Exploring potential mechanisms of an African protective locus for Alzheimer's disease in APOEε4 carriers

Abstract

Introduction: We recently described that the African-specific A allele of rs10423769, which lies in an area of segmental duplications, reduces Alzheimer's disease (AD) risk by ∼ 75% in apolipoprotein E (APOE) ε4 homozygotes.

Methods: Short and long-read sequencing were used to identify the haplotype harboring rs10423769_A and examine DNA methylation and structural variation (SV).

Results: A unique 21 kb haplotype is shared amongst all rs10423769_A carriers (r² > 0.95) and is present in all African ancestry tested. We identified methylation differences between the non-protective and rs10423769_A haplotypes. An expanded variable number tandem repeat (VNTR) containing multiple MEF2-family transcription factor binding motifs in LD with the protective haplotype. Further, rs10423769 is an eQTL for ZNF222.

Discussion: The protective haplotype is unique, with haplotype changes in DNA methylation and SV that could contribute to the protective mechanism of rs10423769_A. The study generates hypothesis for future studies on this important protective mechanism for APOEε4 carriers.

Highlights: We investigated an African-specific haplotype harboring a protective locus for APOEε4 carriers that significantly reduces the risk for Alzheimer Disease. The protective locus lies in an area of segmental duplications 2MB from APOE. We found that the protective haplotype is unique within the segmental duplications. The haplotype is found in all African ancestry tested but not in other ancestries. Long read whole genome sequencing identified an expanded VNTR associated with the protective haplotype containing multiple MEF2 transcription factor binding motifs. Differences in DNA methylation were found between the protective and the reference haplotype.

Keywords: Alzheimer's disease; genetic ancestry; haplotype; long‐read sequencing; methylation; protective variant; structural variation.

FIGURE 1

University of California at Santa Cruz (UCSC) Genome Browser view of surrounding region of rs10423769_A allele (marked by vertical red line). The annotation of segmental duplication includes all non‐allelic intrachromosomal and interchromosomal alignments greater than 1 kb and with more than 90% of sequence identity, excluding common repeats or satellite sequences

FIGURE 2

Haplotype blocks identified in 1962 individuals carrying at least one rs10423769_A allele using Haploview 4.2 and Alzheimer's Disease Sequencing Project (ADSP). Lines between blocks indicate co‐occurrence of adjacent haplotypes in individuals with line thickness representing frequency of co‐occurrence across individuals. Haplotype block frequencies are shown in the right of each block (≥ 0.05). Multiallelic D' is shown on the bottom of crossing areas, which represents the level of recombination between blocks. Blocks with D’ > 0.8 were considered the same haplotype. Rs10423769 is marked by an orange box. The 21 kb (chr19:43099521‐43120243) minimum shared haplotype is marked by a red box and the 11 kb extended haplotype (chr19:43121359‐ 43132912) is marked by blue boxes

FIGURE 3

Population‐specific frequency of the 21 kb minimum shared haplotype harboring the rs10423769_A allele and the 11 kb extended haplotype in the 1000G. Linkage disequilibrium (LD) pruned haplotype across nine blocks determined by Haploview are shown. Frequencies were calculated with the tool LDhap from LDlinkR. ASW, African Ancestry in SW USA; ACB, African Caribbean in Barbados; ESN, Esan in Nigeria; GWD, Gambian in Western Division; LWK, Luhya in Webuye – Kenya; MSL, Mende in Sierra Leone; YRI, Yoruba in Ibadan – Nigeria; PUR, Puerto Rican in Puerto Rico

FIGURE 4

Read the coverage and map of the 21 kb minimum shared haplotype from two representative individuals homozygous for either the rs10423769_A or rs10423769_G alleles. The green star marker represents the rs10423769_A allele, and all other listed markers are in LD > 0.95 with rs10423769_A. Colored lines represent IGV consensus SNPs: A, green; C, blue; T, red; G, orange. IGV, Integrative Genomics Viewer; LD, linkage disequilibrium; SNP, single‐nucleotide polymorphism

FIGURE 5

VNTR length correlation with rs10423769 haplotype (p‐value = 2.9e‐10). The local assembly of the tandem repeat region (chr19:43,131,850‐43,132,621, 772 bp) was done with TREAT and the correlation is based on the length obtained for rs10423769_A versus rs10423769_G haplotypes in 38 individuals submitted to long‐read sequencing. VNTR, variable number tandem repeat

FIGURE 6

Motif analysis of the expanded VNTR allele associated with rs10423769_A. (A) 29 bp Repetitive sequence identified by MEME Suite. (B) MEF2 family of TF binding motifs with FIMO motif finding tool q‐value., VNTR, variable number tandem repeat

FIGURE 7

Allele‐specific differential methylation analysis of the rs10423769_A versus rs10423769_G haplotypes in the 21 kb minimum shared haplotype and surrounding region using five brain samples heterozygous for rs10423769. The dotted line indicates FDR < 0.01. Methylation differences refer to the difference in mean methylation levels between the rs10423769_A and rs10423769_G haplotypes and is shown for DMP with FDR < 0.01. DMP, differentially methylated positions; FDR, false discovery rate