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

常见SPINK1 p.N34S胰腺炎风险单倍型致病机制的研究

• 批准号: 346764549
• 负责人: Professor Dr. Heiko Witt
• 金额: --
• 依托单位: Universitätsklinik und Poliklinik für Innere Medizin I
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/346764549?language=en
• 关键词: Investigation; pathogenic; mechanism; underlying; common

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.

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患者患有遗传决定的胰腺炎，即常染色体显性遗传性CP，或者更常见的是所谓的特发性慢性胰腺炎(ICP)。在过去的研究中，已发现7个与妊娠肝内胆汁淤积症相关的基因存在遗传缺陷，其中编码胰蛋白酶抑制物的SPINK1基因是特发性胰腺炎和热带钙化性胰腺炎的主要遗传危险因素，也是酒精相关性胰腺炎的发病机制之一。在20%至50%的妊娠肝内胆汁淤积症患者中，可以检测到SPINK1突变。一种常见的SPINK1 p.N34S单倍型约占所有疾病相关SPINK1突变的80%，但P.N34S的潜在致病机制仍不清楚。在三项独立的体外研究中，p.N34S不影响SPINK1蛋白水平、分泌或胰酶抑制能力。此外，当评估带有p.N34S单倍型的受试者的内源mRNA水平或使用微型基因系统分析p.N34S及其高密度内含子变体时，没有发现SPINK1的异常剪接。值得注意的是，在评估p.N34S单倍型杂合的PaCa44细胞中的等位基因特异性SPINK1 mRNA转录时，我们发现突变的p.N34S的SPINK1 mRNA水平低于野生型等位基因。利用生物信息学、公共区域表观基因组标记和群体遗传学推断，我们可以将与SPINK1表达调控有关的候选调控变异体的数量从26个减少到2个。对于这两个变异体，我们发现，在欧洲和印度血统的受试者中，TFBS的模块化程度高度保守，与胰腺表观基因组标记重叠，LD到p.N34S都很高，转录活性的等位基因特异性调节，以及EMSA中DNA-蛋白质结合的差异。我们假设这些变体中的一个或两个高LD对编码但无功能的p.N34S变体调节与DNA结合的转录因子和/或辅助因子的亲和力，导致SPINK1转录减少。将在不同的细胞系和原代胰腺器官中鉴定这些调控变异体和等位基因特异性结合蛋白(使用高效蛋白质组学方法)，并深入分析SPINK1的表达是如何调节的(例如使用CRISPR基因组编辑)。SPINK1表达的减弱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。解开p.N34S单倍型的确切机制将有助于我们理解CP的病理生理学。