For genotyping known genetic variants, PCR-RFLP, if available, represents the most simple and rapid method. However, it has an intrinsic disadvantage, that is, it could only detect a "specific" variant, exemplified by the PCR-RFLP analysis of the c.365G>A (CG
C; R122H) variant in PRSS1
]. DGGE is one of the most powerful techniques for mutation detection and screening but, unfortunately, it is time-consuming and technically difficult to implement [5
DHPLC is an automated technology for mutation screening based on the separation of heteroduplexes from homoduplexes on a stationary phase under partialy denaturing conditions [8
]. This technique was initially used for identifying single nucleotide polymorphisms on the Y chromosome [9
] and, thereafter, has been emerging as a sensitive, rapid, low-cost, and reliable method for mutation detection and screening in different genes, including the cystic fibrosis transmembrane conductance regulator gene [10
], the hereditary hemochromatosis gene [11
], and the BRCA1
In this report, DHPLC was attempted to screen the two known mutational events in exon 3 of PRSS1 that result in the most frequent pancreatitis-associated R122H mutation. As shown in Fig , this technique could readily discriminate the two different mutational events in all of our positive control samples. Moreover, it identified new c.365G>A (CGC>CAC; R122H) carriers in our cohort of chronic pancreatitis subjects (data not shown). Further, it found a novel autolysis site mutation: a c.364C>T (CGC>TGC) transition, which is presumed to result in a R122C amino acid change of the human cationic trypsinogen (Fig. ), was detected in a 42 years old women with idiopathic chronic pancreatitis. This new variant was not present in 300 French healthy blood donors evaluated by DHPLC. To date, this c.364C>T (CGC>TGC) variant and the c.365~366GC>AT (CGC>CAT) variant have only been detected once each in our cohort of about 400 French patients with chronic pancreatitis.
Figure 1 Detection of three mutational events in exon 3 of PRSS1 resulting in a disruption of the R122 primary autolysis site of human cationic trypsinogen. Left panel: denaturing high performance liquid chromatography (DHPLC) profile of the mutant compared with (more ...)
Here it might be interersting to note that while the c.365G>A (CGC>CAC; R122H) could be attributable to a spontaneous deamination of 5-methylcytosine to give thymine in the CpG island on the antisense strand, the c.364C>T (CGC>TGC; R122C) could be attributable to such an event on the sense strand. Thus the R122 primary autolysis site seems to be particularly susceptible to mutational events (CpG island and gene conversion-promoting structure) despite of a strong selection pressure (Table ). Nevertheless, the nature of the R122C variant suggests that it is a likely pancreatitis-predisposing mutation: an arginine to cysteine substitution is expected to disrupt the R122 primary autolysis site simply because cysteine could not be cleaved by trypsin, as in the case of the R122H mutation. However, the resistance to autolysis confered by the R122C mutation may be somehow different from that confered by the R122H mutation in a quantitative manner. In parallel functional analysis of the R122H and R122C mutations would certainly help determine genotype/phenotype correlation.
The three different mutational events resulting in a disruption of the R122 primary autolysis site of the human cationic trypsinogen