Double-gradient denaturing gradient gel electrophoresis assay for identification of L-ferritin iron-responsive element mutations responsible for hereditary hyperferritinemia-cataract syndrome: Identification of the new mutation C14G

Background: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date wer e identified by direct sequencing of DNA from probands with hyperferritinem ia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferri tin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis. Methods: The double-gradient denaturing gradient gel electrophoresis (DG-DG CE) method consisted of PCR amplification of the target genomic DNA with GC -clamped oligonucleotides. The sequence encoded the 5' untranslated flankin g region of ferritin L-chain mRNA, which includes an IRE stem-loop structur e. The product was subjected to DG-DGCE (8.5-15% polyacrylamide and 50-95% denaturant) to separate the homo- and heteroduplexes. Results: The method clearly identified all eight accessible mutations, incl uding C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subj ects with serum ferritin >400 mug/L. The new mutation C14C was identified. Conclusions: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large po pulations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized. (C) 20 01 American Association for Clinical Chemistry.