RESUMO
Introducción: La enfermedad por Almacenamiento de Ésteres de Colesterol (CESD; Cholesteryl Ester Storage Disease) es una enfermedad de depósito lisosomal, su presentación es bastante variable y su diagnóstico constituye un desafío. Además, existe un número de anomalías observadas en los pacientes con CESD que se sobreponen a diagnósticos más comunes, siendo probable que sea subdiagnosticada. La mayoría de pacientes relatados hasta el momento son portadores de la mutación E8SJM en el gen LIPA. En este sentido, el auxilio en el diagnóstico es fundamental pues existen opciones de terapia en desarrollo. Diseño: Estudio observacional. Objetivo: Estandarizar la técnica de PCR en tiempo real para la detección de la mutación más frecuente, E8SJM, en muestras de sangre periférica y de biopsia hepática para el auxilio diagnóstico de CESD y futuros estudios de prevalencia de la mutación. Institución: Centro de Terapia Génica del Hospital de Clínicas de Porto Alegre (HCPA), Brasil. Material biológico: Muestras de ADN extraídas de sangre periférica y tejido hepático parafinado. Principales medidas de resultados: Presencia/Ausencia de la mutación E8SJM. Resultados: Se estandarizó la reacción de PCR en tiempo real, la mutación fue detectada correctamente y posteriormente validada por secuenciación de Sanger. La mutación fue analizada en 137 muestras y encontrada en apenas una paciente que ingresó al Servicio de Genética Médica del HCPA con diagnóstico clínico y bioquímico de CESD/Wolman. Conclusiones: La técnica de PCR en tiempo real es ideal para la detección rápida y en gran escala de la mutación frecuente asociada a CESD.
Introduction: Cholesteryl Ester Storage Disease (CESD) is a lysosomal storage disorder, its presentation is highly variable and its diagnosis challenging. In addition, there are several abnormalities observed in patients with CESD who overlap with more common diagnoses and are likely to be underdiagnosed. Most patients reported to date are carriers of the E8SJM mutation in the LIPA gene. In this sense, diagnostic assistance is essential because there are options for therapy in development, as well as mutation prevalence studies. Design: Observational research. Objective: To standardize the real-time PCR technique for the detection of the most frequent mutation, E8SJM, in peripheral blood and liver biopsy specimens for the diagnosis of CESD and future mutation prevalence studies. Institution: Center of Gene Therapy of the Hospital de Clinicas de Porto Alegre (HCPA), Brazil. Biological material: DNA samples extracted from peripheral blood and paraffinembedded liver tissue. Main outcome measures: Presence / Absence of E8SJM mutation. Results: The PCR reaction was standardized in real time; the mutation was correctly detected and validated by Sanger sequencing. The mutation was analyzed in 137 samples and found in only one patient who entered the Medical Genetics Service of the HCPA with clinical and biochemical diagnosis of CESD/Wolman. Conclusions: The real-time PCR technique is ideal for rapid and large-scale detection of the frequent CESD-associated mutation.
RESUMO
INTRODUCTION: Considering the high prevalence of liver tumors and the impact on patient survival, a greater understanding of the biological behavior of those tumors if of great importance. The multidrug resistance gene (MDR1) may present as single nucleotide polymorphism (SNP) which can affect the expression and activity of P-glycoprotein (Pgp), and high expression of Pgp has been associated with a worse prognosis in affected patients. OBJECTIVE: To correlate the C3435T polymorphism in the MDR1 gene with the immunohistochemical expression of Pgp. MATERIAL AND METHODS: A total of 67 samples from patients with diagnosis of hepatocellular carcinoma (HCC), collected in the period from 2000 to 2009, were analyzed. The polymorphism in the MDR1 gene was determined by the technique of allele-specific real time PCR using TaqMan assay, and the expression of protein Pgp was evaluated by immunohistochemistry. RESULTS: Among the samples evaluated, 56 (83.6%) were from male patients and 11 (16.4%) from females. Mean age was 60.6 years (± 8.8), ranging from 37 to 85 years. The etiology of the HCC was related to hepatitis C virus infection (HCV) in 31 (46.3%) of cases, followed by hepatitis C virus infection + alcohol in 24 cases (35.8%), alcohol in 4 cases (6)%, hepatitis B virus (HBV) in 4 cases (6%) and other factors in 4 cases (6%). Liver transplantation was performed in 48 cases (71.6%) and hepatectomia in 19 cases (28.4%). The genotypes CC, CT and TT showed frequencies of 25.4%, 41.8% and 32.8%, respectively, and the allele frequencies were 46.3% for allele C and 53.7% for allele T. The expression of Pgp in over 75% of the cells was significantly more frequent in tumor tissue. On the other hand, a low expression of Pgp, in less than 25% of the cells, was significantly more frequent in non-tumor tissue. The Pgp expression in more than 50% of tumor cells of individuals with genotypes CC, CT and TT was 15.7%, 51.0% and 33.3%, respectively, and was significantly higher when in the presence of allele T (p = 0.002). CONCLUSION: The presence of the polymorphic allele T is related to increased expression of Pgp protein in patients with HCC.
