Padronização de técnica de PCR em tempo real para detecção simultânea das mutações H63D e S65C no gene HFE da hemocromatose

Abstract

Hemochromatosis is the most common genetic disorder in Caucasians. Its geographic distribution is worldwide, but it is seen most commonly in some populations in which it occurs with a prevalence of approximately 1 per 220-250 individuals. It is still underdiagnosed, because it is often considered a rare disorder that is manifested by the clinical findings seen in fully established disease consisting of cirrhosis, diabetes, and skin pigmentation. The diagnosis of hemochromatosis before the development of advanced cirrhosis or other manifestations of this disorder can ensure that steps are taken to maintain the quality of life of the patient. Currently, the guide specialized in the diagnosis and treatment of hemochromatosis recommend the use of genetic testing for HFE gene to aid in decision making in health care of patient. Therefore the development of new methodologies for the identification of mutations is an important step towards providing tools that aid physicians. Our study had the objective to develop a PCR 5 'Nuclease assay to detection in real time simultaneously H63D and S65C mutations. The use of probes in PCR 5 'Nuclease assay able to identify two polymorphisms simultaneously was never been seen before in the literature. The test was planned with the help of bioinformatics tools and then standardized and validated following recommendations of expert guides in the area of molecular genetics.The assay is also suitable validated following recommendations of two guides to good practice for the diagnosis of hemochromatosis. All performance characteristics tested for PCR 5'Nuclease assay for simultaneous detection of the mutations H63D and S65C have been successfully validated and the test can be used in clinical practice.