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Physiopathology
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User name	Nicolas Tchitchek
Email	nicolas.tchitchek@ihes.fr
Phone / Fax number	0033610887604 /
Location	Institut des Hautes Ã‰tudes Scientifiques (Systems Epigenomics Group) - 35, route de Chartres - 91440 Bures-sur-Yvette, FRANCE

Scientific description

Liver failure due to chronic hepatitis C virus infection is a major cause for liver transplantation worldwide. Recurrent infection of the graft is universal in HCV patients following transplant and results in rapid progression to severe fibrosis and end-stage liver disease in one-third of all patients. No single clinical variable, or combination thereof, has so far proven accurate in identifying patients at risk of hepatic decompensation in the transplant setting.  A combination of longitudinal, dimensionality reduction, and categorical analysis of the transcriptome from 111 liver biopsy specimens taken from 57 HCV-infected patients over time identified a molecular signature of gene expression of patients at risk of developing severe fibrosis. Significantly, alterations in gene expression occur prior to histologic evidence of liver disease progression, suggesting that events which occur during the acute phase of infection influence patient outcome. Additionally, a common precursor state for different severe clinical outcomes was identified. Hence, incidence of severe liver disease is a process initiated early during HCV infection of the donor organ. The cellular network at the basis of the initial transition to severe liver disease was identified and characterized.

Technical description

Human Liver Tissue Samples.  Core needle liver biopsies were obtained from liver transplant patients at the University of Washington Medical Center (UWMC).  All patients provided informed consent according to protocols approved by the Human Subject Review Committee at the University of Washington.  No donor organs were obtained from executed prisoners or other institutionalized persons.  Upon collection, biopsies were stored in RNAlater (Life Technologies, Carlsbad, CA) and sent to the Katze lab for RNA isolation and microarray analysis.  Non-identifiable patient data detailed in Table 1 was used to both categorize patients according to clinical outcome.

Total RNA Isolation and mRNA Amplification.  Biopsy specimens were disrupted in TRIzol reagent (Life Technologies, Carlsbad, CA) using a Polytron homogenizer (PowerGene 700; Fisher Scientific, Pittsburgh, PA).  Total RNA was isolated according to the manufacturerâ€™s protocol.  All total RNA samples were amplified using the RiboAmp RNA Amplification Kit (Arcturus/MDS, Mountain View, CA).  The quality of amplified RNA was determined by capillary electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).

Expression Microarray Format. A single experiment comparing two mRNA samples was done with four replicate human 1A (V2) 22K oligonucleotide expression arrays (Agilent Technologies, Palo Alto, CA) using the dye label reverse technique. This allows for the calculation of mean ratios between expression levels of each gene in the analyzed sample pair, standard deviation, and p-values for each experiment. Spot quantitation, normalization, and application of a platform-specific error model were performed using Agilent's Feature Extractor software.

References

Rasmussen AL, Tchitchek N, Susnow NJ, Krasnoselsky AL, Diamond DL, Yeh MM, Proll SC, Korth MJ, Walters KA, Lederer S, Larson AM, Carithers R, Benecke A, Katze MG. 
Early transcriptional programming links progression to hepatitis C virus-induced severe liver disease in transplant patients.

Early transcriptional programming links progression to hepatitis C virus-induced severe liver disease in transplant patients.

Rasmussen AL, Tchitchek N, Susnow NJ, Krasnoselsky AL, Diamond DL, Yeh MM, Proll SC, Korth MJ, Walters KA, Lederer S, Larson AM, Carithers RL, Benecke A, Katze MG.

Hepatology. 2012 Jan 25. doi: 10.1002/hep.25612. [Epub ahead of print]

Pubmed : http://www.ncbi.nlm.nih.gov/pubmed/22278598