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

慢性胰腺炎（CP）是一种以疼痛、消化不良和/或糖尿病为特征的炎症性疾病。在儿科年龄，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7个基因的遗传缺陷，其中，编码胰蛋白酶抑制剂的SPINK 1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CP的发病机制。在20%至50%的ICP和TCP患者中，可以检测到SPINK 1突变。一个常见的SPINK 1 p.N34S单倍型占所有疾病相关的SPINK 1突变的约80%，但p.N34S的潜在致病机制仍然不明确。在三个独立的体外研究中，p.N34S不影响SPINK 1蛋白水平，分泌或胰蛋白酶抑制能力。此外，当评估具有p.N34S单倍型的受试者的内源性mRNA水平时，或者当使用分析高LD中的p.N34S和内含子变体的微型基因系统时，没有发现SPINK 1的异常剪接。值得注意的是，评估p.N34S单倍型杂合的PaCa 44细胞中的等位基因特异性SPINK 1 mRNA转录，我们发现与野生型等位基因相比，突变的p.N34S的SPINK 1 mRNA水平降低。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p.N34S变异的推断，我们可以将LD中可能有助于SPINK 1表达调控的候选调控变异的数量从26个减少到2个。对于这两个变体，我们发现TFBS模块性的高度保守性，与胰腺表观基因组调控区标记重叠，在欧洲和印度血统的受试者中对p.N34S的高LD，转录活性的等位基因特异性调节，我们假设这些变异体中的一个或两个在高LD中对编码但非功能性p.N34S变异体调节DNA-蛋白质结合的亲和力。结合转录因子和/或辅因子，导致SPINK 1转录减少。这些调节变体或变体鉴定，等位基因特异性结合蛋白（使用高效的蛋白质组学方法）和SPINK 1表达如何调节的深入分析（使用例如CRISPR基因组编辑）SPINK 1表达减少将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单倍型的确切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.