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Background: We previously identified a 44‐base pair deletion in (ATP‐binding cassette sub‐family A member 7) (ABCA7) that is significantly associated with Alzheimer's disease (AD) in African Americans (AA), producing a frameshift mutation resulting in a truncated protein (p.Arg578Alafs). ABCA7 is a lipid transporter across cellular membranes. While we have shown the mutant mRNA is present in neurons, whether it is translated into a stable protein is not known due to the lack of antibodies capable of recognizing the N‐terminus of endogenous ABCA7. The abrupt ABCA7 translation due to the deletion may alter the lipid metabolism, which can be investigated using isogenic iPSC and differentiated models. Methods: We analyzed single‐cell RNAseq (scRNAseq) data from spheroid cultures of 12 individuals. We generated a recombinant version of the ABCA7 gene with and without the deletion and bearing an N‐terminal flag tag which were transfected into HEK cells. We utilized CRISPR‐based genome editing using induced pluripotent stem cell (iPSC) lines from three independent AA‐cognitively unimpaired individuals (WT) to introduce the AA‐specific ABCA7 homozygous or heterozygous deletions. These were differentiated using standard protocols in the HIHG iPSC core into monocultures of induced neuron‐like cells. C‐terminal antibodies were used to assess the presence of native ABCA7 in HEK cells. Results: Examination of HEK cells revealed no detectable native ABCA7. The truncated ABCA7‐tagged protein appeared stable and localized in the plasma membrane as seen for the wild‐type protein (Figure 1). ScRNAseq confirmed that expression of ABCA7 is highest in neurons, identifying them as the iPSC differentiated cell of choice. We successfully generated both homozygous and heterozygous deletion isogenic lines in the three WT iPSC lines. Differentiated neurons from these isogenic lines have a normal phenotype, allowing for functional assays and lipidomic studies. Conclusion: The truncated protein p.Arg578Alafs appears to be expressed and stable in HEK cells, and surprisingly located in the plasma membrane despite the absence of most transmembrane domains. Our three isogenic iPSC pairs will be a great resource for studying the pathogenic effects of the ABCA7 truncation in differentiated cells. [ABSTRACT FROM AUTHOR]

Background: Genome‐wide association studies (GWAS) in Alzheimer's disease (AD) are consistently discovering genetic variants linked to the risk of developing this neurodegenerative condition. However, the effect size of the shared associated loci varies across populations as well as each population can have unique associations. This phenomenon could be explained by ancestry‐dependent changes in the genomic regulatory architecture (GRA) influencing the expression of these genes, similar to the effect of different local ancestry on the risk of AD in APOE4 carriers. Thus, understanding of GRA in the context of AD is imperative but currently most GRA data available is predominantly European, limiting our ability to comprehensively interpret the variability associated with AD risk genes across populations. For this study we focused on oligodendroglia, a cell lineage that has been historically overlooked but that is emerging as key players in AD due to their involvement in various pathological processes, including neuroinflammation, oxidative stress, and synaptic dysfunction. Here, we report ancestry‐dependent differences in the GRA of iPSC derived oligodendroglia with African, Amerindian, or European global ancestry. Method: We obtained PBMCs from individuals with Alzheimer's disease (AD) or without cognitive impairment, each with over 85% global ancestry of a specific ancestral background. These cells were then transformed into induced pluripotent stem cells (iPSC) and subsequently differentiated into oligodendroglia‐containing 3D neural cultures. On the 76th day of differentiation, we harvested and lysed the cells to isolate nuclei for Multiomic profiling including Single Cell ATAC and Single Cell RNA‐seq, we analyzed the chromatin accessibility and transcriptomes to identify ancestry‐dependent changes genome‐wide and in AD GWAS hits. Result: We found several AD GWAS hits differentially expressed between ancestries in OPCs and in the more mature oligodendrocyte population (including APP and CLU) and some differentially accessible peaks associated to some of these genes (predominantly PRDM7). Nevertheless, OPCs showed more ancestry‐specific regulation than the more mature oligodendrocytes. Conclusion: Our findings offer ancestry‐specific understanding of oligodendroglia chromatin changes and gene regulation in the context of AD. These results present a comprehensive perspective on the genetic regulatory architecture of oligodendroglia and constitute a resource for gene identification studies in the African American and Hispanic populations. [ABSTRACT FROM AUTHOR]

Background: Annotation of target genes of non‐coding GWAS loci remains a challenge since 1) regulatory elements identified by GWAS can be metabases away from its actual target, 2) one regulatory element can target multiple genes, and 3) multiple regulatory elements can target one gene. AD GWAS in populations with different ancestries have identified different loci, suggesting ancestry‐specific genetic risks. To understand the connection between associated loci (potential regulatory elements) and their target genes, we conducted Hi‐C analysis in frontal cortex of African American (AA) and Non‐Hispanic Whites (NHW) AD patients to map chromatin loops, which often represent enhancer‐promoter (EP) interactions. In our initial analyses, we applied HiC to uncover the regulatory architecture of rs3851179 linked to the embryonic ectoderm development gene (EED) in the ADSP gene verification list. Method: Hi‐C libraries were derived from four AA and four NHW donors matched for age, sex, and APOE genotype. The AA samples had 68∼88% African (AF) genome while NHW samples had >95% European (EU) genome. DeepLoop was used to generate robust maps of chromatin loops. Hi‐C data from each population were pooled to generate reference chromatin loop maps for the AF and EU genomes. Result: rs3851179 (OR = 0.9, P = 3.0 × 10−48) resides between EED and PICALM, about 80 Kb away from both with each gene transcribed in opposite directions. Although PICALM is designated as a causal gene, EED is designated a susceptibility gene by ADSP based on the transcription direction and distance from rs3851179. Hi‐C data from both AF and EU genome in frontal cortex revealed that rs3851179 co‐localizes with a loop anchor, connecting H3K27ac peaks (enhancer) overlapping rs3851179 to the proximal promoter of PICALM, suggesting that PICALM but not EED is the target gene, corroborated by the GTEX e‐QTL data. Conclusion: This initial application of Hi‐C data demonstrates the utility of chromatin regulatory maps in nominating target genes of GWAS hits in non‐coding loci. The generation and eventual application of Hi‐C maps in African and Amerindian genomes (which are part of the AA or American Hispanic genomic admixture) will be key dissecting ancestry‐specific genetic loci and, thereby, broadening diversity in AD research. [ABSTRACT FROM AUTHOR]

Background: The Apoliproprotein E (APOE) e4 allele is the most significant genetic risk factor for late‐onset Alzheimer disease (AD). However, the risk associated with the APOE e4 allele differs across populations with individuals of African ancestry having a reduced risk than individuals of European (EU) ancestry. Further, single‐nuclei RNAseq analysis in autopsy samples from AD APOEε4 homozygotes with EU Local Ancestry (LA) had a significantly increased APOEε4 expression compared to those with African LA, particularly in astrocytes. To test the functional consequences of elevated APOE4 expression on astrocytes, we examined the effect of APOE4 overexpression in inducible pluripotent stem cell (iPSC) derived astrocytes (iAstrocytes). Method: iPSC were derived from European and African LA AD individuals homozygous for APOEε4/ε4. These APOEε4/ε4 iPSC were differentiated into astrocytes (iAstrocytes) which were transduced with a lentiviral construct overexpressing APOEε4 or a control vector. RNAseq analysis was performed to identify differentially expressed genes followed by pathway analyses. Result: iPSC lines were derived, validated for pluripotency, and differentiated into iAstrocytes. Preliminary analysis of transcriptional effects ofApoEe4 overexpression in iAstrocytes showed alterations in gene expression patterns, including the down regulation of the inflammatory markers THBS1 and CLDN11 which had both been shown to be downregulated in AD, the mitochondrial‐related gene NDUFA4L2 shown to be dysregulated in AD, as well as SFRP2 (Wnt modulator involved in TREM2 processing). Upregulated genes included RGMA (accumulates in amyloid plaques), as well as TNC and CECR2. Pathway analysis showed an enrichment for extracellular matrix (ECM)‐receptor interaction, PI3K‐Akt signaling, and TGF‐beta signaling pathways. Interestingly, the transcriptional response to APOEε4 overexpression has an ancestry‐specific component in these iAstrocytes; with some genes dysregulated exclusively in African ancestry iAstrocytes such as ZBTB4 and PRND, and others only in European iAstrocytes, including MYBPHL and VWF. Conclusion: The overexpression of APOEε4 in iAstrocytes resulted in alteration in gene regulatory patterns, including the ECM and key signaling pathways across ancestries (EU and AF). In addition, gene expression differences were identified between ancestries. Further analyses of these ancestry‐specific transcriptional differences may help to elucidate the mechanisms that drive the differential AD risk seen in APOEε4 individuals across ancestries. [ABSTRACT FROM AUTHOR]

Background: Non‐Hispanic White APOE4 carriers have a higher risk of developing AD compared to African American APOE4 carriers. The local ancestry (LA) surrounding the APOE region was previously shown to be the primary factor in this risk difference. APOE4 carriers of European LA (ELA) have been found to have higher APOE4 expression and chromatin accessibility compared to African LA (ALA). We sought to investigate whether the LA around APOE3 has the same effect between ancestries. Differences between alleles could provide insight into ancestry‐specific regulatory areas controlling the APOE4 expression. Method: We identified AD autopsy samples by GSA, all homozygous for APOE3 and LA from 4 ADRC brain banks: Miami, Emory, Duke, and Rush. We performed single nuclei RNA sequencing (snRNA‐seq) on eight frozen frontal cortex (B9 area) samples using 10x Genomics. Result: We performed snRNA‐seq in a total of 51,462 nuclei from eight brains (4 ELA and 4 ALA). We identified 35 distinct cell clusters at a resolution of 0.6. The proportion of cells per cluster between ELA and ALA samples was similar for all clusters, except for cluster 32 (Excitatory Neurons) which had a greater than 2‐fold difference in ALA. Our data shows that APOE3 carriers with ELA have a significantly higher APOE expression in Excitatory Neurons (cluster 7) than those of ALA and contrary to our previous observations in APOE4 carriers (Griswold, A. et al, (2021)), APOE3 carriers of ALA express higher APOE in astrocytes (cluster 2) and Microglia (cluster 6). However, overall, comparison of APOE3 vs APOE4 carriers in this study demonstrated that APOE3 alleles have significantly lower gene expression than APOE4 carriers of the same LA. Conclusion: Our preliminary data suggest that the LA surrounding APOE3 is associated with different effects on APOE expression compared to APOE4 allele. Further, within the same LA, we observed that, overall, the different cell types express less APOE in APOE3 carriers compared to APOE4 carriers. Altogether, this may provide additional insight into the regulatory mechanisms affecting APOE4 expression. [ABSTRACT FROM AUTHOR]

Background: The effect size of APOE4 varies across genetic ancestries with African (AFR) local ancestry conferring a lower risk when compared to other ancestries. Recently, we identified a strong effect of the A allele of rs10423769 (with a minor allele frequency of 0.12 in AFR and 0.003 in Europeans), which lowers the risk of AD by 71% in APOE4 carriers. rs10423769 is 2 MB from APOEe4 in a region of segmental duplications (SD) containing a cluster of pregnancy specific beta‐1 glycoprotein genes (PSGs) and a lncRNA. To gain insights into the mechanism of protection, we characterized the haplotype harboring the variant and investigated differences in methylation and structural variation (SV) between haplotypes. Method: We used the Alzheimer's Disease Sequencing Project (ADSP) short read sequencing database to fine map and extend the protective haplotype using Haploview 4.2. In addition, long read sequencing (LRS) with the Oxford Nanopore PromethION was performed on 16 samples of individuals carrying one or two copies of rs10423769_A. LRS was used for SV calling with Sniffles2 and differential methylation analysis using Nanomethphase. Result: The minimum shared haplotype harboring rs10423769_A allele was ∼21kbp (chr19:43099521‐ 43120243). Though the region is rich in SDs, high quality LRS reads mapped uniquely to the region around the A allele confirming that the haplotype is unique and not duplicated elsewhere in the genome of the sequenced individuals. Moreover, the rs10423769_A haplotype harbors 23 differential CpG sites (fdr<0.05) compared to rs10423769_G, although no differential methylated region was identified. LRS revealed a 140bp insertion of repetitive MEF2 binding motifs. Initial examination demonstrated that the insertion was present in 80% of the A allele carriers. Conclusion: We have identified an AF‐specific haplotype protective against AD risk conferred by APOE ε4. Considering the distance from APOE and the complexity of the highly SD region where the variant is located, we have identified the minimum shared haplotype from AFR origin and are investigating the potential effects of SVs and differential CpG sites. Ongoing analyses include 3D chromatin structure and LRS at higher coverage to assemble and resolve the complex genomic context of the allele. [ABSTRACT FROM AUTHOR]

Background: Previous studies have reported that non‐Hispanic white (NHW) females carrying the APOE ε4 allele differ in risk of developing Alzheimer's disease (AD) when compared to men. Recent studies suggest the association between APOE ε4 and AD risk may be modified by age and its local ancestry in admixed populations. However, there is still scant evidence on how sex could interact with these factors. We aimed to investigate the association of sex, ancestry, APOE, and local ancestry with AD in multiple cohorts. Methods: A pooled case‐control study was undertaken from 50 independent datasets (n = 30,539;age≥55years) in the Alzheimer's Disease Genetics Consortium. Logistic regression models were used to study the associations between disease status and independent variables including age, sex, reported race (as a proxy of ancestry), and APOE genotype. Additionally, mixed models were implemented for the local ancestry APOE haplotype analyses among different ethnicities. A p‐value less than 0.05 was considered statistically significant. Results: Regardless of ancestry, men and women with APOE ε3/ε4 or ε4/ε4 genotypes showed no significant difference in the risk of developing AD. When comparing subjects with APOE ε3/ε4 or ε4/ε4 (vs. ε3/ε3), East‐Asian (EA) males (OR,5.56; 95% CI:4.26‐7.60) and females(OR,5.46:4.34‐6.86) had the strongest ORs; whilst the weakest risk was for South‐Asian (SA) men(OR,1.14:0.99‐2.57) among all races/ethnicities. Age‐stratified analyses showed that NHW females(OR,5.36:4.74‐6.07 vs. males: OR,4.72:4.10‐5.60) had a higher risk of developing AD between ages 66‐75; whereas African American (AA), EA and SA exhibited this pattern at 55‐64. In Hispanics, the relationship is reversed, with males aged 55‐65 years carrying APOE ε3/ε4 or ε4/ε4(OR,4.53:1.67‐12.30: vs. females: OR,3.03:1.59‐5.78) having a stronger association with AD risk than their counterpart. No differences were found when stratified by sex and age for APOE ε2 and AD. Local ancestry (European ‐EU‐; African ‐AF‐; Amerindian ‐AI‐; and EA) analyses are being conducted by ancestry and sex. Preliminary analyses suggest differences by sex and local ancestry for APOE ε4. Conclusions: Our findings suggest differences by sex and reported ancestry in the risk for developing AD in carriers of APOE ε4. Global, local ancestry and sex should be considered in predicting risk due to APOE alleles. [ABSTRACT FROM AUTHOR]

Introduction: European local ancestry (ELA) surrounding apolipoprotein E (APOE) ε4 confers higher risk for Alzheimer's disease (AD) compared to African local ancestry (ALA). We demonstrated significantly higher APOE ε4 expression in ELA versus ALA in AD brains from APOE ε4/ε4 carriers. Chromatin accessibility differences could contribute to these expression changes. Methods: We performed single nuclei assays for transposase accessible chromatin sequencing from the frontal cortex of six ALA and six ELA AD brains, homozygous for local ancestry and APOE ε4. Results: Our results showed an increased chromatin accessibility at the APOE ε4 promoter area in ELA versus ALA astrocytes. This increased accessibility in ELA astrocytes extended genome wide. Genes with increased accessibility in ELA in astrocytes were enriched for synapsis, cholesterol processing, and astrocyte reactivity. Discussion: Our results suggest that increased chromatin accessibility of APOE ε4 in ELA astrocytes contributes to the observed elevated APOE ε4 expression, corresponding to the increased AD risk in ELA versus ALA APOE ε4/ε4 carriers. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is a common neurodegenerative disorder with a significant impact on aging populations. DNA methylation (DNAm) alterations have been implicated in both the aging processes and the development of AD. Given that AD affects more women than men, it is also important to explore DNAm changes that occur in each sex. Method: We created the MIAMI‐AD, a comprehensive database containing manually curated summary statistics from 97 published tables in 37 studies, all of which included at least 100 participants. There are four main sections: Genome‐wide Query, Gene Query, CpG Query, and Epigenetic Clock Query. Result: MIAMI‐AD enables easy browsing, querying, and downloading DNAm associations at multiple levels – at individual CpG, gene, genomic regions, or genome‐wide, in one or multiple studies. Moreover, it also offers tools to perform integrative analyses, such as comparing DNAm associations across different phenotypes or tissues, as well as interactive visualizations. Using several use case examples, we demonstrated that MIAMI‐AD facilitates our understanding of age‐associated CpGs in AD and in revealing sex‐specific DNAm differences in AD. Conclusion: MIAMI‐AD (https://miami‐ad.org/) facilitates integrative explorations to better understand the interplay between DNAm across aging, sex, and AD. This open‐access resource is freely available to the research community, and all the underlying data can be downloaded. [ABSTRACT FROM AUTHOR]

We previously demonstrated that in Alzheimer's disease (AD) patients, European apolipoprotein E (APOE) ε4 carriers express significantly more APOE ε4 in their brains than African AD carriers. We examined single nucleotide polymorphisms near APOE with significant frequency differences between African and European/Japanese APOE ε4 haplotypes that could contribute to this difference in expression through regulation. Two enhancer massively parallel reporter assay (MPRA) approaches were performed, supplemented with single fragment reporter assays. We used Capture C analyses to support interactions with the APOE promoter. Introns within TOMM40 showed increased enhancer activity in the European/Japanese versus African haplotypes in astrocytes and microglia. This region overlaps with APOE promoter interactions as assessed by Capture C analysis. Single variant analyses pinpoints rs2075650/rs157581, and rs59007384 as functionally different on these haplotypes. Identification of the mechanisms for differential regulatory function for APOE expression between African and European/Japanese haplotypes could lead to therapeutic targets for APOE ε4 carriers. [ABSTRACT FROM AUTHOR]

Background: We recently identified a protective interaction between the APOE ε4 allele and an African ancestry‐specific allele (rs10423769_A) significantly reducing the odds ratio for Alzheimer's disease by 75% in APOE ε4 homozygous carriers. rs10423769 is located approximately 2 mB upstream of APOE in a large area of segmental duplication, within a cluster of pregnancy specific beta‐1 glycoproteins (PSG) genes and a long noncoding RNA, lncRNA (ENSG00000282943). Hi‐C analysis in multiple brain cell types showed no direct chromatin interactions between APOE and the rs10423769 region. rs10423769 has been reported to be a splicing quantitative trait locus for the transmembrane protein 145 gene (TMEM145), which has the highest expression in cerebellum. Methods: Since this interaction region is enriched for segmental duplications, long‐read whole genome sequencing was performed on 16 individuals (nine heterozygous and seven homozygous rs10423769_A carriers) using the Oxford Nanopore PromethION24 to more precisely map this variant. Expression of PSG genes (2, 4, 5 and 11), ENSG00000282943, and TMEM145 isoforms were analyzed by qPCR in cerebellum and frontal cortex of carriers and non‐carriers of rs10423769_A. Hi‐C analysis was conducted in these brain samples as well. Results: Nanopore long‐read sequencing showed that rs10423769 is present as a single copy on chromosome 19. In the frontal cortex, preliminary Hi‐C results supported the presence of transcriptionally active chromatin over the PSG region in some individuals but not in others. Both PSG genes and the lncRNA had low expression in the frontal cortex and cerebellum. We observed higher TMEM145 expression in the cerebellum compared to frontal cortex and detected alternative splicing forms of TMEM145. There was a trend of increased expression of TMEM145 in rs10423769_A carriers, which was not statistically significant likely due to sample size. Conclusion: We established that only one copy of rs10423769 exists in the modifying region, which will facilitate future haplotype analysis. The Hi‐C analysis suggested inter‐individual variations on chromatin structure over PSG genes in frontal cortex, although no expression of these genes was detected. Further analysis of regulatory activity of the region is ongoing. This modifier locus appears to represent a novel protective mechanism for APOE ε4. [ABSTRACT FROM AUTHOR]

Background: The APOE4 genotype is the main contributor to the risk of developing Late Onset Alzheimer's Disease (LOAD), while the APOE3 genotype has no effect on LOAD risk. Previously our group has shown that Local Ancestry (LA) surrounding APOE4 regulates APOE expression in adult individuals. As LOAD pathology has been shown to develop decades before the onset of symptoms, we sought to explore if LA has similar or different effect in mitotic cells, and whether the APOE3 genotype modulates the effect of the LA. To answer this question, we generated APOEε3/3 and APOEε4/4 isogenic induced Pluripotent Stem Cells (iPSCs) lines from individuals with either European LA or African LA. Method: PBMCs were derived from APOEε4/4 carrying individuals with either African or European APOE LA. To generate APOE isogenic lines, we formed a ribonucleoprotein (RNP) complex using a previously published APOE sgRNA (Lin et al. 2018) and performed nucleofection using the 4D‐Nucleofector system (Lonza). Homozygous APOEε3/3 edited and APOEε4/4 unedited isogenic clones were selected, and confirmed to lack unwanted off‐target editing. Isogenic clone pluripotency was confirmed by immunocytochemistry (SOX2, NANOG and OCT4a staining). APOE expression in the isogenic clones was tested by quantitative Real‐Time PCR and significance was calculated by Student's T‐test. Result: Isogenic clones were similar in their pluripotency and growth characteristics. Preliminary APOE expression in the iPSC line carrying the African LA was higher (p = 0.0002) in each of the individual APOEε3/3 KI clones (n = 3) compared to each of the individual unedited APOEε4/4 clones (n = 2), showing consistency within each isogenic group. In contrast, the APOE expression in the iPSC line carrying the European LA was not significant in the APOEε3/3 KI clones (n = 2) compared to the parental line. Conclusion: Our data suggests that differences in local ancestry has a direct effect on the expression of APOE isoforms in iPSCs. This finding supports previous reports in frontal cortex suggesting that the regulation of APOE expression differs depending on ancestry. [ABSTRACT FROM AUTHOR]

Background: Genetic risk for Alzheimer Disease (AD) varies across populations with different ancestry. Three‐dimensional (3D) genome architecture regulates gene transcription. Genome‐wide Hi‐C analyses have revealed dynamic 3D genome changes during development and disease progression. The variation of 3D genome architecture on different ancestral background, however, has not been extensively studied, especially at high‐resolution level. Recently, we developed the DeepLoop pipeline enabling robust high‐resolution analysis. We applied DeepLoop to characterize 3D genome architecture in frontal cortex from African American (AA) and European (EU) donors while considering local ancestry (LA) in admixed samples. Methods: Hi‐C libraries were constructed and ∼500 million of paired‐end reads were obtained in 8 samples (4 AA and 4EU) that were sex‐matched and homozygous for APOEε4. We partitioned Hi‐C data into compartment A (open and transcriptionally active chromatin) and compartment B (compacted and transcriptionally silenced chromatin). In total, 6206 compartment bins across the genome were compared at a 500kb resolution. DeepLoop was used to call chromatin loop (often represents promoter‐enhancer interaction) at a 5kb‐resolution. LA was calculated using RFMix with SNP‐array data and a reference panel from 1000 Genomes for EU, African (AF) and Amerindian (AI) ancestry. Results: Most of the A and B compartments were consistent among individuals with only <5% of them switched between AA and EU individuals. The EU samples have slightly (107 bins) more transcriptionally active bins than the AA samples. While many of homozygous LA blocks displayed same chromatin loop pattern between and within ancestries, we did observe ancestry‐specific loop as well as differences between individuals of the same ancestry. Importantly, the presence of heterozygous LA (African/EU) at some loci displayed a composite loop pattern that was present in the same region of homozygous LA of African or EU ancestry. Conclusion: The 3D genome architecture can be variable between different ancestral backgrounds as well as between individuals of the same LA. We hypothesize that admixture of LA can create novel or demolish chromatin interactions observed in the homozygous LA. These data emphasize the importance of LA in identifying the causal loci contributing to AD in admixed population such as AF and Hispanic/Latino. [ABSTRACT FROM AUTHOR]

Background: The APOEε4 confers the highest genetic risk factor for Alzheimer's disease (AD). Specifically, Non‐Hispanic Whites (NHW) are more susceptible to developing AD than African American (AA) individuals (OR∼15 vs 8, respectively). Local ancestry (LA) surrounding the APOE region has been implicated in this risk difference, with APOEε4 carriers of European LA (ELA) having increased chromatin accessibility and higher APOEε4 expression than individuals of African LA (ALA). In contrast, carriers of APOEε3, the most common allele, are not predisposed to AD. We here sought to investigate whether this differential accessibility and gene expression are solely due to APOE LA or whether the APOE genotype also contributes to the different disease risk. Method: We screened 94 brains from individuals self‐identified as either AA or NHW of which we identified a total of 22 AD autopsy samples (18 with ELA and 4 with ALA) by GSA, all homozygous for LA and APOEε3. We performed single nuclei ATAC‐seq and single nuclei RNA sequencing (snRNA‐seq) using frozen frontal cortex using the 10x Genomics. Result: To date, we have performed snRNA‐seq in a total of 16,099 nuclei for four brains (2 ELA and 2 ALA). We identified 27 distinct cell clusters at a resolution of 0.6. The proportion of cells per cluster between ELA and ALA samples was similar for all clusters, except for 8 clusters (primarily neurons) which had a greater than 2‐fold difference in ELA. Preliminary results show that APOEε3 carriers with ELA have a significantly higher APOE expression in astrocytes (cluster 11) than those of ALA similar to previous reports in APOEε4 carriers (Griswold, A. et al, (2021)). Interestingly, we also observed an increase in APOE expression in ALA for one oligodendrocyte cluster and four neuronal clusters (inhibitory and excitatory neurons) independent of those mentioned above. Conclusion: We report novel insights on the effect of APOE LA and genotype and its contributions to Alzheimer's disease risk. Specifically, as APOEε3 allele expression follows a similar pattern of overexpression in ELA vs ALA as observed for APOEε4 carriers, our data support that LA and not allele status contribute to the differential APOE expression observed among different ancestral populations. [ABSTRACT FROM AUTHOR]

Background: We recently identified a protective interaction between the APOE ε4 allele and an African ancestry‐specific allele (rs10423769_A) significantly reducing the odds ratio for Alzheimer's disease by 75% in APOE ε4 homozygous carriers. rs10423769 is located approximately 2 mB upstream of APOE in a large area of segmental duplication, within a cluster of pregnancy specific beta‐1 glycoproteins (PSG) genes and a long noncoding RNA, lncRNA (ENSG00000282943). Hi‐C analysis in multiple brain cell types showed no direct chromatin interactions between APOE and the rs10423769 region. rs10423769 has been reported to be a splicing quantitative trait locus for the transmembrane protein 145 gene (TMEM145), which has the highest expression in cerebellum. Methods: Since this interaction region is enriched for segmental duplications, long‐read whole genome sequencing was performed on 16 individuals (nine heterozygous and seven homozygous rs10423769_A carriers) using the Oxford Nanopore PromethION24 to more precisely map this variant. Expression of PSG genes (2, 4, 5 and 11), ENSG00000282943, and TMEM145 isoforms were analyzed by qPCR in cerebellum and frontal cortex of carriers and non‐carriers of rs10423769_A. Hi‐C analysis was conducted in these brain samples as well. Results: Nanopore long‐read sequencing showed that rs10423769 is present as a single copy on chromosome 19. In the frontal cortex, preliminary Hi‐C results supported the presence of transcriptionally active chromatin over the PSG region in some individuals but not in others. Both PSG genes and the lncRNA had low expression in the frontal cortex and cerebellum. We observed higher TMEM145 expression in the cerebellum compared to frontal cortex and detected alternative splicing forms of TMEM145. There was a trend of increased expression of TMEM145 in rs10423769_A carriers, which was not statistically significant likely due to sample size. Conclusion: We established that only one copy of rs10423769 exists in the modifying region, which will facilitate future haplotype analysis. The Hi‐C analysis suggested inter‐individual variations on chromatin structure over PSG genes in frontal cortex, although no expression of these genes was detected. Further analysis of regulatory activity of the region is ongoing. This modifier locus appears to represent a novel protective mechanism for APOE ε4. [ABSTRACT FROM AUTHOR]

Background: The APOE4 genotype is the main contributor to the risk of developing Late Onset Alzheimer's Disease (LOAD), while the APOE3 genotype has no effect on LOAD risk. Previously our group has shown that Local Ancestry (LA) surrounding APOE4 regulates APOE expression in adult individuals. As LOAD pathology has been shown to develop decades before the onset of symptoms, we sought to explore if LA has similar or different effect in mitotic cells, and whether the APOE3 genotype modulates the effect of the LA. To answer this question, we generated APOEε3/3 and APOEε4/4 isogenic induced Pluripotent Stem Cells (iPSCs) lines from individuals with either European LA or African LA. Method: PBMCs were derived from APOEε4/4 carrying individuals with either African or European APOE LA. To generate APOE isogenic lines, we formed a ribonucleoprotein (RNP) complex using a previously published APOE sgRNA (Lin et al. 2018) and performed nucleofection using the 4D‐Nucleofector system (Lonza). Homozygous APOEε3/3 edited and APOEε4/4 unedited isogenic clones were selected, and confirmed to lack unwanted off‐target editing. Isogenic clone pluripotency was confirmed by immunocytochemistry (SOX2, NANOG and OCT4a staining). APOE expression in the isogenic clones was tested by quantitative Real‐Time PCR and significance was calculated by Student's T‐test. Result: Isogenic clones were similar in their pluripotency and growth characteristics. Preliminary APOE expression in the iPSC line carrying the African LA was higher (p = 0.0002) in each of the individual APOEε3/3 KI clones (n = 3) compared to each of the individual unedited APOEε4/4 clones (n = 2), showing consistency within each isogenic group. In contrast, the APOE expression in the iPSC line carrying the European LA was not significant in the APOEε3/3 KI clones (n = 2) compared to the parental line. Conclusion: Our data suggests that differences in local ancestry has a direct effect on the expression of APOE isoforms in iPSCs. This finding supports previous reports in frontal cortex suggesting that the regulation of APOE expression differs depending on ancestry. [ABSTRACT FROM AUTHOR]

Background: Genetic risk for Alzheimer Disease (AD) varies across populations with different ancestry. Three‐dimensional (3D) genome architecture regulates gene transcription. Genome‐wide Hi‐C analyses have revealed dynamic 3D genome changes during development and disease progression. The variation of 3D genome architecture on different ancestral background, however, has not been extensively studied, especially at high‐resolution level. Recently, we developed the DeepLoop pipeline enabling robust high‐resolution analysis. We applied DeepLoop to characterize 3D genome architecture in frontal cortex from African American (AA) and European (EU) donors while considering local ancestry (LA) in admixed samples. Methods: Hi‐C libraries were constructed and ∼500 million of paired‐end reads were obtained in 8 samples (4 AA and 4EU) that were sex‐matched and homozygous for APOEε4. We partitioned Hi‐C data into compartment A (open and transcriptionally active chromatin) and compartment B (compacted and transcriptionally silenced chromatin). In total, 6206 compartment bins across the genome were compared at a 500kb resolution. DeepLoop was used to call chromatin loop (often represents promoter‐enhancer interaction) at a 5kb‐resolution. LA was calculated using RFMix with SNP‐array data and a reference panel from 1000 Genomes for EU, African (AF) and Amerindian (AI) ancestry. Results: Most of the A and B compartments were consistent among individuals with only <5% of them switched between AA and EU individuals. The EU samples have slightly (107 bins) more transcriptionally active bins than the AA samples. While many of homozygous LA blocks displayed same chromatin loop pattern between and within ancestries, we did observe ancestry‐specific loop as well as differences between individuals of the same ancestry. Importantly, the presence of heterozygous LA (African/EU) at some loci displayed a composite loop pattern that was present in the same region of homozygous LA of African or EU ancestry. Conclusion: The 3D genome architecture can be variable between different ancestral backgrounds as well as between individuals of the same LA. We hypothesize that admixture of LA can create novel or demolish chromatin interactions observed in the homozygous LA. These data emphasize the importance of LA in identifying the causal loci contributing to AD in admixed population such as AF and Hispanic/Latino. [ABSTRACT FROM AUTHOR]

Background: The APOEε4 confers the highest genetic risk factor for Alzheimer's disease (AD). Specifically, Non‐Hispanic Whites (NHW) are more susceptible to developing AD than African American (AA) individuals (OR∼15 vs 8, respectively). Local ancestry (LA) surrounding the APOE region has been implicated in this risk difference, with APOEε4 carriers of European LA (ELA) having increased chromatin accessibility and higher APOEε4 expression than individuals of African LA (ALA). In contrast, carriers of APOEε3, the most common allele, are not predisposed to AD. We here sought to investigate whether this differential accessibility and gene expression are solely due to APOE LA or whether the APOE genotype also contributes to the different disease risk. Method: We screened 94 brains from individuals self‐identified as either AA or NHW of which we identified a total of 22 AD autopsy samples (18 with ELA and 4 with ALA) by GSA, all homozygous for LA and APOEε3. We performed single nuclei ATAC‐seq and single nuclei RNA sequencing (snRNA‐seq) using frozen frontal cortex using the 10x Genomics. Result: To date, we have performed snRNA‐seq in a total of 16,099 nuclei for four brains (2 ELA and 2 ALA). We identified 27 distinct cell clusters at a resolution of 0.6. The proportion of cells per cluster between ELA and ALA samples was similar for all clusters, except for 8 clusters (primarily neurons) which had a greater than 2‐fold difference in ELA. Preliminary results show that APOEε3 carriers with ELA have a significantly higher APOE expression in astrocytes (cluster 11) than those of ALA similar to previous reports in APOEε4 carriers (Griswold, A. et al, (2021)). Interestingly, we also observed an increase in APOE expression in ALA for one oligodendrocyte cluster and four neuronal clusters (inhibitory and excitatory neurons) independent of those mentioned above. Conclusion: We report novel insights on the effect of APOE LA and genotype and its contributions to Alzheimer's disease risk. Specifically, as APOEε3 allele expression follows a similar pattern of overexpression in ELA vs ALA as observed for APOEε4 carriers, our data support that LA and not allele status contribute to the differential APOE expression observed among different ancestral populations. [ABSTRACT FROM AUTHOR]

Background: Growing evidence has demonstrated that DNA methylation (DNAm) plays an important role in Alzheimer's disease and that DNAm differences can be detected in the blood of AD subjects. Because access to human brain tissues is primarily limited to postmortem samples, most studies have correlated blood DNAm with clinical diagnosis of AD in living individuals. However, as the pathophysiological process of AD can begin many years before the onset of clinical symptoms, there is often disagreement between neuropathology in the brain and clinical phenotypes. Thus, the association of blood DNAm with changes in AD neuropathology, rather than with clinical data, would provide more relevant information on AD pathogenesis. Method: CSF biomarkers are well‐established AD endophenotypes and correlate significantly with neuropathology scores measured on postmortem brain samples. In this study, we performed a comprehensive analysis to identify blood DNA methylation associated with CSF pathological biomarkers for AD in the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. Our study included matched samples of whole blood DNA methylation, CSF Aβ42, phosphorylated tau181 (pTau181), and total tau (tTau) biomarkers data, measured on the same subjects and at the same clinical visits from a total of 202 subjects (123 cognitively normal, 79 AD cases). Result: We identified a number of novel associations between blood DNAm and CSF Aβ42, pTau181, and total tau biomarkers, demonstrating that changes in various pathological processes in the CSF are also reflected in the blood epigenome. Overall, the CSF biomarker‐associated DNAm is relatively distinct in cognitively normal (CN) and AD subjects, highlighting the importance of analyzing omics data measured on cognitively normal subjects (which includes preclinical AD subjects) to identify diagnostic biomarkers, and considering disease stages in the development and testing of AD treatment strategies. Moreover, our pathway analysis of CSF biomarker associated‐DNAm from CN subjects revealed that biological processes associated with early brain impairment relevant to AD are marked by DNAm in the blood. Conclusion: Our study provides a valuable resource for future mechanistic and biomarker studies of DNAm in AD. [ABSTRACT FROM AUTHOR]

Background: Previous studies have reported that non‐Hispanic White (NHW) females carrying the APOE ε4 allele have an increased risk for developing AD when compared to men. Recently, analyses showed that the risk for developing AD may be equivalent for males and females but may differ by age. Few studies have been done on this issue in groups other than NHW. We aimed to evaluate the association between sex, age, ancestry, APOE, and AD. Methods: We conducted a pooled case‐control study from 45 independent databases in Alzheimer's Disease Genetics Consortium. Individuals (age≥55 years) were included (n = 31,058). Independent variables were age, sex, ancestry/ethnicity, and APOE genotype (ε3/ε4 vs. ε3/ε3). Logistic, linear, and Cox regression models were performed by sex and ancestry. Results: Regardless of ancestry, men and women with APOE ε3/ε4 showed no significant difference in the risk of developing AD (all age groups combined). When comparing subjects with APOE ε3/ε4 to those with ε3/ε3, East‐Asian (EA) females (OR,4.90; 95% CI: 3.46‐6.95) had the strongest OR, followed by EA males (OR, 4.09: 2.67‐6.27); and NHW females (OR,3.40: 3.14‐3.68) and men (OR,3.33: 3.03‐3.66) among all ethnicities. When stratified by age, NHW females (OR, 3.41: 2.69‐4.32 vs. NHW males: OR,2.57: 1.94‐3.39) and EA women (OR,6.51: 1.31‐32.30 vs. EA men: OR,3.41: 0.66‐17.56) with APOE ε3/ε4 showed a higher risk of developing AD between ages 55‐65; whereas African American (AA) women (OR,4.14: 3.06‐5.60 vs. AA men: OR,2.27: 1.45‐3.55) exhibited this pattern later, at the age of 66‐75 years. In Hispanics, the pattern is reversed, with males aged 55‐65 years carrying APOE ε3/ε4 (OR,6.65; 1.64‐26.89: vs. females: OR,2.27; 1.03‐4.98) had a stronger association with AD risk than their female counterpart. Case‐only and survival analyses also showed that NHW and EA female carriers of APOE ε3/ε4 had an earlier age at the onset of AD than their counterpart in men. Conclusions: Our findings suggest sex differences by age and ethnicity in the risk for developing AD and age at onset of AD for carriers of APOE ε4. Confirming these findings could lead to a better‐personalized risk assessment for AD that accounts for sex differences in APOE. [ABSTRACT FROM AUTHOR]

Background: Previous studies have reported that non‐Hispanic White (NHW) females carrying the APOE ε4 allele have an increased risk for developing AD when compared to men. Recently, analyses showed that the risk for developing AD may be equivalent for males and females but may differ by age. Few studies have been done on this issue in groups other than NHW. We aimed to evaluate the association between sex, age, ancestry, APOE, and AD. Methods: We conducted a pooled case‐control study from 45 independent databases in Alzheimer's Disease Genetics Consortium. Individuals (age≥55 years) were included (n = 31,058). Independent variables were age, sex, ancestry/ethnicity, and APOE genotype (ε3/ε4 vs. ε3/ε3). Logistic, linear, and Cox regression models were performed by sex and ancestry. Results: Regardless of ancestry, men and women with APOE ε3/ε4 showed no significant difference in the risk of developing AD (all age groups combined). When comparing subjects with APOE ε3/ε4 to those with ε3/ε3, East‐Asian (EA) females (OR,4.90; 95% CI: 3.46‐6.95) had the strongest OR, followed by EA males (OR, 4.09: 2.67‐6.27); and NHW females (OR,3.40: 3.14‐3.68) and men (OR,3.33: 3.03‐3.66) among all ethnicities. When stratified by age, NHW females (OR, 3.41: 2.69‐4.32 vs. NHW males: OR,2.57: 1.94‐3.39) and EA women (OR,6.51: 1.31‐32.30 vs. EA men: OR,3.41: 0.66‐17.56) with APOE ε3/ε4 showed a higher risk of developing AD between ages 55‐65; whereas African American (AA) women (OR,4.14: 3.06‐5.60 vs. AA men: OR,2.27: 1.45‐3.55) exhibited this pattern later, at the age of 66‐75 years. In Hispanics, the pattern is reversed, with males aged 55‐65 years carrying APOE ε3/ε4 (OR,6.65; 1.64‐26.89: vs. females: OR,2.27; 1.03‐4.98) had a stronger association with AD risk than their female counterpart. Case‐only and survival analyses also showed that NHW and EA female carriers of APOE ε3/ε4 had an earlier age at the onset of AD than their counterpart in men. Conclusions: Our findings suggest sex differences by age and ethnicity in the risk for developing AD and age at onset of AD for carriers of APOE ε4. Confirming these findings could lead to a better‐personalized risk assessment for AD that accounts for sex differences in APOE. [ABSTRACT FROM AUTHOR]