Scanning mutations of the 5¢UTR regulatory sequence of l-ferritin by denaturing high-performance liquid chromatography: identification of new mutations Laura Cremonesi, 1 Rita Paroni, 2 Barbara Foglieni, 1 Silvia Galbiati, 1 Isabella Fermo, 2 Nadia Soriani, 1 Silvana Belloli, 3 Giuseppina Ruggeri, 3 Giorgio Biasiotto, 3 Mario Cazzola, 4 Francesca Ferrari, 1 Maurizio Ferrari 1,5 and Paolo Arosio 3 1 Istituti di Ricovero e Cura a Carattere Scientifico (IRCCS), H. San Raffaele, Unit of Genetics and Molecular Diagnostics, 2 IRCCS H. San Raffaele, Unit of Separative Techniques, Milan, 3 Section of Chemistry, Faculty of Medicine, University of Brescia, Brescia, 4 Department of Hematology and Department of Internal Medicine and Medical Therapy, University of Pavia Medical School and IRCCS Policlinico S. Matteo, Pavia, and 5 Diagnostica e Ricerca San Raffaele SpA, Milan, Italy Received 26 September 2002; accepted for publication 28 November 2002 Summary. Hereditary hyperferritinaemia cataract syn- drome is an autosomal dominant disorder caused by heterogeneous mutations of the iron regulatory element (IRE) in the ferritin l-chain mRNA. The mutations are rare and fast DNA scanning would facilitate diagnosis. The aim of the study was to compare the analytical performances of two fast DNA scanning techniques: denaturing high-per- formance liquid chromatography (DHPLC) and double-gra- dient denaturing gradient gel electrophoresis (DG-DGGE). We analysed the sequence encoding the 5¢ untranslated flanking region of ferritin l-chain mRNA, which includes an IRE stem loop structure. The two systems unambiguously identified all the 12 accessible mutations in a single run, including the difficult C–G transversions. DHPLC and DG-DGGE identified seven abnormal patterns in DNA sam- ples from 47 subjects with unexplained hyperferritinaemia; all had mutations in the IRE sequence, including two not reported before: C36G and A37G. The scanning of 250 DNA samples from subjects genotyped for HFE led to the identi- fication of four new mutations, all outside the IRE structure: C10T, C16T, C90T and del-T156. We conclude that DHPLC, similar to DG-DGGE, detects all the mutations in the l-ferritin 5‘UTR sequence in a single run, and that various mutations occur outside the IRE structure. Keywords: hereditary hyperferritinaemia–cataract syndrome, ferritin, denaturing HPLC, iron metabolism, DNA variations. Hereditary hyperferritinaemia cataract syndrome [HCS; Online Mendelian Inheritance in Man (OMIM) code 600886] is a genetic, autosomal dominant disorder caused by heterogeneous mutations in the iron responsive element (IRE) of l-ferritin mRNA. The mutations abolish or reduce the affinity binding to the iron regulatory proteins (IRPs) with a constitutive upregulation of ferritin l-chain in serum and all tissues unrelated to body iron stores (Beaumont et al, 1995; Cazzola et al, 1997). The 10–20-fold higher accu- mulation of l-ferritin in the serum and tissues has no evident effects on body iron status, but it is often associated with early bilateral cataract onset, probably caused by aggregation and crystallization of the protein in the lens (Levi et al, 1998; Mumford et al, 2000). Fifteen different mutations have been described to date in more than 20 families (Table I), including one sporadic case (Arosio et al, 1999). The mutations span a 30 nucleotide (nt) section of the IRE structure; some affect the loop that interacts directly with the IRPs, while others affect the stems or the bulge of the structure and modify the conformation of the IRE, leading to lower affinity binding (Allerson et al, 1999). The in vitro effects of the mutations on IRE structures were correlated with the clinical symptoms: the patients with mutations that affect the upper part of the structure (which is involved in IRP binding) had serum ferritin levels of 1000–1500 lg/l and early onset cataract, while those with mutations that affect the distal part of the stems had slightly increased serum ferritin levels (500 lg/l) and subclinical cataract (Allerson et al, 1999). Correspondence: Paolo Arosio, Laboratory of Biotechnology, Sec- tion of Chemistry, Faculty of Medicine, University of Brescia, Via Valsabbina 19, 25123 Brescia, Italy. E-mail: arosio@med.unibs.it British Journal of Haematology, 2003, 121, 173–179 Ó 2003 Blackwell Publishing Ltd 173