In vitro-targeted gene identification in patients with hepatitis C using a genome-wide microarray technology.
Iron in association with reactive oxygen species (ROS) is highly toxic, aggravating oxidative stress reactions. Increased iron not only plays an important role in the progression of hereditary hemochromatosis (HH) but also in common liver diseases such as chronic hepatitis C. The underlying mechanisms of hepatitis C virus (HCV)-mediated iron accumulation, however, are poorly understood. We introduce an in vitro-targeted approach to identify ROS/iron-regulated genes in patients with HCV using a genome-wide DNA microarray. The sensitivity of the 32,231 complementary DNA clone-carrying microarray was approximately 20% as estimated by detecting target genes of the genome-wide transcription factor hypoxia inducible factor 1alpha. Upon in vitro challenge to iron and oxidative stress, 265 iron-related and 1326 ROS-related genes could be identified in HepG2 cells; 233 significantly regulated genes were found in patients with mild (HCV) or severe (HH) iron deposition. Notably, 17 of the in vitro-selected genes corresponded to the genes identified in patients with HCV or HH. Among them, natriuretic peptide precursor B (NPPB) was the only iron-regulated gene identified in vitro that was differentially regulated between HCV and HH. Reverse-transcription polymerase chain reaction confirmed most of the microarray-identified genes in an even larger group of patients (n = 12). In patients with HCV, these included genes that are associated with RNA processing (MED9/NFAT, NSUN2), proliferation, differentiation, hypoxia, or iron metabolism (ISG20, MIG6, HIG2, CA9, NDRG1), whereas none of the nine known iron-related genes showed significant differences between HCV and HH.


CONCLUSIONS
Although high-density microarray technology is less suitable for routine liver diagnosis, its use in combination with prior in vitro selection is a powerful approach to identify candidate genes relevant for liver disease.
Hagist
Susanne
S
Department of Internal Medicine, Salem Medical Center, University of Heidelberg, Heidelberg, Germany.
Sültmann
Holger
H
Millonig
Gunda
G
Hebling
Ulrike
U
Kieslich
Dörthe
D
Kuner
Rupert
R
Balaguer
Sabrina
S
Seitz
Helmut-Karl
HK
Poustka
Annemarie
A
Mueller
Sebastian
S
D000328:Adult; D018076:DNA, Complementary / Q000235:genetics; D018450:Disease Progression; D015894:Genome, Human; D006432:Hemochromatosis / Q000235:genetics; D006526:Hepatitis C / Q000150:complications / Q000235:genetics*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics; D007501:Iron / Q000378:metabolism; D008103:Liver Cirrhosis / Q000453:epidemiology; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D017461:Receptors, Atrial Natriuretic Factor / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000099242:Atrial Natriuretic Factor Receptor B
D018076:DNA, Complementary; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007501:Iron; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D017461:Receptors, Atrial Natriuretic Factor; D000099242:Atrial Natriuretic Factor Receptor B; C497442:HIF1A protein, human