Geneticists have found dozens of loci that associate with Alzheimer’s disease, but the functional variants and even the genes that underlie these associations often remain elusive. Now, researchers led by Alison Goate and Edoardo Marcora at the Icahn School of Medicine, Mount Sinai, New York, describe a method for quickly finding causal genes and variants in risk loci of myeloid cells. Starting with 16 AD risk variants located in enhancers, the DNA regions that influence transcription, the researchers linked each locus to changes in expression of a downstream gene that correlated with AD risk. Nine of the genes had not been associated previously with AD. For eight of the 16 enhancers, the researchers pinpointed the specific genetic change that affected expression. All were point changes that disrupted binding of transcription factors. The paper is available in preprint form on bioRxiv.
- AD risk variants predominantly occur in enhancers of myeloid genes.
- In eight enhancers, the authors pinpointed the functional variant for a GWAS risk locus.
- Altogether, enhancer data nominated 16 causal genes, nine of them new to AD.
“I think this is the first study where we have been able to go from the AD GWAS signal and comprehensively show what genes are affected and how,” Goate told Alzforum. The next step will be to investigate how these changes in gene expression alter microglial function, she added.
Others agreed that the method represents an advance. “It’s exciting that they were able to map a relatively large number of loci associated with AD risk using this approach. This is elegant work that moves the field forward,” Carlos Cruchaga at Washington University in St. Louis told Alzforum (see comment below). He suggested applying the same methodology to other brain cell types such as neurons and oligodendrocytes. Because many putative AD risk genes are highly expressed in microglia, Goate and colleagues limited their investigation to myeloid cells, which comprise microglia, macrophages, and monocytes. “We need to do more of these studies to map GWAS signals to genes, and those genes to pathways,” Cruchaga said.
Chromatin Flags Active Enhancers. Modifications to histone proteins (green and red dots) mark active enhancers, where a transcription complex (colored circles) assembles on a chromosome. DNA then loops over (arrow) to contact an active promoter (gray rectangle) and initiate transcription. [Courtesy of Calo and Wysocka, 2013.]
In the case of myeloid cells, the new genes primarily relate to the endolysosomal system, highlighting the idea that the risk for Alzheimer’s may depend on how effectively microglia respond to amyloid and brain damage and clean up debris (Apr 2019 conference news). “This study is an important step toward extracting meaningful biology from genomic studies of Alzheimer’s disease. The prioritization of variants and genes will accelerate the generation of disease-relevant models, ultimately leading to a better understanding of pathogenesis,” Matthew Hill at Cardiff University, U.K., wrote to Alzforum (full comment below).
Goate and colleagues became interested in the microglial contribution to Alzheimer’s when they identified a polymorphism in AD GWAS data that lowered expression of the microglial master regulator PU.1 and delayed disease onset (Jun 2017 news). Because PU.1 controls the expression of numerous microglial genes, including many linked to AD, the results suggested that wholesale microglial gene expression could influence Alzheimer’s pathogenesis.
If so, AD risk variants might preferentially occur in regulatory regions, such as enhancers and promoters, that are active in these cells, the authors reasoned. To find these regions in myeloid cells, first author Gloriia Novikova examined human datasets such as ENCODE that catalog chromatin modifications in different cell types (ENCODE Project Consortium, 2012; Schmidt et al., 2016). Chromatin modifications point toward open regions of DNA where proteins can bind to initiate transcription. Novikova integrated these structural data with GWAS results from the International Genomics of Alzheimer’s Project (IGAP). She found that active myeloid enhancers contained a disproportionate number of AD risk loci. In particular, AD GWAS hits were associated with binding sites for PU.1 or its partners MAF, SMAD, and USF.
What genes did these enhancers control? The authors used two different approaches to find them, the first based on chromatin structure, the second on expression.
In the first, they made use of the fact that enhancer regions physically bind promoters, the DNA regions that initiate gene transcription (see image above). This brings different parts of the DNA strand together. Those DNA interactions can be teased out with a technique called High-throughput Chromosome Conformation Capture, or Hi-C, which basically cross-links adjacent pieces of DNA, isolates, and sequences them (van Berkum et al., 2010). In this manner, Hi-C can identify the target genes of each enhancer.
Luckily, others have already cataloged Hi-C datasets from various cells, including peripheral blood monocytes and macrophages, but not microglia (Javierre et al., 2016). Novikova and colleagues searched these data for myeloid enhancers that contained AD risk variants that associated with changes in expression of downstream genes. The analysis turned up 14 such genes. Seven of these were previously linked to AD, including SPI1, which makes PU.1, BIN1, MS4A, ABCA7, PTK2B, PILRA, and TREM2. The seven other genes were ZYX, TP53INP1, AP4E1, RIN3, APBB3, RABEP1, and CASS4. All the new genes are highly or exclusively expressed in microglia (Gosselin et al., 2017).
In the second method, the authors investigated whether genetic variants known to affect enhancer activity, or histone modification quantitative trait loci (hQTLs), co-localized with AD risk loci. For those that did, they linked the hQTL to expression changes in a downstream gene, and then examined whether that expression change associated with AD risk. If so, it was likely the causal gene at this risk loci. Again using monocyte data, the authors found 10 genes that fit the bill. Eight had been found by the first method. The two additional genes were CD2AP and GPR141. “We’re encouraged that there’s a lot of overlap between the gene lists that come out of the two approaches,” Goate said.
Notably, most of the new genes pertain to endolysosomal function. For example, ZYX is found in early endosomes and helps assemble actin filaments that propel movement. RIN3 loiters in endosomes along with BIN1, while RABEP1 facilitates endosome trafficking and fusion. AP4E1 sorts APP and other transmembrane proteins from the Golgi to endosomes, and APBB3 helps internalize APP from the cell surface. TP53INP1 is involved in phagocytosis.
While the endolysosomal system has long been of interest in AD research, most previous studies focused on neurons. “They are putting together two things that have never been side by side: microglia and lysosomal dysfunction,” Cruchaga said. Goate believes the findings can guide research into potential therapeutics. If scientists can figure out how risk and protective alleles change the endolysosomal system in microglia, they might be able to develop drugs that protect against AD, she suggested.
Although these approaches identified candidate causal AD genes, they still did not pinpoint the functional genetic variants that directly affected risk. To find these, the authors focused on eight loci where associations between enhancer activity, gene expression, and AD risk would have enough statistical significance to identify the functional variant. For each of the eight loci—SPI1, BIN1, MS4A, ZYX, TP53INP1, AP4E1, RABEP1, and GPR141—the authors found one or more SNPs in the myeloid enhancer region that disrupted transcription factor binding and correlated with differences in gene expression. These SNPs accounted for the entire GWAS signal at each loci, indicating they were the functional variants. That suggests that these genes exert their effect on AD pathogenesis solely through microglia or macrophages, not neurons, Goate noted.
The authors chose one variant to analyze in depth. An SNP in the enhancer region of MS4A disrupted binding of the transcription factor CTCF, abolishing the looping of the DNA strand to engage target genes. CTCF binding compacts chromatin and represses gene expression, so its absence would lead to higher expression of MS4A. The SNP, rs636317-T, boosts the risk of AD. However, other data conflict on whether lower or higher MS4A expression promotes risk (Apr 2019 conference news).
“The data are very exciting, and already pinpoint specific mechanisms altered in myeloid cells. We now need to understand how this translates into a chronic neurodegenerative process, whether the myeloid dysfunction is sufficient to drive disease, and how this relates to the main pathological hallmarks present in the disease, Aβ and tau,” Diego Gómez-Nicola at the University of Southampton, U.K., wrote to Alzforum (full comment below).—Madolyn Bowman Rogers
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- Novikova G, Kapoor M, TCW J, Abud EM, Efthymiou AG, Cheng H, Fullard JF, Bendl J, Roussos P, Poon WW, Hao K, Marcora E, Goate AM. Integration of Alzheimer’s disease genetics and myeloid cell genomics identifies novel causal variants, regulatory elements, genes and pathways. 2019 Jul 6. bioRxiv