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Cancer Research 67, 2611-2616, March 15, 2007. doi: 10.1158/0008-5472.CAN-06-3947
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Association of CYP1B1 Germ Line Mutations with Hepatocyte Nuclear Factor 1{alpha}–Mutated Hepatocellular Adenoma

Emmanuelle Jeannot1,2, Karine Poussin1,2, Laurence Chiche5, Yannick Bacq6, Nathalie Sturm7, Jean-Yves Scoazec8, Catherine Buffet9, Jeanne Tran Van Nhieu10, Christine Bellanné-Chantelot3, Claudia de Toma2, Pierre Laurent-Puig4, Paulette Bioulac-Sage11 and Jessica Zucman-Rossi1,2

1 Inserm, U674, Génomique Fonctionnelle des Tumeurs Solides; 2 Université Paris 7, Denis Diderot, Institut Universitaire d'Hématologie, Centre d'Etude du Polymorphisme Humain; 3 Service de Cytogénétique AP-HP, Saint Antoine, Université Pierre et Marie Curie; 4 Inserm, U775, Université Paris 5, Paris, France; 5 CHU Caen, Service de Chirurgie, Caen Cedex, France; 6 Hôpital Trousseau, Service d'Hépatogastroentérologie, Tours, France; 7 CHU Albert Michallon, Laboratoire de Pathologie Cellulaire, Grenoble, France; 8 Hôpital Edouard Herriot, Service d'Anatomopathologie, Lyon, France; 9 AP-HP, CHU de Bicêtre, Service d'Hépatologie, Le Kremlin-Bicêtre, France; 10 AP-HP, Hôpital Henri-Mondor, Service d'Anatomopathologie, Créteil, France; and 11 Inserm, U889, Université Bordeaux 2, IFR66, CHU Bordeaux, Hôpital Pellegrin, Bordeaux, France

Requests for reprints: Jessica Zucman-Rossi, INSERM U674, 27 rue Juliette Dodu, 75010 Paris, France. Phone: 33-1537-25166; Fax: 33-1537-25158; E-mail: zucman{at}cephb.fr.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Biallelic somatic mutations of TCF1 coding for hepatocyte nuclear factor 1{alpha} (HNF1{alpha}) are found in 50% of the hepatocellular adenoma (HCA) cases usually associated with oral contraception. In rare cases, HNF1{alpha} germ line mutations could also predispose to familial adenomatosis. In order to identify new genetic factors predisposing to HNF1{alpha}-mutated HCA, we searched for mutations in genes involved in the metabolism of estrogen. For 10 genes (CYP1A1, CYP1A2, CYP3A4, CYP3A5, COMT, UGT2B7, NQO1, GSTM1, GSTP1, and GSTT1), we did not find mutations nor differences in the allele distribution among 32 women presenting HNF1{alpha}-mutated adenomas compared with 58 controls. In contrast, we identified a CYP1B1 germ line heterozygous mutation in 4 of 32 women presenting HNF1{alpha}-mutated adenomas compared with none in 58 controls. We confirmed these results with the identification of four additional CYP1B1 mutations in a second series of 26 cases. No mutations were found in the control group, which was extended to 98 individuals, and only a known rare genetic variant was observed in two controls (P = 0.0003). We did an ethoxyresorufin O-deethylase assay to evaluate the functional consequence of the CYP1B1 mutations. We found reduced enzymatic activity in each CYP1B1 variant. In addition, an E229K CYP1B1 mutation was found in a woman with a germ line HNF1{alpha} mutation in a familial adenomatosis context. In this large family, all three patients with adenomatosis bore both HNF1 and CYP1B1 germ line mutations. In conclusion, our data suggested that CYP1B1 germ line–inactivating mutations might increase the incidence of HCA in women with HNF1{alpha} mutations. [Cancer Res 2007;67(6):2611–6]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Use of oral contraception is an important risk factor in the development of hepatocellular adenoma (HCA), a rare benign liver tumor (1, 2). HCA usually occurs as a single tumor, but multiple nodules could be observed and the presence of more than 10 nodules in the liver characterizes liver adenomatosis (3). Most HCAs usually remain stable but could increase in size after withdrawal of oral contraceptives, although they rarely regress (4, 5). The search for genetic alterations in HCA led to the identification of mutations in two genes: (a) biallelic inactivating mutations of the TCF1 gene (chromosomal locus, 12q24.31) coding for the transcription factor hepatocyte nuclear factor 1{alpha} (HNF1{alpha}; ref. 6), and at a lower frequency (b) activating mutations in the CTNNB1 gene coding for ß-catenin (7, 8). Recently, mutation screening of these two genes in 96 HCAs allowed us to identify strong genotype-phenotype correlations and we proposed a new classification of HCA tumors (8). Biallelic mutations of the gene coding for HNF1{alpha} were found in half of the HCA cases. They define a homogeneous group of tumors representing the most usual form of adenoma characterized by a marked steatosis (8). In 84% of these cases, both HNF1{alpha} mutations in tumors are of somatic origin. In the remaining cases, one of the HNF1{alpha} mutations was a germ line mutation, and patients developed adenomatosis. Familial analyses done in four independent germ line adenomatoses showed that all 11 relatives who developed adenomatosis displayed a germ line HNF1{alpha} mutation (9, 10). However, in these families, 16 individuals with a germ line HNF1{alpha} mutation did not develop any liver tumors. These observations suggested that germ line HNF1{alpha} mutations predisposed to liver adenomatosis with an incomplete penetrance and raised the possible existence of modifier genes.

HCA with somatic HNF1{alpha} mutations occur mostly in women (97% of the cases), with 77% using oral contraceptives (8). Among these cases, we identified four women who developed multiple HNF1{alpha}-mutated adenomas. These results raised the question of a potential role for a genetic predisposition to develop HNF1{alpha}-mutated adenomas, possibly related to the metabolism of estrogen.

In order to identify a genetic predisposition in women with somatic HNF1{alpha}-mutated HCA or to find a gene modifying the penetrance of adenomatosis in germ line HNF1{alpha}-mutated patients, we search for alterations in candidate genes involved in the metabolism of estrogen. We screened a first group of 32 women with HNF1{alpha}-mutated HCA for genetic variants by direct sequencing of 11 genes (CYP1A1, CYP1A2, CYP1B1, CYP3A4, CYP3A5, COMT, UGT2B7, NQO1, GSTM1, GSTP1, and GSTT1). The genotypes were compared with those of 58 control women with a HNF1{alpha} nonmutated benign hepatocellular tumor. We validated the identified variants in a second group of 26 women presenting a HNF1{alpha}-mutated HCA and 40 control women.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients. A group of 16 French university hospitals participated in this study. Criteria for patient inclusion were women with confirmed HNF1{alpha}-mutated HCA, and as control, women with non–HNF1{alpha}-mutated benign liver tumors surgically treated in the same hospitals and over the same period. A first group of patients was recruited between 1992 and 2003, and consisted of 32 women with HNF1{alpha}-mutated HCA and 58 women in the control group (25 HCA, 24 focal nodular hyperplasias, and 9 telangiectatic focal nodular hyperplasias). A second group of patients was recruited from 2004 to 2006, and consisted of 26 women with HNF1{alpha}-mutated HCA and 40 women in the control group (23 adenomas, 11 focal nodular hyperplasias, and 6 telangiectatic focal nodular hyperplasias). In the overall series, the mean age at diagnosis was 37 years old (ranging from 14 to 61) in HNF1{alpha}-mutated HCA cases and 38 years old (ranging from 12 to 62) in controls. Among the patients with a HNF1{alpha}-mutated tumor, 72% used oral contraception (12 cases with missing data), in 6 cases, one HNF1{alpha} mutation was a germ line mutation (data was unavailable in 3 cases). In the control group, 89% of the women used oral contraception (11 cases with missing data). Among the 156 women, 38 cases of HNF1{alpha}-mutated adenomas and 31 cases of nonmutated tumors were previously described in Zucman-Rossi et al. and Bioulac-Sage et al., respectively (8, 11). All but four women were of European origin.

A family extensively described in Reznik et al. was also completely genotyped in the present work (10). Briefly, the proband, a 17-year-old female, presented hepatomegaly. She developed early onset diabetes at 32 years of age. Liver nodules were discovered and liver adenomatosis was confirmed by pathologic diagnosis, which led to a familial screening for diabetes and adenomatosis. Germ line HNF1{alpha} mutations were found in 11 individuals and 3 of them were affected by adenomatosis defined by the presence of more than 10 lesions (Fig. 1 ).


Figure 1
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  		Figure 1. Cosegregation of liver adenomatosis and CYP1B1 mutations. Patients are identified by generation numbers (roman numerals, left) and individual number within each generation below the symbol. The second line under the symbols corresponds to the age of patients. Age in brackets corresponds to the age of death. The HNF1{alpha} and CYP1B1 genotypes of each tested patient are indicated on the third and fourth line, respectively (+/–, patients carrying either mutation; –/–, patients not carrying mutations).

 
For all cases, a representative portion of the HCA nodule, as well as of the nontumor liver, was immediately frozen in liquid nitrogen and stored at –80°C until use for molecular studies. All diagnoses were reviewed by a panel of liver pathologists and mutations were searched in the HNF1{alpha} gene as previously described (8). In the present work, gene variants were searched in nontumor DNA and germ line origin was validated by genotyping lymphocytic DNA. All patients were recruited in accordance with French laws and institutional ethical guidelines. The ethical committee of the Saint-Louis Hospital (Paris, France) approved the overall design of the study. Newly identified mutations were also tested in 92 unrelated women from the Centre d'Etude du Polymorphisme Humain families. The E229K CYP1B1 mutation was also tested in 125 independent and Caucasian women from the Human Genome Diversity Project (12).

Mutation screening. Eight genes were screened for mutations or polymorphisms by direct sequencing of PCR products: CYP1A1 (exons 2–7, 3'UTR), CYP1A2 (polymorphism IVS1 + 734G>A and exons 2–7), CYP3A4 and CYP3A5 (exons 1–13), CYP1B1 (exons 1–3), COMT, NQO1, and UGT2B7 (exons 1–6). Sequencing was done as previously described (6). All mutations were confirmed by DNA sequencing from two independent PCR amplifications. The screening for GSTM1, GSTP1, and GSTT1 variants was done according to the protocol described by Cabelguenne et al. (13). Oligonucleotide sequences used for all PCR and programs are provided in Supplemental Table S1.

Screening for the E229K CYP1B1 mutation in the Human Genome Diversity Project population was done by a PCR-RFLP assay. Briefly, a 517-bp fragment was generated with the PCR primers (forward) 5'-TGGCCAACGTCATGAGTGCC-3' and (reverse) 5'ACTCAGCATATTCTGTCTCTAC-3'. The generated fragment contains two EarI sites; the E229K CYP1B1 mutation removes one of the EarI sites.

Mutagenesis. CYP1B1*1 allele into the p-dihydrofolate reductase mammalian expression vector (14) was a generous gift from T. Friedberg, University of Dundee (Dundee, United Kingdom). Wild-type and mutated CYP1B1*1, CYP1B1*2, and CYP1B1*3 alleles were generated using the QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer's conditions. Wild-type CYP1B1*1, CYP1B1*2, and CYP1B1*3 alleles are described in http://www.imm.ki.se/CYPalleles/cyp1b1.htm. The expression of CYP1B1 alleles in mammalian COS-1 cells was done according to Bandiera's conditions using LipofectAMINE reagent (Invitrogen, San Diego, CA) and the cotransfection of a pSV-ß-galactosidase vector (15).

Ethoxyresorufin O-deethylase assay on whole cells. The ethoxyresorufin O-deethylase (EROD) assay was done according to Bandiera's conditions and is briefly described below. After transfection (48 h), cells were incubated with a 400 nmol/L solution of 7-ethoxyresorufin (Sigma-Aldrich, St. Louis, MO) in 1x PBS at 37°C/5% CO2. The fluorescent product resorufin is released from EROD by CYP1B1. Fluorescence was measured in duplicate aliquots of supernatant at 0, 5, and 10 min (excitation, 544 nm; emission, 590 nm). The amount of resorufin formed was calculated by comparison with a standard curve ranging from 30 µmol/L to 3.75 nmol/L (16).

Quantification of protein expression. After resorufin analysis, cells were lysed (lysis reagent as supplied for the ß-Gal Reporter Gene Assay chemiluminescence kit; Roche Molecular Biochemicals, Mannheim, Germany) and the concentration of protein was determined using a bicinchoninic acid protein assay (BCA Protein Assay Kit; Pierce, Rockford, IL). Lysates were subjected to immunoblot analysis employing a polyclonal anti-human CYP1B1 primary antibody (BD Gentest, Bedford, MA) according to the instructions of the manufacturer. The EROD assay was normalized to the transfection efficiency determined with the ß-Gal Reporter Gene Assay chemiluminescence kit (Roche Molecular Biochemicals) according to the instructions of the manufacturer.

Statistical analysis. Statistical analyses were done using the GraphPad prism software version 4 (GraphPad Software, Inc., San Diego, CA). Frequencies of CYP1B1 mutations in patients with HNF1{alpha} mutations and in control populations were compared in contingency tables using a Fisher exact test. The different activities of the mutated and nonmutated alleles of CYP1B1 were compared with a two-tailed, unpaired t test with 95% confidence intervals.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Genotype analysis. We scanned for genetic variants in the CYP1A1, CYP1A2, CYP3A4, CYP3A5, COMT, NQO1, and UGT2B7 genes in the first series of patients including 32 women with HNF1{alpha}-mutated tumors and 58 women presenting non–HNF1{alpha}-mutated benign liver tumor. The allele frequencies of all identified coding single nucleotide polymorphisms in the two patient groups are provided in Table 1 . We did not observe significant differences between the two groups and these frequencies were similar to those from the SNP500 database. Moreover, we did not identify new variants in these genes when compared with the genetic databases available.


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  		Table 1. Allele frequencies of all tested single nucleotide polymorphisms in the two groups of patients

 
Identification of CYP1B1 mutations in patients with HNF1{alpha}-mutated adenoma. Among the first series of 32 women with a HNF1{alpha}-mutated adenoma, we identified a heterozygous CYP1B1 germ line mutation in four individuals. No mutations or rare variants were observed in the 58 women presenting non–HNF1{alpha}-mutated benign liver tumor. Sequencing a second series of 26 cases with a HNF1{alpha}-mutated HCA, we identified four additional women with a CYP1B1 germ line mutation. None of the CYP1B1-identified mutations were found in single nucleotide polymorphism–available databases (NIEHS, SNP500, dbSNP, Panther, Ensembl). No mutations were found in 40 additional women presenting non–HNF1{alpha}-mutated benign liver tumor (Table 2 ). However, among this second control group, we identified two Caucasian individuals with a rare CYP1B1 heterozygous variant leading to an amino acid substitution at codon 81 (Y81N), which was never found in women presenting HNF1{alpha}-mutated HCA (Table 2). The Y81N polymorphism is characterized as a rare variant in the SNP500 database with a 2% allelic frequency in the Caucasian population. Taken together, we found a significant association between CYP1B1 germ line mutation and the occurrence of HNF1{alpha}-mutated adenoma (P = 0.0003, Fisher exact test). The eight mutated individuals identified corresponded with four different nucleotide mutations. An amino acid substitution at codon 229 (E229K) was observed in five cases. Two other amino acid substitutions (P52L and G329S) were found. The last mutation was a small deletion of 13 nucleotides leading to a frameshift (R355fs). We tested a third control group of 92 independent women from the Centre d'Etude du Polymorphisme Humain collection for these mutations and none were found. Because the E229K CYP1B1 mutation was observed in five cases, we genotyped 125 additional Caucasian women from the Human Genome Diversity Project collection. No E229K CYP1B1 mutation was observed in this population and we concluded that the E229K substitution was a mutation.


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  		Table 2. CYP1B1 germ line mutations identified in tumors

 
Among the women with HNF1{alpha}-mutated HCA, there were no significant differences in clinical characteristics (oral contraceptive use, age, number of nodules) when comparing CYP1B1-mutated and nonmutated women. One of the E229K CYP1B1 mutations was found in a woman with a germ line HNF1{alpha} mutation in a familial adenomatosis context. We genotyped 16 members of her family and identified the mutation in eight relatives (Fig. 1). We observed that the three cases with an adenomatosis (II-2, II-6, III-11) had both CYP1B1 and HNF1{alpha} mutations and only a 31-year-old man (III-6) without tumors presented both CYP1B1 and HNF1{alpha} mutations.

Finally, no differences in the frequency of common CYP1B1 polymorphisms and alleles were observed between the 58 patients with HNF1{alpha}-mutated HCA and the 98 control women presenting a nonmutated benign liver tumor (Supplemental Table S2).

Identified mutations decrease CYP1B1 enzymatic activity. The three different CYP1B1 mutations identified change evolutionarily conserved amino acids in mammals (Fig. 2 ) and the modified amino acids were all located at either the hinge region or in some putative substrate recognition sites (17). We therefore tested for the functional consequences of the different mutations using the 7-EROD assay (15). The allelic context of the mutations P52L, E229K, G329S, and R355fs were determined in patients by cloning and sequencing CYP1B1 cDNA from nontumor tissues of the corresponding patient. Interestingly, E229K were located in the same allele *2 in all five mutated patients. Each mutation was introduced in its respective allelic context by site-directed mutagenesis in a p-dihydrofolate reductase plasmid. Wild-type and mutated constructs were transfected in COS-1 cells, which does not express CYP1B1 (15). CYP1B1 expression was assayed by immunoblotting 48 h after transfection. A distinct and comparable signal was obtained by Western blotting in cells transfected with all the CYP1B1 wild-type and mutated alleles. The CYP1B1*1-R355fs mutation was undetectable with the antibody because the variant encodes a protein in which the COOH-terminal which contains the epitope is modified by the frameshift mutation (Fig. 3 ). Using quantitative reverse transcription-PCR, we tested the cells transfected with CYP1B1*1-R355fs and observed that CYP1B1 was expressed at a similar mRNA level when compared with wild-type allele *1 (data not shown). The EROD activity of all normal and mutated alleles was determined after normalization to the transfection efficiency as previously described (15). Because CYP1B1*3 was the most frequently found allele in the populations tested (39% allelic frequency in all patients), we arbitrarily normalized our EROD assay results to the activity of this allele. We did not find any significant differences of activity between the three wild-type alleles, CYP1B1*1, *2, and *3. In contrast, all mutated alleles exhibited a significantly reduced activity when compared with their corresponding wild-type allele (Fig. 3).


Figure 2
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  		Figure 2. Protein diagram showing the locations of the mutations. The transmembrane segment, the hinge region, the meander region, the heme binding domain, and the conserved helixes I, J, K, and L were located according to Mammen et al. (29). The putative CYP1B1 substrate recognition sites, originally determined by Gotoh (30), were located according Watanabe et al. (17). The alignments of amino acids of Human, Rat, Mouse, Opossum, and Drosophila CYP1B1 gene were represented below each substitution.

 

Figure 3
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  		Figure 3. Effect of E229K, G329S, P52L, and R355fs mutations on CYP1B1 catalytic activity. *1, *2 and *3 are the different nonmutated alleles of CYP1B1, defined by three polymorphisms. Each mutation was replaced in its haplotypic context, E229K and G329S in *2 allele, P52L in *3 allele, and R355fs in *1 allele (see Materials and Methods). The EROD activity is expressed as picomoles of resorufin per milligram of protein per minute, and normalized to the total ß-galactosidase activity representing transfection efficiency. Columns, means; bars, SD (n = 4 replicates). EROD activity of the CYP1B1*3 allele was used as a reference and fixed to 1. Corresponding immunoblot analysis using an anti-CYP1B1 primary antibody (arrow).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study identified CYP1B1 germ line heterozygous mutations in 14% of the women presenting a HNF1{alpha}-mutated HCA. All identified CYP1B1 mutations led to a decreased enzymatic activity as tested using the EROD assay. We also identified a rare natural polymorphism (Y81N) in two women from the control group. Curiously, the Y81N CYP1B1 variant exhibited a reduced enzymatic activity as shown using the EROD test (Supplemental Fig. S1). These results suggest that in vivo susceptibility to develop HNF1{alpha}-mutated HCA was not only dependent on the in vitro EROD activity test and should probably be addressed with specific substrates. In fact, the EROD assay estimates the CYP1B1 activity by measuring the rate of cleaved resorufin, but it may only partially reflect the hydroxylation of endogenous substrates. We can hypothesize that, in vivo, the effects of the E229K and P52L CYP1B1 mutations would have a more important effect than the Y81N polymorphism towards endogenous substrates such as estrogens. We propose that CYP1B1 variants affecting specific domains such the hinge region or substrate recognition sites may act as potent genetic factors predisposing to the development of HNF1{alpha}-mutated HCA in sporadic and familial cases.

CYP1B1 mutations were first identified by Stoilov and collaborators in primary congenital glaucoma (PCG; OMIM 231300; ref. 18). PCG is an autosomal recessive disorder that is associated with developmental defects of the anterior chamber (19) with an increase of the intraocular pressure leading to optic nerve damage and blindness. In the families presented by Stoilov et al., the CYP1B1 mutations were homozygous and cosegregated in the affected, but normal, members of the families. As shown in the present work using the EROD assay, the CYP1B1 mutations observed in PCG are expected to inactivate the cytochrome P450 activity (18). However, the precise mechanism by which CYP1B1 inactivation can lead to PCG remains unknown. Two CYP1B1 mutations observed in women with HNF1{alpha}-mutated HCA (E229K and R355fs) have already been reported in PCG. Another CYP1B1 mutation (P52L) observed in a woman with HNF1{alpha} HCA was recently identified in one individual in the Lopez-Garrido et al. control group (20). In contrast, the remaining mutation (G329S) observed in HCA had never been described before.

CYP1B1 is also responsible for hormone metabolism and the hydroxylation of both endogenous and exogenous molecules that could lead to the formation of toxic metabolites [for review, see Sissung et al. (21)]. The up-regulation of CYP1B1 is mediated by some sex hormones and their metabolites in addition to environmental carcinogens, such as polyaromatic hydrocarbons. Once up-regulated, CYP1B1 catalyzes the conversion of steroid hormones and exogenous substrates into hydroxylated metabolites. Depending on the hydroxylated molecule, this step of hydroxylation may increase the genotoxic and oxidative load on the cell and modulate cell signaling. This could further explain the role of CYP1B1 in neoplastic progression. CYP1B1 is responsible for the hydroxylation of estradiol or estrone, and may form chemically reactive catechol estrogens. Among the different catechol estrogens, CYP1B1 mainly catalyzes the formation of 4-OH-estradiol (22), which may directly bind to DNA. A majority of the catechol estrogens are inactivated by COMT, which catalyzes O-methylation. If this inactivation is incomplete, the catechol estrogens may be oxidized to reactive quinone capable of direct formation of DNA adducts. This oxidation step also generates free radical formation.

Several studies showed that CYP1B1 was implicated in the occurrence of tumors. First of all, Liehr and Ricci observed a significant and higher level of 4-hydroxylation of estradiol in breast cancer microsomes compared with a very low level of 4-hydroxylation in normal breast tissue (23). Moreover, immunohistochemical studies of CYP1B1 showed enhanced CYP1B1 in several types of human cancer including breast cancer (24, 25).

Several polymorphisms within the CYP1B1 gene have also been implicated in the risk of different cancers. To date, the association between CYP1B1 polymorphisms and the metabolism of estrogen is unclear [for review, see Sissung et al. (21)]. To our knowledge, this study is the first to associate the decreased activity of CYP1B1 with tumor occurrence in humans. Because CYP1B1 is poorly expressed in the liver (24, 26, 27), we suspect that the reduced CYP1B1 activity observed in our patients might be related to a peripheral effect of the perturbed estrogen metabolism. In this scenario, decreased CYP1B1 activity in extrahepatic organs would lead to a saturation of hepatic cytochrome P450 and potentially disturb the metabolism of estrogen in the liver. We propose a mechanism through an estrogen metabolite genotoxic effect leading to HNF1{alpha} mutations at the origin of HCA. This hypothesis is consistent with the high rate of point mutations, and more precisely, of nucleotide tranversions observed in the spectrum of somatic HNF1{alpha} mutations identified in HCA (8).

In conclusion, CYP1B1 germ line–inactivating mutations seem to be predisposed to the development of HNF1{alpha}-mutated HCA in women. In addition, it seems to modify the penetrance of the liver adenomatosis phenotype in HNF1{alpha} germ line–mutated patients. However, the mechanism by which CYP1B1 inactivation predisposes to the development of HNF1{alpha}-mutated benign liver tumors remains to be elucidated.


   Acknowledgments
 
Grant support: ARC grant no. 5188, INSERM "Réseau de recherche clinique et en santé des populations", Société Nationale Française de Gastroentérologie, and the Fondation de France. E. Jeannot is a recipient of an ARC fellowship.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We warmly thank all the other participants to the GENTHEP (Groupe d'étude Génétique des Tumeurs Hépatiques) network: Charles Balabaud, Michel Beaugrand, Jordi Bruix, Jacques Belghiti, Jean Frédéric Blanc, Pascal Bourlier, Paul Calès, Denis Castaing, Chen Liu, Marie Pierre Chenard-Neu, Daniel Cherqui, Laurence Chiche, Valérie Costes, Thong Dao, Daniel Dhumeaux, Amar Paul Dhillon, Jérôme Dumortier, Olivier Ernst, Monique Fabre, Dominique Franco, Frédéric Gauthier, Jean Gugenheim, Catherine Guettier, Emmanuel Jacquemin, Daniel Jaeck, Christophe Laurent, Brigitte Le bail, Sébastien Lepreux, Emmanuelle Leteurtre, Sophie Michalak, Anne de Muret, Frédéric Oberti, Valérie Paradis, Danielle Pariente, Christian Partensky, François Paye, François-René Pruvost, Alberto Quaglia, Pierre Rousselot, Anne Rullier, Antonio Sa Cunha, Marie Christine Saint-Paul, Jean Saric, Janick Selves, Elie Serge Zafrani, and Dominique Wendum. We thank Cristel Thomas and Philippe Bois for critical reading of this manuscript; Thomas Friedberg and Olivier Bernard, who provided protocols and cell lines; Lucille Mellottee, Sandra Rebouissou, and Hung Bui for their technical help; and all the clinicians, surgeons, and pathologists of the INSERM GENTHEP network "Genetics of Hepatocellular Tumors."


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 10/26/06. Revised 12/ 7/06. Accepted 1/ 8/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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