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CTRC G60G Variant is Common in Children with Acute Recurrent and Chronic Pancreatitis and Associated with Early-Onset Disease
Su2074
Table 1. Demographics of Patients with Disease Causing Mutations vs. VUCS
AGA Abstracts
EZH2 INHIBITION PREVENTS AGE-RELATED SENESCENCE OF INTERSTITIAL CELL OF CAJAL STEM CELLS Yujiro Hayashi, Siva Arumugam Saravanaperumal, Todd A. Kellogg, Gianrico Farrugia, Jeong Heon Lee, Tamas Ordog Background & Aims: The proportion of elderly individuals is rapidly growing worldwide. Aging-related organ dysfunctions also manifest in the stomach, where reduced compliance and accommodation, impaired slow wave activity and variable gastric emptying associate with gastroesophageal reflux, early satiation and consequent reduced food intake. This negatively impacts general health and quality of life. Previously, we reported age-dependent reduction of interstitial cells of Cajal (ICC), electrical pacemaker and neuromodulator cells of the gut, in the gastric muscles of humans and both naturally aged and progeric klotho mice. ICC loss associated with reduced food intake, diminished antral pacemaking and impaired fundal nitrergic control. ICC stem cells (ICC-SC) were also reduced, reflecting Trp53-mediated senescence from overactive Wnt signaling and reduced Kit transcription from increased polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 trimethylation (H3K27me3). Since PRC2 is typically downregulated in cellular stress and senescence, here we aimed to investigate the relationship between Trp53 stabilization and elevated H3K27me3 in ICC-SC senescence during aging. Methods: Old (aged 18-24 months) and young (aged 4-8 weeks) male C57BL6 mice, as well as klotho mice and age-matched wild-type controls (both sexes) were used. Human gastric smooth muscles obtained from bariatric surgeries were also studied. Trp53-mediated senescence was induced by treating ICC-SC (line D2211B) with the Mdm2 antagonist nutlin 3a (30 µM, 72 h; control: nutlin 3b), and assessed by MTS assay, Ki-67 immunostaining and Western blotting. EPZ-6438 and GSK126, FDA-approved inhibitors of the PRC2 histone methyltransferase Ezh2, were applied at 500 nM for 3 days. RNA interference targeting Ezh2 was performed using small interfering RNA (siRNA). Results: Protein expression for PRC2 members Ezh2 and Suz12 and global H3K27me3 levels were increased in gastric muscles of both klotho and naturally aged mice compared to controls. Similarly, there was an increase in the expression of EZH2 and SUZ12 proteins in human samples between 23 and 51 years of age. In cultured ICCSC, Trp53 upregulation with nutlin 3a increased mRNA and protein expression for Ezh2 and Suz12 and global H3K27me3 levels. Both EPZ-6438 and GSK126 prevented nutlin 3ainduced increase in the senescence markers γ-H2A.X and Hp-1α (Cbx5) without affecting Trp53 protein levels. EPZ-6438 and GSK126 also blocked the cytostatic effects of nutlin 3a assessed by MTS assay and Ki-67 immunofluorescence. Similar results were obtained with nutlin 3a-treated ICC-SC transfected with Ezh2 siRNA. Conclusions: Our study shows an unexpected role for PRC2-mediated epigenetic regulation downstream of Trp53 in ICC-SC senescence. Age-related ICC-SC senescence may be delayed by Ezh2 inhibitors already approved for use in humans.
Table 2. Demographics of Patients with CTRC G60G Variant vs. VUCS
Su2076 Su2075 FECAL MICROBIOTA TRANSPLANTATION IN CHILDREN DOES NOT SIGNIFICANTLY ALTER BODY MASS INDEX Dong Xi, Sonia Michail
CTRC G60G VARIANT IS COMMON IN CHILDREN WITH ACUTE RECURRENT AND CHRONIC PANCREATITIS AND ASSOCIATED WITH EARLY-ONSET DISEASE Aliye Uc, Jessica LaRusch, Bridget Zimmerman, Michael Wilschanski, Steven Werlin, David M. Troendle, Uzma Shah, Sarah Jane Schwarzenberg, Sue Rhee, John Pohl, David Piccoli, Emily Perito, Joseph J. Palermo, Chee Y. Ooi, Jaimie D. Nathan, Veronique D. Morinville, Maria R. Mascarenhas, Asim Maqbool, Quin Liu, Tom K. Lin, Sohail Z. Husain, Ryan Himes, Mel Heyman, Tanja Gonska, Matthew J. Giefer, Cheryl E. Gariepy, Steven D. Freedman, Douglas S. Fishman, Melena Bellin, Bradley Barth, Maisam Abu-ElHaija, David C. Whitcomb, Mark Lowe
Fecal microbiota transplantation (FMT) is nowadays a promising therapy for Clostridium difficile infection and a potential treatment for ulcerative colitis. Limited data suggests that FMT donated from healthy individuals is a safe therapy which is not associated with the development of new infections or diseases. However, it is still unclear whether the changes in intestinal microbiome will affect energy homeostasis or metabolism. A recent study demonstrated obese microbiome can be transmitted in animal models. FMT from nonideal donor was found to induce excessive weight gain in a case report. This brings an intriguing question whether FMT from healthy donors affects recipient's body mass index (BMI). In our randomized placebo-controlled pilot study children patients with Clostridium difficile infection (n=8) or ulcerative colitis (n=12) were randomly divided into control and FMT groups. The BMI of both control and FMT groups was recorded at different time points, including pre-transplantation and 1 month, 3 months, 6 months, 12 months post-transplantation. The change in post-FMT BMI percentile compared to pre-FMT was calculated and graphed (Figure). The age range of Clostridium difficile infection cohort was 1 year to 17 years with the average of 8.5 years, while the range was 8 years to 21 years for ulcerative colitis cohort with the average of 15.2 years. Though the sample size was limited for both groups in our study, we successfully found that the BMI percentile was changed by (-0.7), (-1.8), 1.3, 4.6 (%tile) in Clostridium difficile infection, while by 3.6, (-3.3), 3.7, 7.1 (%tile) in ulcerative colitis at 1 month, 3 months, 6 months, 12 months after FMT ("-" means decrease). These changes were confirmed to be not significant compared to control groups (p> 0.05). CONCLUSION: From this randomized pilot study we concluded that FMT from healthy donors does not significantly alter body mass index in children with Clostridium difficile infection and ulcerative colitis over 12 months. Future research will focus on enhancing the study by increasing the cohort size, and minimizing the effects of confounding variables including the medications or the severity of disease.
Introduction: Pancreatitis-associated gene mutations are common in pediatric acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). Chymotrypsin-C (CTRC) G60G variant is found in 16% of adults with CP compared to 11% of healthy controls. It is not known whether CTRC G60G variant or other genetic markers cluster in children and predict earlyonset pancreatitis. Objective: To identify CTRC G60G variant and other genetic disease markers in the well-phenotyped INSPPIRE (INternational Study group of Pediatric Pancreatitis: In search for a cuRE) cohort. Methods: DNA was isolated and Next Gene Sequencing was performed by Ariel Precision Medicine for the following genes: PRSS1, PRSS3, CTRC, SPINK1, CFTR, CPA1, SBDS, UBR1, CEL, and CASR in children with ARP (n=27) or CP (n= 15). Samples were selected for analysis based on the order of enrollment to the INSPPIRE registry. Demographic and clinical information at the time of enrollment were entered into REDCapTM database at INSPPIRE centers. We defined pathogenic or disease-causing mutations and variants of unknown clinical significance (VUCS) using pancreasgenetics.org, CFTR1 and CFTR2 databases. NAPS2 healthy control group served as control for CTRC G60G variant frequency (n=1,107; PMID: 25569187). Differences between groups were analyzed using two-sample t-test for continuous variables and Fisher's exact test for categorical variables. Odds ratio was computed for CTRC in ARP/CP vs. Controls. Results: 27 of 42 patients tested had at least one disease-causing mutation (64%); 7 (17%) PRSS1, 6 (14%) CFTR, 7 (17%) SPINK1, and 18 (43%) CTRC G60G variant; 7 patients with 2 diseasecausing mutations; 2 patients with 3 disease-causing mutations. Compared to NAPS2 control cohort (11%), CTRC G60G variant was significantly more common in children with ARP or CP (OR=2.9, CI=1.5, 5.4 p=0.0006). In 7 (39%) patients with the CTRC G60G variant, at least one disease-causing mutation in other genes was also found. Overall children with VUCS were more often female (93% vs 48%, p=0.004) and older at first diagnosis (12.3 + 4.2 years vs 9.3 + 4 years; p=0.036) compared to children with disease-associated mutations (Table 1). Children with the CTRC G60G variant plus other disease-associated mutations were less likely to be female (43% vs 93%, p=0.021) and presented at a younger age compared to children with VUCS (7.2 + 2.5 years vs 12.3 + 4.2 years; p=0.008) (Table 2). Discussion: CTRC G60G variant is common in children with ARP or CP and associated with early-onset disease if coexistent with other disease-causing mutations. Female gender and older age-onset disease are more frequently associated with variants of unknown clinical significance. Early identification of genetic risk factors may enable risk stratification studies for pediatric ARP and CP.
AGA Abstracts
S-648
Table 1. Demographics of Patients with Disease Causing Mutations vs. VUCS
AGA Abstracts
EZH2 INHIBITION PREVENTS AGE-RELATED SENESCENCE OF INTERSTITIAL CELL OF CAJAL STEM CELLS Yujiro Hayashi, Siva Arumugam Saravanaperumal, Todd A. Kellogg, Gianrico Farrugia, Jeong Heon Lee, Tamas Ordog Background & Aims: The proportion of elderly individuals is rapidly growing worldwide. Aging-related organ dysfunctions also manifest in the stomach, where reduced compliance and accommodation, impaired slow wave activity and variable gastric emptying associate with gastroesophageal reflux, early satiation and consequent reduced food intake. This negatively impacts general health and quality of life. Previously, we reported age-dependent reduction of interstitial cells of Cajal (ICC), electrical pacemaker and neuromodulator cells of the gut, in the gastric muscles of humans and both naturally aged and progeric klotho mice. ICC loss associated with reduced food intake, diminished antral pacemaking and impaired fundal nitrergic control. ICC stem cells (ICC-SC) were also reduced, reflecting Trp53-mediated senescence from overactive Wnt signaling and reduced Kit transcription from increased polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 trimethylation (H3K27me3). Since PRC2 is typically downregulated in cellular stress and senescence, here we aimed to investigate the relationship between Trp53 stabilization and elevated H3K27me3 in ICC-SC senescence during aging. Methods: Old (aged 18-24 months) and young (aged 4-8 weeks) male C57BL6 mice, as well as klotho mice and age-matched wild-type controls (both sexes) were used. Human gastric smooth muscles obtained from bariatric surgeries were also studied. Trp53-mediated senescence was induced by treating ICC-SC (line D2211B) with the Mdm2 antagonist nutlin 3a (30 µM, 72 h; control: nutlin 3b), and assessed by MTS assay, Ki-67 immunostaining and Western blotting. EPZ-6438 and GSK126, FDA-approved inhibitors of the PRC2 histone methyltransferase Ezh2, were applied at 500 nM for 3 days. RNA interference targeting Ezh2 was performed using small interfering RNA (siRNA). Results: Protein expression for PRC2 members Ezh2 and Suz12 and global H3K27me3 levels were increased in gastric muscles of both klotho and naturally aged mice compared to controls. Similarly, there was an increase in the expression of EZH2 and SUZ12 proteins in human samples between 23 and 51 years of age. In cultured ICCSC, Trp53 upregulation with nutlin 3a increased mRNA and protein expression for Ezh2 and Suz12 and global H3K27me3 levels. Both EPZ-6438 and GSK126 prevented nutlin 3ainduced increase in the senescence markers γ-H2A.X and Hp-1α (Cbx5) without affecting Trp53 protein levels. EPZ-6438 and GSK126 also blocked the cytostatic effects of nutlin 3a assessed by MTS assay and Ki-67 immunofluorescence. Similar results were obtained with nutlin 3a-treated ICC-SC transfected with Ezh2 siRNA. Conclusions: Our study shows an unexpected role for PRC2-mediated epigenetic regulation downstream of Trp53 in ICC-SC senescence. Age-related ICC-SC senescence may be delayed by Ezh2 inhibitors already approved for use in humans.
Table 2. Demographics of Patients with CTRC G60G Variant vs. VUCS
Su2076 Su2075 FECAL MICROBIOTA TRANSPLANTATION IN CHILDREN DOES NOT SIGNIFICANTLY ALTER BODY MASS INDEX Dong Xi, Sonia Michail
CTRC G60G VARIANT IS COMMON IN CHILDREN WITH ACUTE RECURRENT AND CHRONIC PANCREATITIS AND ASSOCIATED WITH EARLY-ONSET DISEASE Aliye Uc, Jessica LaRusch, Bridget Zimmerman, Michael Wilschanski, Steven Werlin, David M. Troendle, Uzma Shah, Sarah Jane Schwarzenberg, Sue Rhee, John Pohl, David Piccoli, Emily Perito, Joseph J. Palermo, Chee Y. Ooi, Jaimie D. Nathan, Veronique D. Morinville, Maria R. Mascarenhas, Asim Maqbool, Quin Liu, Tom K. Lin, Sohail Z. Husain, Ryan Himes, Mel Heyman, Tanja Gonska, Matthew J. Giefer, Cheryl E. Gariepy, Steven D. Freedman, Douglas S. Fishman, Melena Bellin, Bradley Barth, Maisam Abu-ElHaija, David C. Whitcomb, Mark Lowe
Fecal microbiota transplantation (FMT) is nowadays a promising therapy for Clostridium difficile infection and a potential treatment for ulcerative colitis. Limited data suggests that FMT donated from healthy individuals is a safe therapy which is not associated with the development of new infections or diseases. However, it is still unclear whether the changes in intestinal microbiome will affect energy homeostasis or metabolism. A recent study demonstrated obese microbiome can be transmitted in animal models. FMT from nonideal donor was found to induce excessive weight gain in a case report. This brings an intriguing question whether FMT from healthy donors affects recipient's body mass index (BMI). In our randomized placebo-controlled pilot study children patients with Clostridium difficile infection (n=8) or ulcerative colitis (n=12) were randomly divided into control and FMT groups. The BMI of both control and FMT groups was recorded at different time points, including pre-transplantation and 1 month, 3 months, 6 months, 12 months post-transplantation. The change in post-FMT BMI percentile compared to pre-FMT was calculated and graphed (Figure). The age range of Clostridium difficile infection cohort was 1 year to 17 years with the average of 8.5 years, while the range was 8 years to 21 years for ulcerative colitis cohort with the average of 15.2 years. Though the sample size was limited for both groups in our study, we successfully found that the BMI percentile was changed by (-0.7), (-1.8), 1.3, 4.6 (%tile) in Clostridium difficile infection, while by 3.6, (-3.3), 3.7, 7.1 (%tile) in ulcerative colitis at 1 month, 3 months, 6 months, 12 months after FMT ("-" means decrease). These changes were confirmed to be not significant compared to control groups (p> 0.05). CONCLUSION: From this randomized pilot study we concluded that FMT from healthy donors does not significantly alter body mass index in children with Clostridium difficile infection and ulcerative colitis over 12 months. Future research will focus on enhancing the study by increasing the cohort size, and minimizing the effects of confounding variables including the medications or the severity of disease.
Introduction: Pancreatitis-associated gene mutations are common in pediatric acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). Chymotrypsin-C (CTRC) G60G variant is found in 16% of adults with CP compared to 11% of healthy controls. It is not known whether CTRC G60G variant or other genetic markers cluster in children and predict earlyonset pancreatitis. Objective: To identify CTRC G60G variant and other genetic disease markers in the well-phenotyped INSPPIRE (INternational Study group of Pediatric Pancreatitis: In search for a cuRE) cohort. Methods: DNA was isolated and Next Gene Sequencing was performed by Ariel Precision Medicine for the following genes: PRSS1, PRSS3, CTRC, SPINK1, CFTR, CPA1, SBDS, UBR1, CEL, and CASR in children with ARP (n=27) or CP (n= 15). Samples were selected for analysis based on the order of enrollment to the INSPPIRE registry. Demographic and clinical information at the time of enrollment were entered into REDCapTM database at INSPPIRE centers. We defined pathogenic or disease-causing mutations and variants of unknown clinical significance (VUCS) using pancreasgenetics.org, CFTR1 and CFTR2 databases. NAPS2 healthy control group served as control for CTRC G60G variant frequency (n=1,107; PMID: 25569187). Differences between groups were analyzed using two-sample t-test for continuous variables and Fisher's exact test for categorical variables. Odds ratio was computed for CTRC in ARP/CP vs. Controls. Results: 27 of 42 patients tested had at least one disease-causing mutation (64%); 7 (17%) PRSS1, 6 (14%) CFTR, 7 (17%) SPINK1, and 18 (43%) CTRC G60G variant; 7 patients with 2 diseasecausing mutations; 2 patients with 3 disease-causing mutations. Compared to NAPS2 control cohort (11%), CTRC G60G variant was significantly more common in children with ARP or CP (OR=2.9, CI=1.5, 5.4 p=0.0006). In 7 (39%) patients with the CTRC G60G variant, at least one disease-causing mutation in other genes was also found. Overall children with VUCS were more often female (93% vs 48%, p=0.004) and older at first diagnosis (12.3 + 4.2 years vs 9.3 + 4 years; p=0.036) compared to children with disease-associated mutations (Table 1). Children with the CTRC G60G variant plus other disease-associated mutations were less likely to be female (43% vs 93%, p=0.021) and presented at a younger age compared to children with VUCS (7.2 + 2.5 years vs 12.3 + 4.2 years; p=0.008) (Table 2). Discussion: CTRC G60G variant is common in children with ARP or CP and associated with early-onset disease if coexistent with other disease-causing mutations. Female gender and older age-onset disease are more frequently associated with variants of unknown clinical significance. Early identification of genetic risk factors may enable risk stratification studies for pediatric ARP and CP.
AGA Abstracts
S-648