Pathogenicity discrimination and genetic test reference for CRX variants based on genotype-phenotype analysis

The cone-rod homeobox (CRX) gene is specifically expressed in developing and mature photoreceptors and is relatively conserved, with limited polymorphisms in coding regions. Rare variants in CRX are usually considered causative for different forms of retinal degeneration, but this might be problematic based on recent data. This study aimed to classify CRX variants based on a genotype-phenotype analysis of our data and the literature. Twenty-four CRX variants, including 14 novel variants, were detected in 37 Chinese families based on exome sequencing data obtained from 4971 Chinese probands with different forms of eye diseases. After detailed phenotypic analysis and cosegregation analysis in families with CRX variants, the 24 variants could be classified into three groups: benign (six), likely benign (six), and pathogenic (12). Somatic mosaicism was identified in a family with unaffected parents (the father had a mutant allele that was detected in approximately 17% of his leukocyte DNA) and two affected sons. Furthermore, a thorough reassessment was systematically performed for all 113 heterozygous variants as well as for their associated phenotypes from our cohort and patients previously reported. Two critical findings on the pathogenicity of CRX variants were obtained based on the genotype-phenotype correlation, family segregation and ensemble predicting methods: 1) approximately half of heterozygous missense variants are likely benign, and 2) heterozygous truncating variants affecting the homeodomain are likely benign. Truncating mutations after the homeodomain are likely associated with a more severe phenotype. Although most heterozygous pathogenic variants in CRX are associated with autosomal dominant retinal degeneration, a homozygous c.268C> T (p.Arg90Trp) substitution and homozygous complete deletion of CRX have been reported to cause Leber congenital amaurosis. In conclusion, many rare missense variants and some truncating variants in CRX are likely benign, although previously, they might have been predicted to be damaging by some online tools. Evaluation of the pathogenicity of a CRX variant should consider both its nature and location. The information obtained in this study is critical in the era of routine clinical genetic test, not only for CRX but also for many other genes with many more variants. Functional studies and additional genotype-phenotype analyses are expected to confirm these associations.