Fig. 3

Mutation analysis in Family B

The nuclear family, shown on the top of Frame A, consists of unaffected, related parents who have an older unaffected son, an affected daughter, and a younger daughter (*) who was predicted by prenatal testing to be clinically unaffected. (A) Heteroduplex analysis of the family members revealed a band with altered mobility in Lanes 1, 2, 4, and 5, compared with normal, unrelated controls (C). The previously affected child (Lane 3) did not show a band of altered mobility due to homozygosity for the mutation; however, when the PCR product from the patient was mixed with a control product (3 + C), a shift similar to those seen in the parents and two other unaffected siblings was noted. (B) Sequencing of the mutant allele, in comparison to normal allele, revealed a C-to-T transition which resulted in substitution of a glutamine codon (CAG) by a premature termination codon (TAG). (C) The presence of the mutation, Q251X, was verified at the genomic level by the loss of an RsaI site. Digestion of PCR-amplified genomic DNA revealed the presence of two bands, 211 and 110 bp (as well as a 46 bp band which is not shown) in normal control individuals (C). The proband (Lane 3) was shown to be homozygous for the loss of the restriction enzyme site, while individuals shown in Lanes 1, 2, 4, and 5 were heterozygous for the mutation.