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Environmental Contribution to Pathogenesis of Cyst Formation in Autosomal-Dominant Polycystic Liver Diseases
GASTROENTEROLOGY 2012;142:e26 – e30
CORRESPONDENCE Readers may submit letters to the editor concerning articles that appeared in GASTROENTEROLOGY within one month of publication. Detailed guidelines regarding the content are included in the Instructions to Authors.
Environmental Contribution to Pathogenesis of Cyst Formation in Autosomal-Dominant Polycystic Liver Diseases Dear Sir:
We read with interest the paper by Janssen et al,1 which elegantly demonstrates that, among patients with polycystic liver disease (PCLD) carrying a heterozygous germline mutation in the PRKCSH gene, somatic mutations (second hits) are highly and widely detectable in the liver cyst epithelia with the loss of heterozygosity being the mechanism underling cyst formation through decreased hepatocystin expression in 67% of the cases. The authors conclude that autosomal-dominant PCLD is recessive at the cellular level and a loss of functional PRKCSH is an important step in cystogenesis.1 Regarding the liver involvement, the autosomaldominantly inherited polycystic diseases PCLD and autosomal-dominant polycystic kidney disease (ADPKD) could be considered clinically homogeneous entities characterized by a massive liver derangement with multiple cysts, the severity being greater in women than in men and correlating with the number of pregnancies and estrogen use.2,3 These observations are consistent with a role of estrogens in the development of PCLDs and support the similarity in the mechanism of liver cyst formation between ADPKD and PCLD.2 The recent findings by Janssen et al, together with several clinical observations, promote the need to elucidate the role of environmental factors in the phenotypic manifestations of inherited autosomal dominant PCLDs. Second somatic mutations underlie the liver cyst formation both in PCLD and in ADPKD, with the loss of heterozygosity being more frequent in PCLD.1,4 Posttrascriptional modifications of polycystins (PC) or hepatocystin could play a pivotal role in development and evolution of the PCLDs. Recently, we demonstrated that in cholangiocytes, 17-estradiol triggers PCs proteolysis associated with a proliferative cell response.5 Estrogens are able to regulate several cell proteolytic complexes. Epithelium lining the hepatic cysts of patients with ADPKD showed a higher expression of receptors for estrogen and insulin-like growth factor 1 with respect to normal cholangiocytes and a strong proliferative response to estrogen administration.3 This evidence provides a scientific basis for clinical observations that in ADPKD the formation and growth of liver cysts are faster in women than in men and are linked to estrogen stimulation. However, the expression of estrogen receptors in PCLD cyst epithelia has been demonstrated not to be
increased with respect to normal cholangiocytes, suggesting a divergence in the mechanisms driving cyst formation between PCLD and ADPKD.6 Because lowering PKD1 expression by 13%–20% of functional transcripts is sufficient to cause polycystic disease, it was proposed that in patients heterozygous for PKD1 (ADPKD patients), reduction of the normal allele products below a critical level, owing to genetic factors, environmental agents and stochastic, can determine the formation of cysts and clinical manifestations of the ADPKD.7 A high mutation rate might explain the high number of cysts observed in PCLD1 and ADPKD.4 The malfunction of PC-1 induces a premature transition from G1 phase to S phase, an increase of proliferation and apoptosis through activation of the mammalian target of rapamycin.8 In addition, we recently demonstrated how a reduction of PC-1 expression of 35% is associated with activation of cholangiocyte proliferation machinery.5 Previous investigations elucidated that, although the pathogenesis of PCLD involves overexpression of growth factor receptors and loss of adhesion, proliferation or deregulated apoptosis do not seem to be implicated.6 However, it has been argued that, in PCLD, cysts grow in a nonlinear fashion.6 Therefore, the pathogenesis of liver cyst development and the cause of the high mutation rate in autosomal-dominant PCLDs are not completely elucidated. On the basis of our recent findings,3,5 we hypothesize that estrogens, by acting on heterozygote susceptible cells of ADPKD patients, reduce the functional levels of PC-1 through posttranscriptional proteolytic modifications below a critical cutoff, thus determining an increased proliferation and finally a high probability of second somatic mutations. Cells undergoing a second mutation can further proliferate and form cysts, determining also a selection of cells sensitive to estrogens through a process of co-segregation of related features.3 Our speculations could apply in the ADPKD pathogenesis, although it is not immediately transposable in PCLD pathogenesis. However, in light of the common pathologic and clinical traits of PCLD and ADPKD, further investigations on posttranscriptional modifications of hepatocystin sustained by environmental stimuli could contribute to elucidate the complex pathogenesis of PCLD. VINCENZO CARDINALE DOMENICO ALVARO University Sapienza of Rome Division of Gastroenterology Department of Medico-Surgical Sciences and Biotechnologies Fondazione Eleonora Lorillard Spencer Cenci Polo Pontino Rome, Italy
March 2012
CORRESPONDENCE
1. 2. 3. 4. 5. 6. 7.
Janssen MJ, et al. Gastroenterology 2011;141:2056 –2063. Qian Q, et al. Hepatology 2003;37:164 –171. Alvaro D, et al. Am J Pathol 2008;172:321–332. Watnick TJ, et al. Mol Cell 1998;2:247–251. Torrice A, et al. Dig Liver Dis 2010;42:377–385. Waanders E, et al. Mod Pathol 2008;21:1293–1302. Lantinga-van Leeuwen IS, et al. Hum Mol Genet 2004;13:3069 – 3077. 8. Weimbs T. Cell Cycle 2006;5:2425–2429.
Conflicts of interests The authors disclose no conflicts. doi:10.1053/j.gastro.2011.10.043
Reply. Vincenzo Cardinale and Domenico Alvaro hypoth-
esize that autosomal-dominant polycystic kidney disease (ADPKD) cholangiocytes heterozygous for PKD1 or PKD2 respond to estrogens with increased cell proliferation and somatic mutations. We would like to reflect on the occurrence of somatic mutations in both polycystic liver disease (PCLD) and ADPKD and propose an alternative model that takes into account the dynamic role of polycystin (PC)-1 levels in the cell. There are several reasons to argue that somatic mutations in PCLD and ADPKD are the cause, rather than the result, of abnormal cell growth and cyst formation. First, the focal nature of renal cyst formation against the background of an otherwise normal renal tubular system in ADPKD suggests that a specific event occurred before the development of a cyst. Second, somatic mutation analysis shows that the cells lining a single cyst are a homogenous cell population sharing the same somatic mutation. This suggests that they developed through clonal cell division from a single cyst stem cell. Finally, if reduced PC-1 in heterozygous PKD1 cells results in greater DNA damage and overall increase of cell proliferation, this would lead to an increased risk for malignant cell growth in all cells expressing low PC-1. However, malignancies are not part of the phenotypical expression of ADPKD. As discussed in our paper, somatic mutations underlying cyst formation are probably not associated with increased mutagenesis, but instead reflect the normal mutation frequency of somatic cells.1 Initial studies reported a relatively low frequency of somatic hit mutations in ADPKD. This raised questions as to whether the presence of somatic mutations is relevant for cyst formation in ADPKD.2 There are a number of technical issues that explain the low frequency of somatic mutations from earlier experiments. PKD1 is difficult to sequence, which hampers somatic mutation analysis. Furthermore, transheterozygous mutations (PKD1-PKD2) do occur in ADPKD, indicating that somatic mutations in other genes within the same pathway can contribute to cyst formation.3,4 Somatic mutation analysis of both PKD1 and PKD2 identified somatic mutations in 34% of the cysts in a PKD1 germline mutation carrier.3 In PKD2 mutant patients, somatic mutations in PKD1 or PKD2 could be detected in 75% of the cysts,4 which is very similar to the 76% that we
e27
describe for PRKCSH.1 This shows that, with better knowledge of the involved genetic pathways and increasingly sensitive detection methods, more somatic mutations can be identified. The finding that transheterozygous mutations in related genes can trigger cyst formation in ADPKD is in line with the finding that reducing the activity of PC-1 is sufficient to affect cell function.5,6 Following this concept, somatic PKD2 mutations could affect PC-1 levels in cells heterozygous for PKD1, triggering cyst formation. In the case of PRKCSH, total loss of functional gene product may be a requisite for cyst formation, which would explain why somatic mutations in these patients are mostly targeted to the PRKCSH wild-type allele. Although we are now beginning to understand how the genes affected in ADPKD play a role in cell signaling and proliferation, the pathways involved in cyst formation in PCLD remain largely unknown. Recent data suggest the presence of a genetic interaction network of the PCLD genes, PRKCSH and SEC63, with the genes involved in ADPKD.7 This suggests overlap between the molecular pathways involved in cyst formation in ADPKD and PCLD. In summary, these data are in line with the notion that somatic mutations are the initiating step in cyst formation in both PCLD and ADPKD. MANOE J. JANSSEN JOOST P. H. DRENTH Department of Gastroenterology and Hepatology Institute for Genetic and Metabolic Disease Radboud University Nijmegen Medical Centre Nijmegen, The Netherlands 1. 2. 3. 4. 5.
Janssen MJ, et al. Gastroenterology 2011;141:2056 –2063. Ong AC, et al. Lancet 1997;349:1039 –1040. Koptides M, et al. Hum Mol Genet 2000;9:447– 452. Watnick TJ, et al. Nat Genet 2000;25:143–144. Lantinga-van Leeuwen IS, et al. Hum Mol Genet 2004;13:3069 – 3077. 6. Torrice A, et al. Dig Liver Dis 2010;42:377–385. 7. Fedeles SV, et al. Nat Genet 2011;43:639 – 647.
Conflicts of interests The authors disclose no conflicts. doi:10.1053/j.gastro.2012.01.025
HLA-Cw*1202-B*5201-DRB1*1502 Haplotype Increases Risk for Ulcerative Colitis but Reduces Risk for Crohn’s Disease Dear Sir:
In the September 2011 issue of GASTROENTEROLOGY, Okada et al1 reported that the HLA-Cw*1202-B*5201DRB1*1502 haplotype increased risk for ulcerative colitis, but reduced risk for Crohn’s disease, based on a genomewide association study of a Japanese population. Recently, a number of genome-wide association studies have identified numerous susceptibility loci for ulcerative colitis and
CORRESPONDENCE Readers may submit letters to the editor concerning articles that appeared in GASTROENTEROLOGY within one month of publication. Detailed guidelines regarding the content are included in the Instructions to Authors.
Environmental Contribution to Pathogenesis of Cyst Formation in Autosomal-Dominant Polycystic Liver Diseases Dear Sir:
We read with interest the paper by Janssen et al,1 which elegantly demonstrates that, among patients with polycystic liver disease (PCLD) carrying a heterozygous germline mutation in the PRKCSH gene, somatic mutations (second hits) are highly and widely detectable in the liver cyst epithelia with the loss of heterozygosity being the mechanism underling cyst formation through decreased hepatocystin expression in 67% of the cases. The authors conclude that autosomal-dominant PCLD is recessive at the cellular level and a loss of functional PRKCSH is an important step in cystogenesis.1 Regarding the liver involvement, the autosomaldominantly inherited polycystic diseases PCLD and autosomal-dominant polycystic kidney disease (ADPKD) could be considered clinically homogeneous entities characterized by a massive liver derangement with multiple cysts, the severity being greater in women than in men and correlating with the number of pregnancies and estrogen use.2,3 These observations are consistent with a role of estrogens in the development of PCLDs and support the similarity in the mechanism of liver cyst formation between ADPKD and PCLD.2 The recent findings by Janssen et al, together with several clinical observations, promote the need to elucidate the role of environmental factors in the phenotypic manifestations of inherited autosomal dominant PCLDs. Second somatic mutations underlie the liver cyst formation both in PCLD and in ADPKD, with the loss of heterozygosity being more frequent in PCLD.1,4 Posttrascriptional modifications of polycystins (PC) or hepatocystin could play a pivotal role in development and evolution of the PCLDs. Recently, we demonstrated that in cholangiocytes, 17-estradiol triggers PCs proteolysis associated with a proliferative cell response.5 Estrogens are able to regulate several cell proteolytic complexes. Epithelium lining the hepatic cysts of patients with ADPKD showed a higher expression of receptors for estrogen and insulin-like growth factor 1 with respect to normal cholangiocytes and a strong proliferative response to estrogen administration.3 This evidence provides a scientific basis for clinical observations that in ADPKD the formation and growth of liver cysts are faster in women than in men and are linked to estrogen stimulation. However, the expression of estrogen receptors in PCLD cyst epithelia has been demonstrated not to be
increased with respect to normal cholangiocytes, suggesting a divergence in the mechanisms driving cyst formation between PCLD and ADPKD.6 Because lowering PKD1 expression by 13%–20% of functional transcripts is sufficient to cause polycystic disease, it was proposed that in patients heterozygous for PKD1 (ADPKD patients), reduction of the normal allele products below a critical level, owing to genetic factors, environmental agents and stochastic, can determine the formation of cysts and clinical manifestations of the ADPKD.7 A high mutation rate might explain the high number of cysts observed in PCLD1 and ADPKD.4 The malfunction of PC-1 induces a premature transition from G1 phase to S phase, an increase of proliferation and apoptosis through activation of the mammalian target of rapamycin.8 In addition, we recently demonstrated how a reduction of PC-1 expression of 35% is associated with activation of cholangiocyte proliferation machinery.5 Previous investigations elucidated that, although the pathogenesis of PCLD involves overexpression of growth factor receptors and loss of adhesion, proliferation or deregulated apoptosis do not seem to be implicated.6 However, it has been argued that, in PCLD, cysts grow in a nonlinear fashion.6 Therefore, the pathogenesis of liver cyst development and the cause of the high mutation rate in autosomal-dominant PCLDs are not completely elucidated. On the basis of our recent findings,3,5 we hypothesize that estrogens, by acting on heterozygote susceptible cells of ADPKD patients, reduce the functional levels of PC-1 through posttranscriptional proteolytic modifications below a critical cutoff, thus determining an increased proliferation and finally a high probability of second somatic mutations. Cells undergoing a second mutation can further proliferate and form cysts, determining also a selection of cells sensitive to estrogens through a process of co-segregation of related features.3 Our speculations could apply in the ADPKD pathogenesis, although it is not immediately transposable in PCLD pathogenesis. However, in light of the common pathologic and clinical traits of PCLD and ADPKD, further investigations on posttranscriptional modifications of hepatocystin sustained by environmental stimuli could contribute to elucidate the complex pathogenesis of PCLD. VINCENZO CARDINALE DOMENICO ALVARO University Sapienza of Rome Division of Gastroenterology Department of Medico-Surgical Sciences and Biotechnologies Fondazione Eleonora Lorillard Spencer Cenci Polo Pontino Rome, Italy
March 2012
CORRESPONDENCE
1. 2. 3. 4. 5. 6. 7.
Janssen MJ, et al. Gastroenterology 2011;141:2056 –2063. Qian Q, et al. Hepatology 2003;37:164 –171. Alvaro D, et al. Am J Pathol 2008;172:321–332. Watnick TJ, et al. Mol Cell 1998;2:247–251. Torrice A, et al. Dig Liver Dis 2010;42:377–385. Waanders E, et al. Mod Pathol 2008;21:1293–1302. Lantinga-van Leeuwen IS, et al. Hum Mol Genet 2004;13:3069 – 3077. 8. Weimbs T. Cell Cycle 2006;5:2425–2429.
Conflicts of interests The authors disclose no conflicts. doi:10.1053/j.gastro.2011.10.043
Reply. Vincenzo Cardinale and Domenico Alvaro hypoth-
esize that autosomal-dominant polycystic kidney disease (ADPKD) cholangiocytes heterozygous for PKD1 or PKD2 respond to estrogens with increased cell proliferation and somatic mutations. We would like to reflect on the occurrence of somatic mutations in both polycystic liver disease (PCLD) and ADPKD and propose an alternative model that takes into account the dynamic role of polycystin (PC)-1 levels in the cell. There are several reasons to argue that somatic mutations in PCLD and ADPKD are the cause, rather than the result, of abnormal cell growth and cyst formation. First, the focal nature of renal cyst formation against the background of an otherwise normal renal tubular system in ADPKD suggests that a specific event occurred before the development of a cyst. Second, somatic mutation analysis shows that the cells lining a single cyst are a homogenous cell population sharing the same somatic mutation. This suggests that they developed through clonal cell division from a single cyst stem cell. Finally, if reduced PC-1 in heterozygous PKD1 cells results in greater DNA damage and overall increase of cell proliferation, this would lead to an increased risk for malignant cell growth in all cells expressing low PC-1. However, malignancies are not part of the phenotypical expression of ADPKD. As discussed in our paper, somatic mutations underlying cyst formation are probably not associated with increased mutagenesis, but instead reflect the normal mutation frequency of somatic cells.1 Initial studies reported a relatively low frequency of somatic hit mutations in ADPKD. This raised questions as to whether the presence of somatic mutations is relevant for cyst formation in ADPKD.2 There are a number of technical issues that explain the low frequency of somatic mutations from earlier experiments. PKD1 is difficult to sequence, which hampers somatic mutation analysis. Furthermore, transheterozygous mutations (PKD1-PKD2) do occur in ADPKD, indicating that somatic mutations in other genes within the same pathway can contribute to cyst formation.3,4 Somatic mutation analysis of both PKD1 and PKD2 identified somatic mutations in 34% of the cysts in a PKD1 germline mutation carrier.3 In PKD2 mutant patients, somatic mutations in PKD1 or PKD2 could be detected in 75% of the cysts,4 which is very similar to the 76% that we
e27
describe for PRKCSH.1 This shows that, with better knowledge of the involved genetic pathways and increasingly sensitive detection methods, more somatic mutations can be identified. The finding that transheterozygous mutations in related genes can trigger cyst formation in ADPKD is in line with the finding that reducing the activity of PC-1 is sufficient to affect cell function.5,6 Following this concept, somatic PKD2 mutations could affect PC-1 levels in cells heterozygous for PKD1, triggering cyst formation. In the case of PRKCSH, total loss of functional gene product may be a requisite for cyst formation, which would explain why somatic mutations in these patients are mostly targeted to the PRKCSH wild-type allele. Although we are now beginning to understand how the genes affected in ADPKD play a role in cell signaling and proliferation, the pathways involved in cyst formation in PCLD remain largely unknown. Recent data suggest the presence of a genetic interaction network of the PCLD genes, PRKCSH and SEC63, with the genes involved in ADPKD.7 This suggests overlap between the molecular pathways involved in cyst formation in ADPKD and PCLD. In summary, these data are in line with the notion that somatic mutations are the initiating step in cyst formation in both PCLD and ADPKD. MANOE J. JANSSEN JOOST P. H. DRENTH Department of Gastroenterology and Hepatology Institute for Genetic and Metabolic Disease Radboud University Nijmegen Medical Centre Nijmegen, The Netherlands 1. 2. 3. 4. 5.
Janssen MJ, et al. Gastroenterology 2011;141:2056 –2063. Ong AC, et al. Lancet 1997;349:1039 –1040. Koptides M, et al. Hum Mol Genet 2000;9:447– 452. Watnick TJ, et al. Nat Genet 2000;25:143–144. Lantinga-van Leeuwen IS, et al. Hum Mol Genet 2004;13:3069 – 3077. 6. Torrice A, et al. Dig Liver Dis 2010;42:377–385. 7. Fedeles SV, et al. Nat Genet 2011;43:639 – 647.
Conflicts of interests The authors disclose no conflicts. doi:10.1053/j.gastro.2012.01.025
HLA-Cw*1202-B*5201-DRB1*1502 Haplotype Increases Risk for Ulcerative Colitis but Reduces Risk for Crohn’s Disease Dear Sir:
In the September 2011 issue of GASTROENTEROLOGY, Okada et al1 reported that the HLA-Cw*1202-B*5201DRB1*1502 haplotype increased risk for ulcerative colitis, but reduced risk for Crohn’s disease, based on a genomewide association study of a Japanese population. Recently, a number of genome-wide association studies have identified numerous susceptibility loci for ulcerative colitis and