title: Discrimination of three mutational events that result in a disruption of the R122 primary autolysis site of the human cationic trypsinogen (PRSS1) by denaturing high performance liquid chromatography.
abstract: BACKGROUND
  R122, the primary autolysis site of the human cationic trypsinogen (PRSS1), constitutes an important "self-destruct" or "fail-safe" defensive mechanism against premature trypsin activation within the pancreas. Disruption of this site by a missense mutation, R122H, was found to cause hereditary pancreatitis. In addition to a c.365G>A (CGC>CAC) single nucleotide substitution, a c.365 through 366GC>AT (CGC>CAT) gene conversion event in exon 3 of PRSS1 was also found to result in a R122H mutation. This imposes a serious concern on the genotyping of pancreatitis by a widely used polymerase chain reaction-restriction fragment length polymorphism assay, which could only detect the commonest c.365G>A variant.
  
  
  METHODS
  DNA samples containing either the known c.365G>A or c.365 through 366GC>AT variant in exon 3 of PRSS1 were used as positive controls to establish a denaturing high performance liquid chromatography (DHPLC) assay.
  
  
  RESULTS
  DHPLC could readily discriminate the two known different mutational events resulting in the R122H mutation. More importantly, under the same experimental conditions, it identified a further mutational event that also occurs in the R122 primary autolysis site but results in a different amino acid substitution: c.364C>T (CGC>TGC; R122C).
  
  
  CONCLUSIONS
  A rapid, simple, and low-cost assay for detecting both the known and new mutations occuring in the R122 primary autolysis site of PRSS1 was established. In addition, the newly found R122C variant represents a likely pancreatitis-predisposing mutation.
authors.affiliation: INSERM-EMI 0115, Génétique Moléculaire et Génétique Epidémiologique, Etablissement Français du Sang-Bretagne, Université de Bretagne Occidentale, Centre Hospitalier Universitaire, Brest, France. cedric.lemarechal@univ-brest.fr
authors.forename: C
authors.initials: C
authors.lastname: Le Maréchal
authors.forename: J M
authors.initials: JM
authors.lastname: Chen
authors.forename: I
authors.initials: I
authors.lastname: Quéré
authors.forename: O
authors.initials: O
authors.lastname: Raguénès
authors.forename: C
authors.initials: C
authors.lastname: Férec
authors.forename: J
authors.initials: J
authors.lastname: Auroux
mesh_terms: D019943:Amino Acid Substitution; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000737:chemistry; D004252:DNA Mutational Analysis / Q000379:methods*; D005785:Gene Conversion; D020022:Genetic Predisposition to Disease; D006801:Humans; D020125:Mutation, Missense; D009691:Nucleic Acid Denaturation; D010195:Pancreatitis / Q000235:genetics*; D014357:Trypsin; D014362:Trypsinogen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D014362:Trypsinogen; D004247:DNA; C489303:PRSS1 protein, human; D014357:Trypsin