studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 1235A
2107
Mutational analysis of ATP7B in Chinese Wilson disease
patients
Shan S. Peng, Lingxia QI, yuanyuan Cui, Wenli Kong, Linyuan
Ma, Yu Pan, Junqi Niu, Rui Hua; Hepatology, The First Hospital,
Jilin University, Changchun, China
Wilson Disease (WD) is an inborn error of copper metabolism
inherited in an autosomal recessive manner caused by
the mutations in the P-type ATPase gene (ATP7B). In this study,
we screen and detect the mutations of the ATP7B gene in unrelated
Chinese WD patients.A total of 62 individuals from ten
provinces of china with WD were recruited. Of them, 41 were
males and 21 were females, and their onset ages were from 5
to 61 years with a median age of 26 years. The full length of
ATP7B gene of the patients was sequenced and aligned to the
referred ATP7B gene sequence. The results suggested that all
the patents carried with mutations and 48 different mutations
were identified, of which 35 were missense, one was nonsense,
two were splicing, and 10 were frameshift (five insertion
and five deletion). Among these mutations, c.2333G>T
(32.3%), c.2975C>T (11.5%), and c.3443T>C (10.4%) were
the most prevalent mutants and c.2310C>G always linked with
c.2333G>T. The eighth, 11 th , and 18 th exons carried more
mutations (6/48, 5/48, and 5/48, respectively). After comparing
with the mutations reported previously, 19 out of the 48
mutations were novel made up of all the splicing and frameshift
mutations and 10 of 35 missense mutations. Two popular algorithms
were used to predict the effects of the novel mutations
on ATP7B function and most of the mutations were unfavorable
(18/21). Our study will broaden our knowledge about ATP7B
mutations in WD patients in China and further analysis need to
examine the relationship between these mutations and clinical
characteristics of WD.
Disclosures:
The following authors have nothing to disclose: Shan S. Peng, Lingxia QI,
yuanyuan Cui, Wenli Kong, Linyuan Ma, Yu Pan, Junqi Niu, Rui Hua
2108
UGT1A regulation by a member of the proto-oncogenic
miR-106b-25 cluster: hsa-miR-25
Sandra Kalthoff, Anja Winkler, Christian P. Strassburg; Department
of Internal Medicine, University Hospital Bonn, Bonn, Germany
Introduction: UDP-glucuronosyltransferases (UGTs) catalyze
the detoxification of xenobiotics including a variety of carcinogens.
Therefore, UGTs play an essential role in defence
mechanisms protecting the organism against the development
of cancer. Recent research has shown that deregulation of
miRNAs can contribute to cancer development. In hepatocellular
carcinoma (HCC) tissues, hsa-miR-25, for example, is
overexpressed and associated with a poor prognosis in HCC
patients. Aim of this study was to examine the influence of hsamiR-25
on UGT1A-expression. Methods: For luciferase assays,
reporter gene constructs containing different UGT1A promoters
(UGT1A1, UGT1A3, UGT1A4, UGT1A7, UGT1A9),
combined with luciferase sequence and the UGT1A 3’untranslated
region (UTR) sequence were generated. Every promoter
was combined with either the UGT1A 3’UTR-wild type(WT)
sequence or the UGT1A 3’UTR-SNP sequence (containing the
3 SNPs rs10929303, rs1042640 and rs8330). The constructs
were transfected together with 5 nM hsa-miR-25 into HepG2
cells. For RNA quantification HepG2 cells were treated with 5
nM hsa-miR-25 and isolated RNA was analyzed by TaqMan
PCR. Results: Transfection of hsa-miR-25 led to a significant
decrease in luciferase expression of the UGT1A1-, UGT1A3-,
UGT1A4- UGT1A7 and UGT1A9 reporter gene constructs (0.4-
0.5 fold). Interestingly, transfection of the construct containing
the UGT1A7 promoter combined with the UGT1A 3’UTR-SNP
sequence, led to a further reduction of luciferase expression
compared to the UGT1A 3 ‘UTR-WT sequence (SNP: 0.21 fold
compared to UGT1A 3’UTR-WT 0.42 fold). RNA analysis of
HepG2 cells revealed that mRNA expression of UGT1A1 (0.47
fold), UGT1A3 (0.49 fold), UGT1A4 (0.68 fold), UGT1A6
(0.6 fold), UGT1A7 (0.38 fold) and UGT1A9 (0.36 fold) were
significantly reduced by treatment with 5 nM hsa-miR-25. Conclusions:
This is the first demonstration of UGT1A regulation
by hsa-miR-25. All UGT1A genes were significantly downregulated
by treatment with 5 nM hsa-miR-25 on mRNA level as
well in reporter gene assays. The presence of 3 SNPs in the
UGT1A 3’untranslated region combined with the UGT1A7
promoter led to a further reduction of luciferase expression
indicating a suppressive effect of theses SNPs on the activity
of the major carcinogen detoxifying UGT1A7 enzyme in the
presence of overexpressed hsa-miR-25. The elucidated significant
reduction of UGT1A expression and the accompanying
affected detoxification capacity mediated by hsa-miR-25 may
represent a possible explanation for the potential association
between hsa-miR-25 and HCC development.
Disclosures:
Christian P. Strassburg - Advisory Committees or Review Panels: Novartis, Roche;
Speaking and Teaching: Novartis, Merz, MSD, Falk Pharma, BMS, Abbvie
The following authors have nothing to disclose: Sandra Kalthoff, Anja Winkler
2109
Wilson disease: gestational methyl group supplementation
affects hepatic oxidative phosphorylation and
movement disorder pathways in fetal mouse liver
Valentina Medici, Noreene Shibata, Charles H. Halsted, Janine M.
LaSalle; University of California Davis, Sacramento, CA
Background & Hypothesis. Wilson Disease (WD) is a condition
with highly variable phenotypic presentation, including
hepatic and neurological involvement. Previously we showed
that methionine metabolism, central in the regulation of methylation
reactions, is altered in the tx-j mouse model of WD and
therefore may influence the expression of genes involved in
liver damage. Since maternal diet is known to affect fetal gene
transcript levels through epigenetic mechanisms, we hypothesize
the phenotype of WD is influenced by maternal methylation
status. Methods. The tx-j mouse model of WD and wildtype
C3H female mice were started on choline-supplemented (choline
36 mmol/Kg of diet) and control diets (choline 8 mmol/
Kg of diet) 2 weeks before mating and through pregnancy
to embryonic day 17. Livers of 6-11 embryos/dam were collected
for RNA-sequencing. Pathway analysis was performed
by DAVID software. An additional group of offspring was
euthanized at 24 weeks to confirm persistence of changes in
gene expression in adult livers. Results. Comparing tx-j and
wildtype fetal livers, pathway analysis revealed differences
in transcript levels of nuclear encoded genes related to oxidative
phosphorylation, which overlapped with genes related to
neurological movement disorders, including Huntington’s and
Parkinson’s disease, both conditions listed in the differential
diagnosis of WD. Maternal choline supplementation was associated
with maintenance of gene transcript levels at control
group levels in fetal livers. Transcript levels of neurexin-1, central
to cell adhesion in the nervous system, was up-regulated
67-fold in fetal livers of tx-j mice compared to wildtype mice,
but were maintained at control levels in offspring of choline
supplemented dams. In 24-week old tx-j offspring mouse livers,
we found persistent changes in transcript levels of nuclear