The genotype at polymorphism rs11200638 is not correlated with the HTRA1 expression level in human retina. Error bars indicate standard error.
The genotype at polymorphism rs11200638 is not correlated with the HTRA1 expression level in human peripheral blood cells (A), and age-related macular degeneration (AMD)–affected status is not correlated with HTRA1 expression (B). Error bars indicate standard error.
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Wang G, Scott WK, Haines JL, Pericak-Vance MA. Genotype at Polymorphism rs11200638 and HTRA1 Expression Level. Arch Ophthalmol. 2010;128(11):1491–1493. doi:10.1001/archophthalmol.2010.256
Genetic factors strongly contribute to age-related macular degeneration (AMD). The loci at chromosomes 1q32 and 10q26 have been repeatedly and consistently linked to the disease. Compared with the successful discovery of the first AMD susceptibility gene CFH at the chromosome 1q32 locus, it has been difficult to identify with certainty the susceptibility variation(s) responsible for linkage and association at the chromosome 10q26 locus. One major reason for the inconclusive findings is that the polymorphisms in genes ARMS2 and HTRA1 are in such strong linkage disequilibrium that their effects are indistinguishable using statistical analysis. HTRA1 was proposed as the susceptibility gene, partly based on the result that the risk allele (A) of the polymorphism rs11200638, which is strongly associated with AMD, was reportedly correlated with a higher level of HTRA1 in lymphocytes and retinal pigment epithelium.1,2 However, this result remains controversial.3,4 We therefore conducted this study to test whether the genotype at rs11200638 is correlated with HTRA1 expression in peripheral blood and retina.
Retinal tissues (including retina, retinal pigment epithelium, and choroid) were punched from the macula of fresh frozen eyes retrieved within 24 hours of death from 24 unrelated white subjects (mean [SD] age, 76.4 [14.5] years) without any known eye diseases, and RNA was extracted with the RNeasy lipid tissue kit (Qiagen Inc, Valencia, California). The RNA was also extracted from whole blood samples of 52 white subjects including 46 cases (mean [SD] age, 79.0 [7.5] years; 50% female) and 6 controls (mean [SD] age, 72.7 [10.5] years; 67% female) using the PAXgene Blood RNA System Kit (PreAnalytiX, Venlo, the Netherlands). Procedures for recruitment, requests for medical records, and consent forms were approved by the University of Miami, Miller School of Medicine Institutional Review Board. Quantitative polymerase chain reaction was performed using an ABI HP7900 (Applied Biosystems, Carlsbad, California). Each sample was repeated 3 times at different locations in the plate. After the polymerase chain reaction run was complete, quantitative gene expression data were acquired and analyzed using the ABI Prism 7900HT Sequence Detection System (RQ manager) (Applied Biosystems). The overall experiment was repeated twice. The t test was applied to compare the difference in the HTRA1 level between the genotypes at rs11200638.
The genotype at rs11200638 of all eye tissue samples was obtained by applying the Taqman assay (Applied Biosystems). Quantitative polymerase chain reaction showed no significant difference in the retinal HTRA1 messenger RNA (mRNA) level between 17 GG samples and 7 GA samples (P = .38) (Figure 1). To compensate for the lack of homozygous risk allele A and the lack of AMD-affected samples, we applied quantitative polymerase chain reaction on RNA samples extracted from peripheral white blood cells. The RNA was extracted from 52 samples that included 26 GG genotypes, 6 AA genotypes, and 20 heterozygous GA samples at rs11200638. No significant difference in the HTRA1 mRNA level was found between the different genotypes at rs11200638 (P = .28) (Figure 2A). No significant difference of the HTRA1 mRNA level was detected between AMD-affected samples and controls either (P = .35) (Figure 2B).
Polymorphism rs11200638 is located in a predicted HTRA1 promoter region. The risk allele of rs11200638 reportedly correlates with a higher level of HTRA1 mRNA and protein in retina.1,5,6 Paradoxically, data have shown that the genotype at rs11200638 is not correlated with the HTRA1 expression level in human retina and white blood cells,3,4 leading to ongoing controversy. Here we further confirm that there are no effects of rs11200638 genotype on HTRA1 expression. We propose that rs11200638 is not likely a functional variant underlying genetic association with AMD, suggesting that potential functional variants such as rs10490924 (Ala69Ser change) in ARMS2 should be the focus of further experiments regarding the susceptibility gene for AMD.
Correspondence: Dr Wang, Dr John T. Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, 1501 NW 10th Ave, Miami, FL 33136 (firstname.lastname@example.org).
Financial Disclosure: Drs Scott, Haines, and Pericak-Vance own a patent licensed by ArcticDx for the use of genetics in AMD diagnosis.
Funding/Support: This research was supported by grant EY12118 from the National Institutes of Health (Drs Haines and Pericak-Vance).
Additional Information: A subset of the participants was ascertained while Dr Pericak-Vance was a faculty member at Duke University, Durham, North Carolina.
Additional Contributions: We thank all the patients, their families, and the controls who participated in the study.