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 were identified by direct sequencing of DNA from probands with hyperferritinemia 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 ferritin 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-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5' untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5-15% polyacrylamide and 50-95% denaturant) to separate the homo- and heteroduplexes.


RESULTS
The method clearly identified all eight accessible mutations, including 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 subjects with serum ferritin >400 microg/L. The new mutation G14C 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 populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.
Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) H. San Raffaele, Unit of Genetics and Molecular Diagnostics, Via Olgettina 58, 20132 Milan, Italy.
L
L
Cremonesi
A
A
Fumagalli
N
N
Soriani
M
M
Ferrari
S
S
Levi
S
S
Belloli
G
G
Ruggeri
P
P
Arosio
D001483:Base Sequence; D002386:Cataract / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005293:Ferritins / Q000097:blood / Q000235:genetics*; D006432:Hemochromatosis / Q000235:genetics*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D020218:Response Elements; D013577:Syndrome
D005293:Ferritins; D007501:Iron