Whole-exome sequencing prioritizes candidate genes for hereditary cataract in the Emory mouse mutant

**Background** The Emory cataract (Em) mouse mutant, first described in 1981, spontaneously developed autosomal dominant lens opacities at 6–8 months of age and has long been proposed as an animal model for human age-related (senile) cataract. Despite extensive biochemical and histological characterization—including increased protein insolubility, calcium accumulation, and proteolytic cleavage of lens membrane proteins—the underlying genetic defect remained unknown. Over 450 genes are known to cause inherited or age-related cataract in humans and mice, but none had been linked to Em. This study aimed to identify the causative mutation(s) in commercially available Em/J mice using whole-exome sequencing.

**Methods** Em/J (homozygous for the Em mutation) and ancestral Carworth Farms White (CFW) mice were obtained from The Jackson Laboratory and Charles River Laboratories, respectively. Lens phenotype was confirmed by slit-lamp examination, dark-field and bright-field microscopy, and spectral-domain optical coherence tomography. Genomic DNA from spleen was subjected to whole-exome capture (SureSelect Mouse All Exon, 50 Mb) and next-generation sequencing (Illumina HiSeq 2000, paired-end 2×101 bp). Reads were aligned to the mouse reference genome (mm10) using NovoalignMPI, and variants were called with SAMtools and Picard. Variants were filtered against RefSeq genes 59 and dbSNP 146, focusing on missense and loss-of-function variants in over 450 known cataract/lens disorder genes (Cat-Map database). Novel variants were validated by PCR-Sanger sequencing or allele-specific PCR. Functional impact was predicted using SIFT (score <0.05 damaging) and PROVEAN (score <−2.5 deleterious).

**Key Results** Em/J mice developed bilateral cortical cataract by 7 months of age, with occasional pupil displacement and misshapen lenses also observed in CFW mice. F1 progeny from an Em/J × C57BL/6J intercross failed to develop cataract by 9–12 months, suggesting reduced penetrance or later onset in heterozygotes. Exome sequencing achieved >86% mapped reads and >98% of exons at ≥10× coverage. After filtering, 43 novel variants in 26 Cat-Map genes were identified. Validation confirmed three novel homozygous variants in Em/J that were absent in CFW and >35 other mouse strains: a missense substitution in Prx (c.499C>T, p.R167C; SIFT score 0.05, PROVEAN −2.31), a missense substitution in Adamts10 (c.2282C>T, p.P761L; SIFT 0.33, PROVEAN 0.96), and a disruptive in-frame deletion/insertion in Abhd12 (c.88_94delCTGGACGinsT, p.L30_A32delinsS; PROVEAN −20.74). The Abhd12 variant was predicted to be strongly damaging, while Prx was borderline and Adamts10 neutral. All three genes are expressed in mouse lens at higher levels than in adjacent ocular tissues.

**Clinical Implications** This study prioritizes Abhd12 as the most promising candidate gene for the Em cataract phenotype. ABHD12 mutations in humans cause PHARC syndrome (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, cataract), an autosomal recessive disorder. The Em/J mouse may therefore serve as a model for the cataract component of PHARC syndrome. PRX and ADAMTS10 are also associated with human cataract (PRX variants of uncertain significance; ADAMTS10 mutations cause Weill-Marchesani syndrome 1, which includes cataract in ~23% of cases), but their predicted functional impacts were less severe. The inability to map Em to a chromosome due to delayed onset/reduced penetrance and the possibility of autosomal recessive inheritance in mice are important caveats. Future gene-editing studies (e.g., CRISPR-Cas9) are needed to confirm whether Abhd12, Prx, or Adamts10 is causative. If excluded, the exome data may help identify a novel cataract gene.