Family study and rearrangement specific PCR design. (A) Family tree. The index case is indicated by ↖. Wt indicates wild-type allele; HFEdel, HFE knocked-down allele. (B) Rearrangement-specific PCR of the patient and her relatives. Primers were designed to amplify across the junctions and give a PCR product of 499 bp (ReaSpe_PCR). Also, a PCR was designed to amplify the wild-type sequence; this targets the HFE exon 2 (HFE_Ex2) and produces a 377 bp DNA fragment. Primer pairs were mixed to create a duplex PCR assay.