Validation of an unlabeled probe high-resolution melt assay for genotyping of HFE C 282 Y, H 63 D, and S 65 C 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. kelli. sumner@aruplab. com Introduction Results H 63 D, S 65 C and C 282 Y 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 Light. Cycler 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 C 282 Y, H 63 D, and S 65 C mutations using the Light. Cycler 480 (LC 480) instrument (Roche Applied Science, Indianapolis, IN) and high-resolution (HR) melting analysis. H 63 D & S 65 C Conclusions Intra-run Variability C 282 Y 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 Extraction Tm SD ranged from 0. 02 ºC to 0. 08 ºC with a ∆Tm SD from 0. 08 ºC to 0. 09 ºC. References DNA was extracted from whole blood using 3 extraction instruments: Radius (Protedyne, Windsor, CT) and Mag. NA Pure (Roche Applied Science, Indianapolis, IN), both with Mag. NA Pure LC DNA Isolation Kit I. The accuracy studies for both the H 63 D and C 282 Y was 100% concordant with the FRET probe genotyped data. PSS SX-96 GC System (Precision System Science, Chiba, Japan) with the PSS Magtration Kit. Primers and Probes Designed by Idaho Technology, Inc. (Salt Lake City, UT). m. RNA reference sequence: NCBI NM_000410. 3 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 SX 96 showed the least inter-run variation while the Mag. NA Pure reagents on the Radius showed the least intra-run variation. A common polymorphism, H 63 H has a melt that is similar to a S 65 C/WT, but can be differentiated using the amplicon melt and a difference plot. This assay for genotyping of HFE C 282 Y, H 63 D, and S 65 C is accurate and reproducible. It is an inexpensive and high-throughput alternative to a FRET probe assay. Åsberg et al. Hereditary Hemochromatosis: The Clinical Significance of the S 65 C Mutation. 2002. Genet. Test. 6(1): 59 -62. Tm SD ranged from 0. 09 ºC to 0. 30 ºC with a ∆Tm SD of 0. 09 ºC to 0. 23 ºC. H 63 H Polymorphism Beutler. The Significance of the 187 G (H 63 D) 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. Inter-run Variability Method 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 LC 480. Analysis For accuracy studies a total of 314 previously genotyped samples were tested using the unlabeled probe for C 282 Y and/or H 63 D separately. In separate runs the common polymorphism, H 63 H (Mag. NA Pure), was characterized. Tm SD ranged from 0. 20 ºC to 0. 64 ºC with a ∆Tm SD from 0. 15 ºC to 0. 65 ºC. Results were analyzed using the LC 480 Software SW 1. 5 (Roche Applied Science, Indianapolis, IN), and the Melting. Wizard HR Melting Analysis v 3. 0 software (University of Utah, Salt Lake City, UT). Validation A sample with the common polymorphism H 63 H was run in duplicate and compared to control samples and a H 63 D sample using the H 63 D probe. The polymorphism can be distinguished from the other genotypes with a difference plot using the LC 480 Gene scanning software. A sample characterized as S 65 C/WT (pink) is set as the baseline. 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. 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. Tm SD ranged from 0. 11 ºC to 0. 50 ºC with a ∆Tm SD from 0. 14 ºC to 0. 36 ºC. Acknowledgements A total of 21 Mag. NA Pure extracted samples with the common polymorphism H 63 H are compared to control samples and a H 63 D sample using the H 63 D probe. H 63 H (blue) is distinguishable from the S 65 C/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. The authors are thankful to Dr. Rong Mao, Kristy Damjanovich, and Cecily Vaughn for editing assistance and Alison Millson for assistance with samples.
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