Linked linear amplification for simultaneous analysis of the two most common hemochromatosis mutations.
BACKGROUND
Two mutations in HFE, G845A (amino acid substitution C282Y) and C187G (H63D), are associated with hereditary hemochromatosis. We developed and validated a novel method, linked linear amplification (LLA), for detection of these two mutations.


METHODS
Two segments of HFE were amplified by a multiplex LLA reaction that generated biotinylated LLA products. Aliquots of the multiplex LLA reaction were captured in microwells by hybridization to immobilized allele-specific oligonucleotides (ASOs). One wild-type and one mutant ASO represented the DNA sequence at each of the two mutation sites. Hybridization was detected by a streptavidin-horseradish peroxidase-based colorimetric method. Genotypes obtained by LLA and PCR-restriction fragment length polymorphism (PCR-RFLP) methods for 320 individuals were compared.


RESULTS
The amplified samples included the following genotypes as determined by PCR-RFLP: wild-type 282 and 63 codons (n = 105), C282Y homozygous mutant (n = 54), C282Y heterozygous (n = 52), H63D homozygous mutant (n = 17), H63D heterozygous (n = 59), and compound H63D and C282Y heterozygous mutant (n = 33). There was complete concordance between the results obtained by LLA and those obtained by PCR-RFLP analysis. The presence of another HFE mutation, A193T (encoding S65C), did not interfere with genotyping at codon 63.


CONCLUSIONS
LLA provides a reliable method to detect the common mutations in HFE that cause hereditary hemochromatosis.
Killeen
Anthony A
AA
Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA. kille001@umn.edu
Breneman
John W
JW
Carillo
Arlene R
AR
Ugozzoli
Luis A
LA
Lowery
Jimmie D
JD
Liu
Jason
J
Hixson
Craig S
CS
D019943:Amino Acid Substitution; D003062:Codon; D005838:Genotype; D006432:Hemochromatosis / Q000235:genetics*; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results
D003062:Codon; C103673:HFE protein, human; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins