Whole-genome sequencing reveals novel ethnicity-specific rare variants associated with Alzheimer’s disease

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder currently lacking effective treatments. While genome-wide association studies (GWAS) have identified common variants associated with AD, these variants typically have small effect sizes. Rare variants, with potentially larger effects, remain understudied. This study aimed to address this gap by using whole-genome sequencing (WGS) to identify rare variants associated with AD in a Japanese population. The researchers hypothesized that WGS would reveal novel rare variants with a larger impact on AD risk than those identified through GWAS. The introduction further highlights the limitations of current GWAS in explaining the substantial heritability of late-onset AD (LOAD), emphasizing the need for WGS to investigate rare variants. The researchers also point out the benefits of WGS over whole-exome sequencing (WES) in detecting a broader range of coding variants, justifying their choice of WGS as the primary method.

The introduction serves as a literature review, citing previous research on AD genetics. It summarizes the established knowledge of AD subtypes (early-onset and late-onset), highlighting the role of known genes (APP, PSEN1, PSEN2) in early-onset AD. It also discusses the limitations of previous GWAS studies in explaining the heritability of late-onset AD, despite their success in identifying several risk loci. The literature review underscores the significant heritability of LOAD (60-80%), with APOE ε4 being the strongest known genetic risk factor, but still accounting for only a fraction of the total heritability. The review establishes the context for the study by emphasizing the need for new approaches, such as WGS, to uncover the missing heritability.

The study involved WGS of 938 samples (140 AD cases and 798 controls) from a Japanese population, supplemented by SNP genotyping data from an independent Japanese case-control cohort (1604 AD cases and 1235 controls). WGS data were analyzed using the Genome Analysis Toolkit (GATK) for variant calling. Stringent quality control (QC) measures were applied to filter variants. The methodology describes the classification of coding variants into rare, medium, and common based on minor allele frequency (MAF). Functional annotation of variants was performed using ANNOVAR. Association studies employed Fisher's exact tests, with significant signals from the discovery set validated in the independent replication cohort via meta-analysis. Logistic regression analysis adjusted for age and sex. The identified variants were validated using multiplex PCR-based assays or Sanger sequencing. Gene-based association tests were conducted to identify genes with multiple causal variants. A network-based meta-analysis using protein-protein interaction (PPI) networks was performed to identify functionally important hub genes. In vitro functional analysis using HEK293 cells and an AD model cell line (APPsw-293 cells) was conducted to assess the functional impact of the identified variants on cell death and Aβ generation, measured via ELISA. Finally, quantitative RT-PCR (qRT-PCR) assessed the expression of hub genes in blood samples from AD and control individuals. The detailed protocols for DNA extraction, library preparation, sequencing, variant calling, QC filters, and statistical analyses are provided in the supplementary materials.

The study identified two rare variants as strong AD candidates: a missense variant in OR5G1 (rs140604166) and a stop-gain variant in MLKL (rs763812068). The OR5G1 variant showed a significant association in both discovery and replication cohorts (Pmeta = 1.36 × 10−3; adjusted Pmeta = 6.82 × 10−7; OR = 2.20). Notably, this variant is predominantly found in East Asian populations. The MLKL stop-gain variant significantly decreased cell death in HEK293 cells and increased the Aβ40/Aβ42 ratio in APPsw-293 cells. Gene-based association tests identified additional candidate genes. Network-based meta-analysis, incorporating these candidates, revealed four hub genes (NCOR2, PLEC, DMD, and NEDD4) with significant functional roles. The qRT-PCR analysis of these hub genes in blood samples did not reveal significant differences in expression between AD and control groups, suggesting these genes may not serve as blood-based biomarkers.

The study's findings support a role for rare variants in AD pathogenesis, particularly ethnicity-specific variants. The identification of the OR5G1 missense variant, predominantly found in East Asians, highlights the importance of considering population-specific genetic factors in AD research. The functional characterization of the MLKL stop-gain variant provides evidence for its involvement in AD pathogenesis through its impact on cell death and Aβ processing. The identification of hub genes like NCOR2, PLEC, DMD, and NEDD4 provides further insights into the complex genetic architecture of AD. The lack of significant differences in the expression of hub genes in blood samples suggests that these genes may not be suitable as blood-based biomarkers for AD prediction; however, this does not negate their potential importance in the disease's development and progression within the brain.

This study successfully identified novel rare variants associated with AD in a Japanese population, emphasizing the importance of population-specific genetic studies. The findings shed light on the role of rare variants in AD pathogenesis, particularly the functional effects of the MLKL stop-gain variant. Further research should explore the interactions between these identified variants and other genetic and environmental factors to gain a more comprehensive understanding of AD etiology. Future work could also focus on validating these findings in other populations and investigating the potential therapeutic implications of targeting these genes or pathways.

The study's limitations include the relatively small sample size for the WGS analysis, which may limit the power to detect other rare variants with smaller effect sizes. The study focused on a Japanese population, limiting the generalizability of the findings to other ethnic groups. Further research with larger, more diverse cohorts is needed to confirm these findings and explore their relevance across different populations. While the study performed extensive in vitro analysis, further in vivo studies are necessary to validate the functional implications of the identified variants.