Missense mutations in the most ancient residues of the PAX6 paired domain underlie a spectrum of human congenital eye malformations

Mutations of the human PAX6gene underlie aniridia (congenital absence of th e iris), a rare dominant malformation of the eye, The spectrum of PAX6 muta tions in aniridia patients is highly biased, with 92% of all reported mutat ions leading to premature truncation of the protein (nonsense, splicing, in sertions and deletions) and just 2% leading to substitution of one amino ac id by another (missense). The extraordinary conservation of the PAX6 protei n at the amino acid level amongst vertebrates predicts that pathological mi ssense mutations should in fact be common even though they are hardly ever seen in aniridia patients. This indicates that there is a heavy ascertainme nt bias in the selection of patients for PAX6 mutation analysis and that th e 'missing' PAX6 missense mutations frequently may underlie phenotypes dist inct from textbook aniridia. Here we present four novel PAX6 missense mutat ions, two in association with atypical phenotypes: ectopia pupillae (displa ced pupils) and congenital nystagmus (searching gaze), and two in associati on with more recognizable aniridia phenotypes. Strikingly, all four mutatio ns are located within the PAX6 paired domain and affect amino acids which a re highly conserved in all known paired domain proteins. Our results suppor t the hypothesis that the under-representation of missense mutations is cau sed by ascertainment bias and suggest that a substantial burden of PAX6-rel ated disease remains to be uncovered.