title: Minigene analysis of intronic variants in common SPINK1 haplotypes associated with chronic pancreatitis.
abstract: OBJECTIVE
  Two common haplotypes of the serine protease inhibitor Kazal type 1 (SPINK1) gene have been shown to increase the risk for chronic pancreatitis. A haplotype comprising the c.101A>G (p.N34S) missense variant and four intronic alterations has been found worldwide, whereas a second haplotype consisting of the c.-215G>A promoter variant and the c.194+2T>C intronic alteration has been observed frequently in Japan.
  
  
  METHODS
  In the present study, the functional significance of the intronic variants in the pathogenic SPINK1 haplotypes was examined by utilising minigenes, which harbour individual introns placed in the appropriate context of the full-length SPINK1 cDNA. Cells transfected with the SPINK1 minigenes secrete active trypsin inhibitor, thereby allowing evaluation of mutational effects simultaneously on transcription, splicing, translation and secretion.
  
  
  RESULTS
  It was found that the c.194+2T>C intronic alteration abolished SPINK1 expression at the mRNA level, with consequent loss of inhibitor secretion, whereas the p.N34S-associated intronic variants had no detectable functional effect.
  
  
  CONCLUSIONS
  Taken together with previous studies, the results indicate that all known variants within the p.N34S-associated haplotype are functionally innocuous, suggesting that an as yet unidentified variant within this haplotype is responsible for the pathogenic effect. The marked negative impact of the c.194+2T>C variant on SPINK1 expression supports the notion that SPINK1 variants increase the risk of chronic pancreatitis by diminishing protective trypsin inhibitor levels.
authors.affiliation: Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts, USA.
authors.forename: E
authors.initials: E
authors.lastname: Kereszturi
authors.forename: O
authors.initials: O
authors.lastname: Király
authors.forename: M
authors.initials: M
authors.lastname: Sahin-Tóth
mesh_terms: D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D020022:Genetic Predisposition to Disease; D006239:Haplotypes; D006367:HeLa Cells; D006801:Humans; D007438:Introns / Q000235:genetics; D050500:Pancreatitis, Chronic / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D014359:Trypsin Inhibitor, Kazal Pancreatic
chemical_list: D002352:Carrier Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; C486642:SPINK1 protein, human; D014359:Trypsin Inhibitor, Kazal Pancreatic