Alzheimer's Disease Sequencing Project Research Articles

MitoH3: Mitochondrial Haplogroup and Homoplasmic/Heteroplasmic Variant Calling Pipeline for Alzheimer's Disease Sequencing Project.

Mitochondrial DNA (mtDNA) is a double-stranded circular DNA and has multiple copies in each cell. Excess heteroplasmy, the coexistence of distinct variants in copies of mtDNA within a cell, may lead to mitochondrial impairments. Accurate determination of heteroplasmy in whole-genome sequencing (WGS) data has posed a significant challenge because mitochondria carrying heteroplasmic variants cannot be distinguished during library preparation. Moreover, sequencing errors, contamination, and nuclear mtDNA segments can reduce the accuracy of heteroplasmic variant calling. To efficiently and accurately call mtDNA homoplasmic and heteroplasmic variants from the large-scale WGS data generated from the Alzheimer's Disease Sequencing Project (ADSP), and test their association with Alzheimer's disease (AD). In this study, we present MitoH3-a comprehensive computational pipeline for calling mtDNA homoplasmic and heteroplasmic variants and inferring haplogroups in the ADSP WGS data. We first applied MitoH3 to 45 technical replicates from 6 subjects to define a threshold for detecting heteroplasmic variants. Then using the threshold of 5% ≤variant allele fraction≤95%, we further applied MitoH3 to call heteroplasmic variants from a total of 16,113 DNA samples with 6,742 samples from cognitively normal controls and 6,183 from AD cases. This pipeline is available through the Singularity container engine. For 4,311 heteroplasmic variants identified from 16,113 samples, no significant variant count difference was observed between AD cases and controls. Our streamlined pipeline, MitoH3, enables computationally efficient and accurate analysis of a large number of samples.

On the effect heterogeneity of established disease susceptibility loci for Alzheimer's disease across different genetic ancestries.

Genome-wide association studies have identified numerous disease susceptibility loci (DSLs) for Alzheimer's disease (AD). However, only a limited number of studies have investigated the dependence of the genetic effect size of established DSLs on genetic ancestry. We utilized the whole genome sequencing data from the Alzheimer's Disease Sequencing Project (ADSP) including 35,569 participants. A total of 25,459 subjects in four distinct populations (African ancestry, non-Hispanic White, admixed Hispanic, and Asian) were analyzed. We found that nine DSLs showed significant heterogeneity across populations. Single nucleotide polymorphism (SNP) rs2075650 in translocase of outer mitochondrial membrane 40 (TOMM40) showed the largest heterogeneity (Cochran's Q=0.00, I2=90.08), followed by other SNPs in apolipoprotein C1 (APOC1) and apolipoprotein E (APOE). Two additional loci, signal-induced proliferation-associated 1 like 2 (SIPA1L2) and solute carrier 24 member 4 (SLC24A4), showed significant heterogeneity across populations. We observed substantial heterogeneity for the APOE-harboring 19q13.32 region with TOMM40/APOE/APOC1 genes. The largest risk effect was seen among African Americans, while Asians showed a surprisingly small risk effect.

Key variants via the Alzheimer's Disease Sequencing Project whole genome sequence data.

Genome-wide association studies (GWAS) have identified loci associated with Alzheimer's disease (AD) but did not identify specific causal genes or variants within those loci. Analysis of whole genome sequence (WGS) data, which interrogates the entire genome and captures rare variations, may identify causal variants within GWAS loci. We performed single common variant association analysis and rare variant aggregate analyses in the pooled population (N cases=2184, N controls=2383) and targeted analyses in subpopulations using WGS data from the Alzheimer's Disease Sequencing Project (ADSP). The analyses were restricted to variants within 100kb of 83 previously identified GWAS lead variants. Seventeen variants were significantly associated with AD within five genomic regions implicating the genes OARD1/NFYA/TREML1, JAZF1, FERMT2, and SLC24A4. KAT8 was implicated by both single variant and rare variant aggregate analyses. This study demonstrates the utility of leveraging WGS to gain insights into AD loci identified via GWAS.

A comparative study of structural variant calling in WGS from Alzheimer's disease families.

Detecting structural variants (SVs) in whole-genome sequencing poses significant challenges. We present a protocol for variant calling, merging, genotyping, sensitivity analysis, and laboratory validation for generating a high-quality SV call set in whole-genome sequencing from the Alzheimer's Disease Sequencing Project comprising 578 individuals from 111 families. Employing two complementary pipelines, Scalpel and Parliament, for SV/indel calling, we assessed sensitivity through sample replicates (N = 9) with in silico variant spike-ins. We developed a novel metric, D-score, to evaluate caller specificity for deletions. The accuracy of deletions was evaluated by Sanger sequencing. We generated a high-quality call set of 152,301 deletions of diverse sizes. Sanger sequencing validated 114 of 146 detected deletions (78.1%). Scalpel excelled in accuracy for deletions ≤100 bp, whereas Parliament was optimal for deletions >900 bp. Overall, 83.0% and 72.5% of calls by Scalpel and Parliament were validated, respectively, including all 11 deletions called by both Parliament and Scalpel between 101 and 900 bp. Our flexible protocol successfully generated a high-quality deletion call set and a truth set of Sanger sequencing-validated deletions with precise breakpoints spanning 1-17,000 bp.

Association Between HSV‐1 and Risk of Alzheimer’s Disease Detected by a Propensity‐Score Matched Case Design

AbstractBackgroundHerpes simplex virus 1 (HSV‐1) is a neurotropic and neuroinvasive species that can enter the brain triggering inflammation and an immune response, increasing the risk of Alzheimer disease (AD). Previously, we found a significant association between HSV‐1 and AD in whole‐exome sequencing (WES) data (OR = 3.69, P = 6.71×10−4) but not whole‐genome sequenced (WGS) data (OR = 0.92, P = 0.27) from the AD Sequencing Project (ADSP).MethodTo understand this discrepancy, and to better control for the numerous covariates and technical differences that influenced viral quantity, we created a novel algorithm using propensity score analysis (PSA) which permits analysis of observational study data comparable to a randomized controlled trial. WGS reads from 6,541 AD cases and 6,855 controls from the ADSP (including 7,395 non‐Hispanic Whites, 2,961 African Americans, and 3,040 Caribbean Hispanics) that did not align to the human genome were mapped to viral reference sequences and quantified. Propensity scores were derived for 7,619 individuals with at least one HSV‐1 read and 5,777 individuals lacking any HSV‐1 reads using a binary treatment indicator for HSV‐1 status. We applied forward stepwise regression including known confounders (APOE ε4 status, sex, age, tissue source (blood or brain), population ancestry, sequencing center, and PCR amplification) to identify other potential confounders and/or interactions and quadratic terms for creating propensity scores. We tried several matching algorithms to generate matched sets of individuals with and without HSV‐1. Utilizing the best quality matched set, we evaluated the association of AD with HSV‐1 status using a weighted generalized linear model.ResultThe final regression model included terms for the covariates mentioned above that were available for most of the original sample (5828 AD cases and 6487 controls). We found a significant association between HSV‐1 and AD (OR = 1.10, P = 0.02).ConclusionResults from WGS data confirm our previous findings obtained from WES data and support the hypothesis that HSV‐1 is associated with an increased AD risk, highlighting the utility of PSA for analyzing association of risk factors with complex disorders like AD. Further studies are needed to replicate this finding in other ancestral populations and explain the lack of association in WGS data.

Identification of rare coding variants associated with Alzheimer’s disease.

AbstractBackgroundPrevious whole exome sequencing (WES) studies of Alzheimer’s disease (AD) have identified genome‐wide significant associations with rare variants in several novel genes and highlighted the need for investigations in larger samples. We combined WES and whole genome sequencing (WGS) data assembled by the Alzheimer Disease Sequencing Project (ADSP) to increase the power for detecting associations of AD with rare variants in gene coding regions.MethodWe developed an efficient computational pipeline to perform both pre‐merging and post‐merging genotype, variant and sample‐level quality control (QC) on WGS data containing 16,905 individuals and WES data for 20,504 individuals. The resultant sample included participants from European (EA, 11,279 AD cases, 8,924 controls), African American (AA, 2,757 AD cases, 4,336 controls), and Caribbean Hispanic (CH 1,438 AD cases, 3,256 controls) ancestries. We employed GENESIS to test association of AD with 250,465 bi‐allelic QC’d variants using a logistic model including covariates for age, sex, exome capture kit, read length, and ancestry principal components (PCs).ResultIn the total sample, variants from APOE and other known AD genes including SORL1 (P = 5.30×10‐7), NECTIN2 (P = 6.94×10‐7), and TREM2 R47H (P = 2.34×10‐13) crossed or neared the study‐wide significance (SWS) threshold of P = 2.00×10‐7 (0.05/250,465). SWS or borderline significant associations were also found with variants in several novel loci including TKTL2 (P = 2.35×10‐8), D2HGDH, (P = 1.09×10‐7), CECR1 (P = 4.02×10‐7), PDHA2 (P = 2.76×10‐7), GOLGA1 (P = 1.72×10‐7), CYLD (P = 1.84×10‐7), and RP11‐243M5.4 (P = 1.37×10‐7). PSEN1 missense mutation G206A (rs63750082) previously associated with early onset AD in the CH group, was significantly associated with late onset AD in the same population (P = 1.58×10‐13). SWS association of AD with APOE was observed in all groups. Significant associations were also observed with variants in SERPINB8 (P = 3.77×10‐9) and FAM171A (P = 7.20×10‐7) in EAs and TLR4 (P = 4.58×10‐7) in CH. Associations with several top‐ranked variants were replicated in the Alzheimer’s Disease Genetics Consortium GWAS dataset that were imputed using the TOPMed reference panel and ADSP 5K WGS dataset.ConclusionWe demonstrated that merging WGS and WES datasets can increase power to detect associations with rare coding variants in genes including ones previously implicated in AD (CYLD and TLR4) and early‐onset stroke (CECR1).

NOTCH3 C201R variant causes cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) that can be confused with early-onset Alzheimer's disease

BackgroundNOTCH3 is the causative gene for autosomal dominant cerebral arteriopathy with subcortical infarctions and leukoencephalopathy (CADASIL) which is associated with both stroke and dementia. When CADASIL presents primarily as dementia it can be difficult to distinguish from Alzheimer's disease (AD) at both the clinical and neuropathological levels. MethodsWe performed exome sequencing of several affected individuals from a large family affected with AD. PCR amplification and direct Sanger sequencing were used to verify variants detected by exome analysis and to screen family members at-risk to carry those variants. Neuropathologic brain evaluation by immunohistochemistry and MRI were performed for the carriers of the NOTCH3 variant. ResultsIn a three-generation family with AD, we found a c.601 T > C p.Cys201Arg variant in the NOTCH3 gene that caused clinical and neuropathological manifestations of CADASIL. These features included earlier onset of dementia accompanied by behavioral abnormalities in the father and son and white matter abnormalities in the asymptomatic grandson. The family is one branch of a large pedigree studied by the Alzheimer's Disease Sequencing Project (ADSP). As part of the ADSP linkage analysis and whole genome sequencing endeavor, an ABCA1 variant, p.Ala937Val, was previously found associated with AD in this pedigree. ConclusionsOur findings, together with other reported pathogenic missense variants of the C201 codon in NOTCH3, support the role of cysteine 201 as a mutation hotspot for CADASIL and highlight the genetic complexity both clinically and pathologically of AD and related dementia.

Admixture mapping implicates 13q33.3 as ancestry-of-origin locus for Alzheimer disease in Hispanic and Latino populations.

Alzheimer disease (AD) is the most common form of senile dementia, with high incidence late in life in many populations including Caribbean Hispanic (CH) populations. Such admixed populations, descended from more than one ancestral population, can present challenges for genetic studies, including limited sample sizes and unique analytical constraints. Therefore, CH populations and other admixed populations have not been well represented in studies of AD, and much of the genetic variation contributing to AD risk in these populations remains unknown. Here, we conduct genome-wide analysis of AD in multiplex CH families from the Alzheimer Disease Sequencing Project (ADSP). We developed, validated, and applied an implementation of a logistic mixed model for admixture mapping with binary traits that leverages genetic ancestry to identify ancestry-of-origin loci contributing to AD. We identified three loci on chromosome 13q33.3 associated with reduced risk of AD, where associations were driven by Native American (NAM) ancestry. This AD admixture mapping signal spans the FAM155A, ABHD13, TNFSF13B, LIG4, and MYO16 genes and was supported by evidence for association in an independent sample from the Alzheimer's Genetics in Argentina-Alzheimer Argentina consortium (AGA-ALZAR) study with considerable NAM ancestry. We also provide evidence of NAM haplotypes and key variants within 13q33.3 that segregate with AD in the ADSP whole-genome sequencing data. Interestingly, the widely used genome-wide association study approach failed to identify associations in this region. Our findings underscore the potential of leveraging genetic ancestry diversity in recently admixed populations to improve genetic mapping, in this case for AD-relevant loci.

The Early-Onset Alzheimer's Disease Whole-Genome Sequencing Project: Study design and methodology.

Sequencing efforts to identify genetic variants and pathways underlying Alzheimer's disease (AD) have largely focused on late-onset AD although early-onset AD (EOAD), accounting for ∼10% of cases, is largely unexplained by known mutations, resulting in a lack of understanding of its molecular etiology. Whole-genome sequencing and harmonization of clinical, neuropathological, and biomarker data of over 5000 EOAD cases of diverse ancestries. A publicly available genomics resource for EOAD with extensive harmonized phenotypes. Primary analysis will (1) identify novel EOAD risk loci and druggable targets; (2) assess local-ancestry effects; (3) create EOAD prediction models; and (4) assess genetic overlap with cardiovascular and other traits. This novel resource complements over 50,000 control and late-onset AD samples generated through the Alzheimer's Disease Sequencing Project (ADSP). The harmonized EOAD/ADSP joint call will be available through upcoming ADSP data releases and will allow for additional analyses across the full onset range. Sequencing efforts to identify genetic variants and pathways underlying Alzheimer's disease (AD) have largely focused on late-onset AD although early-onset AD (EOAD), accounting for ∼10% of cases, is largely unexplained by known mutations. This results in a significant lack of understanding of the molecular etiology of this devastating form of the disease. The Early-Onset Alzheimer's Disease Whole-genome Sequencing Project is a collaborative initiative to generate a large-scale genomics resource for early-onset Alzheimer's disease with extensive harmonized phenotype data. Primary analyses are designed to (1) identify novel EOAD risk and protective loci and druggable targets; (2) assess local-ancestry effects; (3) create EOAD prediction models; and (4) assess genetic overlap with cardiovascular and other traits. The harmonized genomic and phenotypic data from this initiative will be available through NIAGADS.

Variants near X‐Chromosome Genes NLGN4X and PTCHD1 are Significantly Associated with Alzheimer’s Disease Risk

AbstractBackgroundGenome‐wide association studies (GWAS) for AD have identified numerous associated loci but have been focused only on the autosomes. We sought to identify novel AD‐associated genes on X‐Chromosome (X‐Chr).MethodWe evaluated the association of AD with X‐Chr variants in GWAS datasets assembled by the Alzheimer Disease Genetics Consortium including 26,322 non‐Hispanic White (NHW) individuals using logistic regression models with covariates for age, sex and principal components of ancestry. The number of independent SNPs in X‐Chr was calculated by linkage disequilibrium pruning yielding a study‐wide significance threshold of P<7.58×10−7. Genes near SNPs surpassing this threshold were further evaluated by expression quantitative trait locus (eQTL) in the BRAINEAC database and differential gene expression analyses using RNA‐seq data obtained from autopsied brains contained (GEO: GSE44772 and GSE33000). We also tested association of AD with missense or splicing variants with minor allele count (MAC)≥10 in the same genes using GENESIS and whole exome sequencing (WES) data obtained by the Alzheimer Disease Sequencing Project (ADSP) for 11,172 NHWs.ResultWe identified novel associations with variants near NLGN4X (rs34056759; minor allele frequency [MAF] = 0.15, OR = 1.15, P = 5.14×10−7) and PTCHD1 (rs5970663; MAF = 0.30, OR = 1.13, P = 8.26×10−7). The rs5970663 risk allele C was significantly associated with increased expression of PTCHD1 in the medulla region, and PTCHD1 expression was significantly higher in AD cases than in controls in the prefrontal cortex (P<2.1×10−8) and visual cortex (P<2.3×10−4). Rs34056759 was not associated with NLGN4X expression, but NLGN4X expression was significantly lower in AD cases compared to controls in the prefrontal cortex (P<8.7×10−7). A rare PTCHD1 variant (rs201933353; MAF = 0.001) was nominally associated with AD among 6,318 women (OR = 3.72, P = 0.06).ConclusionWe identified novel associations of AD with X‐Chr variants near NLGN4X and PTCHD1, loci that were previously associated with autism spectrum disorder. We plan to replicate, fine‐map, and annotate the candidate loci identified in this study using large whole genome sequencing ADSP datasets.

Association of African Ancestry–Specific APOE Missense Variant R145C With Risk of Alzheimer Disease

Numerous studies have established the association of the common APOE ε2 and APOE ε4 alleles with Alzheimer disease (AD) risk across ancestries. Studies of the interaction of these alleles with other amino acid changes on APOE in non-European ancestries are lacking and may improve ancestry-specific risk prediction. To determine whether APOE amino acid changes specific to individuals of African ancestry modulate AD risk. Case-control study including 31 929 participants and using a sequenced discovery sample (Alzheimer Disease Sequencing Project; stage 1) followed by 2 microarray imputed data sets derived from the Alzheimer Disease Genetic Consortium (stage 2, internal replication) and the Million Veteran Program (stage 3, external validation). This study combined case-control, family-based, population-based, and longitudinal AD cohorts, which recruited participants (1991-2022) in primarily US-based studies with 1 US/Nigerian study. Across all stages, individuals included in this study were of African ancestry. Two APOE missense variants (R145C and R150H) were assessed, stratified by APOE genotype. The primary outcome was AD case-control status, and secondary outcomes included age at AD onset. Stage 1 included 2888 cases (median age, 77 [IQR, 71-83] years; 31.3% male) and 4957 controls (median age, 77 [IQR, 71-83] years; 28.0% male). In stage 2, across multiple cohorts, 1201 cases (median age, 75 [IQR, 69-81] years; 30.8% male) and 2744 controls (median age, 80 [IQR, 75-84] years; 31.4% male) were included. In stage 3, 733 cases (median age, 79.4 [IQR, 73.8-86.5] years; 97.0% male) and 19 406 controls (median age, 71.9 [IQR, 68.4-75.8] years; 94.5% male) were included. In ε3/ε4-stratified analyses of stage 1, R145C was present in 52 individuals with AD (4.8%) and 19 controls (1.5%); R145C was associated with an increased risk of AD (odds ratio [OR], 3.01; 95% CI, 1.87-4.85; P = 6.0 × 10-6) and was associated with a reported younger age at AD onset (β, -5.87 years; 95% CI, -8.35 to -3.4 years; P = 3.4 × 10-6). Association with increased AD risk was replicated in stage 2 (R145C was present in 23 individuals with AD [4.7%] and 21 controls [2.7%]; OR, 2.20; 95% CI, 1.04-4.65; P = .04) and was concordant in stage 3 (R145C was present in 11 individuals with AD [3.8%] and 149 controls [2.7%]; OR, 1.90; 95% CI, 0.99-3.64; P = .051). Association with earlier AD onset was replicated in stage 2 (β, -5.23 years; 95% CI, -9.58 to -0.87 years; P = .02) and stage 3 (β, -10.15 years; 95% CI, -15.66 to -4.64 years; P = 4.0 × 10-4). No significant associations were observed in other APOE strata for R145C or in any APOE strata for R150H. In this exploratory analysis, the APOE ε3[R145C] missense variant was associated with an increased risk of AD among individuals of African ancestry with the ε3/ε4 genotype. With additional external validation, these findings may inform AD genetic risk assessment in individuals of African ancestry.

NIAGADS Alzheimer’s Genomics Database: version GRCh38

AbstractBackgroundThe Alzheimer’s Genomics Database (GenomicsDB) is an interactive knowledgebase for Alzheimer's disease (AD) genetics that offers a platform for data sharing, discovery, and analysis to accelerate research on AD and AD related dementias (ADRD). The resource provides unrestricted access to GWAS summary statistics datasets, variant annotations, and meta‐analysis results deposited at the NIA Genetics of Alzheimer’s Disease Data Storage Site (NIAGADS). Here we introduce an updated version of the GenomicsDB, now mapped against the latest human genome assembly, GRCh38.MethodThe GenomicsDB allows users to interactively mine AD/ADRD summary statistics datasets or visually inspect them and compare with annotated variant tracks and GRCh38 aligned functional genomics tracks from the FILER functional genomics repository (Kuksa et al. 2022). Available tracks include key AD datasets from large‐scale sequencing projects such as IGAP (Lambert et al. 2013; Kunkle et al. 2019) and the ADGC (e.g., Naj et al. 2011) that have been lifted over from earlier genome builds. Available annotated variants include those from the AD Sequencing Project’s (ADSP) Release3 WGS, called from nearly 17k samples. All variants reported in the GenomicsDB are annotated using an assembly specific version of ADSP annotation pipeline (Butkiewicz et al. 2018). The GenomicsDB provides a REST API for programmatic access. Supported queries return complete annotations for single or bulk lookups of GenomicsDB entries (genes, variants, genomic loci) and ranged queries of datasets as JSON documents.ResultThe GenomicsDB currently hosts >70 datasets and reports >150 million annotated variants from the ADSP and 23 AD/ADRD GWAS studies, mapped to GRCh38. Keeping up with community standards allows this resource to provide ongoing support for AD/ADRD research by making it possible to integrate AD/ADRD relevant `omics datasets generated by the ADSP and others who have adopted GRCh38 as their reference genome.ConclusionThe NIAGADS Alzheimer’s Genomics Database v. GRCh38 (www.niagads.org/genomics) provides an up‐to‐date and accessible platform that facilitates unrestricted sharing of genetic knowledge underpinning AD/ADRD. To best serve our user community, NIAGADS will continue to provide the GRCh37.p19 version of the Alzheimer’s Genomics Database (including programmatic access) in archival format.

Using Oligodendrocytes for studies in Alzheimer disease

AbstractBackgroundGenomic regulatory architecture (GRA) has been primarily studied in European ancestry. As part of the functional Consortium of the Alzheimer Disease Sequencing Project, we are determining the GRA in African and Amerindian ancestries. Oligodendrocytes (OLs) are the largest glial population in the adult central nervous system. While their main role is to support neuronal metabolism and connectivity, few studies have examined their importance in Alzheimer’s disease (AD), despite reports of low numbers of oligodendrocytes and reduced myelin in the early stages of AD and a demonstrated role of myelinating oligodendrocytes in learning and memory. Several studies have reported the derivation of OLs from pluripotent cells, such as induced pluripotent stem cells (iPSC) to be challenging. Here, we optimized a protocol to derive oligodendrocytes from iPSC for studies in AD patients with different ancestries and how ancestry‐specific genomic differences drive the onset and pathogenesis of AD.MethodiPSC lines derived from AD patients were cultured and differentiated into oligodendrocytes using different induction media, as well as different seeding densities. The cells in each treatment were compared at multiple time points using immunocytochemistry (ICC) and qRT‐PCR for oligodendroglia lineage markers with the goal of identifying the culture conditions that increase the yield of O4+ cells and myelinating oligodendrocytes.ResultOur results showed that increasing the initial seeding density positively correlates with the number of Olig2+ cells that subsequently transitioned into mature O4+ cells capable of producing myelin. Additionally, we showed that the addition of N2 supplement to the induction media was necessary to maintain the cell viability during the initial stage of differentiation.ConclusionWe have optimized a protocol to derive OLs from human iPSC lines. We determined that both the seeding density and the media supplements used during the initial stage of differentiation directly influence viability and, consequently the amount Olig2+ cells that could be obtained to be terminally differentiated into O4+ and myelinating cells. Our optimized protocol will be used to evaluate the GRA and functionality of potential GWAS driving loci in cultured oligodendrocytes from individuals of African and Amerindian ancestries.

Multiple Viruses Detected in Human DNA are Associated with Alzheimer Disease Risk

AbstractBackgroundMultiple infectious agents, including viruses, bacteria, fungi, and protozoa, have been linked to Alzheimer disease (AD) by independent lines of evidence.MethodWe mapped and quantified DNA sequence reads from whole exome and whole genome sequencing data in 37,000 AD cases and controls from of the Alzheimer Disease Sequencing Project that did not align to the human genome to 318 viral reference sequences. After quality control, the sample included 1,696 samples derived from brain (cases=1,314, controls=382) and 14,588 derived from blood (cases=5,813, controls=8,775). We tested individual and cumulative normalized viral read counts, both free and inserted in the host genome, for association with AD using multiple machine learning (ML) classifiers, selecting the top 27 viral species according to an algorithm based on the species’ predictive rank weighted by the model’s overall predictive accuracy across models. These top 27 viruses were tested for association with AD using logistic regression models adjusted for PCR amplification, sequencing site, sex, APOE genotype, and ancestry. Analyses were stratified by tissue source.ResultWe detected sequence reads from 175 unique viral species, herpes simplex virus 1 (HSV‐1) being the most common. Viral read counts varied significantly by sequence center, tissue source, sex, PCR use, and ancestry in both the WES and WGS datasets. ML results indicated that herpes viruses (1, 2, 4, 5, 6A, 6B, 7, and 8), HIV, Hepatitis C, Molluscum contagiosum, several Torque Teno virus species (TTV), HPV‐71, Coronavirus 229E, Tick‐borne encephalitis, human Mastedenovirus, human polyomavirus 2, variola virus, primate t‐lymphotropic virus 1, human endogenous retrovirus K, and total normalized mapped viral reads were significant predictors of AD. Subsequent meta‐analyses of results stratified by tissue source showed that AD was significantly associated with HSV‐1(OR=4.07, P=2.22x10‐5), Human papillomavirus type 71(OR=3.26, P=0.006), TTV‐10 (OR=22.11, P=0.004) , and cumulative viral read count (OR=7.34, P=0.03). After multiple testing correction, HSV‐1 remains significant.ConclusionThese results support the hypothesis that viral infections, especially HSV‐1, is associated with AD risk and demonstrate the potential of using deep sequencing technology to detect microbial agents in multiple tissues and discerning here‐to‐for unrecognized associations of infectious agents and AD.

Ancestral Analysis of the Presenilin‐1 G206A Variant Reveals it as a Founder Event on an African Haplotype in the Puerto Rican Population

AbstractBackgroundVariants in the presenilin‐1 gene (PSEN1) are known to be pathogenic for Alzheimer disease (AD). The change of glycine at amino acid 206 to alanine (G206A) in PSEN1 has been identified in AD Caribbean Hispanic families from Puerto Rico with variable ages of onset and incomplete segregation (Athan et.al., 2001). Here we set out to confirm the role of G206A in the genetic etiology of AD in Puerto Rico while investigating its ancestral history and founding haplotype.MethodWe performed genotyping and whole genome sequencing (WGS) of 43 AD families (N = 182: 87 AD, 41 MCI, and 44 cognitively unimpaired) and 272 AD, 145 MCI, and 297 cognitively unimpaired unrelated individuals of Puerto Rican background and identified carriers of G206A. Genotyping data were phased using SHAPEIT to identify local ancestry of the PSEN1 haplotype followed by RFMix to estimate the genetic ancestral background (African, European, or Amerindian). Haplotype modeling was performed using MERLIN software.ResultWe identified 19 carriers of G206A among individuals with sequencing data in eight of the 43 families (14 AD, two MCI, one neuropsychiatric disorder, and two cognitively unimpaired individuals under 65 years old). G206A did not completely explain AD in these eight families as six other AD cases in the families did not carry the variant. In the unrelated cohort, we identified 13 carriers (nine AD, one MCI, one Pick’s disease, and two cognitively unimpaired under 65 years old). Local ancestry indicated that the mutation arose on an African ancestral haplotype. However, in screening WGS of individuals of primarily African ancestry from Ibadan, Nigeria (63 AD and 648 controls) and African Americans part of the Alzheimer Disease Sequencing Project (1347 AD, 2290 controls) no other carriers were identified.ConclusionOur results support that G206A contributes to AD in the Puerto Rican population, but in AD families does not completely explain the genetic risk. We also show that this variant occurs on a common haplotype across carriers representing a founder event on an African haplotype background in Puerto Rico.

MATS: a novel multi-ancestry transcriptome-wide association study to account for heterogeneity in the effects of cis-regulated gene expression on complex traits.

The Transcriptome-Wide Association Study (TWAS) is a widely used approach which integrates gene expression and Genome Wide Association Study (GWAS) data to study the role of cis-regulated gene expression (GEx) in complex traits. However, the genetic architecture of GEx varies across populations, and recent findings point to possible ancestral heterogeneity in the effects of GEx on complex traits, which may be amplified in TWAS by modeling GEx as a function of cis-eQTLs. Here, we present a novel extension to TWAS to account for heterogeneity in the effects of cis-regulated GEx which are correlated with ancestry. Our proposed Multi-Ancestry TwaS (MATS) framework jointly analyzes samples from multiple populations and distinguishes between shared, ancestry-specific and/or subject-specific expression-trait associations. As such, MATS amplifies power to detect shared GEx associations over ancestry-stratified TWAS through increased sample sizes, and facilitates the detection of genes with subgroup-specific associations which may be masked by standard TWAS. Our simulations highlight the improved Type-I error conservation and power of MATS compared with competing approaches. Our real data applications to Alzheimer's disease (AD) case-control genotypes from the Alzheimer's Disease Sequencing Project (ADSP) and continuous phenotypes from the UK Biobank (UKBB) identify a number of unique gene-trait associations which were not discovered through standard and/or ancestry-stratified TWAS. Ultimately, these findings promote MATS as a powerful method for detecting and estimating significant gene expression effects on complex traits within multi-ancestry cohorts and corroborates the mounting evidence for inter-population heterogeneity in gene-trait associations.

Multiple gene variants linked to Alzheimer's-type clinical dementia via GWAS are also associated with non-Alzheimer's neuropathologic entities

The classic pathologic hallmarks of Alzheimer's disease (AD) are amyloid plaques and neurofibrillary tangles (AD neuropathologic changes, or ADNC). However, brains from individuals clinically diagnosed with “AD-type” (amnestic) dementia usually harbor heterogeneous neuropathologies in addition to, or other than, ADNC. We hypothesized that some AD-type dementia associated genetic single nucleotide variants (SNVs) identified from large genomewide association studies (GWAS) were associated with non-ADNC neuropathologies. To test this hypothesis, we analyzed data from multiple studies with available genotype and neuropathologic phenotype information. Clinical AD/dementia risk alleles of interest were derived from the very large GWAS by Bellenguez et al. (2022) who reported 83 clinical AD/dementia-linked SNVs in addition to the APOE risk alleles. To query the pathologic phenotypes associated with variation of those SNVs, National Alzheimer's disease Coordinating Center (NACC) neuropathologic data were linked to AD Sequencing Project (ADSP) and AD Genomics Consortium (ADGC) data. Separate data were obtained from the harmonized Religious Orders Study and the Rush Memory and Aging Project (ROSMAP). A total of 4811 European participants had at least ADNC neuropathology data and also genotype data available; data were meta-analyzed across cohorts. As expected, a subset of dementia-associated SNVs were associated with ADNC risk in Europeans—e.g., BIN1, PICALM, CR1, MME, and COX7C. Other gene variants linked to (clinical) AD dementia were associated with non-ADNC pathologies. For example, the associations of GRN and TMEM106B SNVs with limbic-predominant age-related TDP-43 neuropathologic changes (LATE-NC) were replicated. In addition, SNVs in TNIP1 and WNT3 previously reported as AD-related were instead associated with hippocampal sclerosis pathology. Some genotype/neuropathology association trends were not statistically significant at P < 0.05 after correcting for multiple testing, but were intriguing. For example, variants in SORL1 and TPCN1 showed trends for association with LATE-NC whereas Lewy body pathology trended toward association with USP6NL and BIN1 gene variants. A smaller cohort of non-European subjects (n = 273, approximately one-half of whom were African-Americans) provided the basis for additional exploratory analyses. Overall, these findings were consistent with the hypothesis that some genetic variants linked to AD dementia risk exert their affect by influencing non-ADNC neuropathologies.

A Fast and Robust Strategy to Remove Variant-Level Artifacts in Alzheimer Disease Sequencing Project Data.

Background and ObjectivesExome sequencing (ES) and genome sequencing (GS) are expected to be critical to further elucidate the missing genetic heritability of Alzheimer disease (AD) risk by identifying rare coding and/or noncoding variants that contribute to AD pathogenesis. In the United States, the Alzheimer Disease Sequencing Project (ADSP) has taken a leading role in sequencing AD-related samples at scale, with the resultant data being made publicly available to researchers to generate new insights into the genetic etiology of AD. To achieve sufficient power, the ADSP has adapted a study design where subsets of larger AD cohorts are collected and sequenced across multiple centers, using a variety of sequencing platforms. This approach may lead to variable variant quality across sequencing centers and/or platforms. In this study, we sought to implement and evaluate filters that can be applied fast to robustly remove variant-level artifacts in the ADSP data.MethodsWe implemented a robust quality control procedure to handle ADSP data. We evaluated this procedure while performing exome-wide and genome-wide association analyses on AD risk using the latest ADSP whole ES (WES) and whole GS (WGS) data releases (NG00067.v5).ResultsWe observed that many variants displayed large variation in allele frequencies across sequencing centers/platforms and contributed to spurious association signals with AD risk. We also observed that sequencing platform/center adjustment in association models could not fully account for these spurious signals. To address this issue, we designed and implemented variant filters that could capture and remove these center-specific/platform-specific artifactual variants.DiscussionWe derived a fast and robust approach to filter variants that represent sequencing center-related or platform-related artifacts underlying spurious associations with AD risk in ADSP WES and WGS data. This approach will be important to support future robust genetic association studies on ADSP data, as well as other studies with similar designs.

Molecular Quantitative Trait Locus Mapping in Human Complex Diseases.

Mapping quantitative trait loci (QTLs) for molecular traits from chromatin to metabolites (i.e., xQTLs) provides insight into the locations and effect modes of genetic variants that influence these molecular phenotypes and the propagation of functional consequences of each variant. xQTL studies indirectly interrogate the functional landscape of the molecular basis of complex diseases, including the impact of non-coding regulatory variants, the tissue specificity of regulatory elements, and their contribution to disease by integrating with genome-wide association studies (GWAS). We summarize a variety of molecular xQTL studies in human tissues and cells. In addition, using the Alzheimer's Disease Sequencing Project (ADSP) as an example, we describe the ADSP xQTL project, a collaborative effort across the ADSP Functional Genomics Consortium (ADSP-FGC). The project's ultimate goal is a reference map of Alzheimer's-related QTLs using existing datasets from multiple omics layers to help us study the consequences of genetic variants identified in the ADSP. xQTL studies enable the identification of the causal genes and pathways in GWAS loci, which will likely aid in the discovery of novel biomarkers and therapeutic targets for complex diseases. © 2022 Wiley Periodicals LLC.

An association test of the spatial distribution of rare missense variants within protein structures identifies Alzheimer's disease-related patterns.

More than 90% of genetic variants are rare in most modern sequencing studies, such as the Alzheimer's Disease Sequencing Project (ADSP) whole-exome sequencing (WES) data. Furthermore, 54% of the rare variants in ADSP WES are singletons. However, both single variant and unit-based tests are limited in their statistical power to detect an association between rare variants and phenotypes. To best use missense rare variants and investigate their biological effect, we examine their association with phenotypes in the context of protein structures. We developed a protein structure–based approach, protein optimized kernel evaluation of missense nucleotides (POKEMON), which evaluates rare missense variants based on their spatial distribution within a protein rather than their allele frequency. The hypothesis behind this test is that the three-dimensional spatial distribution of variants within a protein structure provides functional context to power an association test. POKEMON identified three candidate genes (TREM2, SORL1, and EXOC3L4) and another suggestive gene from the ADSP WES data. For TREM2 and SORL1, two known Alzheimer's disease (AD) genes, the signal from the spatial cluster is stable even if we exclude known AD risk variants, indicating the presence of additional low-frequency risk variants within these genes. EXOC3L4 is a novel AD risk gene that has a cluster of variants primarily shared by case subjects around the Sec6 domain. This cluster is also validated in an independent replication data set and a validation data set with a larger sample size.