title: Microelectronic DNA chip for hereditary hyperferritinemia cataract syndrome, a model for large-scale analysis of disorders of iron metabolism.
abstract: Hereditary hyperferritinemia cataract syndrome (HHCS) is caused by mutations in the regulatory iron responsive element (IRE) in the 5'UTR of the L-ferritin transcript that reduce binding affinity to the iron regulatory proteins (IRPs) and lead to a constitutive upregulation of the protein in tissue and serum. Twenty-nine mutations have been reported within the L-ferritin (FTL) IRE sequence, 21 of which were available to us. In addition, we included in this study three new mutations. Thus, we analyzed 24 mutations spanning over a DNA stretch of 48 nucleotides, including four deletions 2-29 nucleotides long and 20 substitutions, seven of which were conservative transversions. With this unique experimental model we developed a microchip diagnostic platform for identifying known molecular defects in the L-ferritin IRE structure with a microelectronic array approach, which we optimized after studying the effects of various parameters. The system enables electronic deposition of biotinylated amplicons to selected pads. Under optimized conditions, no cross-hybridization was found, even for mutations that affected the same or adjacent nucleotide positions. The same cartridge could be serially hybridized with all the 24 reporter probe sets, which allowed correct genotyping right up until the end of the analysis. Extensive validation on 200 samples in a blinded fashion gave total concordance of results. This pilot study represents a first step toward developing a diagnostic microchip for large-scale analyses for epidemiological studies and screening of mutations associated with iron disorders.
authors.affiliation: Unit of Genomics for Diagnosis of Human Pathologies, IRCCS H. San Raffaele, Milan, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Ferrari
authors.forename: Barbara
authors.initials: B
authors.lastname: Foglieni
authors.forename: Paolo
authors.initials: P
authors.lastname: Arosio
authors.forename: Clara
authors.initials: C
authors.lastname: Camaschella
authors.forename: Filomena
authors.initials: F
authors.lastname: Daraio
authors.forename: Sonia
authors.initials: S
authors.lastname: Levi
authors.forename: José Antonio
authors.initials: JA
authors.lastname: García Erce
authors.forename: Carole
authors.initials: C
authors.lastname: Beaumont
authors.forename: Mario
authors.initials: M
authors.lastname: Cazzola
authors.forename: Maurizio
authors.initials: M
authors.lastname: Ferrari
authors.forename: Laura
authors.initials: L
authors.lastname: Cremonesi
mesh_terms: D020121:5' Untranslated Regions; D001052:Apoferritins; D001483:Base Sequence; D019921:Biotinylation; D002386:Cataract / Q000235:genetics*; D005293:Ferritins / Q000097:blood*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D035925:Iron-Regulatory Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D013577:Syndrome
chemical_list: D020121:5' Untranslated Regions; D035925:Iron-Regulatory Proteins; D005293:Ferritins; D001052:Apoferritins; D007501:Iron