Development of a Genotyping Assay for Age-Related Macular Degeneration: The EYE-RISK Consortium

Abstract

Purpose : Current genetic tests for age-related macular degeneration (AMD) are limited to a low number of genetic variants and vary widely in their risk assessment capacity. We aimed to develop an AMD genotyping assay that covers all currently known genetic variants and that can detect novel rare coding variants in AMD-related and AMD-mimicking genes, in order to establish genetic risk scores for AMD, to assess associations of genetic variants with AMD, and to establish a validated genotyping assay for AMD.

Methods : We developed a genotyping assay based on single molecule molecular inversion probes (smMIPs) and next generation sequencing (NGS) covering 87 single nucleotide polymorphisms (SNPs), and the coding and flanking regions of 10 AMD-related genes (ARMS2, C3, C9, CD46, CFB, CFH, CFI, HTRA1, TIMP3, SLC16A8) and 3 AMD-mimicking genes (ABCA4, CTNNA1, PRPH2). 4,718 DNA samples from five different European cohorts (EUGENDA, CES, MARS, CORRBI, GAIN) were available for genotyping. Reads were mapped using BWA and variant calling was performed by GATK and SAMtools. Quality control steps were applied to ensure high quality genotyping data.

Results : In total we genotyped 4,718 samples of 2,428 AMD patients and 1,297 control individuals. Seventy SNPs were successfully genotyped, while seventeen SNPs were discarded due to coverage or mapping problems, deviation from Hardy-Weinberg expectations (P < 10^-4), low genotype concordance with other genotyping platforms (< 95%) or a high proportion of missing genotypes per SNP (>10%). For the 70 successfully genotyped SNPs we observed a high concordance rate between our platform and other genotyping platforms (96.02% - 99.96%). Allele frequencies of the SNPs and odds ratios for association with AMD were similar to previously reported data. Sequencing of the coding and flanking regions of 10 AMD-related and 3 AMD-mimicking genes identified 2,830 different rare coding or splice site variants, which will be further analyzed for their involvement in AMD by single variant association tests, gene burden tests, and genotype-phenotype correlations.

Conclusions : We developed a comprehensive AMD genotyping assay which successfully genotyped 70 SNPs and the coding regions of 13 AMD-related and AMD-mimicking genes. After further optimization this assay will be part of an AMD prediction website for risk assessment of AMD development and AMD progression.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.