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1.
Genet Test Mol Biomarkers ; 16(7): 656-60, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22364140

RESUMO

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.


Assuntos
Sondas de DNA/genética , Técnicas de Genotipagem/métodos , Hemocromatose/genética , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Sondas de DNA/química , Feminino , Hemocromatose/diagnóstico , Proteína da Hemocromatose , Heterozigoto , Homozigoto , Humanos , Masculino
2.
Genet Test Mol Biomarkers ; 15(4): 207-13, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21254846

RESUMO

The Factor V Leiden mutation (FVL; c.1601G>A, 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.1598G>A 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.1598G>A variant was indistinguishable from the heterozygous FVL control. The c.1598G>A 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.


Assuntos
Fator V/genética , Testes Genéticos/métodos , Mutação , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/métodos , Análise Custo-Benefício , Sondas de DNA , Genótipo , Ensaios de Triagem em Larga Escala , Humanos , Reação em Cadeia da Polimerase/instrumentação , Trombofilia/genética , Temperatura de Transição
3.
Am J Clin Pathol ; 128(3): 482-90, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17709323

RESUMO

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.


Assuntos
Algoritmos , Deficiência de alfa 1-Antitripsina/diagnóstico , alfa 1-Antitripsina/sangue , alfa 1-Antitripsina/genética , Alelos , Bases de Dados como Assunto , Predisposição Genética para Doença , Genótipo , Humanos , Reprodutibilidade dos Testes , Risco
6.
Clin Chem ; 50(7): 1156-64, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15229148

RESUMO

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 (A, prothrombin 20210G>A, methylenetetrahydrofolate reductase (MTHFR) 1298A>C, hemochromatosis (HFE) 187C>G, and beta-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 (T(m)s) that differ by 0.8-1.4 degrees C. However, in approximately 16% of SNPs, the bases only switch strands and preserve the base pair, producing very small T(m) differences between homozygotes (<0.4 degrees C). Although most of these cases can be genotyped by T(m), one-fourth (4% of total SNPs) show nearest-neighbor symmetry, and, as predicted, the homozygotes cannot be resolved from each other. In these cases, adding 15% of a known homozygous genotype to unknown samples allows melting curve separation of all three genotypes. This approach was used for the HFE 187C>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.


Assuntos
Polimorfismo de Nucleotídeo Único , Fator V/genética , Genótipo , Hemocromatose/genética , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Mutação , Reação em Cadeia da Polimerase , Protrombina/genética
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