A Candidate Gene Association Study Identifies DAPL1 as a Female-Specific Susceptibility Locus for Age-Related Macular Degeneration (AMD)

Abstract

Age-related macular degeneration (AMD) is the leading cause of blindness among white caucasians over the age of 50 years with a prevalence rate expected to increase markedly with an anticipated increase in the life span of the world population. To further expand our knowledge of the genetic architecture of the disease, we pursued a candidate gene approach assessing 25 genes and a total of 109 variants. Of these, synonymous single nucleotide polymorphism (SNP) rs17810398 located in death-associated protein-like 1 (DAPL1) was found to be associated with AMD in a joint analysis of 3,229 cases and 2,835 controls from five studies [combined PADJ = 1.15 × 10(-6), OR 1.332 (1.187-1.496)]. This association was characterized by a highly significant sex difference (Pdiff = 0.0032) in that it was clearly confined to females with genome-wide significance [PADJ = 2.62 × 10(-8), OR 1.541 (1.324-1.796); males: PADJ = 0.382, OR 1.084 (0.905-1.298)]. By targeted resequencing of risk and non-risk associated haplotypes in the DAPL1 locus, we identified additional potentially functional risk variants, namely a common 897-bp deletion and a SNP predicted to affect a putative binding site of an exonic splicing enhancer. We show that the risk haplotype correlates with a reduced retinal transcript level of two, less frequent, non-canonical DAPL1 isoforms. DAPL1 plays a role in epithelial differentiation and may be involved in apoptotic processes thereby suggesting a possible novel pathway in AMD pathogenesis.

Fig. 1

Association with AMD of imputed and typed variants at the DAPL1 locus. Association signals of markers are shown by the log-P value from a logistic regression model (additive model adjusted for age and sex; y-axis) and are plotted against their physical position (x-axis). Stage 1 results (GER1 sample) are marked by filled (genotyped) and open triangles (imputed). Association signals of rs17810398 and rs17810816 in the pooled samples (3,229 cases and 2,835 controls) are indicated by blue and green diamonds, respectively

Fig. 2

Subgroup analysis in the combined study of candidate SNPs rs17810398 and rs17810816 in the DAPL1 gene. OR and corresponding 95 % confidence intervals are given with the size of each rectangles representing the respective number of cases. AMD phenotypic subgroups comprise patients with geographic atrophy (GA), and neovascular AMD (NV) and both late stage forms (GA&NV)

Fig. 3

Functional consequences for isoform expression of DAPL1 variants. a Exon/intron structure of four frequent DAPL1 isoforms (gene orientation is from left to right). SNP positions are marked by vertical dotted lines. b Frequency of the four isoforms as determined from sequencing 1,200 cDNA clones that were obtained after 3′-RACE of four unrelated RPE/retina tissue samples either homozygous (ID_16, ID_17) or heterozygous (ID_13, ID_14) for the non-risk alleles of rs17810398 and rs17810816. c Distribution of rs17810398 alleles in heterozygous RPE/retina tissue samples ID_13 and ID_14. Statistically significant deviations from the reference transcript (i.e., isoform 1) are indicated by asterisks (P < 0.0001)

Fig. 4

Semi-quantitative cDNA sequencing of eight RPE/retina tissue samples heterozygous for synonymous coding SNP rs17810398:C>T. The chromatograms of the variant nucleotide at rs17810398 flanked by ±3 bp are shown for isoforms 1 & 2, 3, and 4. Isoforms were specifically amplified by three different exon-spanning primer combinations. Genotypes of the four candidate variants are given above the chromatograms. The sample heterozygous for rs17810398 but homozygous for the non-risk alleles of rs6416986, rs17810816, and rs144087548 is highlighted in red