Simultaneous detection of C282Y and H63D hemochromatosis mutations by dual-color probes.
BACKGROUND
Hemochromatosis is a common genetic disease, affecting one in every 200 individuals in the United States. A PCR assay was designed using fluorescent melting curve analysis to simultaneously detect the G845-->A (C282Y) and C187-->G (H63D) mutations. The G845-->A and C187-->G loci are distinguished by color, and mutant alleles are distinguished from wild type by probe melting temperature (Tm).


RESULTS
The probe sets used two fluorophore pairs, fluorescein with LCRed 640 for G845-->A and fluorescein with LCRed 705 for C187-->G. The probes, complementary to the mutant allele, dissociate from the product at specific Tms. Wild-type alleles form mismatches with the probes, reducing the Tms by 6 degrees C (G845-->A) and 10 degrees C (C187-->G). One of 133 samples had a Tm shift 4 degrees C less than the wild-type Tm for the G845-->A locus. Sequencing confirmed the sample to be homozygous for G845-->A and heterozygous for a C-->A substitution at position 842 (C842-->A), substituting lysine for threonine.


CONCLUSIONS
Multiplexing by color and Tm allows for simultaneous genotyping of each mutation. A novel base-pair alteration was detected in cis with a G845-->A mutation.
Phillips
M
M
Associated and Regional University Pathologist Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA.
Meadows
C A
CA
Huang
M Y
MY
Millson
A
A
Lyon
E
E
D004247:DNA / Q000032:analysis / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes; D005838:Genotype; D006432:Hemochromatosis / Q000235:genetics*; D006801:Humans; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic / Q000235:genetics; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
D005456:Fluorescent Dyes; D004247:DNA