Browsing by Subject "Secretagogins"
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Item Missense Mutation in SCGN (Secretagogin) as a Possible Cause of Ulcerative Colitis in Three Siblings(2014-02-04) Llano, Ernesto; Burstein, Ezra; Chan, Lilliene; Harrison, Steven M.; Li, Haiying; Sifuentes-Dominguez, Luis; Park, Jason Y.; Cohen, Jonathan C.; Baker, Linda A.Inflammatory bowel disease (IBD) consists of two disorders, Crohn's disease and ulcerative colitis (UC), which affect 1.4 million Americans. Genetic factors contribute to the development of these disorders but not all culprit genes have been identified. To further our understanding of the genetic causes of IBD, we examined a consanguinous family with a high incidence of childhood-onset UC. We performed exome sequencing of five siblings, three of which were diagnosed with severe UC before the age of 10. Given the parents' consanguinity, areas of homozygosity were examined using a SNP array. Two areas of homozygosity, in chromosomes 6 and 12, were shared by the three affected probands but not by their two unaffected siblings. Given the inheritance pattern in the pedigree, we speculated that a homozygous recessive mutation in a gene contained within these intervals should be responsible for the phenotype. Thus, a total of 140 potential genes were implicated. Variants found by exome sequencing were prioritized if they affected the three probands and not their unaffected siblings, and if they were rare in the general population according to their frequency in the 1000 genomes database. Afterwards, we cross-referenced the identified potential culprit variants found in the exome sequence analysis against the unique areas of homozygosity shared by the probands. The top candidate change was in SCGN, which had a coding variant (c. 433G>A/p.R77H) not previously found in the 1000 genomes database or in the dbSNP database. Sanger sequencing confirmed that both parents were carriers, the three probands were homozygous, and one sibling was a carrier while the other was wild-type. SCGN encodes a 276 amino acid, calcium-binding protein, secretagogin. The protein is expressed in tissues of neuroendocrine lineage, such as pancreatic β-cells and intestinal enteroendocrine cells. The coding change found in these patients is located in one of the calcium binding domains (EF hand 2). Immunohistochemical analyses of the patients' colon biopsies did not demonstrate a decrease of the presence of secretagogin when compared to controls, suggesting that the mutation does not affect expression, but may have a functional effect yet to be determined. We are building a molecular and cellular model of the R77H mutation to identify if this coding change alters the function of the protein. If a functional impairment of SCGN is confirmed, it could represent the first description of enteroendocrine dysfunction playing a role in IBD pathophysiology.Item Mutant Secretagogin, a Potential Cause of Ulcerative Colitis, Exhibits Reduced Affinity for SNARE Complex Protein SNAP-25(2016-01-19) Norris, Nicholas; Sifuentes-Dominguez, Luis; Burstein, EzraINTRODUCTION: Inflammatory Bowel Diseases (IBD) encompass a set of poorly understood multifactorial inflammatory disorders including Chrohn's disease (CD) and Ulcerative Colitis (UC). To date, around 200 genetic variants associated with increased risk of IBD have been identified through genome-wide association studies. Recently, our lab identified three siblings with UC from consanguineous parents who were all homozygous for a rare hypomorphic variant in the gene SCGN which encodes the calcium sensing protein secretagogin. Within the GI tract, secretagogin is localized specifically to enteroendocrine cells (EECs) suggesting an unforeseen role of EECs in IBD pathogenesis. This disease associated variant results in an amino acid substitution (R77H) in the Ca2+ binding region of secretagogin, which has been shown to interact with SNARE proteins in a Ca2+ dependent manner to mediate membrane fusion and exocytosis. Therefore, we examined whether this SCGN mutation impairs its Ca2+ dependent binding to the SNARE complex component SNAP-25 in vitro. METHODS: A GST pull-down assay was performed using recombinant GST fusion proteins of human secretagogin (WT or R77H) immobilized to glutathione-agarose beads. These beads were used to affinity purify SNAP-25 from whole cell lysates of STC-1 cells, a mouse enteroendocrine tumor cell line. Because SNAP-25 interactions with secretagogin are Ca2+ dependent, whole cell lysates were prepared in either Ca2+ rich or Ca2+ free conditions. The beads were washed and bound proteins were detected by SDS-PAGE and western blot analysis using appropriate antibodies. RESULTS: Under Ca2+ conditions, SNAP-25 was successfully bound to secretagogin, but the amount of SNAP-25 bound to mutant R77H secretagogin was significantly less. As expected, SNAP-25 did not bind secretagogin under Ca2+ free conditions. CONCLUSIONS: We confirm secretagogin interacts with SNAP-25 in a Ca2+ dependent fashion and the R77H mutation, located in the Ca2+ binding pocket of secretagogin, disrupts this interaction representing a hypomorphic mutation. This finding is consistent with the recessive nature of this mutation and strengthens its physiological relevance. Furthermore, it suggests that impaired exocytosis and hormone release from EECs contributes to the disease pathogenesis. Further study to elucidate which (if any) EEC secretory products play key roles in IBD prevention is needed. Understanding potentially aberrant paracrine mechanisms underlying IBD could open the door to novel treatments like hormone replacement therapy.