title: A polymorphism that delays fibrosis in hepatitis C promotes alternative splicing of AZIN1, reducing fibrogenesis.
abstract: Among several single-nucleotide polymorphisms (SNPs) that correlate with fibrosis progression in chronic HCV, an SNP in the antizyme inhibitor (AzI) gene is most strongly associated with slow fibrosis progression. Our aim was to identify the mechanism(s) underlying this observation by exploring the impact of the AzI SNP on hepatic stellate cell (HSC) activity. Seven novel AZIN1 splice variants ("SV2-8") were cloned by polymerase chain reaction from the LX2 human HSC line. Expression of a minigene in LX2 containing the AZIN1 slow-fibrosis SNP yielded a 1.67-fold increase in AZIN1 splice variant 2 (AZIN1 SV2) messenger RNA (mRNA) (P = 0.05). In healthy human leukocytes, the SNP variant also correlated with significantly increased SV2 mRNA. Cells (293T) transfected with short hairpin RNA (shRNA) complementary to the exonic splicing chaperone SRp40 expressed 30% less SRp40 (P = 0.044) and 43% more AzI SV2 (P = 0.021) than control shRNA-expressing cells, mimicking the effect of the sequence variant. LX2 cells transfected with AZIN1 full-length complementary DNA expressed 35% less collagen I mRNA (P = 0.09) and 18% less α-smooth muscle actin mRNA (P = 0.09). Transient transfection of AZIN1 SV2 complementary DNA into LX2 cells reduced collagen I gene expression by 64% (P = 0.001) and α-smooth muscle actin by 43% (P = 0.005) compared to vector-transfected controls, paralleling changes in protein expression. Both AZIN1 and AZIN-SV2 mRNAs are detectable in normal human liver and reduced in HCV cirrhotic livers. The AZIN1-SV2 acts via a polyamine-independent pathway, as it neither interacts with antizyme nor affects the ability of AZIN1 lacking this variant to neutralize antizyme.
  
  
  CONCLUSIONS
  An SNP variant in the AZIN1 gene leads to enhanced generation of a novel alternative splice form that modifies the fibrogenic potential of HSCs.
authors.affiliation: Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Paris
authors.forename: Zohar
authors.initials: Z
authors.lastname: Snapir
authors.forename: Cindy D
authors.initials: CD
authors.lastname: Christopherson
authors.forename: Shirley Y
authors.initials: SY
authors.lastname: Kwok
authors.forename: Ursula E
authors.initials: UE
authors.lastname: Lee
authors.forename: Zahra
authors.initials: Z
authors.lastname: Ghiassi-Nejad
authors.forename: Peri
authors.initials: P
authors.lastname: Kocabayoglu
authors.forename: John J
authors.initials: JJ
authors.lastname: Sninsky
authors.forename: Josep M
authors.initials: JM
authors.lastname: Llovet
authors.forename: Chaim
authors.initials: C
authors.lastname: Kahana
authors.forename: Scott L
authors.initials: SL
authors.lastname: Friedman
mesh_terms: D000328:Adult; D017398:Alternative Splicing; D002352:Carrier Proteins / Q000235:genetics*; D024042:Collagen Type I / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005260:Female; D055166:Hepatic Stellate Cells / Q000378:metabolism; D019698:Hepatitis C, Chronic / Q000150:complications / Q000235:genetics*; D006801:Humans; D008103:Liver Cirrhosis / Q000378:metabolism / Q000517:prevention & control*; D008297:Male; D008875:Middle Aged; D009955:Ornithine Decarboxylase / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D014162:Transfection
chemical_list: C484011:AZIN1 protein, human; D002352:Carrier Proteins; D024042:Collagen Type I; D004791:Enzyme Inhibitors; D009955:Ornithine Decarboxylase