a group of 14 university hospitals participated in this study and 72 patients were recruited between 1992 and 2006 . individuals with confirmed hnf1a - mutated hca who underwent curative resection of the tumor(s ) were eligible for inclusion in this study .
the mean age of the patients was 37.5 years ( ranging from 14 to 66 years ) , and there were only seven male patients . among
the women studied , 71% ( 34 of 48 ) had a history of oral contraception use , and 17 patients were missing this data .
there were 89 hnf1a - mutated hca samples from 72 patients , although 44 samples had been reported previously ( 8) . in 10 patients with multiple and sporadic tumors
, different somatic hnf1a mutations were observed in the genotyped nodules . for all patients , a representative part of the hca nodule , as well as of the nontumor liver when it was available ,
was immediately frozen in liquid nitrogen and stored at 80c until used for molecular studies .
the ethical committee of the saint - louis hospital ( paris , france ) approved the overall design of the study . in all 89 tumors , we sequenced the hnf1a gene using direct sequencing of the exons on genomic dna or rt - pcr to identify mutation , as previously described ( 5 ) .
all identified mutations ( tables 1 and 2 ) were confirmed by two independent pcr amplifications of genomic dna from tumor , and corresponding nontumor tissues were systematically sequenced to search for germline mutations . in samples with mutations affecting splicing sites , we characterized the transcripts by sequence analysis of the mrna transcripts . in 37 h - hca , the coding exons of myh ( exons 116 ) and ogg1 ( exons 17 ) were screened for mutations by direct sequencing of pcr products .
oligonucleotides used for all pcr reactions and experimental conditions are listed in supplementary tables 1 and 2 available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/db09-1819/dc1 .
allelic frequency of an ogg1 polymorphism observed in h - hca was compared with those obtained in a control group of non - hnf1a - mutated hepatocellular tumors ( 58 samples ) as previously described ( 13 ) and presented in supplementary table 3 ( available in an online appendix ) .
biallelic hnf1 mutations identified in hca cases of hca were previously described in * bluteau et al .
mody3 mutations were previously described in ellard and colclough ( 25 ) or bellann - chantelot et al .
del , deletion ; fs , frameshift ; ivs , intervening sequence ; loh , loss of heterozygosity ; ins , insertion ; nm , nonmutated .
germline hnf1 mutations in monoallelic mutated hca cases of hca were previously described in * bluteau et al .
mody3 mutations were previously described in ellard and colclough ( 25 ) or bellann - chantelot et al .
total rna was extracted using the rneasy kit ( qiagen , valencia , ca ) from all frozen samples of available hca and nontumor liver tissues .
rna isolated from 16 h - hca and 24 nontumor tissue samples was deemed to be of acceptable quality for quantitative rt - pcr experiments ( 23 ) .
specific assays for myh ( hs01014866_g1 ) , ogg1 ( hs00213454m1 ) , ape1 ( hs00172396_m1 ) , pol ( hs00160263_m1 ) , fabp1 ( hs00155026_m1 ) , ugt2b7 ( hs00426592_m1 ) , and r18s ( rrna , hs99999901_s1 ) were obtained from applied biosystems ( foster city , ca ) .
the relative amount of target gene mrnas was determined using the 2 method ( 24 ) .
the values obtained for fabp1 and ugt2b7 were expressed as the n - fold ratio of the gene expression in a tested sample as compared with the mean of 11 nontumor tissues .
only six nontumor tissues from patients with a h - hca were used with the myh , ogg1 , ape1 , and pol assays .
the values obtained were compared with the mean of seven nontumor tissues from patients with a non - hnf1a  mutated hca .
western blot analyses were performed as described ( 9 ) using two primary goat polyclonal anti - hnf1 antibodies ( santa cruz biotechnology ) , one detecting the amino terminus , and the other detecting the carboxy terminus of the protein ( sc-6548 and sc-6547 , respectively ) , used at the dilution 1:500 .
after centrifugation at 8,000 g for 10 min , the supernatant was assessed for reduced and total glutathione content with an apogsh glutathione colorimetric assay kit ( biovision , mountain view , ca ) , following the manufacturer 's protocol .
formalin - fixed , paraffin - embedded sections ( 5 m ) were mounted on glass slides .
sections were deparaffinized in xylene , rehydrated in a series of graded alcohol concentrations , and placed in pbs with 0.1% tween 20 .
immunostaining was performed using a dako envision system horseradish peroxidase ( dako cytomation , carpinteria , ca ) kit using primary antibody ( 1:2,000 dilution in pbs containing 1% bsa , incubated overnight at 4c ) against 4-hydroxynonenal protein adducts ( alpha diagnostics , san antonio , tx ) .
statistical analysis was performed using graphpad prism software ( version 4 , graphpad software , san diego , ca ) .
allelic frequencies of the ogg1 polymorphism in h - hca and the group control were compared using contingency tables with a fisher exact test .
in all 89 tumors , we sequenced the hnf1a gene using direct sequencing of the exons on genomic dna or rt - pcr to identify mutation , as previously described ( 5 ) .
all identified mutations ( tables 1 and 2 ) were confirmed by two independent pcr amplifications of genomic dna from tumor , and corresponding nontumor tissues were systematically sequenced to search for germline mutations . in samples with mutations affecting splicing sites , we characterized the transcripts by sequence analysis of the mrna transcripts . in 37 h - hca , the coding exons of myh ( exons 116 ) and ogg1 ( exons 17 ) were screened for mutations by direct sequencing of pcr products .
oligonucleotides used for all pcr reactions and experimental conditions are listed in supplementary tables 1 and 2 available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/db09-1819/dc1 .
allelic frequency of an ogg1 polymorphism observed in h - hca was compared with those obtained in a control group of non - hnf1a - mutated hepatocellular tumors ( 58 samples ) as previously described ( 13 ) and presented in supplementary table 3 ( available in an online appendix ) .
biallelic hnf1 mutations identified in hca cases of hca were previously described in * bluteau et al .
mody3 mutations were previously described in ellard and colclough ( 25 ) or bellann - chantelot et al .
del , deletion ; fs , frameshift ; ivs , intervening sequence ; loh , loss of heterozygosity ; ins , insertion ; nm , nonmutated .
germline hnf1 mutations in monoallelic mutated hca cases of hca were previously described in * bluteau et al .
mody3 mutations were previously described in ellard and colclough ( 25 ) or bellann - chantelot et al .
total rna was extracted using the rneasy kit ( qiagen , valencia , ca ) from all frozen samples of available hca and nontumor liver tissues .
rna isolated from 16 h - hca and 24 nontumor tissue samples was deemed to be of acceptable quality for quantitative rt - pcr experiments ( 23 ) .
specific assays for myh ( hs01014866_g1 ) , ogg1 ( hs00213454m1 ) , ape1 ( hs00172396_m1 ) , pol ( hs00160263_m1 ) , fabp1 ( hs00155026_m1 ) , ugt2b7 ( hs00426592_m1 ) , and r18s ( rrna , hs99999901_s1 ) were obtained from applied biosystems ( foster city , ca ) .
the relative amount of target gene mrnas was determined using the 2 method ( 24 ) .
the values obtained for fabp1 and ugt2b7 were expressed as the n - fold ratio of the gene expression in a tested sample as compared with the mean of 11 nontumor tissues .
only six nontumor tissues from patients with a h - hca were used with the myh , ogg1 , ape1 , and pol assays .
the values obtained were compared with the mean of seven nontumor tissues from patients with a non - hnf1a  mutated hca .
western blot analyses were performed as described ( 9 ) using two primary goat polyclonal anti - hnf1 antibodies ( santa cruz biotechnology ) , one detecting the amino terminus , and the other detecting the carboxy terminus of the protein ( sc-6548 and sc-6547 , respectively ) , used at the dilution 1:500 .
after centrifugation at 8,000 g for 10 min , the supernatant was assessed for reduced and total glutathione content with an apogsh glutathione colorimetric assay kit ( biovision , mountain view , ca ) , following the manufacturer 's protocol .
formalin - fixed , paraffin - embedded sections ( 5 m ) were mounted on glass slides .
sections were deparaffinized in xylene , rehydrated in a series of graded alcohol concentrations , and placed in pbs with 0.1% tween 20 .
immunostaining was performed using a dako envision system horseradish peroxidase ( dako cytomation , carpinteria , ca ) kit using primary antibody ( 1:2,000 dilution in pbs containing 1% bsa , incubated overnight at 4c ) against 4-hydroxynonenal protein adducts ( alpha diagnostics , san antonio , tx ) .
statistical analysis was performed using graphpad prism software ( version 4 , graphpad software , san diego , ca ) .
allelic frequencies of the ogg1 polymorphism in h - hca and the group control were compared using contingency tables with a fisher exact test .
among 89 hca samples with hnf1a alteration , mutations and deletion were biallelic in 84 ( 94% ) samples . to further analyze the spectrum of hnf1a mutations
, we included 151 hnf1a alterations with a firm somatic origin identified in 75 different hcas ; in contrast , all samples with a proven or highly suspected germline mutation were excluded from this first analysis ( fig .
. a : spectrum of 136 somatic hnf1a mutations observed in 75 hca samples ( top ) and in 364 mody3 individuals ( bottom ) ( data from ellard and colclough ) .
 , in - frame deletion ;  , in - frame duplication ;  , mutation in splicing site .
hca (  ) and mody3 (  ) histograms represent the percentage of total mutations observed in each domain ; substitutions and stops are represented in the upper part and the lower part , respectively .
significant differences between hca and mody3 individuals are indicated : * 0.05 > p > 0.01 ; * * 0.01 > p > 0.001 ; * * * p < 0.001 .
c : comparison of the number of stops in the transactivation domain ( 281631 ) vs. the rest of the protein observed in individuals with hca (  ) and mody3 (  ) . * * * significant difference ( p < 0.001 ) between the two populations .
d : spectrum of eight germline hnf1a mutations identified in h - hca ( top ) and five germline hnf1a mutations identified in monoallelic mutated hca ( bottom ) .
somatic alterations , 90% were point mutations , and the remaining 10% ( 15 of 151 ) were gene deletions as evidenced by the loss of heterozygosity at a hnf1a single nucleotide polymorphism(s ) compared with the corresponding nontumor tissues .
among the mutations , 57 were missense , 62 were frameshift and nonsense , and 12 were mutations affecting a splicing site .
next , we compared the spectrum of somatic hnf1a mutations identified in hca with 364 germline mutations previously described in individuals with mody3 without hca ( fig . 1a , lower panel , supplementary table 4 , available in an online appendix ) and by ellard and colclough ( 25 ) .
in both hca and mody3 , a hotspot mutation at codon 291 was present , whereas a hotspot mutation at codon 206 was specifically identified in hca ( fig .
as previously reported , codon 291 mutation is located in a polyc-8 tract , whereas codon 206 mutation is not located in a repeated sequence motif .
because codon 291 mutations represented 20% of the mutations in both spectra , we did not include them in the following analysis .
there was no significant difference ( p = 0.16 ) in the proportion of missense and truncating ( frameshift , nonsense , and mutations affecting a splicing site ) mutations in mody3 and hca spectra .
thus , there were 63 and 55% of missense mutations in mody3 and hca , respectively , and 37 and 45% of truncating mutations in mody3 and hca , respectively .
in contrast , the distribution of the mutations was significantly different in hca compared with the mody3 spectrum .
although almost all of the missense mutations were restricted to the pou - h domain ( p < 0.001 ) in hca , they were distributed in all hnf1 domains in individuals with mody3 ( fig .
also , in hca , truncating mutations were localized predominantly in the first 280 amino acids ( p < 0.001 ) , whereas they were distributed equally between the first 280 amino acids and the transactivation domain in the individuals with mody3 ( fig .
1c ) . in a second step , we analyzed the distribution of the 13 germline hnf1a mutations identified in patients with adenomas . in eight patients
, we identified a mutation of the second hnf1a allele in the corresponding tumor leading to a biallelic inactivation of hnf1 ( table 1 ) .
the corresponding germline mutations fit the distribution of the somatic hnf1a mutations : six of them were nonsense or frameshift occurring before codon 292 , and the remaining two mutations were missense affecting codon 272 , an amino acid found to be mutated by a somatic event ( fig .
in contrast , in four hca samples [ 372 ( r583q ) , 769 ( t525s ) , 966 ( l389v ) , and 998 ( r278q ) ] , we observed a germline hnf1a mutation without alteration of the second hnf1a allele .
all of these germline mutations were missense ; three of them localized in the transactivation domain , and the last one was at the end of the pou - h domain .
none of these mutations were described in the spectrum of somatic hnf1a mutations ; however , three of them ( r583q , t525s , and l389v ) have been previously reported in individuals with mody3 ( 21,25 ) .
thus , these four germline mutations did not fit the characteristic distribution of the somatic one .
moreover , the corresponding adenomas were classified in different molecular subgroups of hca : one of them was -catenin activated ( case 372 ) , two were inflammatory adenomas with or without a gp130 activating mutation ( cases 769 and 966 ) , and the last one remained unclassified ( case 998 ) .
most of the somatic mutations found in hca are predicted to inactivate the protein because they are almost all biallelic and frequently nonsense or missense affecting the pou - h domain that is essential for dna binding .
all of these mutations resulted in aberrant splicing , the outcome of which is either an aberrant protein with an early stop ( fig .
2a , b , and d ) , or a protein with an amino acid replacement and a deletion of five amino acids in the dna - binding domain ( fig .
then we assessed the protein level of mutant hnf1 in hca and compared it with that in nontumor tissue .
samples with mutations leading to an early stop contained little detectable hnf1 protein ( supplementary fig .
1 , available in an online appendix ; samples identified with arrows and data on gradient migration , not shown ) .
additionally , some samples harboring missense mutations exhibited a dramatic increase in the expression of hnf1 compared with normal liver ( supplementary fig .
consequences of the mutations at different splice sites in hnf1a . a : ivs12 a > t or ivs11 g > t mutations .
b : ivs2 + 1 g > a or ivs2 + 1 del 13 bp mutations .
the hnf1 transcription factor regulates expression of several key liver genes , including ugt2b7 and fabp1 ( 16,18 ) .
indeed , a shutdown of mrna expression for these target genes was observed in all patients with hcas tested with biallelic hnf1a mutations , as previously reported ( 4 ) .
an effect of monoallelic hnf1 mutants on expression of ugt2b7 and fabp1 has not been previously examined in liver tissues .
our data show that monoallelic mutations did not affect expression of fabp1 and ugt2b7 , which is not in favor of a dominant - negative effect ( fig .
however , these results can not completely exclude a subtle dosage effect or a low dominant effect .
in contrast to other solid tumors , point mutations were much more frequent in hca than were chromosome deletions .
the latter were found in only 10% of the patients with hcas . among the point mutations in hca
, we analyzed the proportion of different hnf1a nucleotide changes and found that g - to - t transversions , accounting for 36% of the cases , were the most common type ( fig .
4a ) . moreover , the distribution of nucleotide substitution subtypes in h - hca was strikingly different from that in mody3 ( fig .
4b ) but very similar to a spectrum of mutations known to be induced by genotoxic events , that is , in tp53-mutated lung cancers in smokers ( fig .
4c ) ( 26 ) . in addition , when the hnf1a nucleotide substitutions were partitioned between the two dna strands , we observed that g - to - t transversions were significantly more frequent on a nontranscribed strand ( p = 0.01 ) ( fig .
4d ) . taking these results together , we suggest that hnf1a somatic mutations showed a typical spectrum of mutations induced by the genotoxic exposure targeting a specific nucleotide sequence .
comparison of the mutation profiles in h - hca ( 67 transversions and transitions , a ) , mody3 ( 229 transversions and transitions , b ) , and tp53-mutated lung cancers ( 311 transversions and transitions , c ) and smokers ( 26 ) ( d ) .
repartitioning of transition and transversion mutations between the transcribed and the nontranscribed strands in h - hca .
black and white histograms indicate the number of nucleotide substitutions on the nontranscribed and the transcribed strand , respectively . *
significant ( p = 0.01 ) elevation in g - to - t transversions on the nontranscribed strand .
because both direct dna damage by an environmental agent and oxidative stress are known to result in g - to - t transversion mutations ( 27,28 ) , we also assessed oxidative stress markers in nontumor liver from six patients with h - hca and seven patients with non - hnf1a  mutated hca ( supplementary fig .
we observed no difference in total or reduced glutathione content , expression of four base excision dna repair genes , markers of oxidative dna damage ( 29 ) , or 4-hydroxynonenal protein adducts , a marker of lipid peroxidation ( supplementary fig .
these data showed a lack of evidence for elevated oxidative stress in livers of patients with h - hca as a potential source of dna mutations .
in addition , it is possible that a large number of g - to - t transversions could be caused by inefficient repair of dna damage due to mutations or polymorphisms in myh and ogg1 , genes involved in the repair of 8-hydroxyguanine ( 3032 ) . thus , we sequenced myh and ogg1 dna in 37 subjects with an hnf1a - mutated hca , and no mutations were detected in either gene . a known functional polymorphism in ogg1 ( s326c )
was detected in hnf1a - mutated hca subjects , albeit there was no difference in the frequency of this polymorphism compared with that in control subjects ( supplementary table 3 , available in an online appendix ) .
among 89 hca samples with hnf1a alteration , mutations and deletion were biallelic in 84 ( 94% ) samples . to further analyze the spectrum of hnf1a mutations
, we included 151 hnf1a alterations with a firm somatic origin identified in 75 different hcas ; in contrast , all samples with a proven or highly suspected germline mutation were excluded from this first analysis ( fig .
. a : spectrum of 136 somatic hnf1a mutations observed in 75 hca samples ( top ) and in 364 mody3 individuals ( bottom ) ( data from ellard and colclough ) .
 , in - frame deletion ;  , in - frame duplication ;  , mutation in splicing site .
hca (  ) and mody3 (  ) histograms represent the percentage of total mutations observed in each domain ; substitutions and stops are represented in the upper part and the lower part , respectively .
significant differences between hca and mody3 individuals are indicated : * 0.05 > p > 0.01 ; * * 0.01 > p > 0.001 ; * * * p < 0.001 .
c : comparison of the number of stops in the transactivation domain ( 281631 ) vs. the rest of the protein observed in individuals with hca (  ) and mody3 (  ) . * * * significant difference ( p < 0.001 ) between the two populations .
d : spectrum of eight germline hnf1a mutations identified in h - hca ( top ) and five germline hnf1a mutations identified in monoallelic mutated hca ( bottom ) .
somatic alterations , 90% were point mutations , and the remaining 10% ( 15 of 151 ) were gene deletions as evidenced by the loss of heterozygosity at a hnf1a single nucleotide polymorphism(s ) compared with the corresponding nontumor tissues .
among the mutations , 57 were missense , 62 were frameshift and nonsense , and 12 were mutations affecting a splicing site .
next , we compared the spectrum of somatic hnf1a mutations identified in hca with 364 germline mutations previously described in individuals with mody3 without hca ( fig . 1a , lower panel , supplementary table 4 , available in an online appendix ) and by ellard and colclough ( 25 ) .
in both hca and mody3 , a hotspot mutation at codon 291 was present , whereas a hotspot mutation at codon 206 was specifically identified in hca ( fig .
as previously reported , codon 291 mutation is located in a polyc-8 tract , whereas codon 206 mutation is not located in a repeated sequence motif .
because codon 291 mutations represented 20% of the mutations in both spectra , we did not include them in the following analysis .
there was no significant difference ( p = 0.16 ) in the proportion of missense and truncating ( frameshift , nonsense , and mutations affecting a splicing site ) mutations in mody3 and hca spectra .
thus , there were 63 and 55% of missense mutations in mody3 and hca , respectively , and 37 and 45% of truncating mutations in mody3 and hca , respectively .
in contrast , the distribution of the mutations was significantly different in hca compared with the mody3 spectrum .
although almost all of the missense mutations were restricted to the pou - h domain ( p < 0.001 ) in hca , they were distributed in all hnf1 domains in individuals with mody3 ( fig .
also , in hca , truncating mutations were localized predominantly in the first 280 amino acids ( p < 0.001 ) , whereas they were distributed equally between the first 280 amino acids and the transactivation domain in the individuals with mody3 ( fig .
in a second step , we analyzed the distribution of the 13 germline hnf1a mutations identified in patients with adenomas . in eight patients , we identified a mutation of the second hnf1a allele in the corresponding tumor leading to a biallelic inactivation of hnf1 ( table 1 ) .
the corresponding germline mutations fit the distribution of the somatic hnf1a mutations : six of them were nonsense or frameshift occurring before codon 292 , and the remaining two mutations were missense affecting codon 272 , an amino acid found to be mutated by a somatic event ( fig .
in contrast , in four hca samples [ 372 ( r583q ) , 769 ( t525s ) , 966 ( l389v ) , and 998 ( r278q ) ] , we observed a germline hnf1a mutation without alteration of the second hnf1a allele .
all of these germline mutations were missense ; three of them localized in the transactivation domain , and the last one was at the end of the pou - h domain .
none of these mutations were described in the spectrum of somatic hnf1a mutations ; however , three of them ( r583q , t525s , and l389v ) have been previously reported in individuals with mody3 ( 21,25 ) .
thus , these four germline mutations did not fit the characteristic distribution of the somatic one .
moreover , the corresponding adenomas were classified in different molecular subgroups of hca : one of them was -catenin activated ( case 372 ) , two were inflammatory adenomas with or without a gp130 activating mutation ( cases 769 and 966 ) , and the last one remained unclassified ( case 998 ) .
most of the somatic mutations found in hca are predicted to inactivate the protein because they are almost all biallelic and frequently nonsense or missense affecting the pou - h domain that is essential for dna binding .
all of these mutations resulted in aberrant splicing , the outcome of which is either an aberrant protein with an early stop ( fig .
2a , b , and d ) , or a protein with an amino acid replacement and a deletion of five amino acids in the dna - binding domain ( fig .
then we assessed the protein level of mutant hnf1 in hca and compared it with that in nontumor tissue .
samples with mutations leading to an early stop contained little detectable hnf1 protein ( supplementary fig .
1 , available in an online appendix ; samples identified with arrows and data on gradient migration , not shown ) .
additionally , some samples harboring missense mutations exhibited a dramatic increase in the expression of hnf1 compared with normal liver ( supplementary fig .
consequences of the mutations at different splice sites in hnf1a . a : ivs12 a > t or ivs11 g > t mutations .
b : ivs2 + 1 g > a or ivs2 + 1 del 13 bp mutations .
the hnf1 transcription factor regulates expression of several key liver genes , including ugt2b7 and fabp1 ( 16,18 ) .
indeed , a shutdown of mrna expression for these target genes was observed in all patients with hcas tested with biallelic hnf1a mutations , as previously reported ( 4 ) .
an effect of monoallelic hnf1 mutants on expression of ugt2b7 and fabp1 has not been previously examined in liver tissues .
our data show that monoallelic mutations did not affect expression of fabp1 and ugt2b7 , which is not in favor of a dominant - negative effect ( fig .
, these results can not completely exclude a subtle dosage effect or a low dominant effect .
in contrast to other solid tumors , point mutations were much more frequent in hca than were chromosome deletions .
the latter were found in only 10% of the patients with hcas . among the point mutations in hca
, we analyzed the proportion of different hnf1a nucleotide changes and found that g - to - t transversions , accounting for 36% of the cases , were the most common type ( fig .
4a ) . moreover , the distribution of nucleotide substitution subtypes in h - hca was strikingly different from that in mody3 ( fig .
4b ) but very similar to a spectrum of mutations known to be induced by genotoxic events , that is , in tp53-mutated lung cancers in smokers ( fig .
4c ) ( 26 ) . in addition , when the hnf1a nucleotide substitutions were partitioned between the two dna strands , we observed that g - to - t transversions were significantly more frequent on a nontranscribed strand ( p = 0.01 ) ( fig .
4d ) . taking these results together , we suggest that hnf1a somatic mutations showed a typical spectrum of mutations induced by the genotoxic exposure targeting a specific nucleotide sequence . comparison of the mutation profiles in h - hca ( 67 transversions and transitions , a ) , mody3 ( 229 transversions and transitions , b ) , and tp53-mutated lung cancers ( 311 transversions and transitions , c ) and smokers ( 26 ) ( d ) .
repartitioning of transition and transversion mutations between the transcribed and the nontranscribed strands in h - hca .
black and white histograms indicate the number of nucleotide substitutions on the nontranscribed and the transcribed strand , respectively .
* significant ( p = 0.01 ) elevation in g - to - t transversions on the nontranscribed strand .
because both direct dna damage by an environmental agent and oxidative stress are known to result in g - to - t transversion mutations ( 27,28 ) , we also assessed oxidative stress markers in nontumor liver from six patients with h - hca and seven patients with non - hnf1a  mutated hca ( supplementary fig .
2 , available in an online appendix ) . we observed no difference in total or reduced glutathione content , expression of four base excision dna repair genes , markers of oxidative dna damage ( 29 ) , or 4-hydroxynonenal protein adducts , a marker of lipid peroxidation ( supplementary fig .
these data showed a lack of evidence for elevated oxidative stress in livers of patients with h - hca as a potential source of dna mutations .
in addition , it is possible that a large number of g - to - t transversions could be caused by inefficient repair of dna damage due to mutations or polymorphisms in myh and ogg1 , genes involved in the repair of 8-hydroxyguanine ( 3032 ) . thus , we sequenced myh and ogg1 dna in 37 subjects with an hnf1a - mutated hca , and no mutations were detected in either gene . a known functional polymorphism in ogg1 ( s326c )
was detected in hnf1a - mutated hca subjects , albeit there was no difference in the frequency of this polymorphism compared with that in control subjects ( supplementary table 3 , available in an online appendix ) .
in this study we analyzed the spectrum of hnf1a mutations in hca and showed a significant difference in pattern in comparison with individuals with mody3 , both at the nucleotide and amino acid levels . in hca ,
location of the mutations is very restricted because almost all of the truncating mutations led to the loss of the transactivation domain and the missense mutations altered mainly the pou - h domain .
when we take into account the two largest series of hnf1a screening in mody3 ( 21,25 ) , only 48% of the germline mutations ( 117 of 720 are missense mutations in pou - h , and 227 of 720 are truncating mutations localized in the first 291 amino acids ) fit the features of the hnf1a somatic mutations .
this observation suggests that only a part of hnf1a mutations that are associated with diabetes could predispose to the development of a h - hca .
previous analysis of the mody3 mutations showed that the age at onset of diabetes is modulated according to the position of the mutation relative to the hnf1a isoforms : missense mutations located in exons 7 to 10 that affect only a or b isoforms of hnf1a are associated with the late onset of diabetes ( 21,22 ) .
interestingly , these mutations are predicted to have a less severe effect on the hnf1 function and are not found in hca .
similarly , we can hypothesize that frequent mody3 missense mutations located outside the pou - h domain and mutations truncating hnf1 after codon 291 are possibly less inactivating than the mutations found in hca .
in contrast to mody3 , in hca the lack of mutations leading to amino acid substitution in pou - s suggests that the pou - s and pou - h domains are functionally different .
chi et al . ( 20 ) found that the pou - s domain interacts with the pou - h domain in the recognition and binding on promoter sequences ; however , the pou - s domain is not conserved in homeobox transcription factors outside the pou family .
thus , based on the profile of the hnf1a mutations that occur in hca , it may be of interest to reanalyze clinical severity of the diabetes and putative associated phenotypes in patients with mody3 , taking into the account only a premature stop before codon 291 , missense restricted at pou - h , and mutations affecting splicing sites from exon 1 to 7 . interestingly , among the five hcas with a monoallelic germline hnf1a mutation , three of them were inflammatory .
we previously noted that inflammatory adenomas are associated with obesity ( 4 ) , and the present observation raised the question of an association with mody3 and possibly with other subtypes of adenomas .
we can hypothesize that according to the nature of their hnf1a germline mutation , patients with mody3 could be at risk of different subtypes of hca : mutations leading to a severe impaired hnf1 function predispose to the development of h - hca , whereas germline mutation with a mild functional consequence could predispose to inflammatory or  - catenin  activated hcas without a familial context . in consideration of the high risk of malignant transformation of the -catenin - activated hca , this observation could have important clinical consequences .
our analysis of the expression of mutant hnf1 proteins in liver tissue did not reveal a detectable level of truncated proteins , and most of the mutated proteins were not expressed .
moreover , mutations of both hnf1a alleles are required to observe a downregulation of the genes physiologically regulated by hnf1. these observations suggest that if a dominant - negative effect exists , as suggested by the in vitro analyses for particular hnf1a mutants ( 15 ) , it is not sufficient to shut down activity of hnf1 in vivo . accordingly
, other researchers ( 33,34 ) , including harries et al . ( 35 ) showed that hnf1a transcripts harboring a premature termination codon ( ptc ) were less expressed in lymphoblastoid cells than the normal allele resulting from a  nonsense - mediated mrna decay ,  which detects and degrades the transcripts with a ptc to prevent the synthesis of truncated proteins . in the present study
, we observed a decrease of mrna expression for 16 of 21 different mutants leading to ptc ( data not shown ) .
altogether , in hepatocytes , a dominant - negative effect of an hnf1 mutant is unlikely in vivo .
this study not only confirms , in a large series of adenomas , that the spectrum of hnf1a mutations in hca is different from that in individuals with mody3 , it also supports the hypothesis that hnf1a mutations in hca arise because of a genotoxic mechanism .
first , we observed a consistent hotspot mutation at the codon 206 of hnf1a in hca .
hotspots of transversion identified in tumors without a repeated nucleotide sequence context were previously suggested to be a hallmark of an exposure to a genotoxic compound , such as an r249s mutation in the tp53 gene in hepatocellular carcinoma ( hcc ) associated with an exposure to the aflatoxin b1 mycotoxin ( 36,37 ) .
second , hainaut and pfeifer ( 26 ) showed that there is a significant difference in the pattern of tp53 mutations in lung cancers between smokers and nonsmokers , with a 30% frequency of g - to - t transversions in smoking - related lung cancer vs. 10% in non - smoking  related lung cancer .
the overabundance of g - to - t transversions in smoking - related lung cancers was consistent with the studies of dna damage and mutations due to benzo[a]pyrene and other cigarette smoke - derived carcinogens and their metabolites ( 38,39 ) .
our observation of an uneven distribution among hnf1a nucleotide substitutions in hca and high frequency of g - to - t transversions is in many ways similar to that in lung cancer caused by tobacco ( fig .
4a ) . third , denissenko et al . ( 40 ) showed that the repair of benzo[a]pyrene diol epoxide adducts in the nontranscribed strand was slower than that in the transcribed strand and may explain the strand bias of transversions in cancer .
similarly , we observed that g - to - t transversions were significantly localized to the nontranscribed strand in h - hca , an additional argument in favor of a genotoxic effect and , possibly , a presence of bulky dna adducts .
we can hypothesize that it could be related to a particular genetic susceptibility and/or to an exposure to a specific toxic . according to the first hypothesis
, we searched for mutations in ogg1 and myh , both genes specifically involved in the repair of 8-hydroxyguanine , a product of oxidative dna damage that can also lead to g - to - t transversions ( 3032 ) .
in addition , we did not observe a higher level of oxidative stress in the livers of patients with a h - hca .
these results strongly suggest that exposure to an environmental agent appears to be the most likely event to explain the elevated rate of g - to - t transversions in h - hca .
because hca occurrence is highly associated with oral contraception , we can hypothesize that estrogen exposure could play a role in this mechanism .
this last hypothesis is supported by the facts that 1 ) the genotoxic activity of estrogen metabolites has been shown , particularly for catechol estrogens that may be oxidized to a reactive quinone capable of direct formation of dna adducts ( 41,42 ) ; and 2 ) germline heterozygous mutations of cyp1b1 , a key metabolism enzyme responsible for the formation of hydroxylated and genotoxic metabolites of estrogen , contribute to the development of h - hca in women using hormonal contraception ( 13 ) . in conclusion ,
when analyzing a large series of individuals with hcas , we observed a significant differences between hnf1a somatic mutations identified in those with hcas and the germline mutations identified in individuals with mody3 .
somatic mutations in hca predict a more inactivating profile , resulting in a complete loss of function of the protein when biallelic .
moreover , we showed that the origin of the mutations might be the result of dna damage caused by a genotoxic agent , possibly resulting from the metabolism of estrogen that could also be associated with a genetic susceptibility that remains to be identified .
 objectivematurity onset diabetes of the young type 3 ( mody3 ) is a consequence of heterozygous germline mutation in hnf1a . 
 a subtype of hepatocellular adenoma ( hca ) is also caused by biallelic somatic hnf1a mutations ( h - hca ) , and rare hca may be related to mody3 . to better understand a relationship between the development of mody3 and hca , we compared both germline and somatic spectra of hnf1a mutations.research design and methodswe compared 151 somatic hnf1a mutations in hca with 364 germline mutations described in mody3 . 
 we searched for genotoxic and oxidative stress features in hca and surrounding liver tissue.resultsa spectrum of hnf1a somatic mutations significantly differed from the germline changes in mody3 . in hca , we identified a specific hot spot at codon 206 , nonsense and frameshift mutations mainly in the nh2-terminal part , and almost all amino acid substitutions were restricted to the pou - h domain . 
 the high frequency of g - to - t tranversions , predominantly found on the nontranscribed dna strand , suggested a genotoxic mechanism . 
 however , no features of oxidative stress were observed in the nontumor liver tissue . 
 finally , in a few mody3 patients with hnf1a germline mutation leading to amino acid substitutions outside the pou - h domain , we identified a different subtype of hca either with a gp130 and/or ctnnb1 activating mutation.conclusionsgermline hnf1a mutations could be associated with different molecular subtypes of hca . 
 h - hca showed mutations profoundly inactivating hepatocyte nuclear factor-1 function ; they are associated with a genotoxic signature suggesting a specific toxicant exposure that could be associated with genetic predisposition . 
RESEARCH DESIGN AND METHODS
Mutation screening.
Quantitative RT-PCR.
Western blotting.
Determination of reduced and total glutathione levels.
Immunohistochemistry.
Statistical analysis.
RESULTS
Spectrum of 
Germline 
Functional consequences of 
HNF1A mutations in HCA are likely to arise due to a genotoxic event.
HNF1A spectrum of mutation is not related to measurable increase in oxidative stress in liver.
DISCUSSION
Supplementary Material