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Mutational analysis of the autophagy-related 16 like 1 (ATG16L1) gene in Japanese patients with chronic pancreatitis
S160
Abstracts / Pancreatology 16 (2016) S1eS192
1
Department of Gastroenterology, Tohoku University, Japan Department of Medical Genetics, Tohoku University, Japan 3 Division of Cell Proliferation, Tohoku University, Japan 2
Aim: Factors underlying genetic predisposition for development of alcoholic pancreatitis are largely unknown. Whole exome sequenceing (WES) focusing on only the coding regions of the genome, has become in a few years the choice strategy to identify a coding allelic variant for genetic disorder. The aim of this study was to identify a new candidate gene responsible for alcoholic pancreatitis. Methods: Genomic DNA was prepared from a total of 560 patients with pancreatitis (chronic pancreatitis, 288；acute pancreatitis, 272) and 439 control subjects. The SureSelect Human All Exon kit was used for the analysis of all exons in 16 patients with alcoholic pancreatitis. DNA libraries were constructed and sequenced using paired-end protocol with 101-base reads on Illumina HiSeq2500. Mutational analysis of the RNF113B gene was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis. This study was approved by the Ethics Committee of Tohoku University School of Medicine. Results: WES resulted in each samples having greater than 61 million reads. About 70% of all coding regions were covered at 50x or more. Mean depth was over 70x. There were about 1,340,000 variants in total of the 16 samples. After removing synonymous variant, we compared frequency of the residual variants from that of control samples by Human Genetic Variation Browser. Of these, there were four candidate genes that might be associated with pancreatitis. Statistically significant difference was observed in the frequency of p.S89R variant in ring finger protein 113B between patients with alcoholic pancreatitis (14.3%；31/217) and control subjects (6.6%；29/410) (OR 2.4 p¼0.002). The frequency of this variant in patients with non-alcoholic pancreatitis (8.2%；28/343) was not statistically different from that in controls. Conclusion: Next generation sequencing might become the new strategy to identify the candidate genes for alcoholic pancreatitis.
P-257. Mutational analysis of the autophagy-related 16 like 1 (ATG16L1) gene in Japanese patients with chronic pancreatitis Eriko Nakano 1, Atsushi Masamune 1, Tetsuya Niihori 2, Kiyoshi Kume 1, Shin Hamada 1, Yoko Aoki 2, Tooru Shimosegawa 1 1 Division of Gastroenterology, Tohoku University Graduate School of Medicine, Japan 2 Department of Medical Genetics, Tohoku University Graduate School of Medicine, Japan
Background: To date, several pancreatitis susceptibility genes have been identified, including PRSS1, SPINK1, CTRC and CPA1 genes. However, in a nationwide study in Japan about 30% of families with hereditary pancreatitis do not carry mutations in any of the known pancreatitis susceptibility genes, suggesting that other yet unidentified genes might be involved. Recent studies using knockout mice have suggested that impaired autophagy might be involved in the pathogenesis of pancreatitis (Li N, et al. J Clin Invest 2013). This study aimed to clarify whether variants in the autophagy related 16 like 1 (ATG16L1) gene are associated with chronic pancreatitis (CP) in Japan by targeted next generation sequencing. Methods: 282 patients with CP (112 idiopathic, 157 alcoholic, and 13 hereditary/familial) were enrolled in this study. We designed the HaloPlex Target Enrichment System (Agilent Technologies) targeting all of the exons and adjacent intronic regions of the known pancreatitis susceptibility genes and ATG16L1 gene. All samples were sequenced on the Illumina Miseq platform with paired-end 151-bp reads. SIFT and PolyPhen-2 were used to predict whether an amino acid substitution would affect the structure and function of a protein. The variant frequencies in the Japanese control population were obtained from the integrative Japanese Genome Variation Database (https://ijgvd.megabank.tohoku.ac.jp). This study was approved by the Ethics Committee of Tohoku University School of Medicine (article#：2015-1-613).
Results: We could identify 5 non-synonymous variants including 2 known [c.409A＞G (p.T137A), c.1160C＞T (p.A387V)], 3 novel ones [c.11G＞T (p.G4L), c.76G＞C (p.V26L) and c.581A＞G (p.E194A)] and 2 synonymous variants including one novel variant in the exonic regions. p.G4L, p.V26L, p.T137A, and p.E194A were probably damaged based on the SIFT and/or the PolyPhen-2 prediction. The frequencies of these variants were not different between all patients with CP and controls. Conclusions: Variants in the ATG16L1 gene are not associated with CP in Japan.
P-258. Chronic exercise improve s disrupt ion of pancreatic B-cells morphology in WBN/Kob-Fatty rats Kumiko Minato 1, Yoko Shiroya 1, Hideki Yamauchi 2 1
Faculty of Life Science, Wayo Women's University, Japan Department of Molecular Physiology, Jikei University School of Medicine, Japan 2
Aim: Leptin receptor-deficient WBN/Kob-Fatty (WKF) rat was developed for a model of chronic pancreatitis and diabetes with obesity. We recently indicated that pancreatic dysfunction in the genesis of chronic pancreatitis with obesity could be improved by adequate exercise habit. The purpose of this study was to investigate whether chronic exercise with restricted diet can improve early lesions of pancreatic B-cells in WKF rats. Methods: Male WKF rats (age, 6 weeks) were divided into a FattyObese (FOB；n¼10), a Fatty-Diet Restriction (FDR；n¼8), and a Fatty-Exercise (FEX；n¼9) groups. WBN/Kob (WK；lean) rats were used as Control (C； n¼6). The C and the FOB rats had free access to food, the FDR and the FEX rats had food intake restricted to 69% and 70% of the FOB level, respectively. The FEX rats voluntarily ran on a rotary wheel ergometer for 6 weeks. Results: Mean body weight, serum glucose and triglyceride, HOMA-IR, pancreatic IL6 and XBP1 (parameter of Endoplasmic Reticulum stress) protein expression in the FOB were significantly higher than in the WK, whereas chronic exercise improved these parameters. Hyperlipidemia and insulin resistance were more improved in the FEX than in the FDR. Histopathological examination of the pancreatic islets of FOB rats demonstrated enlarged and abnormal appearance showing fibrosis, while nearly normal islets were found in both the C and FEX rats. Electron micrographs of pancreatic B-cells in FOB rats revealed markedly reduced secretory granules and obvious dilated endoplasmic reticulum. These ultrastructural abnormalities were improved in the FEX rats. Discussion: Male 12wks WKF rats had symptoms of pancreatitis and diabetes with early morphological lesion of B cells. Chronic exercise beneficially affected early lesion of B cells in WKF rats which may be by ameliorating inflammation and ER stress in pancreatic islets.
P-259. Influence of Helicobacter pylori (Hp) infection on the severity of chronic alcoholic pancreatitis (CAP) Nadiya Byelyayeva, Natalya Gubergrits Department of Internal Medicine, Donetsk National Medical University, Ukraine The role of this infection is more studied upon autoimmune pancreatitis. The aim of research is to study the influence of Hp on the severity of CAP. Materials and methods: The study included 68 patients with CAP. They were divided into two groups：Hp+ (36 patients) and Hp- (32 patients). We studied the severity of clinical manifestations, activity of a-amylase, blood and urine pancreatic isoamylase, blood lipase. Fecal elastase test, sonography with ultrasound histography of the pancreas were performed. All the
Abstracts / Pancreatology 16 (2016) S1eS192
1
Department of Gastroenterology, Tohoku University, Japan Department of Medical Genetics, Tohoku University, Japan 3 Division of Cell Proliferation, Tohoku University, Japan 2
Aim: Factors underlying genetic predisposition for development of alcoholic pancreatitis are largely unknown. Whole exome sequenceing (WES) focusing on only the coding regions of the genome, has become in a few years the choice strategy to identify a coding allelic variant for genetic disorder. The aim of this study was to identify a new candidate gene responsible for alcoholic pancreatitis. Methods: Genomic DNA was prepared from a total of 560 patients with pancreatitis (chronic pancreatitis, 288；acute pancreatitis, 272) and 439 control subjects. The SureSelect Human All Exon kit was used for the analysis of all exons in 16 patients with alcoholic pancreatitis. DNA libraries were constructed and sequenced using paired-end protocol with 101-base reads on Illumina HiSeq2500. Mutational analysis of the RNF113B gene was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis. This study was approved by the Ethics Committee of Tohoku University School of Medicine. Results: WES resulted in each samples having greater than 61 million reads. About 70% of all coding regions were covered at 50x or more. Mean depth was over 70x. There were about 1,340,000 variants in total of the 16 samples. After removing synonymous variant, we compared frequency of the residual variants from that of control samples by Human Genetic Variation Browser. Of these, there were four candidate genes that might be associated with pancreatitis. Statistically significant difference was observed in the frequency of p.S89R variant in ring finger protein 113B between patients with alcoholic pancreatitis (14.3%；31/217) and control subjects (6.6%；29/410) (OR 2.4 p¼0.002). The frequency of this variant in patients with non-alcoholic pancreatitis (8.2%；28/343) was not statistically different from that in controls. Conclusion: Next generation sequencing might become the new strategy to identify the candidate genes for alcoholic pancreatitis.
P-257. Mutational analysis of the autophagy-related 16 like 1 (ATG16L1) gene in Japanese patients with chronic pancreatitis Eriko Nakano 1, Atsushi Masamune 1, Tetsuya Niihori 2, Kiyoshi Kume 1, Shin Hamada 1, Yoko Aoki 2, Tooru Shimosegawa 1 1 Division of Gastroenterology, Tohoku University Graduate School of Medicine, Japan 2 Department of Medical Genetics, Tohoku University Graduate School of Medicine, Japan
Background: To date, several pancreatitis susceptibility genes have been identified, including PRSS1, SPINK1, CTRC and CPA1 genes. However, in a nationwide study in Japan about 30% of families with hereditary pancreatitis do not carry mutations in any of the known pancreatitis susceptibility genes, suggesting that other yet unidentified genes might be involved. Recent studies using knockout mice have suggested that impaired autophagy might be involved in the pathogenesis of pancreatitis (Li N, et al. J Clin Invest 2013). This study aimed to clarify whether variants in the autophagy related 16 like 1 (ATG16L1) gene are associated with chronic pancreatitis (CP) in Japan by targeted next generation sequencing. Methods: 282 patients with CP (112 idiopathic, 157 alcoholic, and 13 hereditary/familial) were enrolled in this study. We designed the HaloPlex Target Enrichment System (Agilent Technologies) targeting all of the exons and adjacent intronic regions of the known pancreatitis susceptibility genes and ATG16L1 gene. All samples were sequenced on the Illumina Miseq platform with paired-end 151-bp reads. SIFT and PolyPhen-2 were used to predict whether an amino acid substitution would affect the structure and function of a protein. The variant frequencies in the Japanese control population were obtained from the integrative Japanese Genome Variation Database (https://ijgvd.megabank.tohoku.ac.jp). This study was approved by the Ethics Committee of Tohoku University School of Medicine (article#：2015-1-613).
Results: We could identify 5 non-synonymous variants including 2 known [c.409A＞G (p.T137A), c.1160C＞T (p.A387V)], 3 novel ones [c.11G＞T (p.G4L), c.76G＞C (p.V26L) and c.581A＞G (p.E194A)] and 2 synonymous variants including one novel variant in the exonic regions. p.G4L, p.V26L, p.T137A, and p.E194A were probably damaged based on the SIFT and/or the PolyPhen-2 prediction. The frequencies of these variants were not different between all patients with CP and controls. Conclusions: Variants in the ATG16L1 gene are not associated with CP in Japan.
P-258. Chronic exercise improve s disrupt ion of pancreatic B-cells morphology in WBN/Kob-Fatty rats Kumiko Minato 1, Yoko Shiroya 1, Hideki Yamauchi 2 1
Faculty of Life Science, Wayo Women's University, Japan Department of Molecular Physiology, Jikei University School of Medicine, Japan 2
Aim: Leptin receptor-deficient WBN/Kob-Fatty (WKF) rat was developed for a model of chronic pancreatitis and diabetes with obesity. We recently indicated that pancreatic dysfunction in the genesis of chronic pancreatitis with obesity could be improved by adequate exercise habit. The purpose of this study was to investigate whether chronic exercise with restricted diet can improve early lesions of pancreatic B-cells in WKF rats. Methods: Male WKF rats (age, 6 weeks) were divided into a FattyObese (FOB；n¼10), a Fatty-Diet Restriction (FDR；n¼8), and a Fatty-Exercise (FEX；n¼9) groups. WBN/Kob (WK；lean) rats were used as Control (C； n¼6). The C and the FOB rats had free access to food, the FDR and the FEX rats had food intake restricted to 69% and 70% of the FOB level, respectively. The FEX rats voluntarily ran on a rotary wheel ergometer for 6 weeks. Results: Mean body weight, serum glucose and triglyceride, HOMA-IR, pancreatic IL6 and XBP1 (parameter of Endoplasmic Reticulum stress) protein expression in the FOB were significantly higher than in the WK, whereas chronic exercise improved these parameters. Hyperlipidemia and insulin resistance were more improved in the FEX than in the FDR. Histopathological examination of the pancreatic islets of FOB rats demonstrated enlarged and abnormal appearance showing fibrosis, while nearly normal islets were found in both the C and FEX rats. Electron micrographs of pancreatic B-cells in FOB rats revealed markedly reduced secretory granules and obvious dilated endoplasmic reticulum. These ultrastructural abnormalities were improved in the FEX rats. Discussion: Male 12wks WKF rats had symptoms of pancreatitis and diabetes with early morphological lesion of B cells. Chronic exercise beneficially affected early lesion of B cells in WKF rats which may be by ameliorating inflammation and ER stress in pancreatic islets.
P-259. Influence of Helicobacter pylori (Hp) infection on the severity of chronic alcoholic pancreatitis (CAP) Nadiya Byelyayeva, Natalya Gubergrits Department of Internal Medicine, Donetsk National Medical University, Ukraine The role of this infection is more studied upon autoimmune pancreatitis. The aim of research is to study the influence of Hp on the severity of CAP. Materials and methods: The study included 68 patients with CAP. They were divided into two groups：Hp+ (36 patients) and Hp- (32 patients). We studied the severity of clinical manifestations, activity of a-amylase, blood and urine pancreatic isoamylase, blood lipase. Fecal elastase test, sonography with ultrasound histography of the pancreas were performed. All the