Investigation of the pathogenic mechanism underlying the common SPINK1 p.N34S pancreatitis risk haplotype

• Applicant: Professor Dr. Heiko Witt
• Subject Area: Pediatric and Adolescent Medicine ; Gastroenterology
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 346764549

Chronic pancreatitis (CP) is an inflammatory disease characterized by agonizing pain, maldigestion and/or diabetes mellitus. In the paediatric age, most patients with CP suffer from genetically determined pancreatitis i.e. autosomal dominant hereditary CP or - more common - from so-called idiopathic chronic pancreatitis (ICP). In the past, genetic defects in seven genes associated to ICP have been identified.Of these genes, SPINK1, encoding a trypsin inhibitor, represents the major genetic risk factor for idiopathic CP as well as tropical calcific pancreatitis (TCP) and also contributes to the pathogenesis of alcohol-related CP. In 20% to 50% of ICP and TCP patients a SPINK1 mutation can be detected. A common SPINK1 p.N34S haplotype accounts for approximately 80% of all disease associated SPINK1 mutations, but the underlying pathogenic mechanism of p.N34S remains elusive.p.N34S did not to affect SPINK1 protein levels, secretion or trypsin inhibitory capacity in three independent in vitro studies. Moreover, no aberrant splicing of SPINK1 was found when assessing endogenous mRNA levels of subjects with the p.N34S haplotype or when using a mini gene system analysing p.N34S and the intronic variants in high LD.Notably, assessing allele-specific SPINK1 mRNA transcription in PaCa44 cells heterozygous for the p.N34S haplotype, we found decreased SPINK1 mRNA levels of the mutated p.N34S compared to the wild-type allele. This strongly indicates that impaired gene expression is the underlying mechanism of the p.N34S SPINK1 risk haplotype.Using bioinformatics, public domain epigenomic mark and population genetics inferences on variants in LD with p.N34S, we could reduce the number of candidate regulatory variants in LD, which may contribute to regulation of SPINK1 expression, from 26 to 2. For these two variants, we find high conservation of TFBS modularity, overlap to pancreas epigenomic regulatory region marks, high LD to p.N34S in both, subjects of European and Indian ancestry, allele-specific modulation of transcriptional activity, and differential DNA-protein binding in EMSA.We hypothesize that one or both of these variants in high LD to the coding but non-functional p.N34S variant modulate the affinity of DNA-binding transcription factors and/or co-factors, resulting in reduced SPINK1 transcription. Identification of these regulatory variant or variants, of the allele-specific binding proteins (using highly efficient proteomics methodology) and the in-depth analysis of how SPINK1 expression is modulated (using e.g. CRISPR genome editing) will be performed in different cell lines and primary pancreatic organoids.Diminished SPINK1 expression would directly impact the control of pancreatic trypsin activity and might thereby explain the observed CP phenotype. Unravelling the precise underlying mechanism of the p.N34S haplotype will greatly contribute to our understanding of CP pathophysiology.

• DFG Programme: Research Grants
• Co-Investigator: Dr. Helmut Laumen