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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).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.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.

Hereditary hemochromatosis is an inherited disorder of iron metabolism, characterized by high absorption of iron by the gastrointestinal tract leading to a toxic accumulation of iron in various organs and impaired organ function. Three variants in the HFE gene (p.C282Y, p.H63D, and p.S65C) are commonly associated with the development of the disease. Of these, p.C282Y homozygotes are at the highest risk. Compound heterozygotes of p.C282Y along with p.H63D or p.S65C have reduced penetrance. Furthermore, p.H63D homozygotes are not at an increased risk and little is known about the risk associated with homozygocity for p.S65C. Our current clinical assay for the three common HFE variants utilizes the LightCycler platform and paired probes employing fluorescent resonance energy transfer. To increase throughput and decrease costs, we developed a method whereby automated extraction was combined with unlabeled probes and differential melt profiles to detect these variants using the LightCycler 480 instrument. Using this approach, 43 samples extracted with three different extraction platforms were correctly genotyped. These data demonstrate that the newly developed assay to genotype the HFE mutations p.C282Y, p.H63D, and p.S65C, combined with high-throughput extraction platforms, is accurate and reproducible and represents an alternative to previously described tests.

The Factor V Leiden mutation (FVL; c.1601GA, p.Arg534Gln), the most common aberration underlying activated Protein C resistance, results in disruption of a major anticoagulation pathway and is a leading cause of inherited thrombophilia. A high-throughput assay for FVL mutation detection was developed using a single unlabeled probe on a high-resolution platform, the 96-well Roche 480 LightCycler (LC480) instrument. This method replaced the U.S. Food and Drug Administration-approved Roche Factor V Leiden kit assay on the LightCycler PCR instrument, decreasing total cost by 48%. The analytical sensitivity and specificity of the LC480 high-resolution assay approached 100% for the FVL mutation. Factor V mutations in proximity to the FVL locus may influence probe binding efficiency and melt characteristics. One out of three very rare variants tested in a separate study, 1600delC, was not distinguishable from FVL using the described high-resolution assay. However, a c.1598GA variant, which changes the amino acid sequence from arginine to lysine at position 533, was detected by this high-resolution assay and confirmed by bidirectional sequencing. In the labeled probe LightCycler assay, the c.1598GA variant was indistinguishable from the heterozygous FVL control. The c.1598GA variant has not been described previously and its clinical significance is uncertain. In conclusion, the LC480 FVL assay is cost effective in a high-throughput setting, with capability to detect both previously described and novel FV variants.

An integrative diagnostic algorithm for alpha1-antitrypsin (AAT) deficiency testing in the clinical laboratory was developed and evaluated. A novel rapid LightCycler (Roche, Indianapolis, IN) molecular assay was used to detect the common S and Z deficiency allelic variants. However, use of such molecular assays for these variants also can result in the misclassification of significant numbers of "at-risk" patient samples containing other rare AAT deficiency alleles. In the diagnostic algorithm presented herein, patient samples with selected genotypes that exhibit abnormally low AAT concentrations by immunoassay are phenotyped by isoelectric focusing. To test the efficacy of this algorithm, we retrospectively evaluated a data set of 50,020 serum samples for which protein phenotype and AAT concentration had been determined. This algorithm can successfully detect the majority of at-risk samples containing rare deficiency alleles.

With a frequency of 1 in 1000 bp, single nucleotide polymorphisms (SNPs) are used to study complex inherited diseases (1,2). A universal concern in genotyping techniques is that rare variants may interfere. Melting analysis addresses this concern and is a homogeneous and simple method for genotyping (3,4). For example, the 2-probe/2-fluorophore system HybProbe

High-resolution melting was recently introduced as a technique to genotype single-nucleotide polymorphisms (SNPs) within small amplicons (1). This closed-tube method (including rapid-cycle PCR) can be completed in

Background: High-resolution melting of PCR amplicons with the DNA dye LCGreen™ I was recently introduced as a homogeneous, closed-tube method of genotyping that does not require probes or real-time PCR. We adapted this system to genotype single-nucleotide polymorphisms (SNPs) after rapid-cycle PCR (12 min) of small amplicons (≤50 bp).Methods: Engineered plasmids were used to study all possible SNP base changes. In addition, clinical protocols for factor V (Leiden) 1691G>A, prothrombin 20210G>A, methylenetetrahydrofolate reductase (MTHFR) 1298A>C, hemochromatosis (HFE) 187C>G, and β-globin (hemoglobin S) 17A>T were developed. LCGreen I was included in the reaction mixture before PCR, and high-resolution melting was obtained within 2 min after amplification.Results: In all cases, heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves. Approximately 84% of human SNPs involve a base exchange between A::T and G::C base pairs, and the homozygotes are easily genotyped by melting temperatures (Tms) that differ by 0.8–1.4 °C. However, in ∼16% of SNPs, the bases only switch strands and preserve the base pair, producing very small Tm differences between homozygotes (G protocol, but, as predicted from the sequence changes, was not needed for the other four clinical protocols.Conclusions: SNP genotyping by high-resolution melting analysis is simple, rapid, and inexpensive, requiring only PCR, a DNA dye, and melting instrumentation. The method is closed-tube, performed without probes or real-time PCR, and can be completed in less than 2 min after completion of PCR.

Hemochromatosis is the most common genetic illness in people of Northern European descent. This autosomal recessive disorder of iron metabolism occurs with a frequency of approximately 0.5% in Caucasian populations (1). Hemochromatosis leads to organ failure due to iron accumulation and can be misdiagnosed as heart or liver disease. Serious complications include cirrhosis, hepatomas, diabetes, cardiomyopathy, arthritis and hypogonadotrophic hypogonadism (2–4). A cysteine to tyrosine amino acid substitution at codon 282 (C282Y), caused by a G to A transition at nucleotide position 845, is found on 85% to 100% of disease chromosomes from patients of northern European ancestry who meet well defined clinical criteria for iron overload (5–8). Another mutation (H63D) is created by a C to G transversion at nucleotide position 187. This substitution has an estimated penetrance between 0.44 and 1.5% of the homozygous C282Y genotype and is considered pathogenic only when inherited in trans with a C282Y mutation (compound heterozygous; 5–7).

We evaluated the MagNA Pure LC System (Roche Applied Science) for automated DNA extraction for use in mutation detection and allele quantification. The system uses magnetic silica beads that bind DNA to a silica surface and transfer DNA through various steps of the extraction process. This system can be linked to LightCycler assays for further automation. The LightCycler is an enclosed thermocycler with continuous monitoring for PCR quantification and allele discrimination (1)(2)(3). Combined MagNA Pure/LightCycler automation of sample preparation, amplification, and detection has the potential to reduce human error in sample tracking and to standardize DNA extraction and inoculation into the PCR mixture. The MagNA Pure/LightCycler system has been evaluated for infectious disease applications, detecting PCR product by crossing point (4)(5). In this study we compared the MagNA Pure LC DNA extraction with the Qiagen 9604 BioRobot (Qiagen) for real-time mutation detection and allele quantification. We evaluated 280 samples with the MagNA Pure System and Qiagen 9604 BioRobot under an Internal Review Board-approved protocol for instrument validation. Genomic DNA was extracted from 200 μL of EDTA-anticoagulated whole blood by use of the MagNA Pure LC DNA Isolation Kit I (Roche Diagnostics), with its high-performance DNA protocol, and by the QIAamp 9604 DNA Blood BioRobot Kit (Qiagen) according to manufacturer’s instructions. Both methods initially use red blood cell lysis and a proteinase …