Exome Sequencing In Multiplex AMD Families

Abstract

Purpose: : A substantial portion of the genetic risk for age-related macular degeneration (AMD) can be attributed to alterations in either the CFH or the ARMS2 locus. However, some individuals still develop severe phenotypes (neovascular AMD) despite being negative for the associated risk variants in CFH and ARMS2. The purpose of this study is to use next generation sequencing to identify variants contributing to disease in patients without the strongest genetic risk factors.

Methods: : From our data set of over 2,000 subjects, we screened all individuals with neovascular AMD; smoking status was assessed and they were all genotyped for the main known genetic risk factors CFH, ARMS2/HTRA1, C3, CFB/CC2. We selected 15 white non-Hispanic siblings (12 cases and 3 controls) in 4 multiplex families based on having in the family affected individuals with bilateral neovascular AMD without CFH402H and ARMS269S risk alleles. Affected individuals developed severe AMD earlier in life with a mean age of 70.9 at recruitment. Exome capture was performed using Agilent SureSelect and sequenced on the Illumina HiSeq 2000 platform. Base calling, sequence alignment, variant calling and filtering used Illumina, MAQ and Seattle Seq programs as well as an in-house bioinformatics pipeline. Allele sharing among affected individuals from each family was used to determine possible disease associated variants; candidate alleles were also evaluated in unaffected relatives. Variants were filtered to eliminate common and previously reported variants (using dbSNP and Hapmap databases) assuming these multiplex families have novel and possibly very family-specific variants in association to AMD.

Results: : By focusing on families with multiple affected individuals with severe AMD without the strongest known genetic risk factors, we identified the most potentially informative individuals to screen for novel variants using exome sequencing analysis. Evaluating allele sharing among affected individuals within these 4 multiplex families facilitated the discovery of rare variants not previously associated with AMD that may potentially have an influence on disease pathogenesis.

Conclusions: : These results will contribute to our understanding of the remaining genetic factors contributing to AMD.

Keywords: age-related macular degeneration • genetics • gene screening