Scanning mutations of the 5′UTR regulatory sequence of l‐ferritin by denaturing high‐performance liquid chromatography: identification of new mutations

• Published in: British journal of haematology Vol. 121; no. 1; pp. 173 - 179
• Main Authors: Cremonesi, Laura, Paroni, Rita, Foglieni, Barbara, Galbiati, Silvia, Fermo, Isabella, Soriani, Nadia, Belloli, Silvana, Ruggeri, Giuseppina, Biasiotto, Giorgio, Cazzola, Mario, Ferrari, Francesca, Ferrari, Maurizio, Arosio, Paolo
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-04-2003
• Subjects: denaturing HPLC; DNA variations; ferritin; hereditary hyperferritinaemia–cataract syndrome; iron metabolism
• ISSN: 0007-1048; 1365-2141
• DOI: 10.1046/j.1365-2141.2003.04253.x

Hereditary hyperferritinaemia cataract syndrome 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‐performance liquid chromatography (DHPLC) and double‐gradient 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 samples 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 identification 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.