Disease-causing missense variants generally alter amino-acid residues that are more highly conserved during evolution than polymorphisms. Thus, we ranked the 983 missense variants according to a score that reflects the conservation of each amino acid (see Methods). Scrutiny of the top ranking variants from this analysis highlighted CASK. Only two missense variants in CASK were identified in the primary sequencing screen, and these are positioned second and third in the ranking (Fig. 1c and Supplementary Table 5 online). In silico and RT-PCR analyses indicate that one of these, 2129A>G (D710G), introduces a splice site that removes 27 bp of the coding sequence and thus nine amino acids of the CASK protein. Two further missense variants in CASK were found in a screen of 150 additional families with XLMR, both of which are at highly conserved amino acids and would score second and sixth in the ranking of missense variants. We did not find any missense variants in the complete coding region of CASK in 390 control X chromosomes. Mental retardation was mild to moderate in the four families with missense
