Restriction analysis of the proband and family members. (A) The presence of the -206C>G mutation was detected by mismatch PCR and restriction analysis with DdeI as described in “Patients and methods.” The PCR product in the presence of the mutation had an additional DdeI restriction site. Lanes 1, 2, and 3 showed the restriction fragments from healthy individuals (128, 75, 66, and 58 bp). Lane 4 shows the restricted PCR products of the heterozygous proband (128, 105, 75, 66, 58, and 23 bp), while lane 5 shows the restriction fragments from her hemizygous son (105, 75, 66, 58, and 23 bp). The 23-bp fragment is not resolved on this gel. (B) Lanes 1 and 5 show a normal pattern for Sau3AI digestion of the PCR-amplified region of the His63Asp mutation of the HFE gene (138 and 70 bp; the weak 208 bp band is due to incomplete digestion). Lanes 2 to 4 demonstrate that 3 family members are heterozygous for the His63Asp mutation (208, 138, and 70 bp).