Global Sequencing Approach for Characterizing the Molecular Background of Hereditary Iron Disorders

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 53; no. 12; pp. 2060 - 2069
• Main Authors: Cunat, Severine, Giansily-Blaizot, Muriel, Bismuth, Michael, Blanc, Francois, Dereure, Olivier, Larrey, Dominique, Quellec, Alain Le, Pouderoux, Philippe, Rose, Christian, Raingeard, Isabelle, Renard, Eric, Schved, Jean-Francois, Aguilar-Martinez, Patricia, CHU Montpellier AOI 2004 Working Group
• Format: Journal Article
• Language: English
• Published: Washington, DC Am Assoc Clin Chem 01.12.2007; American Association for Clinical Chemistry; Oxford University Press
• Subjects: Adult; Aged; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Child; Disease; Endocrinology and metabolism; Female; Ferritins; Ferritins - blood; Fundamental and applied biological sciences. Psychology; Genes; Genetic counseling; Genotype & phenotype; Hemochromatosis; Hemochromatosis - genetics; Human health and pathology; Humans; Investigative techniques, diagnostic techniques (general aspects); Iron; Iron Metabolism Disorders; Iron Metabolism Disorders - genetics; Life Sciences; Male; Medical sciences; Middle Aged; Molecular Diagnostic Techniques; Mutation; Nucleic Acid Amplification Techniques; Patients; Sequence Analysis, DNA; Sequence Analysis, DNA - methods; Nucleotide sequence; Clinical biology; Biochemistry; Iron; Molecular biology; Hereditary; Sequencing
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2007.090605

New genetic forms of hereditary hemochromatosis (HH) or hereditary hyperferritinemia (HF) have been identified over the last few years, and abnormalities of various genes may interact in a single patient. This study aimed to develop a rapid automated method for sequencing the main genes involved. We used a standard 96-well microplate with a single PCR condition in an adaptation of the SCAIP (single-condition amplification with internal primer) method to sequence the HFE (hemochromatosis), HAMP (hepcidin antimicrobial peptide), HFE2/HJV [hemochromatosis type 2 (juvenile)], SLC40A1 (ferroportin), and TFR2 (transferrin receptor 2) genes, and the 5' untranslated region of the FTL (ferritin, light polypeptide) gene. To further simplify the method, we adjusted PCR conditions to avoid the use of an internal primer and applied this single-condition amplification method to 38 selected, unrelated patients. We tailored the genetic investigation according to the clinical picture, with the patients falling into 2 groups. Group 1 consisted of patients with hyperferritinemia and high transferrin saturation (TS) (classic adult and juvenile HH forms, groups 1A and 1B, respectively), and group 2 consisted of patients with hyperferritinemia and low, typical, or slightly increased TS, with or without iron overload (groups 2A and 2B, respectively). With this strategy we identified single-gene and multigene abnormalities, including 6 previously undescribed abnormalities in HFE (c.794dupA), HFE2 (c.-89-4dupT), and SLC40A1 (c.262A>G, c.533G>A, c.1468G>A, and c.-59_-45del). This method is a simple approach for investigating hereditary iron overload or HF and allows rapid evaluation of patients.