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Simultaneous Detection of C282Y and H63D Hemochromatosis Mutations by Dual-color Probes

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Abstract

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).

Methods and 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°C (G845→A) and 10°C (C187→G). One of 133 samples had a Tm shift 4°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.

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Authors and Affiliations

  1. Associated and Regional University Pathologist Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA

    Marec Phillips, Ming Y. Huang MS, Alison Millson MT (ASCP) & Elaine Lyon PhD

  2. Associated and Regional University Pathologist Laboratories, Salt Lake City, Utah, USA

    Cindy A. Meadows BS CLSp(MB)

  3. Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT

    Elaine Lyon PhD

Authors
  1. Marec Phillips
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  2. Cindy A. Meadows BS CLSp(MB)
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  3. Ming Y. Huang MS
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  4. Alison Millson MT (ASCP)
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  5. Elaine Lyon PhD
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Correspondence to Elaine Lyon PhD.

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Phillips, M., Meadows, C.A., Huang, M.Y. et al. Simultaneous Detection of C282Y and H63D Hemochromatosis Mutations by Dual-color Probes. Molecular Diagnosis 5, 107–116 (2000). https://doi.org/10.1007/BF03262029

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