High resolution melting for the identification of mutations in the iron responsive element of the ferritin light chain gene.
BACKGROUND
Among the causes of hyperferritinemia, hereditary hyperferritinemia cataract syndrome (HHCS) is an autosomal dominant disease characterized by distinctive cataracts and high serum ferritin. It is caused by mutations in the iron responsive element (IRE) of the ferritin light chain gene (FTL).


METHODS
To speed up and simplify mutational scanning in this genomic region, we developed a protocol based on high-resolution melting (HRM) analysis.


RESULTS
Validation was carried out using 18 wild-type and 14 DNA samples carrying different mutations, each analyzed in replicates of 20. The method allowed for correct identification and genotyping of all mutant samples, and each variant generated a specific profile distinguishable from the wild type. A 5.5% proportion of false positive results were obtained. In addition, in two patients with HHCS, two new mutations were identified by HRM based on an altered melting profile. These mutations were subsequently characterized by direct sequencing (7C>G+40A>G and 49A>C).


CONCLUSIONS
The high reliability of HRM in detecting known and new DNA variations indicate that this could be an effective and sensitive method for molecular scanning of mutations in the IRE of the FTL gene in patients presenting with either HHCS or unexplained hyperferritinemia.
Castiglioni
Emanuela
E
San Raffaele Scientific Institute, Center for Genomics, Bioinformatics and Biostatistics, Genomic Unit for the Diagnosis of Human Pathologies, Milan, Italy.
Soriani
Nadia
N
Girelli
Domenico
D
Camaschella
Clara
C
Spiga
Ivana
I
Della Porta
Matteo G
MG
Ferrari
Maurizio
M
Cremonesi
Laura
L
D001052:Apoferritins / Q000235:genetics*; D002386:Cataract / Q000235:genetics; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D007501:Iron / Q000494:pharmacology*; D019189:Iron Metabolism Disorders / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009691:Nucleic Acid Denaturation; D020218:Response Elements / Q000235:genetics*; D012680:Sensitivity and Specificity; D013577:Syndrome
C507433:FTL protein, human; D004247:DNA; D001052:Apoferritins; D007501:Iron