Is the SPINK1 p.Asn34Ser Missense Mutation Per se the True Culprit within its Associated Haplotype?

Although SPINK1, which encodes the pancreatic secretory trypsin inhibitor (PSTI), has been firmly established as a chronic pancreatitis-predisposing gene, the causal variant within the most common c.101A>G (p.Asn34Ser)-containing haplotype remains to be identified. The low penetrance of this haplotype implies a minor effect on gene expression and/or protein function; such a minor effect may not have been readily detectable in the previous studies. Here, we focused on the putative functional effect of the p.Asn34Ser haplotype on pre-mRNA splicing and mRNA stability. Two measures were taken to maximize the relevance and accuracy of our in vitro analysis. First, all five cis-linked variants were analyzed together within a single expression plasmid. Second, a real-time quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) assay with high accuracy and reproducibility was developed. The exclusion of a minor effect of the p.AsnN34Ser haplotype on gene expression prompted us to reconsider the possible role of the p.Asn34Ser missense mutation in trypsin inhibition. A survey of previous studies that analyzed the inhibitory activity of purified recombinant wild-type and p.Asn34Ser mutant PSTI peptides revealed that a minor effect of the p.Asn34Ser missense mutation on trypsin inhibition appeared to be overlooked. Moreover, the reduced inhibition of trypsin by the p.Asn34Ser mutant peptide is consistent with findings from analyses of PSTI’s 3D structures and with the presence of an increase in p.Asn34Ser mutants in the urine of p.Asn34Ser heterozygotes. A combination of these diverse lines of evidence strongly suggests that the p.Asn34Ser missense mutation is the true culprit within its associated haplotype. The clarification of this issue is pivotal for developing targeted therapies for pancreatitis.