Computational protocol: Association of Matrix Metalloproteinase 9 (MMP9) Variants with Primary Angle Closure and Primary Angle Closure Glaucoma

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Protocol publication

[…] We selected six tag SNPs that captured >95% of alleles at r2 greater than 0.8 across the MMP9 genomic region, including all exons, introns, the 5’- and 3’-UTR, and the 5-kb proximal promoter region (). Tag SNPs were selected according to the HapMap CHB+JPT data (version 2, release 21) using Haploview (version 4.2) []. The minimum minor allele frequency for checking SNPs was set to 0.1. Genotyping was performed by TaqMan assays (Applied Biosystems [ABI], Foster City, CA) according to the manufacturer’s instructions. [...] The analysis of CCT, ACD and AXL was performed using the average measurements from both eyes. IOP and VCDR were analyzed using the eye with greater values. Chi-squared test was used to compare the difference in sex between cases and controls. Mann-Whitney U test was used to compare the differences in age, IOP, VCDR, ACD, AXL and CCT between cases and controls.The linkage disequilibrium (LD) plot was generated using Haploview (version 4.2) [], where squared Pearson correlation coefficient (r2) was used to measure LD. Hardy-Weinberg equilibrium was assessed by the chi-squared test. The association analysis was performed using PLINK (version 1.07) [] for overall PAC/PACG, the subgroups (PAC, PACG, acute PAC/PACG and chronic PAC/PACG), and the subphenotypes (age at diagnosis, IOP at diagnosis, maximum IOP, VCDR, ACD, AXL and CCT). Logistic or linear regression was used to adjust for age and sex in the association analysis. Multiple comparisons were corrected for the number of SNPs for each analysis using the Bonferroni method.As described in detail previously [], haplotype analysis was performed with the standard Expectation-Maximization algorithm and the chi-squared test. P values were obtained from the haplotype-specific test and the omnibus test. The odds ratio (OR) and 95% confidence interval (CI) were calculated for each of individual haplotypes.Meta-analysis was performed using the Mantel-Haenszel method, assuming fixed or random effects based on the heterogeneity test results. The heterogeneity between datasets was evaluated using the heterogeneity index (I2) and the Cochran’s Q statistic []. The forest plot was generated using Inkscape (Release 0.91, The Inkscape Team, 2015, based on the output from the Review Manager software (RevMan, version 5.3; Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2014).Power analysis for association of MMP9 SNPs with PAC/PACG was performed using the Genetic Power Calculator []. The disease prevalence was set as 0.4% for the non-Chinese datasets [] and 1.1% for the Chinese datasets []. The risk allele frequency was set to the same as the marker allele frequency, with 0.38 and 0.34 for the non-Chinese and Chinese datasets based on the allele frequencies of rs17576 in the 1000 Genomes Project []. Linkage disequilibrium between the marker and the risk allele was set at D’ = 1.0. The genotypic relative risks for heterozygous (Aa)/high risk homozygous (AA) genotypes were set as 1.78/3.17 and 1.23/1.51 respectively for the non-Chinese and Chinese datasets based on the results of meta-analyses for rs17576, assuming an additive risk model [].Power analysis for association of MMP9 SNPs with subphenotypes in PAC/PACG cases was performed using R scripts [], assuming a linear model. The phenotypic variance explained by additive effects at the marker of interest was set as 1%-5%. […]

Pipeline specifications

Software tools Haploview, PLINK, Inkscape
Applications Miscellaneous, GWAS
Organisms Homo sapiens
Diseases Glaucoma, Angle-Closure