Simon Hsu

Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease

Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimers disease (LOAD). These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case–control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer’s disease in seven independent case–control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3, reveal that several variants in this gene increase risk for Alzheimer’s disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer’s disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer’s disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-β precursor protein (APP) and extracellular Aβ42 and Aβ40 (the 42- and 40-residue isoforms of the amyloid-β peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aβ42 and Aβ40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex traits.

The DAF-16 FOXO Transcription Factor Regulates natc-1 to Modulate Stress Resistance in Caenorhabditis elegans, Linking Insulin/IGF-1 Signaling to Protein N-Terminal Acetylation

The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance.

Functional characterization improves associations between rare non-synonymous variants in CHRNB4 and smoking behavior

Smoking is the leading cause of preventable death worldwide. Accordingly, effort has been devoted to determining the genetic variants that contribute to smoking risk. Genome-wide association studies have identified several variants in nicotinic acetylcholine receptor genes that contribute to nicotine dependence risk. We previously undertook pooled sequencing of the coding regions and flanking sequence of the CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 genes and found that rare missense variants at conserved residues in CHRNB4 are associated with reduced risk of nicotine dependence among African Americans. We identified 10 low frequency (<5%) non-synonymous variants in CHRNB4 and investigated functional effects by co-expression with normal α3 or α4 subunits in human embryonic kidney cells. Voltage-clamp was used to obtain acetylcholine and nicotine concentration–response curves and qRT-PCR, western blots and cell-surface ELISAs were performed to assess expression levels. These results were used to functionally weight genetic variants in a gene-based association test. We find that there is a highly significant correlation between carrier status weighted by either acetylcholine EC50 (β = −0.67, r2 = 0.017, P = 2×10−4) or by response to low nicotine (β = −0.29, r2 = 0.02, P = 6×10−5) when variants are expressed with the α3 subunit. In contrast, there is no significant association when carrier status is unweighted (β = −0.04, r2 = 0.0009, P = 0.54). These results highlight the value of functional analysis of variants and the advantages to integrating such data into genetic studies. They also suggest that an increased sensitivity to low concentrations of nicotine is protective from the risk of developing nicotine dependence.

Discovery and validation of autosomal dominant Alzheimer’s disease mutations

BackgroundAlzheimer’s disease (AD) is a neurodegenerative disease that is clinically characterized by progressive cognitive decline. Mutations in amyloid-β precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2) are the pathogenic cause of autosomal dominant AD (ADAD). However, polymorphisms also exist within these genes.MethodsIn order to distinguish polymorphisms from pathogenic mutations, the DIAN Expanded Registry has implemented an algorithm for determining ADAD pathogenicity using available information from multiple domains, including genetic, bioinformatic, clinical, imaging, and biofluid measures and in vitro analyses.ResultsWe propose that PSEN1 M84V, PSEN1 A396T, PSEN2 R284G, and APP T719N are likely pathogenic mutations, whereas PSEN1 c.379_382delXXXXinsG and PSEN2 L238F have uncertain pathogenicity.ConclusionsIn defining a subset of these variants as pathogenic, individuals from these families can now be enrolled in observational and clinical trials. This study outlines a critical approach for translating genetic data into meaningful clinical outcomes.

The MS4A gene cluster is a key regulator of soluble TREM2 and Alzheimer disease risk

Soluble triggering receptor expressed on myeloid cells 2 (sTREM2) levels in the cerebrospinal fluid (CSF) have been associated with Alzheimer disease (AD) status. TREM2 plays a critical role in microglial activation, survival, and phagocytosis; however, the pathophysiological role of sTREM2 in AD is not well understood. Understanding the role of sTREM2 in AD may help reveal biological mechanisms underlying AD and identify novel therapeutic targets. We performed a genome-wide association study (GWAS) to identify genetic modifiers of CSF sTREM2 levels. Common variants in the membrane-spanning 4-domains subfamily A (MS4A) gene region were associated with higher CSF sTREM2 levels (rs1582763; P = 1.15×10−15) and replicated in independent datasets. The variants associated with increased levels of sTREM2 are also associated with reduced AD risk and delayed age-at-onset. Rs1582763 influences expression of MS4A4A and MS4A6A in multiple tissues, suggesting that one or both of these genes are important for regulating sTREM2. MS4A genes encode transmembrane proteins that may play a role in intracellular protein trafficking in microglia. We used human macrophages to begin to test the relationship between MS4A4A and TREM2 and found that they co-localize intracellularly and that antibody-mediated targeting of MS4A4A reduces sTREM2. Thus, genetic, molecular, and cellular findings suggest that MS4A4A regulates sTREM2. These findings also provide a mechanistic explanation of the original GWAS signal in the MS4A locus for AD risk and indicate that TREM2 is involved in sporadic AD risk in general, not only in TREM2 risk-variant carriers.

Human fibroblast and stem cell resource from the Dominantly Inherited Alzheimer Network

BackgroundMutations in amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) cause autosomal dominant forms of Alzheimer disease (ADAD). More than 280 pathogenic mutations have been reported in APP, PSEN1, and PSEN2. However, understanding of the basic biological mechanisms that drive the disease are limited. The Dominantly Inherited Alzheimer Network (DIAN) is an international observational study of APP, PSEN1, and PSEN2 mutation carriers with the goal of determining the sequence of changes in presymptomatic mutation carriers who are destined to develop Alzheimer disease.ResultsWe generated a library of 98 dermal fibroblast lines from 42 ADAD families enrolled in DIAN. We have reprogrammed a subset of the DIAN fibroblast lines into patient-specific induced pluripotent stem cell (iPSC) lines. These cells were thoroughly characterized for pluripotency markers.ConclusionsThis library represents a comprehensive resource that can be used for disease modeling and the development of novel therapeutics.

Precision genome-editing with CRISPR/Cas9 in human induced pluripotent stem cells

Genome engineering in human induced pluripotent stem cells (iPSCs) represent an opportunity to examine the contribution of pathogenic and disease modifying alleles to molecular and cellular phenotypes. However, the practical application of genome-editing approaches in human iPSCs has been challenging. We have developed a precise and efficient genome-editing platform that relies on allele-specific guideRNAs (gRNAs) paired with a robust method for culturing and screening the modified iPSC clones. By applying an allele-specific gRNA design strategy, we have demonstrated greatly improved editing efficiency without the introduction of additional modifications of unknown consequence in the genome. Using this approach, we have modified nine independent iPSC lines at five loci associated with neurodegeneration. This genome-editing platform allows for efficient and precise production of isogenic cell lines for disease modeling. Because the impact of CRISPR/Cas9 on off-target sites remains poorly understood, we went on to perform thorough off-target profiling by comparing the mutational burden in edited iPSC lines using whole genome sequencing. The bioinformatically predicted off-target sites were unmodified in all edited iPSC lines. We also found that the numbers of de novo genetic variants detected in the edited and unedited iPSC lines were similar. Thus, our CRISPR/Cas9 strategy does not specifically increase the mutational burden. Furthermore, our analyses of the de novo genetic variants that occur during iPSC culture and genome-editing indicate an enrichment of de novo variants at sites identified in dbSNP. Taken together, we propose that this enrichment represents regions of the genome more susceptible to mutation. Herein, we present an efficient and precise method for allele-specific genome-editing in iPSC and an analyses pipeline to distinguish off-target events from de novo mutations occurring with culture.

LINKAGE AND WHOLE GENOME SEQUENCE ANALYSIS OF ALZHEIMER'S DISEASE RESILIENCE AND RISK

P2-158 LINKAGE ANDWHOLE GENOME SEQUENCE ANALYSIS OFALZHEIMER’S DISEASE RESILIENCE AND RISK Keoni Kauwe, Ivan Arano, Jose T. Bras, Lisa Cannon-Albright, Carlos Cruchaga, Alison M. Goate, Josue D. Gonzalez Murcia, Rita Guerreiro, John Hardy, Simon Hsu, Celeste Karch, Ronald G. Munger, Maria C. Norton, Perry G. Ridge, Celeste Sassi, Andrew Singleton, Craig Teerlink, JoAnn Tschanz, Steven G. Younkin, Christopher Corcoran, AESG (Alzheimer’s Exome Sequencing Group), Brigham Young University, Provo, UT, USA; UCL Institute of Neurology, London, United Kingdom; University of Utah School of Medicine, Salt Lake City, UT, USA; 4 Washington University in St. Louis, St. Louis, MO, USA; Icahn School of Medicine at Mount Sinai, New York, NY, USA; UCL Institute of Neurology, London, United Kingdom; 7 Washington University in St. Louis, St. Louis, MO, USA; 8 Utah State University, Logan, UT, USA; 9 University College London, London, United Kingdom; National Institute of Aging, Bethesda, MD, USA; Mayo Clinic, Jacksonville, FL, USA. Contact e-mail: kauwe@byu.edu

Phospholipase d3 contributes to Alzheimer’s disease risk via disruption in app trafficking and Aβ generation

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Modeling tauopathies in human pluripotent stem cells

Background: Tauopathies are a class of neurodegenerative diseases that are characterized by hyperphosphorylated tau aggregates in the brain. In a subset of tauopathies, genetic changes in MAPT, the gene encoding the tau protein, are sufficient to initiate a cascade of events that leads to tau aggregation and death of neuronal populations in the brain. Increasing evidence suggests that tau aggregates spread along neuronal networks in the brain via tau release and tau uptake. However, the cascade of events that leads to disease remains poorly understood.Methods:We sought to develop a stem cell model of tauopathies that captures the genetic complexities of the MAPT gene and the phenotypic complexities of the human neuron. We generated human induced pluripotent stem cell (iPSC) using nonintegrating Sendai virus from dermal fibroblasts carryingMAPTmutations (P301L, IVS10+16, V337M, R406W). We measured tau phosphorylation, release and aggregation in iPSC-derived neurons from MAPT mutation carriers and controls. To determine if correction of MAPT mutations restores normal tau metabolism, we developed a pipeline for efficient, seamless modification of point mutations using CRISPR/Cas9 technology. Applying this pipeline to a MAPT P301L iPSC line, we generated isogenic, iPSC clones that are corrected toWTor that are modified toMAPTP301S.Results: Consistent with our findings in immortalized cell models, we found that tau is actively released from human iPSC-derived neurons via the unconventional secretory pathway and that calcium signaling plays an important role in tau release. We found that the rate of tau release was modified in iPSC-derived neurons fromMAPTmutation carriers compared with controls. Reversion of the MAPT P301L to WT using CRISPR/Cas9 restored the tau release rate to the rates observed in unrelated control lines. Additionally, human iPSCderived neurons endogenously expressingMAPTmutations produced multimeric tau species that were absent in unrelated and isogenic control lines. Conclusions:Together, these stem cell models capture key pathological hallmarks of Alzheimer’s disease and other tauopathies and, thus, are beginning to define the cascade of events that lead to neurodegenerative tauopathies, providing avenues for advancement in therapeutic intervention.