Detection of IL28B SNP DNA from buccal epithelial cells, small amounts of serum, and dried blood spots.

BACKGROUND & AIMS: Point mutations in the coding region of the interleukin 28 gene (rs12979860) have recently been identified for predicting the outcome of treatment of hepatitis C virus infection. This polymorphism detection was based on whole blood DNA extraction. Alternatively, DNA for genetic diagnosis has been derived from buccal epithelial cells (BEC), dried blood spots (DBS), and genomic DNA from serum. The aim of the study was to investigate the reliability and accuracy of alternative routes of testing for single nucleotide polymorphism allele rs12979860CC. METHODS: Blood, plasma, and sera samples from 200 patients were extracted (400 µL). Buccal smears were tested using an FTA card. To simulate postal delay, we tested the influence of storage at ambient temperature on the different sources of DNA at five time points (baseline, 48 h, 6 days, 9 days, and 12 days). RESULTS: There was 100% concordance between blood, plasma, sera, and BEC, validating the use of DNA extracted from BEC collected on cytology brushes for genetic testing. Genetic variations in HPTR1 gene were detected using smear technique in blood smear (3620 copies) as well as in buccal smears (5870 copies). These results are similar to those for whole blood diluted at 1/10. A minimum of 0.04 µL, 4 µL, and 40 µL was necessary to obtain exploitable results respectively for whole blood, sera, and plasma. No significant variation between each time point was observed for the different sources of DNA. IL28B SNPs analysis at these different time points showed the same results using the four sources of DNA. CONCLUSION: We demonstrated that genomic DNA extraction from buccal cells, small amounts of serum, and dried blood spots is an alternative to DNA extracted from peripheral blood cells and is helpful in retrospective and prospective studies for multiple genetic markers, specifically in hard-to-reach individuals.

The natural history of hepatitis B virus cirrhosis after the first hepatic decompensation in Tunisia.

AIM: To define the natural long term course of viral B cirrhosis after the onset of hepatic decompensation and to determine the predictive factors of death. METHODS: Retrospective longitudinal study including 77 cases of viral B cirrhosis among 192 consecutive patients with cirrhosis, hospitalized between 1997 and 2005 for the first hepatic decompensation. All those patients were followed- up until death or until December 2006. The probability of survival after the first hepatic decompensation was calculated using the Kaplan Meier method. The predictive factors of death were determined through univariate and multivariate analyses with the Cox regression model. RESULTS: Fifty four men and 23 women with an average age of 54±14.9 years were hospitalized for the first decompensation of the viral B cirrhosis. The 77 patients had been under observation for an average period of 24.2 ±21.1 months. During that time 64% among them died. The probability of survival after decompensation was 47% in 2 years and 22 % in 5 years. During follow- up, ascites was the most frequent decompensation (85%) followed by hepatic encephalopathy (38 %), variceal hemorrhage (34 %), jaundice (30%), hepato renal syndrome (27%), hepatocellular carcinoma (21%), and spontaneous bacterial peritonitis (14%). At univariate analysis four factors were predictive of death: Child Pugh C score (p=0.009), hepatocellular carcinoma (p=0.01), rate of serum gammaglobulin superior to18g / l (p=0.008) and prothrombin time inferior to 50 % (p=0.02). According to the multivariate analysis only the rate of serum gammaglobulin superior to 18g /l was an independent predictive factor of mortality (p=0,001) with IC (95 %) [1.623 - 5.88]. CONCLUSION: In Tunisia, the prognosis of viral B cirrhosis after the first decompensation is bad, because a patient on 5 only was able to survive beyond 5 years. Ascites is the most frequent decompensation. Only the rate of serum gammaglobulin superior to 18g / l is an independent predictive factor of mortality.