Associating Genes and Protein Complexes with Disease via Network Propagation

A fundamental challenge in human health is the identification of disease-causing genes. Recently, several studies have tackled this challenge via a network-based approach, motivated by the observation that genes causing the same or similar diseases tend to lie close to one another in a network of protein-protein or functional interactions. However, most of these approaches use only local network information in the inference process and are restricted to inferring single gene associations. Here, we provide a global, network-based method for prioritizing disease genes and inferring protein complex associations, which we call PRINCE. The method is based on formulating constraints on the prioritization function that relate to its smoothness over the network and usage of prior information. We exploit this function to predict not only genes but also protein complex associations with a disease of interest. We test our method on gene-disease association data, evaluating both the prioritization achieved and the protein complexes inferred. We show that our method outperforms extant approaches in both tasks. Using data on 1,369 diseases from the OMIM knowledgebase, our method is able (in a cross validation setting) to rank the true causal gene first for 34% of the diseases, and infer 139 disease-related complexes that are highly coherent in terms of the function, expression and conservation of their member proteins. Importantly, we apply our method to study three multi-factorial diseases for which some causal genes have been found already: prostate cancer, alzheimer and type 2 diabetes mellitus. PRINCE's predictions for these diseases highly match the known literature, suggesting several novel causal genes and protein complexes for further investigation

Let My People Stay: Irregular Migrants' Struggle for Rights and Recognition

Thesis (Ph.D.)--University of Washington, 2018In January 2014, a mass protest of 40,000 African migrants, demanding rights, recognition and a fair asylum process took place in Tel-Aviv. Their demonstration was unprecedented in its nature and magnitude offering a unique and interesting puzzle: how a foreign-born community without resources or familiarity with the country’s authorities, culture, or tolerance for protest, successfully mobilized and why did the high cost of engaging in protest did not deter the participants? Exiting literature often treats mobilization as an engagement with traditional political institutions, those however, are mostly irrelevant for people without legal status. Scholarship on the issue tends to separate the analysis of the macro level (structure and institutions) from that of the micro level (identity and culture). This gap creates a disconnect between the structural conditions and the ways in which they are experienced and understood at the individual and community levels. My hypothesis is that mass organizing by irregular migrants is dependent on their ability to draw on their lived experiences and skill-sets to navigate an alien political structure and utilize the support of local allies to make legal claims and political demands. To test it, I used a mixed methods approach combining content analysis of articles and reports with fieldwork conducted in Israel in 2016-17 featuring interviews with asylum seekers, activists and NGO workers. The case of asylum seekers in Israel demonstrates their ability to self-organize as a diasporic group with collective interests as well the cooperation they built with local human-rights NGOs and other aid groups as the basis for their successful collective operation. Their struggle raised important questions about Israel’s migration regime, workforce dependability, and the balance between adherence to democratic norms and securing a Jewish majority, while capturing the attention of global media and international actors. Despite the inability to achieve their stated policy goals, their success is articulated by their own admission in their ability to mobilize and spread the language of rights in their communities. By examining the similarities in collective action practices and discourses adopted by migrant movements in Washington State I discuss the struggle of asylum seekers in Israel in a wider context and offer a comparative perspective on social movements and political engagement of unrecognized members of society

African and European local ancestry surrounding Apolipoprotein E has a differential biological effect upon acute amyloid beta exposure in iPSC‐differentiated astrocytes

Background Studies have shown that the lower risk associated with the ε4 allele for African ancestry is associated with the local ancestry (LA) surrounding the ApoE gene. Previous studies have shown differences between ApoE3 and ApoE4 alleles in isogenic induced pluripotent stem cell (iPSC) models when exposed to Aβ. We hypothesized that ApoE4 individuals with African LA would respond differently to Aβ compared to European ApoE3 and European ApoE4 LA lines. However, as we cannot produce isogenic lines to test LA, we used RNA expression changes to Aβ exposure to increase our sensitivity to potential differences. Method We differentiated European LA ε4/ε4 and African LA ε4/ε4 allele astrocytes from iPSC lines. Astrocytes were exposed to exogenous Aβ and RNA was obtained at 0 and 24 hours. We performed bioinformatic analyses with the STAR algorithm and differential expression calculation using linear models implemented in edgeR. Pathway enrichment analysis for Gene Ontology Biological Processes, KEGG and Reactome pathways was performed using Metascape. Result Twenty‐four hours following Aβ exposure, 524 and 671 genes were deferentially expressed from baseline in African and European LA lines respectively. Analysis of the unregulated genes in the two different ancestries revealed markedly different pathways. The unregulated genes in African LA astrocytes were enriched for Ribosome Biogenesis and RNA modification processes while the upregulated genes in the European LA astrocytes were enriched for Cell Cycle and DNA modification processes. In the European LA astrocytes, downregulated genes were enriched for Synaptic Assembly and Kainate Receptor Activity while in the African LA astrocytes downregulated genes enriched for Extracellular Matrix‐related processes. Conclusion Our initial results suggest that the two ancestries respond differently to Aβ exposure. Whether this is due to global or local ancestry differences is unclear. Further studies including astrocytes bearing African LA ε3/ε3 are needed to clarify that question. Both ribosomal dysfunction and astrocyte‐neuronal and astrocyte‐microglia synaptic assembly have been implicated in Aβ clearance and/or AD. A potential link between LA and the regulation of these processes due to Aβ exposure could pave the path to a better understanding of LOAD pathology

Harnessing Chromatin 3D interactions to Understand Ancestry‐Specific Alzheimer Disease (AD) risk

Background Ancestry‐specific Alzheimer Disease (AD) genetic risk is well recognized. Chromatin 3D interaction map in AD‐relevant cells with different ancestries is lacking but needed to understand the genetic/epigenetic basis for AD in diverse populations. We have shown that the local ancestry (LA) block (∼2Mb in size) surrounding APOE confers differential genetic risk for APOE ε4 carriers in Non‐Hispanic Whites (NHW) and African Americans (AA). Recently, we reported that NHW APOE ε4 carriers have a higher APOE ε4 expression and higher number of astrocytes compared to their AA counterparts in the frontal cortex. We therefore sought to characterize chromatin 3D interactions in astrocytes within different APOE LA. Method Induced pluripotent stem cells (iPSCs) with African or European LA block (N=2 individuals for each) were differentiated into astrocytes. Chromatin Hi‐C libraries were constructed with 4‐cutter enzyme and sequenced targeting 500 million pair‐end reads per library. Spatial genome structure was examined at compartment, topological associated domain (TAD), and loop levels. To better define enhancer‐promoter interaction (EPI), high‐resolution contact matrices were built using the HiCorr and DeepLoop algorithms. Result Each library generated over 250 million uniquely mapped, non‐redundant reads for Hi‐C analysis. Compartment and TAD had limited variability among samples (correlation co‐efficiency = 0.62∼0.90 for compartment, 0.84∼0.94 for TAD). Chromatin loop, which usually represents EPI, displays higher variability among samples (correlation co‐efficiency = 0.38∼0.75). In the 5kb‐contact‐matrix analysis, a chromatin interactive event involving APOE was detected, which is supported by Capture‐C data. The higher‐resolution 500bp‐contact‐matrix analysis revealed multiple better‐defined interactions within the event. Of particular interest, an interaction between the 5’end of TOMM40 and CLPTM1 (about 100 kb apart from each other) surrounding APOE is only observed on European but not African LA background. Conclusion Chromatin loop displays more inter‐individual variations compared to other spatial genome structures and therefore is more informative in elucidating the epigenetic basis for inter‐ancestry differences in gene expression regulation. While identifying chromatin loop from Hi‐C data at higher resolution is challenging, it has the potential to delimitate EPI and offers insights on the ancestry‐specific AD genetic risk by providing functional mechanisms underlying AD‐associated genetic variants

ATAC-seq on iPSC derived astrocytes to assess chromatin accessibility differences between African and European local ancestry

Local ancestry (LA) surrounding the APOE4 allele is associated with an increased risk for Alzheimer Disease (AD) in European (EU) LA compared to African (AF) LA. We recently demonstrated that APOE4 has significantly higher expression in astrocytes in brain from EU compared to AF APOE4 LA carriers, but the specific molecular mechanism leading to this difference is not known. We investigated whether evaluating chromatin accessibility and gene expression profiles of inducible pluripotent stem cell (iPSC) derived astrocytes in the LA region in both AF and EU LA could lead us to identify potential regulatory factors affecting chromatin remodeling and expression of APOE. We assayed for Transposase Accessible Chromatin (ATAC-seq), and RNA-seq in three iPSC derived astrocytes from two AF LA and one EU LA individuals with AD. We processed the ATAC-seq data with the ENCODE ATAC-seq pipeline and called peaks using MACS2. An average of 692 peaks were detected in the LA region with no statistical difference between EU and AF LA. Interestingly, an African APOE4 homozygote LA astrocyte line with high APOE expression had an exclusive ATAC peak at the APOE promoter. Amongst the transcription factors (TFs) suggested to bind this differentially accessible peak in APOE are HNF4A and TEAD4. Both TFs had significantly increased expression in this astrocyte line compared to the other lines. Additionally, differential accessibility in this line include peaks in an intragenic enhancer of PVRL2 and the promoter of CBLC, both shown to be bound by HNF4A and both overexpressed in this cell line and one in the promoter of CYP2S1 with TEAD4 binding, which was also overexpressed. This study is one of the first studies to compare chromatin accessibility in AF and EU ancestries. It also represents initial efforts to investigate potential mechanisms and factors, using iPSC-derived cell lines, that could contribute to the differential expression we have previously reported in APOE4 between AF and EU LA brains. Our data suggest that elevated astrocytic expression of APOE4 is associated with rs

Intragenic loci within TOMM40 enhances APOE expression in human microglia

Background Previously, we demonstrated that the ancestry‐related risk for Late Onset Alzheimer’s Disease (LOAD) is driven by a local genomic region (termed Local Ancestry; LA) around APOEε4. Furthermore, we showed that in the brain of individuals bearing European LA there is higher expression of APOEε4 compared to those with African LA. In a follow‐up study, utilizing reporter assays and Capture‐C data we located two intronic regions within the European LA, both in the TOMM40 gene (named B10 and B13), that increased APOE expression in microglia and astrocytes. In this study, we sought to validate their regulatory role in APOE expression using CRISPR interference/activation (CRISPRi/a). Method Human Microglial Clone 3 (HMC3) CRISPRi/a lines were produced by transducing inducible dCas9‐VP64 (Activation), dCas9‐KRAB (Interference) or dCas9 (control) using lentiviral vectors. To direct the dCas9 constructs to our regions of interest, we generated multiplex vectors that encode 4 short‐guide RNAs (sgRNAs) targeting either B10 or B13. We used 4 different sgRNAs in each case to ensure full‐length coverage of the tested regions (∼850bp size). An empty multiplex vector was used as a control. We then transduced either of the multiplex vectors into the HMC3 CRISPRi/a lines. We induced expression of the dCas9 constructs for 2 or 6 days with Doxycycline (2ug/ml). RNA was extracted and the expression of APOE and TOMM40 was measured by qRT‐PCR. Result APOE expression significantly increased when targeting B10 or B13 (p=0.001; p=0.003 respectively) with dCas9‐VP64 after 2 days of Doxycycline treatment. Six days after treatment the significance persisted only when targeting B10 (p=0.01). No significant changes in APOE expression were observed in the cells bearing the dCas9‐KRAB presumably due to low endogenous APOE levels. Expression of TOMM40 did not vary under any treatment. Conclusion These preliminary results support our previous findings that regions B10 and B13 may act as regulators for APOE expression as demonstrated by the elevation of ApoE expression when targeting an activator to these regions. The expression of TOMM40 did not vary across cell lines in the evaluated time points supporting that the effect observed is specific for APOE

Ancestry-specific intronic variants on the APOEɛ4 haplotype influence enhancer activity and interaction with APOE promoter

The risk for late-onset Alzheimer disease (AD) in APOEε4 carriers differs between ancestral groups, where APOEε4's odds ratio for AD risk is lower in African (AFR) homozygous carriers than in non-Hispanic White (NHW) or Japanese (JPT) carriers (odds ratio ∼2-5 vs >15). Local ancestry (LA) analyses in APOEε4 carrier populations have shown the protective effect in AFR relative to EUR/JPT is due to noncoding factors lying in the LA surrounding APOEε4. Thus, regulatory differences between risk and protective LA haplotypes are most likely involved in the differential risk effect seen for APOEε4 on different backgrounds. We identified 56 significant sequence differences between AFR and EUR/JPT APOEε4 haplotypes from the 1000 genomes in the immediate topologically associated domain surrounding APOE. We performed two different Massively Parallel Reporter Assay (MPRA) designs; one assessing small haplotype (∼900bp) effects and one based upon single variant effects. We supplemented these results with single fragment luciferase reporter assays. All assays were performed in at least duplicate in HMC3 (microglia), U118 (astrocytes) and SH-SY5Y (neurons) cell lines. Additionally, we integrated chromatin interaction information from promoter capture C chromatin conformation assays in the same cell types. We identified a region in the first introns of TOMM40 with increased EUR/JPT enhancer activity, supported by both MPRA analyses and APOE promoter interaction in astrocytes and microglia. Two additional regions with differential enhancer activity in neurons, but no promoter interaction, were identified; downstream of APOE and in PVRL2 introns upstream of APOE presenting with higher EUR or higher JPT haplotype variant enhancer activity compared to AFR, respectively. Our results indicate several areas of differential regulation in this LA region on APOEε4 haplotypes dependent on cell type. As APOE is mostly expressed in glial cells, the data in TOMM40 introns points to this region as having the biggest impact on APOE expression in our study and thus highly supports the involvement of this region in the differential risk effects seen for APOEε4. Follow-up of the identified regulatory regions is currently ongoing using in-house iPSC derived cell lines