Here we report that SPINK1 alterations which cause a complete block in secretion are associated with hereditary pancreatitis in heterozygotes. This is the first clear genetic-biochemical demonstration that severe loss-of-function SPINK1
alterations are associated with a high risk of chronic pancreatitis, therefore, these alterations should be regarded as disease-causing rather than disease-modifiers. Potential examples of other severe SPINK1
alterations in association with hereditary pancreatitis have been described previously (Witt et al., 2000
; Le Maréchal et al., 2004
; Masson et al., 2006
). The results presented here support the notion that this group of SPINK1 alterations should be classified as “severe” and should be differentiated from “mild” alterations, particularly from the relatively frequent, archetypal N34S variant (Witt et al., 2001a
; Le Maréchal et al., 2004
; Masson et al., 2006
). Mild alterations confer a smaller risk of pancreatitis, are typically found in association with idiopathic pancreatitis, and additional genetic and environmental risk factors are believed to influence their expression to a significant degree.
The L14R alteration is a novel variant, which we identified in two unrelated families, a Bulgarian family living in France, and a German family originating from the northern part of Germany. In both families, the alteration is inherited through several generations, thus establishing the diagnosis of autosomal dominant hereditary pancreatitis. The penetrance of the L14R alteration is high but incomplete, which is in agreement with the typical penetrance of dominant PRSS1
(cationic trypsinogen) alterations associated with classic hereditary pancreatitis (Whitcomb et al., 1996
; Le Maréchal et al., 2006
). The newly described L14R alteration affects the same amino acid as the previously reported L14P alteration (Witt et al., 2000
). The L14P alteration was originally identified in a 5-year old boy without a family history of pancreatitis. Interestingly, since that time, the mother, who also carried the alteration, developed pancreatitis. In addition to the two disease-causing alterations within the SPINK1 signal peptide, we also found a frequent polymorphic variant, L12F, in pancreatitis patients of African origin as well as in African controls, but not in white subjects. This variant served as an important control, demonstrating that not all signal peptide alterations are associated with pancreatitis and a direct relationship exists between the functional effects of the alterations and disease-association.
Functional analysis using transient expression in HEK 293T and CHO cells demonstrated that although mRNA expression was unaffected, both L14P and L14R abolished SPINK1 secretion, whereas L12F was secreted normally. The SPINK1 signal peptide conforms to the general paradigm of secretory signal peptides and comprises three functionally important regions; a positively charged N-terminal domain (5 amino acids), a hydrophobic central core (12 amino acids) and a polar C-terminal region (6 amino acids) including the signal-peptidase cleavage site (von Heijne G, 1990
). The alterations studied here all disrupt the hydrophobic core region and thus are expected to block translocation of preSPINK1 into the lumen of the endoplasmic reticulum. Alterations that affect the hydrophobic core of the signal peptide have been described and characterized in association with various human diseases. As shown in , the Leu→Pro change is a relatively frequent occurrence and a Leu→Arg alteration has also been detected. Functional analyses invariably demonstrated that these variations block secretion by inhibiting translocation of the preprotein into the endoplasmic reticulum. Therefore, signal peptide alterations that disrupt the hydrophobic core result in a loss of protein expression and consequently a loss of function.
Mutations that affect the hydrophobic core of the signal peptide in human diseases. The OMIM database numbers and literature references are also indicated.
It is arguable whether the constitutive secretory pathway of HEK or CHO cells used in our experiments adequately model the regulated secretory pathway of the pancreatic acinar cells. Unfortunately, there are no human acinar cell lines available which we could have used to characterize regulated secretion. The widely used rat acinar cell line AR42J (Jessop and Hay, 1980
) exhibits diminished secretory function and its utility as a model to study acinus-related secretory phenomena is questionable. Given the difficulty in testing the SPINK1 variants in authentic acinar cells, we utilized epithelial cell lines that carry out constitutive secretion. In these cell lines, the alterations clearly impair secretion of SPINK1, which we interpret as a defect in targeting to the endoplasmic reticulum. Because targeting and translocation of secretory proteins to the endoplasmic reticulum is a universally conserved mechanism, we posit that the same defect is expected to occur in pancreatic acinar cells, eventually leading to decreased SPINK1 secretion through the regulated pathway as well. Direct measurements of SPINK1 levels from pancreatic juice sample of L14R or L14P carriers would provide strong corroborating evidence for our findings. However, juice samples from the patients described in this study were not available for analysis.
In summary, the present study demonstrates that the p.L14R and p.L14P signal peptide alterations result in essentially complete loss of SPINK1 secretion. Clinically, these loss-of-function alterations are associated with autosomal dominant hereditary pancreatitis, confirming the notion that severe SPINK1 alterations represent strong risk factors for chronic pancreatitis.