Several studies have shown previously strong association of multiple single nucleotide polymorphisms (SNPs) in chromosome 10q26 encompassing PLEKHA1
, and HTRA1
with advanced AMD 
. From our previous study 
, we identified a common disease haplotype TAT tagged by rs10490924, rs11200638 and rs2293870 that is significantly associated with a risk of AMD (P
), as well as a haplotype GGG that is modestly, yet significantly, associated with protection from AMD (P
0.003). Next, to define the extent of the haplotype structures and capture all variants in the region, we undertook a re-sequencing effort of a region spanning ~100 kb, starting in the 3′-UTR of PLEKA
and ending ~20kb downstream of the 3′ UTR of HTRA1
in six individuals with a homozygous risk haplotype and six individuals with a homozygous protective haplotype, followed by assessment of association of each discovered variant with AMD in 200 AMD cases and 200 normal controls (, Table S1
, Table S2
Association study for SNPs in 10q26 region in 200 cases and 200 controls in the Utah cohort.
In agreement with recent work 
, we discovered that both risk and protective haplotypes span 21.5kb in the ARMS2
region, starting from upstream of LOC387715
to rs58077526, in intron 1 of HTRA1
(, , Table S1
). We confirmed recent data 
, according to which 22 SNPs tagged a risk and a protective haplotype including previously reported SNPs rs10490924, rs3750848, an in/del, rs3793917, and rs11200638 ( and ). Moreover, the reported 54 base pair insertion and 443 base pair deletion (in/del) at the 3′ end of LOC387715
resides exclusively on the disease risk haplotype that is strongly associated with a risk of AMD (P
) in a Utah case-control cohort (705 cases, 650 controls) ( and ), as well as an independent replication cohort of northern European ancestry (442 cases, 434 controls ) ( and ) and a third replication cohort of Han Chinese in China (138 cases, 591 controls ) ( and ) 
Genes and the main SNPs in the AMD 10q26 region.
Association of rs2736911, rs10490924, in/del/Wt, and rs11200638 in the three independent cohorts.
Genotyping results of rs2736911, rs10490924, in/del/Wt, and rs11200638 in the three independent cohorts.
In contrast to the loss of function role of in/del in LOC387715
, the T allele of SNP rs2736911, a non-synonymous coding SNP leading to a predicted premature stop (R38X) in LOC387715
is associated with a protective haplotype T-G-Wt-G (P
, and and ) defined by rs2736911, rs10490924, in/del/Wt and rs11200638. We replicated these findings twice and found that this haplotype is clearly not associated with AMD risk; rather it is (conservatively) neutral and possibly protective with regard to the disorder (P
0.001 in the second replication cohort; P
0.007 in third Han Chinese cohort, ).
The haplotype block in D' of rs2736911, rs10490924, in/del/Wt, and rs11200638 in the Utah case-control cohort.
Haplotype structures generated by the Hapview program using rs2736911, rs10490924, in/del/Wt, and rs11200638.
These findings present a paradox. Recent studies have shown the in/del to cause destabilization of LOC387715
, suggesting that loss of function at that locus might confer risk to AMD. However, the introduction of the R38X mutation, which is also predicted to give rise to loss of LOC387715
message due to nonsense-mediated mRNA decay (NMD), is mildly protective. We considered two plausible alternatives; either that effect of R38X and the in/del have different effects on the transcript, or that loss of LOC387715
is insufficient to cause AMD. The first possibility is unlikely. Upon quantification of LOC387715
mRNA levels in seven placentas homozygous for the major disease haplotype, we found a 4.7-fold reduction in endogenous LOC387715
expression (), in agreement with recent published data 
. However, we were also able to examine LOC387715
mRNA levels in five patients heterozygous for the R38X mutation, where we observed a 50% reduction (), suggesting that each of the R38X and in/del have the same mode of action.
Endogenous expression studies comparing effects of genotype on HTRA1 and LOC387715.
Given these data, we considered the potential role of HTRA1
, the only other locus encompassed by the major risk haplotype, in the pathogenesis of AMD. We have shown previously a three-fold increase expression of HTRA1
in the retinal pigmented epithelium (RPE) of patients with the risk haplotype 
, while others have also observed increased HTRA1
expression in AMD eyes in humans 
. In addition, a functional SNP in the promoter region of HTRA1
is associated with increased HTRA1
expression in non-human primates with AMD-like phenotype 
. We therefore expanded our analyses of mRNA levels in human placentas to determine the effect of a haplotype on endogenous expression. We found that, in addition to the observed decrease of LOC387715
message, the disease haplotype is also associated with a 2.7-fold increase in HTRA1
expression (). These data confirmed that the in/del or another component of the common risk haplotype affects LOC387715
mRNA stability. At the same time, it likely has an effect on HTRA1 expression. Although caution should be exercised when translating findings from one tissue to another, these data from placenta should serve as an indication for the effects of different haplotypes on the gene expression profiles in the eye.
To test this hypothesis further, we generated expression constructs that model the effect of the risk and protective haplotype on the HTRA1
promoter using a luciferase reporter assay on cultured human retinal pigment epithelial cells (). We observed a two-fold increase in luciferase expression in constructs that modeled the disease haplotype encompassing the in/del and the A allele of SNP rs11200638 (MT(L+in/del), ). We detected no increase in luciferase expression from constructs that contained a short disease haplotype including the A risk allele of SNP rs11200638 but did not contain the in/del (MT(S), ). These findings are consistent with Fritsche et al. and Kanda et al 
, who reported an inability to verify that the A allele of SNP rs11200638 by itself alters transcriptional activity in heterologous cell systems. We did not observe increased luciferase expression either when we placed the in/del or A allele of SNP rs11200638 on a construct containing a protective haplotype, suggesting that the in/del or A allele of SNP rs11200638 by itself is insufficient to drive HTRA1 expression (WT(L+in/del), WT(L-A), ).
Heterologous Luciferase assays in vitro and in vivo.
Because of the potentially ambiguous nature of in vitro
luciferase assays, we repeated these experiments in an in vivo
system, whereby the constructs containing the disease haplotype including the in/del and the protective haplotype described earlier () were electroporated into the RPE of wild-type adult mice. Luciferase activity was assayed four days after transfection. Consistent with the in vitro
data, we observed a significant increase in normalized luciferase activity in a construct bearing the disease haplotype tagged by the in/del and A allele of SNP rs11200638 (P
0.032) (), whereas no increased luciferase activity was observed in a construct with the in/del on a protective haplotype (P