U6 is unsuitable for normalization of serum miRNA levels in patients with sepsis or liver fibrosis

MicroRNA (miRNA) levels in serum have recently emerged as potential novel biomarkers for various diseases. miRNAs are routinely measured by standard quantitative real-time PCR (qPCR); however, the high sensitivity of qPCR demands appropriate normalization to correct for nonbiological variation. Presently, RNU6B (U6) is used for data normalization of circulating miRNAs in many studies. However, it was suggested that serum levels of U6 themselves might differ between individuals. Therefore, no consensus has been reached on the best normalization strategy in 'circulating miRNA'. We analyzed U6 levels as well as levels of spiked-in SV40-RNA in sera of 44 healthy volunteers, 203 intensive care unit patients and 64 patients with liver fibrosis. Levels of U6 demonstrated a high variability in sera of healthy donors, patients with critical illness and liver fibrosis. This high variability could also be confirmed in sera of mice after the cecal ligation and puncture procedure. Most importantly, levels of circulating U6 were significantly upregulated in sera of patients with critical illness and sepsis compared with controls and correlated with established markers of inflammation. In patients with liver fibrosis, U6 levels were significantly downregulated. In contrast, levels of spiked-in SV40 displayed a significantly higher stability both in human cohorts (healthy, critical illness, liver fibrosis) and in mice. Thus, we conclude that U6 levels in the serum are dysregulated in a disease-specific manner. Therefore, U6 should not be used for data normalization of circulating miRNAs in inflammatory diseases and previous studies using this approach should be interpreted with caution. Further studies are warranted to identify specific regulatory processes of U6 levels in sepsis and liver fibrosis.

Relevalence of hepatitis B virus genome variability in organ transplantation

Hepatitis B virus [HBV] can cause acute and chronic hepatitis in humans, with the latter possibly leading to liver cirrhosis and hepatocellular carcinoma. The clinical course of HBV infection is critically dependent on genetic and immune features of the host as well as on virological factors. In situations of immune suppression, e.g. in patients after organ transplantation with chronic HBV infection, severe progression of liver disease can occur, due to direct effects of immunosuppressive regimens on HBV's hepatotoxicity or replication and due to the selection of HBV mutants. HBV variants are commonly found in chronically infected patients because of the lack of proofreading capacity of the HBV reverse transcriptase. Examples of relevant HBV mutations include precore or basal core promoter mutants with increased replication capacity, escape mutants with alterations in the 'a-determinant' immune epitope, core gene deletions after renal transplantation, antiviral drug resistant strains and accumulation of complex HBV variants after long-term immune suppression. In this review, we present the virological background of HBV genetic variability, discuss frequent mutations observed in transplanted patients and address the effects of HBV genetic variability on the clinical outcome in solid organ transplant recipients