Evaluation of a Nucleic Acid-based Cross-Linking Assay to Screen for Hereditary Hemochromatosis in Healthy Blood Donors

[摘要] Hereditary hemochromatosis (HH) is a common autosomal recessive disorder (frequency, 1 in 300–500 in the Northern European population) characterized by overabsorption of iron with consequent multiorgan failure secondary to iron overload (1)(2). Because early diagnosis and therapy can entirely prevent clinical complications, HH presents a model system for presymptomatic detection at the molecular level. HFE , the disease-causing gene of HH, encodes a 343-amino acid protein with high structural similarity to MHC class I molecules (3)(4). The primary disease-causing mutation is a single G-to-A transition at position 845, encoding a protein with a Cys282Tyr amino acid substitution (3).Current HH genotyping techniques include restriction fragment length polymorphism (RFLP) analysis (5) and heteroduplex analysis (6), both PCR based. We evaluated a nucleic acid-based test with cross-linkable DNA probes to screen for the Cys282Tyr mutation in a total of 101 presumably healthy blood donors. The assay uses oligonucleotide probes modified with photo-activatable cross-linker molecules (7) and has been used to detect the factor V Leiden mutation (8). Two sets of allele-specific cross-linkable DNA probes were prepared that detect either the wild-type or mutant Cys282Tyr gene sequences. Samples prepared from donor blood were assayed with each probe set, and the genotype of each individual was determined by comparison of the fluorescent signals obtained. All blood samples were also assayed by a PCR-RFLP test.Two capture probes that hybridized preferentially to the wild-type or mutant HFE gene sequence, respectively, were synthesized for the cross-linking assay: …

[学科分类] 过敏症与临床免疫学