deeper quantification of intestinal inflammation, which could augment current clinical assessments used for disease monitoring and have potential for predicting therapeutic response and patients at higher risk of disease flares.

De novo mutations in schizophrenia implicate synaptic networks.

Inherited alleles account for most of the genetic risk for schizophrenia. However, new (de novo) mutations, in the form of large chromosomal copy number changes, occur in a small fraction of cases and disproportionally disrupt genes encoding postsynaptic proteins. Here we show that small de novo mutations, affecting one or a few nucleotides, are overrepresented among glutamatergic postsynaptic proteins comprising activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-d-aspartate receptor (NMDAR) complexes. Mutations are additionally enriched in proteins that interact with these complexes to modulate synaptic strength, namely proteins regulating actin filament dynamics and those whose messenger RNAs are targets of fragile X mental retardation protein (FMRP). Genes affected by mutations in schizophrenia overlap those mutated in autism and intellectual disability, as do mutation-enriched synaptic pathways. Aligning our findings with a parallel case-control study, we demonstrate reproducible insights into aetiological mechanisms for schizophrenia and reveal pathophysiology shared with other neurodevelopmental disorders.

A next generation sequencing based approach to identify extracellular vesicle mediated mRNA transfers between cells.

BACKGROUND: Exosomes and other extracellular vesicles (EVs) have emerged as an important mechanism of cell-to-cell communication. However, previous studies either did not fully resolve what genetic materials were shuttled by exosomes or only focused on a specific set of miRNAs and mRNAs. A more systematic method is required to identify the genetic materials that are potentially transferred during cell-to-cell communication through EVs in an unbiased manner. RESULTS: In this work, we present a novel next generation of sequencing (NGS) based approach to identify EV mediated mRNA exchanges between co-cultured adipocyte and macrophage cells. We performed molecular and genomic profiling and jointly considered data from RNA sequencing (RNA-seq) and genotyping to track the "sequence varying mRNAs" transferred between cells. We identified 8 mRNAs being transferred from macrophages to adipocytes and 21 mRNAs being transferred in the opposite direction. These mRNAs represented biological functions including extracellular matrix, cell adhesion, glycoprotein, and signal peptides. CONCLUSIONS: Our study sheds new light on EV mediated RNA communications between adipocyte and macrophage cells, which may play a significant role in developing insulin resistance in diabetic patients. This work establishes a new method that is applicable to examining genetic material exchanges in many cellular systems and has the potential to be extended to in vivo studies as well.

Trunk mutational events present minimal intra- and inter-tumoral heterogeneity in hepatocellular carcinoma.

BACKGROUND & AIMS: According to the clonal model of tumor evolution, trunk alterations arise at early stages and are ubiquitous. Through the characterization of early stages of hepatocarcinogenesis, we aimed to identify trunk alterations in hepatocellular carcinoma (HCC) and study their intra- and inter-tumor distribution in advanced lesions. METHODS: A total of 151 samples representing the multistep process of hepatocarcinogenesis were analyzed by targeted-sequencing and a single nucleotide polymorphism array. Genes altered in early lesions (31 dysplastic nodules [DNs] and 38 small HCCs [sHCC]) were defined as trunk. Their distribution was explored in: a) different regions of large tumors (43 regions, 21 tumors), and b) different nodules of the same patient (39 tumors, 17 patients). Multinodular lesions were classified as intrahepatic metastases (IMs) or synchronous tumors based on chromosomal aberrations. RESULTS: TERT promoter mutations (10.5%) and broad copy-number aberrations in chromosomes 1 and 8 (3-7%) were identified as trunk gatekeepers in DNs and were maintained in sHCCs. Trunk drivers identified in sHCCs included TP53 (23%) and CTNNB1 (11%) mutations, and focal amplifications or deletions in known drivers (6%). Overall, TERT, TP53 and CTNNB1 mutations were the most frequent trunk events and at least one was present in 51% of sHCCs. Around 90% of mutations in these genes were ubiquitous among different regions of large tumors. In multinodular HCCs, 35% of patients harbored IMs; 85% of mutations in TERT, TP53 and/or CTNNB1 were retained in primary and metastatic tumors. CONCLUSIONS: Trunk events in early stages (TERT, TP53, CTNNB1 mutations) were ubiquitous across different regions of the same tumor and between primary and metastatic nodules in >85% of cases. This concept supports the knowledge that single biopsies would suffice to capture trunk mutations in HCC. LAY SUMMARY: Trunk alterations arise at early stages of cancer and are shared among all malignant cells of the tumor. In order to identify trunk alterations in HCC, we characterized early stages of hepatocarcinogenesis represented by dysplastic nodules and small lesions. Mutations in TERT, TP53 and CTNNB1 genes were the most frequent. Analyses in more advanced lesions showed that mutations in these same genes were shared between different regions of the same tumor and between primary and metastatic tumors, suggesting their trunk role in this disease.

Clinicopathological indices to predict hepatocellular carcinoma molecular classification.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the second most lethal cancer caused by lack of effective therapies. Although promising, HCC molecular classification, which enriches potential responders to specific therapies, has not yet been assessed in clinical trials of anti-HCC drugs. We aimed to overcome these challenges by developing clinicopathological surrogate indices of HCC molecular classification. METHODS: Hepatocellular carcinoma classification defined in our previous transcriptome meta-analysis (S1, S2 and S3 subclasses) was implemented in an FDA-approved diagnostic platform (Elements assay, NanoString). Ninety-six HCC tumours (training set) were assayed to develop molecular subclass-predictive indices based on clinicopathological features, which were independently validated in 99 HCC tumours (validation set). Molecular deregulations associated with the histopathological features were determined by pathway analysis. Sample sizes for HCC clinical trials enriched with specific molecular subclasses were determined. RESULTS: Hepatocellular carcinoma subclass-predictive indices were steatohepatitic (SH)-HCC variant and immune cell infiltrate for S1 subclass, macrotrabecular/compact pattern, lack of pseudoglandular pattern, and high serum alpha-foetoprotein (>400 ng/ml) for S2 subclass, and microtrabecular pattern, lack of SH-HCC and clear cell variants, and lower histological grade for S3 subclass. Macrotrabecular/compact pattern, a predictor of S2 subclass, was associated with the activation of therapeutically targetable oncogene YAP and stemness markers EPCAM/KRT19. BMP4 was associated with pseudoglandular pattern. Subclass-predictive indices-based patient enrichment reduced clinical trial sample sizes from 121, 184 and 53 to 30, 43 and 22 for S1, S2 and S3 subclass-targeting therapies respectively. CONCLUSIONS: Hepatocellular carcinoma molecular subclasses can be enriched by clinicopathological indices tightly associated with deregulation of therapeutically targetable molecular pathways.

A genomic and clinical prognostic index for hepatitis C-related early-stage cirrhosis that predicts clinical deterioration.

OBJECTIVE: The number of patients with HCV-related cirrhosis is increasing, leading to a rising risk of complications and death. Prognostic stratification in patients with early-stage cirrhosis is still challenging. We aimed to develop and validate a clinically useful prognostic index based on genomic and clinical variables to identify patients at high risk of disease progression. DESIGN: We developed a prognostic index, comprised of a 186-gene signature validated in our previous genome-wide profiling study, bilirubin (>1 mg/dL) and platelet count (<100,000/mm(3)), in an Italian HCV cirrhosis cohort (training cohort, n=216, median follow-up 10 years). The gene signature test was implemented using a digital transcript counting (nCounter) assay specifically developed for clinical use and the prognostic index was evaluated using archived specimens from an independent cohort of HCV-related cirrhosis in the USA (validation cohort, n=145, median follow-up 8 years). RESULTS: In the training cohort, the prognostic index was associated with hepatic decompensation (HR=2.71, p=0.003), overall death (HR=6.00, p<0.001), hepatocellular carcinoma (HR=3.31, p=0.001) and progression of Child-Turcotte-Pugh class (HR=6.70, p<0.001). The patients in the validation cohort were stratified into high-risk (16%), intermediate-risk (42%) or low-risk (42%) groups by the prognostic index. The high-risk group had a significantly increased risk of hepatic decompensation (HR=7.36, p<0.001), overall death (HR=3.57, p=0.002), liver-related death (HR=6.49, p<0.001) and all liver-related adverse events (HR=4.98, p<0.001). CONCLUSIONS: A genomic and clinical prognostic index readily available for clinical use was successfully validated, warranting further clinical evaluation for prognostic prediction and clinical trial stratification and enrichment for preventive interventions.

How do students react to analyzing their own genomes in a whole-genome sequencing course?: outcomes of a longitudinal cohort study.

PURPOSE: Health-care professionals need to be trained to work with whole-genome sequencing (WGS) in their practice. Our aim was to explore how students responded to a novel genome analysis course that included the option to analyze their own genomes. METHODS: This was an observational cohort study. Questionnaires were administered before (T3) and after the genome analysis course (T4), as well as 6 months later (T5). In-depth interviews were conducted at T5. RESULTS: All students (n = 19) opted to analyze their own genomes. At T5, 12 of 15 students stated that analyzing their own genomes had been useful. Ten reported they had applied their knowledge in the workplace. Technical WGS knowledge increased (mean of 63.8% at T3, mean of 72.5% at T4; P = 0.005). In-depth interviews suggested that analyzing their own genomes may increase students' motivation to learn and their understanding of the patient experience. Most (but not all) of the students reported low levels of WGS results-related distress and low levels of regret about their decision to analyze their own genomes. CONCLUSION: Giving students the option of analyzing their own genomes may increase motivation to learn, but some students may experience personal WGS results-related distress and regret. Additional evidence is required before considering incorporating optional personal genome analysis into medical education on a large scale.

A multiprotein complex necessary for both transcription and DNA replication at the ß-globin locus.

DNA replication, repair, transcription and chromatin structure are intricately associated nuclear processes, but the molecular links between these events are often obscure. In this study, we have surveyed the protein complexes that bind at ß-globin locus control region, and purified and characterized the function of one such multiprotein complex from human erythroleukemic K562 cells. We further validated the existence of this complex in human CD34+ cell-derived normal erythroid cells. This complex contains ILF2/ILF3 transcription factors, p300 acetyltransferase and proteins associated with DNA replication, transcription and repair. RNAi knockdown of ILF2, a DNA-binding component of this complex, abrogates the recruitment of the complex to its cognate DNA sequence and inhibits transcription, histone acetylation and usage of the origin of DNA replication at the ß-globin locus. These results imply a direct link between mammalian DNA replication, transcription and histone acetylation mediated by a single multiprotein complex.

A large gene network in immature erythroid cells is controlled by the myeloid and B cell transcriptional regulator PU.1.

PU.1 is a hematopoietic transcription factor that is required for the development of myeloid and B cells. PU.1 is also expressed in erythroid progenitors, where it blocks erythroid differentiation by binding to and inhibiting the main erythroid promoting factor, GATA-1. However, other mechanisms by which PU.1 affects the fate of erythroid progenitors have not been thoroughly explored. Here, we used ChIP-Seq analysis for PU.1 and gene expression profiling in erythroid cells to show that PU.1 regulates an extensive network of genes that constitute major pathways for controlling growth and survival of immature erythroid cells. By analyzing fetal liver erythroid progenitors from mice with low PU.1 expression, we also show that the earliest erythroid committed cells are dramatically reduced in vivo. Furthermore, we find that PU.1 also regulates many of the same genes and pathways in other blood cells, leading us to propose that PU.1 is a multifaceted factor with overlapping, as well as distinct, functions in several hematopoietic lineages.