Validation of an unlabeled probe high-resolution melt assay for genotyping of HFE C282Y, H63D, and S65C. Introduction. Results. Conclusions. H63D, S65C and C282Y Mutations. Intra-run Variability.
H63D, S65C and C282Y Mutations
Hereditary hemochromatosis is an inherited disorder of iron metabolism, characterized by high absorption of iron by the gastrointestinal tract leading to iron accumulation in various organs and impaired organ function. Our current assay for the common mutations in the HFE gene uses a multiplexed LightCycler assay and fluorescent resonance energy transfer (FRET) hybridization probes. To increase throughput and decrease costs, automated extraction combined with unlabeled probes were used to detect the C282Y, H63D, and S65C mutations using the LightCycler 480 (LC480) instrument (Roche Applied Science, Indianapolis, IN) and high-resolution (HR) melting analysis.
A total of 314 samples from three different extraction methods were correctly genotyped. The inter-run and intra-run variation were within the acceptable range for a clinical assay, although the Magtration reagents on the PSS SX96 showed the least inter-run variation while the MagNA Pure reagents on the Radius showed the least intra-run variation. A common polymorphism, H63H has a melt that is similar to a S65C/WT, but can be differentiated using the amplicon melt and a difference plot. This assay for genotyping of HFE C282Y, H63D, and S65C is accurate and reproducible. It is an inexpensive and high-throughput alternative to a FRET probe assay.
H63D & S65C
The Tm standard deviations (SD) ranged from 0.00 ºC to 0.19 ºC with a ∆Tm SD from 0.03 ºC to 0.11 ºC.
Materials and Methods
Tm SD ranged from 0.02 ºC to 0.08 ºC with a ∆Tm SD from 0.08 ºC to 0.09 ºC.
DNA was extracted from whole blood using 3 extraction instruments:
Radius (Protedyne, Windsor, CT) and MagNA Pure (Roche Applied Science, Indianapolis, IN), both with MagNA Pure LC DNA Isolation Kit I.
PSS SX-96GC System (Precision System Science, Chiba, Japan) with the PSS Magtration Kit.
Designed by Idaho Technology, Inc. (Salt Lake City, UT). mRNA reference sequence: NCBI NM_000410.3
Following an asymmetric amplification using 1 µM forward primer, 10 µM reverse primer and 10 µM probe, PCR products were subjected to HR melting analysis in the LC480.
Results were analyzed using the LC480 Software SW1.5 (Roche Applied Science, Indianapolis, IN), and the MeltingWizard HR Melting Analysis v3.0 software (University of Utah, Salt Lake City, UT).
For accuracy studies a total of 314 previously genotyped samples were tested using the unlabeled probe for C282Y and/or H63D separately. In separate runs the common polymorphism, H63H (MagNA Pure), was characterized.
Precision was measured by melting temperature (Tm) and the Tm differences between alleles (∆Tm). Intra-run (triplicate) and inter-run (5 runs different days) variability were performed.
K. Sumner*, G. Pont-Kingdon*, S. Mitchell, T. Wayman*, C. Meadows*, S. Dobrowolski*, R. Prior††, E. Lyon*†
ARUP Institute for Clinical and Experimental Pathology *, Salt Lake City, UT, Department of Pathology †, Department of Internal Medicine ††, University of Utah, Salt Lake City.
Åsberg et al. Hereditary Hemochromatosis: The Clinical Significance of the S65C Mutation. 2002. Genet. Test. 6(1): 59-62.
Beutler. The Significance of the 187G (H63D) Mutation in Hemochromatosis. 1997. Am J Hum Genet. 61:762-764.
Beutler et al. A Previously Undescribed Nonsense Mutation of the HFE Gene. 2002. Clin Genet. 61: 40-42.
De Villiers et al. Spectrum of Mutations in the HFE Gene Implicated in Haemochromatosis and porphyria. 1999. Hum Molec Gen 8: 1517-1522.
Wittwer et al. High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen, 2003. Clin Chem. 49(6): 853–860.
Zhou et al., Closed-Tube Genotyping with Unlabeled Oligonucleotide Probes and a Saturating DNA Dye. 2004. Clin Chem. 50(8): 1328–1335.
The accuracy studies for both the H63D and C282Y was 100% concordant with the FRET probe genotyped data.
Primers and Probes
Tm SD ranged from 0.09 ºC to 0.30 ºC with a ∆Tm SD of 0.09 ºC to 0.23 ºC.
A sample with the common polymorphism H63H was run in duplicate and compared to control samples and a H63D sample using the H63D probe. The polymorphism can be distinguished from the other genotypes with a difference plot using the LC480 Gene scanning software. A sample characterized as S65C/WT (pink) is set as the baseline.
Tm SD ranged from 0.20 ºC to 0.64 ºC with a ∆Tm SD from 0.15 ºC to 0.65 ºC.
Tm SD ranged from 0.11 ºC to 0.50 ºC with a ∆Tm SD from 0.14 ºC to 0.36 ºC.
The authors are thankful to Dr. Rong Mao, Kristy Damjanovich, and Cecily Vaughn for editing assistance and Alison Millson for assistance with samples.
A total of 21 MagNA Pure extracted samples with the common polymorphism H63H are compared to control samples and a H63D sample using the H63D probe. H63H (blue) is distinguishable from the S65C/WT sample (green) in the difference plot.
Tm SD ranged from 0.06 ºC to 0.35 ºC with a ∆Tm SD from 0.08 ºC to 0.24 ºC.