Fig. 6. Mutation in a highly conserved Sema3C immunoglobulin domain. Family 53 pups with PTA are homozygous for a T to C substitution (m/m sequencing trace files, right) in the highly conserved immunoglobulin (Ig) domain of Sema3C, which caused a leucine to proline substitution, and also resulted in the loss of a PstI restriction site and the gain of an AciI site. PCR amplification using primers spanning the Sema3C^L605P mutation, followed by PstI digestion was used to genotype a litter of fetuses obtained from intercrossing two heterozygous Sema3C^L605P mutants (see bottom gel). Three homozygous Sema3C^L605Pmutants showed PTA, whereas one heterozygous fetus had DORV.