Gene conversion between functional trypsinogen genes PRSS1 and PRSS2 associated with chronic pancreatitis in a six-year-old girl

Abstract

Gene conversion--the substitution of genetic material from another gene--is recognized as the underlying cause of a growing number of genetic diseases. While in most cases conversion takes place between a normal gene and its pseudogene, here we report an occurrence of disease-associated gene conversion between two functional genes. Chronic pancreatitis in childhood is frequently associated with mutations of the cationic trypsinogen gene (serine protease 1; PRSS1). We have analyzed PRSS1 in 1106 patients with chronic pancreatitis, and identified a novel conversion event affecting exon 2 and the subsequent intron. The recombination replaced at least 289 nucleotides with the paralogous sequence from the anionic trypsinogen gene (serine protease 2; PRSS2), and resulted in the PRSS1 mutations c.86A > T and c.161A > G, causing the amino acid substitutions N29I and N54S, respectively. Analysis of the recombinant N29I-N54S double mutant cationic trypsinogen revealed increased autocatalytic activation, which was solely due to the N29I mutation. In conclusion, we have demonstrated that gene conversion between two functional paralogous trypsinogen genes can occur and cause genetically determined chronic pancreatitis.

Figure 1

Schematic representation of the conversion mutation within the human trypsinogen gene family at the TCR-beta locus on chromosome 7q35. Each gene represents a tandem 10-kb repeat (Rowen et al., 1996). A region comprising parts of exon 2 and intron 2 of the PRSS1 acceptor gene is converted by the PRSS2 donor gene. The PRSS2 gene remains unaltered (adapted from Chen et al., 2001).

Figure 2

Alignment of the PRSS1 gene between c.41–148 (IVS1-148) and c.200+499 (IVS2+499) with the corresponding PRSS2 sequence. Exon 2 is shown in capital letters. Positions of the forward sequencing primers and reverse PCR primers B and D are underlined. Forward PCR primers A and C are not shown, as these anneal upstream of the sequence region displayed here. The maximal converted region is highlighted in gray; the minimal converted region is framed.

Figure 3

Autocatalytic activation of wild type (PRSS1), single-mutant (N29I) and double-mutant (N29I-N54S) cationic trypsinogen. 2 μM trypsinogen (final concentration, in a final volume of 100 μL) was incubated at 37 ^oC, in 0.1 M Tris-HCl (pH 8.0, upper panel) or 0.1 M Na-acetate buffer (pH 5.0, lower panel), in the presence of 2 mg/mL bovine serum albumin. Aliquots of 2 μL were withdrawn from reaction mixtures at indicated times and trypsin activity was determined with 0.14 mM N-CBZ-Gly-Pro-Arg-p-nitroanilide (final concentration). Activity was expressed as percentage of the potential total activity, which was determined on similar zymogen samples activated with bovine enterokinase at 22 ^oC in 0.1 M Tris-HCl (pH 8.0), 2 mg/mL bovine serum albumin and 1 mM CaCl₂.