Clinical, morphological and functional aspects of patients suffering from pancreatitis associated with mutations of CFTR and SPINK1 genes

Published on May 1, 2013in Pancreatology3.629
· DOI :10.1016/J.PAN.2012.11.235
Giulia De Marchi6
Estimated H-index: 6
(University of Verona),
Antonio Amodio13
Estimated H-index: 13
(University of Verona)
+ 7 AuthorsLuca Frulloni58
Estimated H-index: 58
(University of Verona)
s / Pancreatology 13 (2013) S2–S98 S10 sequenced using next generation sequencing (Illumina GAIIx) in 5 pancreatitis and 5 control patients, heterozygous for the p.N34S variant. Identified sequence variants were then filtered based on their increased frequency within chronic pancreatitis patients, revealing 7 haplotype segments. Seven variants (Single Nucleotide Polymorphisms), one selected within each haplotype segment, were then screened in 38 pancreatitis patients and 20 controls (both groups heterozygous for p.N34S). Results: 29 haplotypes were identified; only haplotype 13 was significantly associated with pancreatitis (p1⁄40.0009, hapscore: -3.31). Conclusion: It is possible that a high-risk haplotype rather than a simple base variant is responsible for SPINK1 associated pancreatitis. O-22 Abstract id: 306. Copy number variants of carboxyl ester lipase – A role in pancreatic disease? Karianne Fjeld , Stefan Johansson , Bente Berg Johansson , Erling Tjora , P al Rasmus Njolstad , Anders Molven . 1 KG Jebsen Center for Diabetes Research, Department of Clinical Medicine, University of Bergen, Norway 2 Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway Department of Pediatrics, Haukeland University Hospital, Bergen, Norway 4 The Gade Institute, University of Bergen, Norway Introduction: We have previously described a syndrome of exocrine and endocrine pancreatic dysfunction caused by single-base mutations in the highly polymorphic carboxyl ester lipase (CEL) gene. Copy number variations (CNV) of the CEL gene have also been reported. Aims:We aimed to characterize the structure of CEL CNV alleles and to examine their potential functional properties. Materials & methods: PCR, fragment analysis and sequencing were performed for genotyping of CEL CNV alleles. For functional analysis, we did transfection of human embryonic kidney (HEK293) and mouse acinar (266-6) cells, protein deglycosylation, Western blotting, pulse-chase analysis, enzyme activity measurements, immunostaining, and confocal microscopy. Results: We identified and fine-mapped three recombined CEL CNV alleles, two with gene duplication and one with a deletion. These alleles have all probably arisen from non-allelic, homologous recombination events between CEL and the neighbouring CEL pseudogene (CELP). The deletion allele encodes a CEL proteinwith a truncated C-terminal end. This protein showed reduced glycosylation, secretion and enzyme activity as compared with the CEL wild-type protein. In addition, the truncated protein exhibited a tendency to accumulate inside the cell, and confocal microscopy indicated that it localized to intracellular bodies in HEK293 cells. Conclusion: We have determined the structure of three recombined CEL alleles. One of them encodes a truncated CEL protein, which showed impaired functional properties in transfected cells. Further investigations are now needed to examine whether this protein variant has a role in pancreatic disease. O-23 Abstract id: 245. Clinical, morphological and functional aspects of patients suffering from pancreatitis associated with mutations of CFTR and SPINK1 genes Giulia De Marchi, Antonio Amodio, Francesco Vitali, Chiara Cristofori, Tiziana Tumelero, Mattia Pellicciari, Armando Gabbrielli, Luigi Benini, Italo Vantini, Luca Frulloni. Department of Medicine, University of Verona, Italy Introduction: Sporadic pancreatitis may be associated with mutations of CFTR and SPINK genes. Few data are present in literature about clinical, instrumental and functional profiles of pancreatitis associated with CFTR GM compared to SPINK1 GM. Aims: To evaluate pancreatitis associated with CFTR Vs SPINK1GM Patients & methods: Data from patients suffering from pancreatitis associated with GM were studied. The diagnosis of GM was gathered by investigation on 35 CFTR GM or complete gene sequencing and the 2 main SPINK1 GM(N34S and P55I). Patients were divided in 3 groups: CFTRS(single CFTR GM), CFTR-D(compound CFTR GM) and SPINK1(single or double SPINK1 GM). Results: 114 pts (59 M, 45 F) were studied, 72(63.2%) in CFTR-S group(45 M, 27 F, mean age 31.1 14.6 yrs), 23(20.2%) in CFTR-D group(12 M, 11 F, mean age 29.3 16.4 yrs) and 19(16.7%) in SPINK1 group(12 M, 7 F, mean age 30.5 18.3 yrs). 118 CFTR GM were found in 95 pts and 21 N34S SPINK1 GM in 20 pts. 17 pts(15%) suffered from painless pancreatitis. No differences were observed in episodes of pancreatitis, need for an endoscopic approach, evolution toward pancreatic insufficiency among groups. However, a diagnosis of chronic pancreatitis(p1⁄40.05) and onset of calcifications(p1⁄40.01) were more frequently observed in SPINK1 group. 24 pts(21%) underwent surgery, 12 derivative-type and 12 demolitive-type. 6 pts developed a pancreatic neoplasia(4 adenocarcinoma and 2 IPMNs) at a mean age of 58 yrs(range 48-72), and 4 patients died for pancreatic adenocarcinoma. Conclusion: Pancreatitis associated with SPINK1 gene mutations seems to differ from that associated with CFTR gene mutations. O-24 Abstract id: 176. Epigenetic regulation of autophagosome formation in pancreatic cancer cells by the methyltransferase G9a Alexander K€ onig , Amaia Artal-Martinez des Narvajas , Timothy Gomez , Jin-San Zhang , Thomas Gress , Volker Ellenrieder , Daniel Billadeau . 1 Phillipps-Universit€at Marburg, Germany Mayo Clinic, Rochester, MN, United States Introduction: Macroautophagy is a highly conserved cellular process involved in the clearance of proteins and organelles, impacting several cellular and developmental functions including cell growth, homeostasis, and immunity. Due to its integral function in these vital processes it is not surprising that macroautophagy has been implicated in diseases such as cancer and neurodegenerative disorders. Aims: Although the cytoplasmic network leading to macroautophagy induced by starvation, hypoxia, or receptor stimulation is widely studied, and recent publications have identified transcription factors involved in the induction of autophagy, the mechanisms regulating chromatin reorganization that initiate and maintain the macroautophagy process have not been elucidated. Materials & methods: We performed ChIP, Array, qRT-PCR, WB, and IF in cancer cells, MEFs, and naive human T-cells. Results: Here we provide experimental evidence that modulation of the chromatin landscape in pancreatic cancer cells facilitated by the methyltransferase G9a regulates the expression of key players in autophagosome formation, a crucial step in the process of macroautophagy.We show that G9a associates with, and epigenetically represses theMAP1LC3B (refers to LC3B), WIPI1 and TP53INP2 (refers to Diabetes and Obesity Regulated DOR) gene promoters through histone methylation under normal conditions, but G9a repressive histone marks are removed during starvation and T-cell stimulation, two processes that induce macroautophagy. Interestingly, pharmacological inhibition or RNA-mediated suppression of G9a induces autophagosome formation, but is not sufficient to complete the autophagic flux, which requires an additional signal following mTOR inhibition. Conclusion: Together these findings provide evidence that epigenetic control in pancreatic cancer represents an important mechanism during the regulation of macroautophagy.
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