A variant of mitochondrial protein LOC387715/ARMS2, not HTRA1, is strongly associated with age-related macular degeneration

Abstract

Genetic variants at chromosomes 1q31-32 and 10q26 are strongly associated with susceptibility to age-related macular degeneration (AMD), a common blinding disease of the elderly. We demonstrate, by evaluating 45 tag SNPs spanning HTRA1, PLEKHA1, and predicted gene LOC387715/ARMS2, that rs10490924 SNP alone, or a variant in strong linkage disequilibrium, can explain the bulk of association between the 10q26 chromosomal region and AMD. A previously suggested causal SNP, rs11200638, and other examined SNPs in the region are only indirectly associated with the disease. Contrary to previous reports, we show that rs11200638 SNP has no significant impact on HTRA1 promoter activity in three different cell lines, and HTRA1 mRNA expression exhibits no significant change between control and AMD retinas. However, SNP rs10490924 shows the strongest association with AMD (P = 5.3 x 10(-30)), revealing an estimated relative risk of 2.66 for GT heterozygotes and 7.05 for TT homozygotes. The rs10490924 SNP results in nonsynonymous A69S alteration in the predicted protein LOC387715/ARMS2, which has a highly conserved ortholog in chimpanzee, but not in other vertebrate sequences. We demonstrate that LOC387715/ARMS2 mRNA is detected in the human retina and various cell lines and encodes a 12-kDa protein, which localizes to the mitochondrial outer membrane when expressed in mammalian cells. We propose that rs10490924 represents a major susceptibility variant for AMD at 10q26. A likely biological mechanism is that the A69S change in the LOC387715/ARMS2 protein affects its presumptive function in mitochondria.

Fig. 1.

Association analysis of the 10q26 chromosomal region. Shown are P values for single SNP association tests comparing unrelated cases and controls. The genes in the indicated region are PLEKHA1, LOC387715/ARMS2, HTRA1, and DMBT1. rs10490924, the SNP showing strongest association in the region, is colored in red. Markers in strong association are colored in blue (r² > 0.5) or green (r² > 0.3).

Fig. 2.

Graphical overview of linkage disequilibrium (46) among 45 SNPs. The plot summarizes the linkage disequilbrium (D′) between all pairs of SNPs in the region. Pairs of SNPs showing strong linkage disequilibrium (≈0.70 or greater) are colored in red and orange. Intermediate levels of disequilibrium (≈0.30–0.70) are colored in green, and lower levels of disequilibrium are colored in blue.

Fig. 3.

Analysis of the HTRA1 promoter region and the previously reported AMD-associated SNP rs11200638. (A) Schematic representation of the human and mouse HTRA1 upstream promoter region and of luciferase reporter constructs used in the transactivation assays. The gray boxes indicate the genomic regions conserved between human and mouse, and the arrow indicates the position of rs11200638 SNP. HTRA1 promoter fragments (L-3.7 kb, M-0.83 kb, and S-0.48 kb) were cloned into pGL3-basic plasmid with the luciferase reporter gene. (B) Three different lengths of HTRA1 WT promoter-luciferase constructs [WT-long (WT-L), WT-medium (WT-M), and WT-short (WT-S)] and two mutant constructs [SNP-long (SNP-L) and SNP-medium (SNP-M)] were transfected into HEK293 cells. Promoterless vector pGL3 was used as a negative control, and the value of luciferase activity was set to 1. (C and D) The same as B, except that ARPE-19 or Y79 cells were transfected with the promoter constructs. (E) Sequence comparison between human and mouse HTRA1 upstream promoter region spanning rs11200638 (gray box) using rVISTA (47). Predicted transcription factor binding sites are shown. The bold line indicates the oligonucleotide that was used as a probe for EMSA. (F) EMSA for rs11200638-spanning region. The ³²P-labeled WT (lanes 1–6 and 10) or SNP (lanes 7–8) oligonucleotide probe was incubated with bovine retina nuclear extracts (BRNE). Competition experiments were performed with the unlabeled 50X specific (lane 3) or 50X nonspecific (lane 4) oligonucleotide to validate the specificity of the band shift. EMSA experiments were also performed in the presence of the antibody against activating enhancer-binding protein-2α (AP-2α) (lanes 5 and 8), stimulating protein 1 (SP-1) (lanes 6 and 9), and neural retina leucine zipper protein (NRL) (lane 10). NRL antibody represents a negative control here. The arrow shows the position of a specific DNA–protein binding complex.

Fig. 4.

Amino acid sequence and expression of the LOC387715/ARMS2 protein. (A) Amino acid sequence alignment and secondary structure analysis. Line 1, amino acid sequence of the predicted human LOC387715/ARMS2 protein; line 2, chimpanzee LOC387715/ARMS2 sequence; line 3, WT LOC387715/ARMS2 secondary structure prediction (H, helix; E, strand; C, the rest); line 4, secondary structure of LOC387715/ARMS2 altered by the A69S variation (dot, same as WT). The gray box shows Ala codon 69 that is altered by the SNP rs10490924. (B) RT-PCR analysis of LOC387715/ARMS2 transcripts in cultured cell lines and in the retina of control and AMD subjects. HPRT was used as a control to evaluate RNA quality and to normalize for the quantity. All PCR products were confirmed by sequencing. (C) Immunoblot analysis of COS-1 whole-cell extracts expressing human LOC387715/ARMS2 protein with N-terminal Xpress-tag. The expressed LOC387715/ARMS2 protein was detected using anti-LOC387715/ARMS2 (anti-LOC) or anti-Xpress (anti-Xp) antibody. (D) Fractionation of COS-1 cell extracts expressing LOC387715/ARMS2. Un+Nu, unbroken cells and nuclear fraction; Mt, mitochondria fraction; Sol, soluble fraction. (E) Proteinase K (ProK) treatment of the mitochondria. The mitochondrial fractions from transfected COS-1 were treated with increasing concentrations of Proteinase K. The antibodies used for immunoblot analysis are indicated.

Fig. 5.

Subcellular localization of the LOC387715/ARMS2 protein. Human LOC387715/ARMS2 cDNA was cloned in pcDNA4 vector and transiently expressed in COS-1 cells. The cells were stained with anti-Xpress (green) and an organelle-specific marker (red) as follows. (A and B) MitoTracker (A) and anti-COX IV antibody (B) were used for mitochondria. (C) Anti-PDI antibody was used for endoplasmic reticulum. (D) Anti-Giantin antibody was used for Golgi. (E) LysoTracker was used for for lysosome. Bisbenzimide was used to stain the nuclei (blue). (Scale bar, 25 μm.)