Evaluation of LOXL1 polymorphisms in exfoliation syndrome in a Chinese population

Purpose: To evaluate the association profiles of the lysyl oxidase-like 1 (LOXL1) gene polymorphisms with exfoliation syndrome in a Chinese population. Methods: Fifty unrelated patients with exfoliation syndrome and 125 control subjects were included. Genotypes of the three single nucleotide polymorphisms (SNPs) of LOXL1 (rs1048661, rs3825942, and rs2165241) were analyzed by direct sequencing, and a case-control association study was performed. Results: The three SNPs were significantly associated with exfoliation syndrome (XFS) and exfoliation glaucoma (XFG) individually. After controlling for rs3825942 and rs2165241, the association between rs1048661 and XFS/XFG remained significant (p=3.6x10(-7)). At this SNP, the T allele and TT genotype conferred a 7.59-(95% confidence interval [CI]: 3.87-14.89, p=6.95x10(-11)) and 8.69-(95% CI: 4.15-18.20, p<1.00x10(-7)) fold increased risk to the disease. The alleles of T at rs1048661 and C at rs2165241 were found to be risk alleles in Chinese subjects, which were opposite to Caucasian individuals. The haplotypes T-G, defined by SNPs rs1048661 and rs3825942, and T-C by SNPs rs1048661 and rs2165241, were also significantly associated with the disorder. However when the genotypic or allelic frequencies of the three SNPs were compared between XFS and XFG, no significant difference was detected. Conclusions: LOXL1 is a susceptibility gene of XFS/XFG in the Chinese population, and the association is mainly attributed to SNP rs1048661. The risk alleles of rs1048661 and rs2165241 in Chinese subjects were found to be opposite to that of Caucasians. The genotypic and allelic distributions of these SNPs are similar between XFS and XFG.Biochemistry & Molecular BiologyOphthalmologySCI(E)30ARTICLE250-522349-23571

Genetic and functional dissection of ARMS2 in age-related macular degeneration and polypoidal choroidal vasculopathy.

Age-related maculopathy susceptibility 2(ARMS2) was suggested to be associated with neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) in multiple genetic studies in Caucasians and Japanese. To date, no biological properties have been attributed to the putative protein in nAMD and PCV. The complete genes of ARMS2 and HTRA1 including all exons and the promoter region were assessed using direct sequencing technology in 284 unrelated mainland northern Chinese individuals: 96 nAMD patients, 92 PCV patients and 96 controls. Significant associations with both nAMD and PCV were observed in 2 polymorphisms of ARMS2 and HTRA1 rs11200638, with different genotypic distributions between nAMD and PCV (p<0.001). After adjusting for rs11200638, ARMS2 rs10490924 remained significantly associated with nAMD and PCV (p<0.001). Then we overexpressed wild-type ARMS2 and ARMS2 A69S mutation (rs10490924) in RF/6A cells and RPE cells as in vitro study model. Cell proliferation, attachment, migration and tube formation were analyzed for the first time. Compare with wild-type ARMS2, A69S mutation resulted in a significant increase in proliferation and attachment but inhibited cell migration. Moreover, neither wild-type ARMS2 nor A69S mutation affected tube formation of RF/6A cells. There is a strong and consistent association of the ARMS2/HTRA1 locus with both nAMD and PCV, suggesting the two disorders share, at least partially, similar molecular mechanisms. Neither wild-type ARMS2 nor A69S mutation had direct association with neovascularisation in the pathogenesis of AMD

Logistic regression analysis of SNPs in ARMS2 and HTRA1 between AMD and PCV.

<p>P value and OR (95%CI) of rs10490924 and rs11200638 after adjusting for the following SNPs.</p

Characteristics of the study population.

<p>Characteristics of the study population.</p

Summary of flow cytomery data of apoSptosis measured by Annexin V and PI.

<p>Summary of flow cytomery data of apoSptosis measured by Annexin V and PI.</p

Effect of wild-type ARMS2 and rs10490924 on the apoptosis of human RPE cells.

<p>Apoptosis was quantified by flow cytometry measured by Annexin V and PI staining. Data are presented as mean±SEM.Each experiment was repeated at least three independent times. DMEM+10%FBS control was set to 100%.*P<0.05. UR: late apoptotic cells; LR: early apoptotic cells, UR+LR: apoptotic cells. Abbreviations: wild-type ARMS2 plasmid-treated cells (WT); rs10490924 plasmid -treated cells (G270T); pReceiver-M29-Basic plasmid-treated cells (Vector).</p

Effect of wild-type ARMS2 and rs10490924 on the tube formation of RF/6A cells.

<p>PReceiver-M29-Basic plasmid-treated RF/6A cells (A), wild-type ARMS2 plasmid-treated cells RF/6A cells (B) and rs10490924 plasmid -treated RF/6A cells (C) were plated on Matrigel as described in Methods. After 24 h of incubation, the three groups cells formed well organized capillary-like structures. Values are the means±SD of at least three independent experiments. There are no significant differenence during the three groups (D, p>0.05). Abbreviations: wild-type ARMS2 plasmid-treated cells (WT); rs10490924 plasmid -treated cells (G270T); pReceiver-M29-Basic plasmid-treated cells (Vector).</p

Rs10490924 (A69S in a coding change) does not affect mRNA, protein, or surface expression of ARMS2.

<p>A)Direct sequencing verified the wild-type (WT) and G270T ARMS2 plasmids. <i>B</i>) RT-PCR showed no significant difference between wild-type and G270T ARMS2 mRNA expression. <i>C</i>) Real-time RT-PCR confirmed the mRNA level finding. <i>D</i>) Western blot showed that the levels and migrations of the SNP mutant proteins were comparable with wild type. All experiments were repeated >3 times.</p

Polymorphisms in the <i>ARMS2</i> gene lesion: Distribution and Genotypes in neovascular Age-Related Macular Degeneration (nAMD), Polypoidal Choroidal Vasculopathy, and Controls in the northern Chinese Population.

*<p>: <i>p</i>-Value <0.05 is considered to be statistically significant and they are shown in bold.</p>**<p>: OR(95%CI): Odds ratios are given for the risk allele compared with the wildtype allele.</p

Effect of wild-type ARMS2 and rs10490924 on the proliferation of RF/6A and human RPE cells.

<p>RF/6A (<b>A</b>) and ARPE-19 (<b>B</b>) cell proliferation was measured with an MTT assay at 24 h, 48 h, 72 h, 96 h. Values are the means±SD of at least three independent experiments. Asterisks denote values significantly different from those of cells treated with wild-type ARMS2 and rs10490924 compared to negative control (p<0.01). (<b>C</b>)The time course of the ARMS2 protein expression profile mirrored that for ARMS2 protein expression levels after transfection in ARPE-19 cells. Abbreviations: wild-type ARMS2 plasmid-treated cells (WT); rs10490924 plasmid -treated cells (G270T); pReceiver-M29-Basic plasmid-treated cells (Vector) (*P<0.05, **P<0.01).</p