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

Background: 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. Objective: 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). Methods: 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. Results: 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. Conclusions: Our streamlined pipeline, MitoH3, enables computationally efficient and accurate analysis of a large number of samples.

Generalizability of Tau and Amyloid Plasma Biomarkers in Alzheimer's Disease Cohorts of Diverse Genetic Ancestries.

Introduction: Plasma phosphorylated threonine-181 of Tau and amyloid beta are biomarkers for differential diagnosis and preclinical detection of Alzheimer disease (AD). Given differences in AD risk across diverse populations, generalizability of existing biomarker data is not assured. Methods: In 2,086 individuals of diverse genetic ancestries (African American, Caribbean Hispanic, and Peruvians) we measured plasma pTau-181 and Aß42/Aß40. Differences in biomarkers between cohorts and clinical diagnosis groups and the potential discriminative performance of the two biomarkers were assessed. Results: pTau-181 and Aß42/Aß40 were consistent across cohorts. Higher levels of pTau181 were associated with AD while Aß42/Aß40 had minimal differences. Correspondingly, pTau-181 had greater predictive value than Aß42/Aß40, however, the area under the curve differed between cohorts. Discussion: pTau-181 as a plasma biomarker for clinical AD is generalizable across genetic ancestries, but predictive value may differ. Combining genomic and biomarker data from diverse individuals will increase understanding of genetic risk and refine clinical diagnoses.

Longitudinal Evaluation of the Distribution of Intraretinal Hyper-Reflective Foci in Eyes with Intermediate Age-Related Macular Degeneration.

PURPOSE: Intraretinal hyper-reflective foci (IHRF) are optical coherence tomography (OCT) risk factors for progression of age-related macular degeneration (AMD). In this study we assess the change in the number and distribution of IHRF over two years. METHODS: The axial distribution of IHRF were quantified in eyes with intermediate AMD (iAMD) at baseline and 24 months, using a series of 5 sequential equidistant en face OCT retinal slabs generated between the outer border of the internal limiting membrane (ILM) and the inner border of the retinal pigment epithelium (RPE). Following thresholding and binarization, IHRF were quantified in each retinal slab using ImageJ. The change in IHRF number in each slab between baseline and month 24 was calculated. RESULTS: Fifty-two eyes showed evidence of IHRF at baseline, and all continued to show evidence of IHRF at 24 months (M24). The total average IHRF count/eye increased significantly from 4.67 ± 0.63 at baseline to 11.62 ± 13.86 at M24 (p < 0.001) with a mean increase of 6.94 ± 11.12 (range: - 9 to + 60). Overall, at M24, 76.9% eyes showed an increase in IHRF whereas 15.4% of eyes showed a decrease (3 eyes [5.7%] showed no change). There was a greater number of IHRF and a greater increase in IHRF over M24 in the outer slabs. CONCLUSIONS: IHRF are most common in the outer retinal layers and tend to increase in number over time. The impact of the distribution and frequency of these IHRF on the overall progression of AMD requires further study.

Alterations of the Ganglion Cell Complex in Age-Related Macular Degeneration: an AMISH Eye Study Analysis.

PURPOSE: To compare the Ganglion Cell Complex (GCC) thickness in eyes with age-related macular degeneration (AMD) versus healthy controls in an elderly Amish population DESIGN: Cross-sectional study METHODS: This is a post hoc analysis of the family-based prospective study of Amish subjects. Study subjects were imaged by the Cirrus HD-OCT (Carl Zeiss Meditec Inc, Dublin, CA) using a macular cube protocol of 512 × 128 scans (128 horizontal B-scans, each compromising 512 A-scans) over a 6 mm x 6 mm region centered on the fovea. The ganglion cell analysis algorithm calculated the GCC thickness by segmenting the outer boundaries of the retinal nerve fiber layer (RNFL) and inner plexiform layer (IPL) in all B-scans of the volume, with the region between these boundaries representing the combined thickness of the GCL and the IPL layer. A number of parameters were used to evaluate the GCC thickness: the average GCC thickness, minimum (lowest GCC thickness at a single meridian crossing the elliptical annulus), and sectoral (within each of six sectoral areas: superior, superotemporal, superonasal, inferior, inferonasal, and inferotemporal). The stage of AMD was graded on color fundus photographs in accordance with the Beckman Initiative for Macular Research classification system. RESULTS: Of 1339 subjects enrolled in the Amish eye study, a total of 1294 eyes of twelve hundred and ninety-four subjects had all required imaging studies of sufficient quality and were included in the final analysis, and of these 798 (62%) were female. Following age adjustment, the average GCC thickness was significantly (p<0.001) thinner in AMD subjects (73.71 ± SD; 13.77 µm) compared to normals (77.97 ± 10.42 µm). Independent t test showed that, early (75.03 ± 12.45 µm), and late AMD (61.64 ± 21.18 µm) groups (among which GA eyes had the lowest thickness of 58.10 ± 20.27 microns) had a statistically significant lower GCC thickness compared to eyes without AMD. There was no significant difference in average GCC thickness between early AMD and intermediate AMD (76.36 ± 9.25 µm) eyes. CONCLUSIONS: The GCC thickness in AMD eyes is reduced compared to normal eyes, but the relationship is complex with the greatest reduction in late AMD eyes (particularly GA eyes) but no difference between early and intermediate AMD eyes.

Artificial intelligence and open science in discovery of disease-modifying medicines for Alzheimer's disease.

The high failure rate of clinical trials in Alzheimer's disease (AD) and AD-related dementia (ADRD) is due to a lack of understanding of the pathophysiology of disease, and this deficit may be addressed by applying artificial intelligence (AI) to "big data" to rapidly and effectively expand therapeutic development efforts. Recent accelerations in computing power and availability of big data, including electronic health records and multi-omics profiles, have converged to provide opportunities for scientific discovery and treatment development. Here, we review the potential utility of applying AI approaches to big data for discovery of disease-modifying medicines for AD/ADRD. We illustrate how AI tools can be applied to the AD/ADRD drug development pipeline through collaborative efforts among neurologists, gerontologists, geneticists, pharmacologists, medicinal chemists, and computational scientists. AI and open data science expedite drug discovery and development of disease-modifying therapeutics for AD/ADRD and other neurodegenerative diseases.

African Ancestry Individuals with Higher Educational Attainment Are Resilient to Alzheimer's Disease Measured by pTau181.

Background: Cognitive and functional abilities in individuals with Alzheimer's disease (AD) pathology (ADP) are highly variable. Factors contributing to this variability are not well understood. Previous research indicates that higher educational attainment (EA) correlates with reduced cognitive impairments among those with ADP. While cognitive and functional impairments are correlated, they are distinguishable in their manifestations. Objective: To investigate whether levels of education are associated with functional impairments among those with ADP. Methods: This research involved 410 African American (AA) individuals (Institutional Review Boards 20070307, 01/27/2023) to ascertain whether EA correlates with functional resilience and if this effect varies between APOE ɛ4 carriers and non-carriers. Utilizing EA as a cognitive reserve proxy, CDR-FUNC as a functional difficulties measure, and blood pTau181 as an ADP proxy, the non-parametric Mann-Whitney U test assessed the relationship between EA and CDR-FUNC in individuals with advanced pTau181 levels. Results: The results showed that EA correlated with functional difficulties in AA individuals with high levels of pTau181, such that individuals with high EA are more likely to have better functional ability compared to those with lower EA (W = 730.5, p = 0.0007). Additionally, we found that the effect of high EA on functional resilience was stronger in ɛ4 non-carriers compared to ɛ4 carriers (W = 555.5, p = 0.022). Conclusion: This study extends the role of cognitive reserve and EA to functional performance showing that cognitive reserve influences the association between ADP burden and functional difficulties. Interestingly, this protective effect seems less pronounced in carriers of the strong genetic risk allele ɛ4.

Attitudes and Perceptions about Brain Donation Among African Americans: Implications for Recruitment into Alzheimer's Disease Research.

The objective of this study was to investigate attitudes toward brain donation and perceptions of medical research that influence brain donation among African Americans. Cross-sectional surveys were administered to African American community members (n = 227). Findings indicate that only 27% of respondents were willing to donate their brain. As medical mistrust was not found to be a significant barrier to research participation, there may be opportunity to increase brain donation by providing information about Alzheimer's disease and brain donation to potential donors and their families so that informed decisions about participating in research can be made.

Generation of an induced pluripotent stem cell line (UMi043-A) from an African American patient with Alzheimer's disease carrying an ABCA7 deletion (p.Arg578Alafs).

The ATP-binding cassette, subfamily A (ABC1), member 7 (ABCA7) gene is associated with Alzheimer's disease (AD) risk in populations of African, Asian, and European ancestry1-5. Numerous ABCA7 mutations contributing to risk have been identified, including a 44 base pair deletion (rs142076058) specific to individuals of African ancestry and predicted to cause a frameshift mutation (p.Arg578Alafs) (Cukier et al., 2016). The UMi043-A human induced pluripotent stem cell line was derived from an African American individual with AD who is heterozygous for this deletion and is a resource to further investigate ABCA7 and how this African-specific deletion may influence disease pathology.

Report of the APOE4 National Institute on Aging/Alzheimer Disease Sequencing Project Consortium Working Group: Reducing APOE4 in Carriers is a Therapeutic Goal for Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder and one of the leading causes of disability worldwide. The apolipoprotein E4 gene (APOE4) is the strongest genetic risk factor for AD. In 2023, the APOE4 National Institute on Aging/Alzheimer's Disease Sequencing Project working group came together to gather data and discuss the question of whether to reduce or increase APOE4 as a therapeutic intervention for AD. It was the unanimous consensus that cumulative data from multiple studies in humans and animal models support that lowering APOE4 should be a target for therapeutic approaches for APOE4 carriers. ANN NEUROL 2024;95:625-634.

Human whole-exome genotype data for Alzheimer's disease.

The heterogeneity of the whole-exome sequencing (WES) data generation methods present a challenge to a joint analysis. Here we present a bioinformatics strategy for joint-calling 20,504 WES samples collected across nine studies and sequenced using ten capture kits in fourteen sequencing centers in the Alzheimer's Disease Sequencing Project. The joint-genotype called variant-called format (VCF) file contains only positions within the union of capture kits. The VCF was then processed specifically to account for the batch effects arising from the use of different capture kits from different studies. We identified 8.2 million autosomal variants. 96.82% of the variants are high-quality, and are located in 28,579 Ensembl transcripts. 41% of the variants are intronic and 1.8% of the variants are with CADD > 30, indicating they are of high predicted pathogenicity. Here we show our new strategy can generate high-quality data from processing these diversely generated WES samples. The improved ability to combine data sequenced in different batches benefits the whole genomics research community.

Genome-wide association studies identify novel loci in rapidly progressive Alzheimer's disease.

INTRODUCTION: Recent data suggest that distinct prion-like amyloid beta and tau strains are associated with rapidly progressive Alzheimer's disease (rpAD). The role of genetic factors in rpAD is largely unknown. METHODS: Previously known AD risk loci were examined in rpAD cases. Genome-wide association studies (GWAS) were performed to identify variants that influence rpAD. RESULTS: We identified 115 pathology-confirmed rpAD cases and 193 clinical rpAD cases, 80% and 69% were of non-Hispanic European ancestry. Compared to the clinical cohort, pathology-confirmed rpAD had higher frequencies of apolipoprotein E (APOE) ε4 and rare missense variants in AD risk genes. A novel genome-wide significant locus (P < 5×10-8 ) was observed for clinical rpAD on chromosome 21 (rs2832546); 102 loci showed suggestive associations with pathology-confirmed rpAD (P < 1×10-5 ). DISCUSSION rpAD constitutes an extreme subtype of AD with distinct features. GWAS found previously known and novel loci associated with rpAD. Highlights Rapidly progressive Alzheimer's disease (rpAD) was defined with different criteria. Whole genome sequencing identified rare missense variants in rpAD. Novel variants were identified for clinical rpAD on chromosome 21.

Assessment of intraretinal hyperreflective foci using multimodal imaging in eyes with age-related macular degeneration.

PURPOSE: This study aimed to investigate the correspondence between intraretinal hyperreflective foci (IHRF) identified on optical coherence tomography (OCT) B-scans with hyperpigmentation on colour fundus photography (CFP) or hyperreflectivity on infrared reflectance (IR) images in eyes with age-related macular degeneration (AMD). METHODS: Flash CFP, IR images and OCT B-scans obtained at the same visit were evaluated. Individual IHRF identified on OCT B-scans were assessed for the qualitative presence or absence of a hypotransmission tail into the choroid. The corresponding IR image obtained at the time of OCT acquisition was analysed for the presence or absence of hyperreflectivity in this region. The IR images were manually registered to the CFP image, and CFP images were inspected for the presence or absence of hyperpigmentation at the location of IHRF. RESULTS: From 122 eyes, a total of 494 IHRF were evaluated. For the primary analysis of qualitative presence or absence of hyperpigmentation on CFP and hyperreflectivity on IR at the locations corresponding to IHRF on OCT, 301 (61.0%) of the IHRFs demonstrated evidence of hyperpigmentation on CFP, while only 115 (23.3%) showed evidence of hyperreflectivity on IR. The qualitative determination of the presence or absence of an abnormality on CFP or IR were significantly different (p < 0.0001). 327 (66.2%) of the IHRF showed hypotransmission, and 80.4% of these IHRF showed hyperpigmentation on CFP, though only 23.9% (p < 0.0001) demonstrated hyperreflectivity on IR. CONCLUSIONS: Less than two-thirds of IHRF evident on OCT manifest as hyperpigmentation on colour photos, though IHRF with posterior shadowing are more likely to be evident as pigment. IR imaging appears to be even more poorly sensitive for visualizing IHRF.

DNA from multiple viral species is associated with Alzheimer's disease risk.

INTRODUCTION: Multiple infectious agents, including viruses, bacteria, fungi, and protozoa, have been linked to Alzheimer's disease (AD) risk by independent lines of evidence. We explored this association by comparing the frequencies of viral species identified in a large sample of AD cases and controls. METHODS: DNA sequence reads that did not align to the human genome in sequences were mapped to viral reference sequences, quantified, and then were tested for association with AD in whole exome sequences (WES) and whole genome sequences (WGS) datasets. RESULTS: Several viruses were significant predictors of AD according to the machine learning classifiers. Subsequent regression analyses showed that herpes simplex type 1 (HSV-1) (odds ratio [OR] = 3.71, p = 8.03 × 10-4) and human papillomavirus 71 (HPV-71; OR = 3.56, p = 0.02), were significantly associated with AD after Bonferroni correction. The phylogenetic-related cluster of Herpesviridae was significantly associated with AD in several strata of the data (p < 0.01). DISCUSSION: Our results support the hypothesis that viral infection, especially HSV-1, is associated with AD risk.

Sex-specific genetic architecture of late-life memory performance.

BACKGROUND: Women demonstrate a memory advantage when cognitively healthy yet lose this advantage to men in Alzheimer's disease. However, the genetic underpinnings of this sex difference in memory performance remain unclear. METHODS: We conducted the largest sex-aware genetic study on late-life memory to date (Nmales  = 11,942; Nfemales  = 15,641). Leveraging harmonized memory composite scores from four cohorts of cognitive aging and AD, we performed sex-stratified and sex-interaction genome-wide association studies in 24,216 non-Hispanic White and 3367 non-Hispanic Black participants. RESULTS: We identified three sex-specific loci (rs67099044-CBLN2, rs719070-SCHIP1/IQCJ-SCHIP), including an X-chromosome locus (rs5935633-EGL6/TCEANC/OFD1), that associated with memory. Additionally, we identified heparan sulfate signaling as a sex-specific pathway and found sex-specific genetic correlations between memory and cardiovascular, immune, and education traits. DISCUSSION: This study showed memory is highly and comparably heritable across sexes, as well as highlighted novel sex-specific genes, pathways, and genetic correlations that related to late-life memory. HIGHLIGHTS: Demonstrated the heritable component of late-life memory is similar across sexes. Identified two genetic loci with a sex-interaction with baseline memory. Identified an X-chromosome locus associated with memory decline in females. Highlighted sex-specific candidate genes and pathways associated with memory. Revealed sex-specific shared genetic architecture between memory and complex traits.

Longitudinal change in memory performance as a strong endophenotype for Alzheimer's disease.

INTRODUCTION: Although large-scale genome-wide association studies (GWAS) have been conducted on AD, few have been conducted on continuous measures of memory performance and memory decline. METHODS: We conducted a cross-ancestry GWAS on memory performance (in 27,633 participants) and memory decline (in 22,365 participants; 129,201 observations) by leveraging harmonized cognitive data from four aging cohorts. RESULTS: We found high heritability for two ancestry backgrounds. Further, we found a novel ancestry locus for memory decline on chromosome 4 (rs6848524) and three loci in the non-Hispanic Black ancestry group for memory performance on chromosomes 2 (rs111471504), 7 (rs4142249), and 15 (rs74381744). In our gene-level analysis, we found novel genes for memory decline on chromosomes 1 (SLC25A44), 11 (BSX), and 15 (DPP8). Memory performance and memory decline shared genetic architecture with AD-related traits, neuropsychiatric traits, and autoimmune traits. DISCUSSION: We discovered several novel loci, genes, and genetic correlations associated with late-life memory performance and decline. HIGHLIGHTS: Late-life memory has high heritability that is similar across ancestries. We discovered four novel variants associated with late-life memory. We identified four novel genes associated with late-life memory. Late-life memory shares genetic architecture with psychiatric/autoimmune traits.

Genetic analyses in multiplex families confirms chromosome 5q35 as a risk locus for Alzheimer's Disease in individuals of African Ancestry.

There is a paucity of genetic studies of Alzheimer Disease (AD) in individuals of African Ancestry, despite evidence suggesting increased risk of AD in the African American (AA) population. We performed whole-genome sequencing (WGS) and multipoint linkage analyses in 51 multi-generational AA AD families ascertained through the Research in African American Alzheimer Disease Initiative (REAAADI) and the National Institute on Aging Late Onset Alzheimer's disease (NIA-LOAD) Family Based Study. Variants were prioritized on minor allele frequency (<0.01), functional potential of coding and noncoding variants, co-segregation with AD and presence in multi-ancestry ADSP release 3 WGS data. We identified a significant linkage signal on chromosome 5q35 (HLOD=3.3) driven by nine families. Haplotype segregation analysis in the family with highest LOD score identified a 3'UTR variant in INSYN2B with the most functional evidence. Four other linked AA families harbor within-family shared variants located in INSYN2B's promoter or enhancer regions. This AA family-based finding shows the importance of diversifying population-level genetic data to better understand the genetic determinants of AD on a global scale.

Longitudinal evaluation of the distribution of intraretinal hyper-reflective foci in eyes with intermediate age-related macular degeneration.

Purpose: Intraretinal hyper-reflective foci (IHRF) are optical coherence tomography (OCT) risk factors for progression of age-related macular degeneration (AMD). In this study we assess the change in the number and distribution of IHRF over two years. Methods: The axial distribution of IHRF were quantified in eyes with intermediate AMD (iAMD) at baseline and 24 months, using a series of 5 sequential equidistant en face OCT retinal slabs generated between the outer border of the internal limiting membrane (ILM) and the inner border of the retinal pigment epithelium (RPE). Following thresholding and binarization, IHRF were quantified in each retinal slab using ImageJ. The change in IHRF number in each slab between baseline and month 24 was calculated. Results: Fifty-two eyes showed evidence of IHRF at baseline, and all continued to show evidence of IHRF at 24 months (M24). The total average IHRF count/eye increased significantly from 4.67 ± 0.63 at baseline to 11.62 ± 13.86 at M24 (p<0.001) with a mean increase of 6.94 ± 11.12 (range: - 9 to + 60). Overall, at M24, 76.9% eyes showed an increase in IHRF whereas 15.4% of eyes showed a decrease (4 eyes [7.6%] showed no change). There was a greater number of IHRF and a greater increase in IHRF over M24 in the outer slabs. Conclusions: IHRF are most common in the outer retinal layers and tend to increase in number over time. The impact of the distribution and frequency of these IHRF on the overall progression of AMD requires further study.

Founder population-specific weights yield improvements in performance of polygenic risk scores for Alzheimer disease in the Midwestern Amish.

Alzheimer disease (AD) is the most common type of dementia and is estimated to affect 6 million Americans. Risk for AD is multifactorial, including both genetic and environmental risk factors. AD genomic research has generally focused on identification of risk variants. Using this information, polygenic risk scores (PRSs) can be calculated to quantify an individual's relative disease risk due to genetic factors. The Amish are a founder population descended from German and Swiss Anabaptist immigrants. They experienced a genetic bottleneck after arrival in the United States, making their genetic architecture different from the broader European ancestry population. Prior work has demonstrated the lack of transferability of PRSs across populations. Here, we compared the performance of PRSs derived from genome-wide association studies (GWASs) of Amish individuals to those derived from a large European ancestry GWAS. Participants were screened for cognitive impairment with further evaluation for AD. Genotype data were imputed after collection via Illumina genotyping arrays. The Amish individuals were split into two groups based on the primary site of recruitment. For each group, GWAS was conducted with account for relatedness and adjustment for covariates. PRSs were then calculated using weights from the other Amish group. PRS models were evaluated with and without covariates. The Amish-derived PRSs distinguished between dementia status better than the European-derived PRS in our Amish populations and demonstrated performance improvements despite a smaller training sample size. This work highlighted considerations for AD PRS usage in populations that cannot be adequately described by basic race/ethnicity or ancestry classifications.

Spatial Distribution of Missense Variants within Complement Proteins Associates with Age Related Macular Degeneration.

Purpose: Genetic variants in complement genes are associated with age-related macular degeneration (AMD). However, many rare variants have been identified in these genes, but have an unknown significance, and their impact on protein function and structure is still unknown. We set out to address this issue by evaluating the spatial placement and impact on protein structureof these variants by developing an analytical pipeline and applying it to the International AMD Genomics Consortium (IAMDGC) dataset (16,144 AMD cases, 17,832 controls). Methods: The IAMDGC dataset was imputed using the Haplotype Reference Consortium (HRC), leading to an improvement of over 30% more imputed variants, over the original 1000 Genomes imputation. Variants were extracted for the CFH , CFI , CFB , C9 , and C3 genes, and filtered for missense variants in solved protein structures. We evaluated these variants as to their placement in the three-dimensional structure of the protein (i.e. spatial proximity in the protein), as well as AMD association. We applied several pipelines to a) calculate spatial proximity to known AMD variants versus gnomAD variants, b) assess a variant's likelihood of causing protein destabilization via calculation of predicted free energy change (ddG) using Rosetta, and c) whole gene-based testing to test for statistical associations. Gene-based testing using seqMeta was performed using a) all variants b) variants near known AMD variants or c) with a ddG >|2|. Further, we applied a structural kernel adaptation of SKAT testing (POKEMON) to confirm the association of spatial distributions of missense variants to AMD. Finally, we used logistic regression on known AMD variants in CFI to identify variants leading to >50% reduction in protein expression from known AMD patient carriers of CFI variants compared to wild type (as determined by in vitro experiments) to determine the pipeline's robustness in identifying AMD-relevant variants. These results were compared to functional impact scores, ie CADD values > 10, which indicate if a variant may have a large functional impact genomewide, to determine if our metrics have better discriminative power than existing variant assessment methods. Once our pipeline had been validated, we then performed a priori selection of variants using this pipeline methodology, and tested AMD patient cell lines that carried those selected variants from the EUGENDA cohort (n=34). We investigated complement pathway protein expression in vitro , looking at multiple components of the complement factor pathway in patient carriers of bioinformatically identified variants. Results: Multiple variants were found with a ddG>|2| in each complement gene investigated. Gene-based tests using known and novel missense variants identified significant associations of the C3 , C9 , CFB , and CFH genes with AMD risk after controlling for age and sex (P=3.22×10 -5 ;7.58×10 -6 ;2.1×10 -3 ;1.2×10 -31 ). ddG filtering and SKAT-O tests indicate that missense variants that are predicted to destabilize the protein, in both CFI and CFH, are associated with AMD (P=CFH:0.05, CFI:0.01, threshold of 0.05 significance). Our structural kernel approach identified spatial associations for AMD risk within the protein structures for C3, C9, CFB, CFH, and CFI at a nominal p-value of 0.05. Both ddG and CADD scores were predictive of reduced CFI protein expression, with ROC curve analyses indicating ddG is a better predictor (AUCs of 0.76 and 0.69, respectively). A priori in vitro analysis of variants in all complement factor genes indicated that several variants identified via bioinformatics programs PathProx/POKEMON in our pipeline via in vitro experiments caused significant change in complement protein expression (P=0.04) in actual patient carriers of those variants, via ELISA testing of proteins in the complement factor pathway, and were previously unknown to contribute to AMD pathogenesis. Conclusion: We demonstrate for the first time that missense variants in complement genes cluster together spatially and are associated with AMD case/control status. Using this method, we can identify CFI and CFH variants of previously unknown significance that are predicted to destabilize the proteins. These variants, both in and outside spatial clusters, can predict in-vitro tested CFI protein expression changes, and we hypothesize the same is true for CFH . A priori identification of variants that impact gene expression allow for classification for previously classified as VUS. Further investigation is needed to validate the models for additional variants and to be applied to all AMD-associated genes.

Association of Common and Rare Variants with Alzheimer's Disease in over 13,000 Diverse Individuals with Whole-Genome Sequencing from the Alzheimer's Disease Sequencing Project.

Alzheimer's Disease (AD) is a common disorder of the elderly that is both highly heritable and genetically heterogeneous. Here, we investigated the association between AD and both common variants and aggregates of rare coding and noncoding variants in 13,371 individuals of diverse ancestry with whole genome sequence (WGS) data. Pooled-population analyses identified genetic variants in or near APOE, BIN1, and LINC00320 significantly associated with AD (p < 5×10-8). Population-specific analyses identified a haplotype on chromosome 14 including PSEN1 associated with AD in Hispanics, further supported by aggregate testing of rare coding and noncoding variants in this region. Finally, we observed suggestive associations (p < 5×10-5) of aggregates of rare coding rare variants in ABCA7 among non-Hispanic Whites (p=5.4×10-6), and rare noncoding variants in the promoter of TOMM40 distinct of APOE in pooled-population analyses (p=7.2×10-8). Complementary pooled-population and population-specific analyses offered unique insights into the genetic architecture of AD.