Figure 2. Evaluation of two novel CRB1 missense mutations. Multiple alignments using Clustal W and amino acid conservation of three novel missense sequence variants were performed. The alignment results showed that cysteine at codon 1154 and glutamic acid at codon 1403 were fully conserved through all species. The predicted effect of the mutation on the protein was estimated with the online prediction programs SIFT and PolyPhen-2, which are shown at the bottom. For each mutation, a high pathogenicity score for the altered amino acid was obtained.