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

慢性胰腺炎（CP）是一种以剧烈疼痛、消化不良和/或糖尿病为特征的炎症性疾病。在儿童时代，大多数CP患者患有遗传性胰腺炎，即常染色体显性遗传性CP或更常见的所谓特发性慢性胰腺炎（ICP）。在过去，已经确定了与ICP相关的七个基因的遗传缺陷。在这些基因中，编码胰蛋白酶抑制剂的SPINK1是特发性CP和热带钙化性胰腺炎（TCP）的主要遗传危险因素，也有助于酒精相关性CP的发病机制。在20%至50%的ICP和TCP患者中可以检测到SPINK1突变。常见的SPINK1 p.N34S单倍型约占所有疾病相关的SPINK1突变的80%，但p.N34S的潜在致病机制尚不清楚。在三个独立的体外研究中，N34S没有影响SPINK1蛋白水平、分泌或胰蛋白酶抑制能力。此外，在评估pn34s单倍型受试者的内源mRNA水平时，以及在分析pn34s单倍型受试者和高lds的intronic变异体时，均未发现SPINK1剪接异常。值得注意的是，在pn34s单倍型杂合PaCa44细胞中评估等位基因特异性SPINK1 mRNA转录时，我们发现突变的pn34s的SPINK1 mRNA水平比野生型等位基因低。这强烈表明基因表达受损是p.N34S SPINK1风险单倍型的潜在机制。利用生物信息学、公共领域表观基因组标记和群体遗传学对LD中含有p.N34S的变异的推断，我们可以将LD中可能参与调控SPINK1表达的候选调控变异的数量从26个减少到2个。对于这两种变体，我们发现TFBS模块化高度保守，与胰腺表观基因组调控区域标记重叠，对p.N34S的高LD，欧洲和印度血统的受试者，转录活性的等位基因特异性调节，以及EMSA中dna -蛋白结合的差异。我们假设，与编码但无功能的p.N34S变体高LD的一个或两个变体调节dna结合转录因子和/或辅助因子的亲和力，导致SPINK1转录减少。将在不同细胞系和初级胰腺类器官中鉴定这些或多个调节变体、等位基因特异性结合蛋白（使用高效蛋白质组学方法）和深入分析SPINK1表达是如何被调节的（例如使用CRISPR基因组编辑）。SPINK1表达的减少会直接影响胰腺胰蛋白酶活性的控制，从而可能解释观察到的CP表型。揭示p.N34S单倍型的精确潜在机制将极大地有助于我们对CP病理生理学的理解。

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.