- Email: [email protected]
30 LOW EFFICACY OF CURRENT NUCLEIC ACID TESTING (NAT) ASSAYS FOR THE IDENTIFICATION OF OCCULT HBV INFECTION (OBI) AND CONSEQUENCES FOR SAFETY OF BLOOD SUPPLY
ORAL PRESENTATIONS 28 EFFECT OF HEPATITIS C COINFECTION ON CAUSE-SPECIFIC MORTALITY FOLLOWING HIV SEROCONVERSION IN THE PRE-CART AND CART ERAS J. van der Helm1 , R. Geskus1,2 , C. Sabin3 , L. Meyer4 , J. del Amo5 , G. Chene ` 6 , M. Dorrucci7 , R. Muga8 , K. Porter9 , M. Prins1,10 , on behalf of CASCADE Collaboration in EuroCoord. 1 Public Health Service Amsterdam, 2 Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 3 Research Department of Infection and Population Health, Division of Population Health, UCL Medical School, London, UK; 4 Service d’Epid´emiologie et de Sant´e Publique, Hˆ opital de Bicˆetre, AP-HP, INSERM U1018, Univ Paris Sud, Paris, France; 5 Instituto de Salud Carlos III, Madrid, Spain; 6 Univ. Bordeaux S´egalen, INSERM U897 & CIC-EC7, CHU de Bordeaux, Bordeaux, France; 7 Istituto Superiore di Sanit` a, Rome, Italy; 8 Department of Internal Medicine, Hospital Universitari Germans Trias i Pujol, Badalona, Spain; 9 MRC Clinical Trials Unit, London, UK; 10 Academic Medical Center, University of Amsterdam, CINIMA, Amsterdam, The Netherlands E-mail: [email protected] Background and Aims: Hepatitis C coinfection (HCV) is frequent among HIV-infected individuals but its impact on HIV disease progression remains unknown. The CASCADE collaboration provides a unique opportunity to study the impact of HCV coinfection on both HIV/AIDS and hepatitis/liver-related mortality, adjusting for duration of HIV infection. Methods: Analysis was restricted to the 16 CASCADE cohorts that collect data on cause of death and HCV infection. A competing risks proportional subdistribution hazards model was used to evaluate the effect of HCV infection on four specific causes of death: HIV/AIDS-related, hepatitis/liver-related, natural and nonnatural causes. The year 1997 was used to differentiate between the pre-combination antiretroviral therapy (pre-cART) and cART eras. Analyses were adjusted for the possible confounders sex, risk group and age. Results: Of 9164 HIV seroconverters, 2332 (25.4%) were HCV coinfected. Of 718 deaths observed, 395 were due to HIV/AIDS and 39 to hepatitis/liver-related causes. In multivariable analysis, the risk of HIV/AIDS-related mortality was higher for coinfected individuals in all risk groups in the cART era: injecting drug users (IDU) (aHR = 2.43, 95% CI=1.14–5.20), heterosexuals/those with haemophilia (aHR = 3.43, 95% CI=1.70–6.93) and men who have sex with men (aHR = 3.11, 95% CI=1.49–6.48), compared to HIV monoinfected individuals of the same risk group. Compared to HIV monoinfected individuals, coinfected individuals had a higher risk of hepatitis/liver-related mortality in the pre-cART era (aHR 21.8, 95% CI=6.26–75.9 for IDU and aHR 27.9, 95% CI=8.39–92.6 for all other risk groups combined) and the cART era (aHR 7.86, 95% CI=2.56–24.1 for IDU and aHR = 10.0, 95% CI 3.14–32.2 for all other groups). Among coinfected individuals, the risk of death from HIV/AIDS-related causes and hepatitis/liver-related causes was significantly lower in the cART era than in the pre-cART era. Conclusions: Importantly, HCV coinfection was associated with HIV/AIDS-related mortality as well as with the risk of hepatitis/liver-related death in the cART era, highlighting the importance of early diagnosis and HCV treatment in coinfected individuals.
29 BODY FAT DISTRIBUTION AND RISK FACTORS FOR FIBROSIS IN PATIENTS WITH ALCOHOLIC LIVER DISEASE 1 S. Naveau1,2,3 , A.-S. Dobrin1,3 , A. Balian1 , M. Njike-Nakseu ´ , P. Nohra1 , 3,4 A. Asnacios1 , S. Prevot ´ , G. Perlemuter1,2,3 . 1 H´epatogastroenterologie, AP-HP Hˆ opital Antoine B´ecl`ere, 2 INSERM U996, 3 Univ Paris-Sud, 4 Anatomie Pathologique, AP-HP Hˆ opital Antoine B´ecl`ere, Clamart, France E-mail: [email protected] Introduction: Only a small proportion of alcoholic patients develop advanced liver disease, suggesting that factors other than alcohol intake may influence alcoholic liver disease progression. We have shown that body mass index (BMI) is an independent risk factor for fibrosis in alcohol-induced liver disease and that adipose tissue inflammation is correlated with liver lesions in alcoholic patients. Aim: The aim of this study was to determine whether visceral adipose tissue, as assessed by abdominal height measurement, affected individual susceptibility to fibrosis in alcoholic patients. Patients and Methods: We included 127 consecutive alcoholic patients with abnormal liver test findings for whom liver histology data were available. Visceral abdominal fat was determined by measuring abdominal height using a Holtain-Kahn abdominal caliper; abdominal height measured by this method is indeed highly correlated with visceral fat assessed by computed tomograpy. We carried out univariate comparisons followed by multivariate regression analysis, to investigate the relationship between abdominal height and fibrosis score. Results: Abdominal height (23±0.4 vs 21±0.5 cm; p < 0.005), waist circumference (94±1.6 vs 88±1.9 cm; p < 0.05), fasting blood glucose concentration (5.8±0.15 vs 5.3±0.2 mmol/L; p < 0.05), serum triglyceride concentration (2.4±0.75 versus 1.1±0.86 g/L; p < 0.05) and BMI (25±0.6 vs 23±0.7; p = 0.05) were higher, whereas HDL cholesterol levels (1.7±0.1 versus 2.1±0.1 mmol/L; p < 0.01) were lower in the 72 patients with advanced (F2-F4) fibrosis than in the 55 patients with F0-F1 fibrosis. In multivariate regression analysis, only abdominal height (b = 6.99, p < 0.003) was independently and positively correlated with fibrosis score, which was also negatively correlated with HDL cholesterol level (b = −1.07, p < 0.05). Conclusion: We provide the first demonstration that abdominal height may be a predictor of significant fibrosis in patients with alcoholic liver disease. Our findings support a role for visceral fat accumulation, independent of BMI and of metabolic syndrome criteria, in the onset of alcoholic liver damage. 30 LOW EFFICACY OF CURRENT NUCLEIC ACID TESTING (NAT) ASSAYS FOR THE IDENTIFICATION OF OCCULT HBV INFECTION (OBI) AND CONSEQUENCES FOR SAFETY OF BLOOD SUPPLY M. Spreafico, B. Foglieni, A. Berzuini, L. Raffaele, I. Guarnori, D. Prati. Ospedale A. Manzoni, Lecco, Italy E-mail: ma.spreafi[email protected] Background: OBI can be transmitted by blood, therefore HBV-DNA or anti-HBc screening of donations are added to HBsAg to prevent post-transfusion hepatitis. In Italy and other countries with high anti-HBc prevalence, HBV-DNA is adopted to avoid the exclusion of high numbers of donations. This is considered safe, as there is no current evidence that HBV infection can be transmitted in the absence of detectable HBV-DNA. However, we recently observed a case of acute hepatitis B following transfusion of a red cell unit from a NAT negative donor who was later found to be an OBI carrier. This case of possible transmission was investigated by sequencing. In addition, taking advantage from the availability of a large donor/recipient biorepository developed at our under a EU grant, we conducted a retrospective analysis of stored NAT negative samples from all the OBI carriers identified since the
Journal of Hepatology 2012 vol. 56 | S1–S20
S13
ORAL PRESENTATIONS implementation of the current HBV-DNA screening assay, as well as a look-back of blood recipients. Methods: Sequence analysis revealed identical HBV sequences in donor and recipient. Back-up samples testing negative by NAT minipool at previous donations were individually tested by more sensitive assays (Roche Individual NAT, and home-made real-time PCR of S region). When available, pre and post-transfusion samples of recipients were tested for HBV markers. Sequence analyses were conducted by ABIPrism 3100. Results: 28 back-up samples (1–3 per patient) from 13 OBI carriers (2 anti-HBc only, 2 anti-HBs only, 8 anti HBs/antiHBc, 1 without any marker) were available. Overall, 62% tested positive with at least one of the two assays. Of them, 42% were positive by Individual NAT, and 58% by home-made protocol. Six donors gave at least one unit to 4 recipients, for whom both pre- and post-transfusion samples were available. One recipient acquired infection, as indicated by seroconversion and ALT elevation, two had pre-existing immunity to HBV, and one remained seronegative. Conclusions: A substantial proportion of donations containing HBV DNA are not identified by current NAT minipool assays. These donations can cause acute infections in recipients. Current approaches are highly ineffective, and alternative strategies based on more sensitive protocols or anti-HBc testing should be developed.
Parallel Session: BILIARY PATHOPHYSIOLOGY 31 MONOCYTE/MACROPHAGE PERIBILIARY RECRUITMENT BY PKHD1-DEFECTIVE CHOLANGIOCYTES INDUCES EXPRESSION OF avb6 INTEGRIN ON BILIARY CYSTS VIA TGF-b1 AND TNF-a IN CONGENITAL HEPATIC FIBROSIS L. Fabris1 , L. Locatelli2 , C. Spirlì3 , M. Cadamuro1 , S. Lecchi2 , C. Morell2 , Y. Popov4 , D. Schuppan4,5 , M. Strazzabosco2,3 . 1 Department of Surgical and Gastroenterological Sciences, University of Padua, Padova, 2 Department of Clinical Medicine and Prevention, University of Milano-Bicocca, Monza, Italy; 3 Department of Medicine and Liver Center, Yale University, New Haven, CT, 4 Division of Gastroenterology Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 5 Division of Molecular and Translational Medicine, Johannes Gutenberg University, Mainz, Germany E-mail: [email protected] Background and Aims: Congenital Hepatic Fibrosis (CHF) is caused by mutations in PKHD1, a gene encoding for fibrocystin, a protein of unknown function, expressed in cholangiocyte cilia. In CHF, biliary dysgenesis is accompanied by severe progressive portal fibrosis and portal hypertension. The mechanisms responsible for portal fibrosis in CHF are unclear. The avb6 integrin mediates local activation of TGFb1 and is expressed by reactive cholangiocytes during cholestasis. To understand the mechanisms of fibrosis in CHF we studied the expression of avb6 integrin and its regulation in fibrocystin-defective mice. Methods: In Pkhd1 KO mice (Pkhd1del4/del4 ) we studied, at different ages (1–12 months): a) portal fibrosis (Sirius Red); b) avb6 mRNA and protein expression (RT-PCR, IHC); c) a-SMA, TGFb1, TGFb2 mRNA expression (RT-PCR); d) portal inflammatory infiltrate (IHC for CD45[leukocytes] and NIMP-R14[neutrophils], FACS analysis of whole liver infiltrate); e) cytokines secretion from cultured monolayers of primary cholangiocytes (Luminex assay); f) TGFb1, TGFb2, and TNFa effects on b6 integrin mRNA expression by cultured cholangiocytes before and after inhibition of the TGFb receptor type II (TGFbRII). S14
Results: Pkhd1del4/del4 mice showed a progressive increase in avb6 integrin expression on biliary cyst epithelia. Expression of avb6 correlated with portal fibrosis (r = 0.94, p < 0.02) and with enrichment of a CD45+ve cell infiltrate in the portal space (r = 0.97, p < 0.01). Gene expression of TGFb1/2 showed a similar age-dependent increase. FACS analysis showed that 81% of the CD45+ve cells were macrophages (CD45/CD11b/F4/80+ve). Cultured polarized Pkhd1del4/del4 cholangiocytes secreted IP-10(48x), LIF(20x), and IL13(17x), potent monocyte chemoattractants. Expression of b6 mRNA by cultured Pkhd1del4/del4 cholangiocytes was potently stimulated by TNFa (2.3× vs WT) and TGFb1 (2×). Inhibition of TGFbRII abolished TGFb1 but not TNFa effects on b6 mRNA. Microbiological analysis showed that Pkhd1del4/del4 livers were sterile; gut decontamination had no effect on liver fibrosis. Conclusions: Pkhd1-deficient cholangiocytes secrete cytokines able to recruit inflammatory mononuclear cells and macrophages into the peribiliary microenvironment. In turn, TGFb1 and TNFa released by macrophages up-regulate avb6 integrin. avb6 integrin activates latent TGFb1, further increasing the fibrogenic stimulus. Interestingly, TNFa up-regulates avb6 integrin expression independently from TGFb, a mechanism that may be important in the early phases of the disease. 32 GENOME WIDE ANALYSIS IDENTIFIES MITOTIC RECOMBINATION AS CAUSE OF SOMATIC LOSS OF HETEROZYGOSITY IN CYSTS FROM POLYCYSTIC LIVER DISEASE PATIENTS M. Janssen1 , R. Pfundt2 , J.P.H. Drenth1 . 1 Gastroenterology & Hepatology, 2 Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands E-mail: [email protected] Background and Aims: Somatic mutations play an important role in the progression of many diseases, including inheritable cancers, and benign disorders such as polycystic liver disease (PCLD). We recently found in PCLD patients that carry a PRKCSH germline mutation, that 76% of the liver cysts had lost the wild type PRKCSH allele through loss of heterozygosity (LOH). The aim of this study was to determine the nature and the extent of the underlying somatic genomic changes. Methods: We isolated cyst epithelial cells from liver tissue which was obtained during laporoscopic cyst fenestration from a PCLD patient carrying a c.292+1G>C PRKCSH germline mutation. Cells were isolated from adjacent liver tissue using EDTA and trypsin. Identity of the cells was confirmed using CK19 staining. We performed a genome wide cytogenetic array analysis (Affymetrix CytoScanTM HD with 2.7 Million probes of which 750.000 SNP probes) for high resolution imaging of both copy number variations and LOH regions from 2 cysts. Results: Using this approach we found that the LOH region leading to loss of the wild type PRKCSH allele was in both cysts the result of terminal copy number neutral LOH on the short arm of chromosome 19. We could identify in each cyst a single breakpoint from where the LOH continued to the end of the p arm, covering a region of 14 Mb (megabases) in the first and 18 Mb in the second cyst. The location of the breakpoint was different in each cyst, thereby indicating these cysts developed independently and resulted from a different somatic event. No other genomic abnormalities were found. Conclusions: In conclusion, these data shows that a single breakpoint in the p arm of chromosome 19 is responsible for the LOH which leads to loss of the wide type PRKCSH allele in these cysts. As the LOH is not caused by a deletion, a mitotic recombination event must have occurred leading to copy number neutral LOH. These data provide important insights in the mechanisms behind somatic mutations in a benign disorder.
Journal of Hepatology 2012 vol. 56 | S1–S20
29 BODY FAT DISTRIBUTION AND RISK FACTORS FOR FIBROSIS IN PATIENTS WITH ALCOHOLIC LIVER DISEASE 1 S. Naveau1,2,3 , A.-S. Dobrin1,3 , A. Balian1 , M. Njike-Nakseu ´ , P. Nohra1 , 3,4 A. Asnacios1 , S. Prevot ´ , G. Perlemuter1,2,3 . 1 H´epatogastroenterologie, AP-HP Hˆ opital Antoine B´ecl`ere, 2 INSERM U996, 3 Univ Paris-Sud, 4 Anatomie Pathologique, AP-HP Hˆ opital Antoine B´ecl`ere, Clamart, France E-mail: [email protected] Introduction: Only a small proportion of alcoholic patients develop advanced liver disease, suggesting that factors other than alcohol intake may influence alcoholic liver disease progression. We have shown that body mass index (BMI) is an independent risk factor for fibrosis in alcohol-induced liver disease and that adipose tissue inflammation is correlated with liver lesions in alcoholic patients. Aim: The aim of this study was to determine whether visceral adipose tissue, as assessed by abdominal height measurement, affected individual susceptibility to fibrosis in alcoholic patients. Patients and Methods: We included 127 consecutive alcoholic patients with abnormal liver test findings for whom liver histology data were available. Visceral abdominal fat was determined by measuring abdominal height using a Holtain-Kahn abdominal caliper; abdominal height measured by this method is indeed highly correlated with visceral fat assessed by computed tomograpy. We carried out univariate comparisons followed by multivariate regression analysis, to investigate the relationship between abdominal height and fibrosis score. Results: Abdominal height (23±0.4 vs 21±0.5 cm; p < 0.005), waist circumference (94±1.6 vs 88±1.9 cm; p < 0.05), fasting blood glucose concentration (5.8±0.15 vs 5.3±0.2 mmol/L; p < 0.05), serum triglyceride concentration (2.4±0.75 versus 1.1±0.86 g/L; p < 0.05) and BMI (25±0.6 vs 23±0.7; p = 0.05) were higher, whereas HDL cholesterol levels (1.7±0.1 versus 2.1±0.1 mmol/L; p < 0.01) were lower in the 72 patients with advanced (F2-F4) fibrosis than in the 55 patients with F0-F1 fibrosis. In multivariate regression analysis, only abdominal height (b = 6.99, p < 0.003) was independently and positively correlated with fibrosis score, which was also negatively correlated with HDL cholesterol level (b = −1.07, p < 0.05). Conclusion: We provide the first demonstration that abdominal height may be a predictor of significant fibrosis in patients with alcoholic liver disease. Our findings support a role for visceral fat accumulation, independent of BMI and of metabolic syndrome criteria, in the onset of alcoholic liver damage. 30 LOW EFFICACY OF CURRENT NUCLEIC ACID TESTING (NAT) ASSAYS FOR THE IDENTIFICATION OF OCCULT HBV INFECTION (OBI) AND CONSEQUENCES FOR SAFETY OF BLOOD SUPPLY M. Spreafico, B. Foglieni, A. Berzuini, L. Raffaele, I. Guarnori, D. Prati. Ospedale A. Manzoni, Lecco, Italy E-mail: ma.spreafi[email protected] Background: OBI can be transmitted by blood, therefore HBV-DNA or anti-HBc screening of donations are added to HBsAg to prevent post-transfusion hepatitis. In Italy and other countries with high anti-HBc prevalence, HBV-DNA is adopted to avoid the exclusion of high numbers of donations. This is considered safe, as there is no current evidence that HBV infection can be transmitted in the absence of detectable HBV-DNA. However, we recently observed a case of acute hepatitis B following transfusion of a red cell unit from a NAT negative donor who was later found to be an OBI carrier. This case of possible transmission was investigated by sequencing. In addition, taking advantage from the availability of a large donor/recipient biorepository developed at our under a EU grant, we conducted a retrospective analysis of stored NAT negative samples from all the OBI carriers identified since the
Journal of Hepatology 2012 vol. 56 | S1–S20
S13
ORAL PRESENTATIONS implementation of the current HBV-DNA screening assay, as well as a look-back of blood recipients. Methods: Sequence analysis revealed identical HBV sequences in donor and recipient. Back-up samples testing negative by NAT minipool at previous donations were individually tested by more sensitive assays (Roche Individual NAT, and home-made real-time PCR of S region). When available, pre and post-transfusion samples of recipients were tested for HBV markers. Sequence analyses were conducted by ABIPrism 3100. Results: 28 back-up samples (1–3 per patient) from 13 OBI carriers (2 anti-HBc only, 2 anti-HBs only, 8 anti HBs/antiHBc, 1 without any marker) were available. Overall, 62% tested positive with at least one of the two assays. Of them, 42% were positive by Individual NAT, and 58% by home-made protocol. Six donors gave at least one unit to 4 recipients, for whom both pre- and post-transfusion samples were available. One recipient acquired infection, as indicated by seroconversion and ALT elevation, two had pre-existing immunity to HBV, and one remained seronegative. Conclusions: A substantial proportion of donations containing HBV DNA are not identified by current NAT minipool assays. These donations can cause acute infections in recipients. Current approaches are highly ineffective, and alternative strategies based on more sensitive protocols or anti-HBc testing should be developed.
Parallel Session: BILIARY PATHOPHYSIOLOGY 31 MONOCYTE/MACROPHAGE PERIBILIARY RECRUITMENT BY PKHD1-DEFECTIVE CHOLANGIOCYTES INDUCES EXPRESSION OF avb6 INTEGRIN ON BILIARY CYSTS VIA TGF-b1 AND TNF-a IN CONGENITAL HEPATIC FIBROSIS L. Fabris1 , L. Locatelli2 , C. Spirlì3 , M. Cadamuro1 , S. Lecchi2 , C. Morell2 , Y. Popov4 , D. Schuppan4,5 , M. Strazzabosco2,3 . 1 Department of Surgical and Gastroenterological Sciences, University of Padua, Padova, 2 Department of Clinical Medicine and Prevention, University of Milano-Bicocca, Monza, Italy; 3 Department of Medicine and Liver Center, Yale University, New Haven, CT, 4 Division of Gastroenterology Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 5 Division of Molecular and Translational Medicine, Johannes Gutenberg University, Mainz, Germany E-mail: [email protected] Background and Aims: Congenital Hepatic Fibrosis (CHF) is caused by mutations in PKHD1, a gene encoding for fibrocystin, a protein of unknown function, expressed in cholangiocyte cilia. In CHF, biliary dysgenesis is accompanied by severe progressive portal fibrosis and portal hypertension. The mechanisms responsible for portal fibrosis in CHF are unclear. The avb6 integrin mediates local activation of TGFb1 and is expressed by reactive cholangiocytes during cholestasis. To understand the mechanisms of fibrosis in CHF we studied the expression of avb6 integrin and its regulation in fibrocystin-defective mice. Methods: In Pkhd1 KO mice (Pkhd1del4/del4 ) we studied, at different ages (1–12 months): a) portal fibrosis (Sirius Red); b) avb6 mRNA and protein expression (RT-PCR, IHC); c) a-SMA, TGFb1, TGFb2 mRNA expression (RT-PCR); d) portal inflammatory infiltrate (IHC for CD45[leukocytes] and NIMP-R14[neutrophils], FACS analysis of whole liver infiltrate); e) cytokines secretion from cultured monolayers of primary cholangiocytes (Luminex assay); f) TGFb1, TGFb2, and TNFa effects on b6 integrin mRNA expression by cultured cholangiocytes before and after inhibition of the TGFb receptor type II (TGFbRII). S14
Results: Pkhd1del4/del4 mice showed a progressive increase in avb6 integrin expression on biliary cyst epithelia. Expression of avb6 correlated with portal fibrosis (r = 0.94, p < 0.02) and with enrichment of a CD45+ve cell infiltrate in the portal space (r = 0.97, p < 0.01). Gene expression of TGFb1/2 showed a similar age-dependent increase. FACS analysis showed that 81% of the CD45+ve cells were macrophages (CD45/CD11b/F4/80+ve). Cultured polarized Pkhd1del4/del4 cholangiocytes secreted IP-10(48x), LIF(20x), and IL13(17x), potent monocyte chemoattractants. Expression of b6 mRNA by cultured Pkhd1del4/del4 cholangiocytes was potently stimulated by TNFa (2.3× vs WT) and TGFb1 (2×). Inhibition of TGFbRII abolished TGFb1 but not TNFa effects on b6 mRNA. Microbiological analysis showed that Pkhd1del4/del4 livers were sterile; gut decontamination had no effect on liver fibrosis. Conclusions: Pkhd1-deficient cholangiocytes secrete cytokines able to recruit inflammatory mononuclear cells and macrophages into the peribiliary microenvironment. In turn, TGFb1 and TNFa released by macrophages up-regulate avb6 integrin. avb6 integrin activates latent TGFb1, further increasing the fibrogenic stimulus. Interestingly, TNFa up-regulates avb6 integrin expression independently from TGFb, a mechanism that may be important in the early phases of the disease. 32 GENOME WIDE ANALYSIS IDENTIFIES MITOTIC RECOMBINATION AS CAUSE OF SOMATIC LOSS OF HETEROZYGOSITY IN CYSTS FROM POLYCYSTIC LIVER DISEASE PATIENTS M. Janssen1 , R. Pfundt2 , J.P.H. Drenth1 . 1 Gastroenterology & Hepatology, 2 Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands E-mail: [email protected] Background and Aims: Somatic mutations play an important role in the progression of many diseases, including inheritable cancers, and benign disorders such as polycystic liver disease (PCLD). We recently found in PCLD patients that carry a PRKCSH germline mutation, that 76% of the liver cysts had lost the wild type PRKCSH allele through loss of heterozygosity (LOH). The aim of this study was to determine the nature and the extent of the underlying somatic genomic changes. Methods: We isolated cyst epithelial cells from liver tissue which was obtained during laporoscopic cyst fenestration from a PCLD patient carrying a c.292+1G>C PRKCSH germline mutation. Cells were isolated from adjacent liver tissue using EDTA and trypsin. Identity of the cells was confirmed using CK19 staining. We performed a genome wide cytogenetic array analysis (Affymetrix CytoScanTM HD with 2.7 Million probes of which 750.000 SNP probes) for high resolution imaging of both copy number variations and LOH regions from 2 cysts. Results: Using this approach we found that the LOH region leading to loss of the wild type PRKCSH allele was in both cysts the result of terminal copy number neutral LOH on the short arm of chromosome 19. We could identify in each cyst a single breakpoint from where the LOH continued to the end of the p arm, covering a region of 14 Mb (megabases) in the first and 18 Mb in the second cyst. The location of the breakpoint was different in each cyst, thereby indicating these cysts developed independently and resulted from a different somatic event. No other genomic abnormalities were found. Conclusions: In conclusion, these data shows that a single breakpoint in the p arm of chromosome 19 is responsible for the LOH which leads to loss of the wide type PRKCSH allele in these cysts. As the LOH is not caused by a deletion, a mitotic recombination event must have occurred leading to copy number neutral LOH. These data provide important insights in the mechanisms behind somatic mutations in a benign disorder.
Journal of Hepatology 2012 vol. 56 | S1–S20