The Mutation Distribution And Clinical Significance Of The Main Pathogenic Gene Of Chronic Pancreatitis In China

Part Ⅰ: SPINK1,PRSS1,CTRC and CFTR Genotypes Influence Disease Onset and Clinical Outcomes in Chronic PancreatitisChronic pancreatitis(CP)is a chronic inflammatory process of the pancreas that leads to irreversible morphological changes and progressive impairment of both exocrine and endocrine functions.The disease is associated with a poor quality of life,confers an increased risk of pancreatic cancer,and represents a major cause of morbidity.Over the past two decades,it has been increasingly appreciated that ICP has a strong genetic basis,to which rare pathogenic variants in the SPINK1,PRSS1,CTRC and CFTR genes have been strongly associated with a risk of developing chronic pancreatitis(CP).However,their potential impact on the age of disease onset and clinical outcomes,as well as their potential interactions with environmental risk factors,remain unclear.These issues are addressed here in a large Chinese CP cohort.We performed targeted next-generation sequencing of the four CP-associated genes in 1061 Han Chinese CP patients and 1196 controls.To evaluate gene–environment interactions,the patients were divided into three subgroups,idiopathic CP(ICP;n?=?715),alcoholic CP(ACP;n?=?206),and smoking-associated CP(SCP;n?=?140).The potential impact of rare pathogenic variants on the age of onset of CP and clinical outcomes was evaluated using the Kaplan–Meier model.As the fact that the SPINK1 c.194+2T>C variant has been increasingly recognized as one of the endogenous factors for CP,we conducted a systematic review and meta-analysis on its contribution in different ethnicities and CP etiologies.We identified rare pathogenic genotypes involving the SPINK1,PRSS1,CTRC and CFTR genes in 535(50.42%)CP patients but in only 71(5.94%)controls(odds ratio?=?16.12;P?<?0.001).Mutation-positive patients had significantly earlier median ages at disease onset and at diagnosis of pancreatic stones,diabetes mellitus and steatorrhea than mutationnegative ICP patients.Pathogenic genotypes were present in 57.1,39.8,and 32.1% of the ICP,ACP,and SCP patients,respectively,and influenced age at disease onset and clinical outcomes in all subgroups.The SPINK1 c.194+2T>C variant is an important risk factor for CP and had the highest correlation in East Asian patients with ICP.In summary,we provide evidence that rare pathogenic variants in the SPINK1,PRSS1,CTRC and CFTR genes significantly influence the age of onset and clinical outcomes of CP.Extensive gene–environment interactions were also identified.Part Ⅱ: Toward a clinical diagnostic pipeline for SPINK1 intronic variantsWith the extensive development of high-throughput target sequencing,the distribution and clinical significance of known gene mutations in chronic pancreatitis(CP)are becoming more and more clear.However,nearly half of the CP patients in the Chinese population are still unable to find the disease-causing mutations.SPINK1 intronic variants continue to be reported in the literature for the attention of scientists.In contrast,the data of the Chinese CP population has been still lacking.The clinical relevance of canonical splice site variants,nonsense mutations,or largescale genomic deletions in the SPINK1 gene is generally clear.By contrast,the clinical relevance of SPINK1 promoter and enhancer variants,missense variants,or intronic variants occurring outwith the canonical splice sites has often had to be ascertained by in vitro functional analysis.The clinical significance of SPINK1 intronic variants in CP has been previously assessed by various approaches including a cell culture-based full-length gene assay.A close correlation between the results of this assay and in silico splicing prediction was apparent.However,until now,a clinical diagnostic pipeline specifically designed to classify SPINK1 intronic variants accurately and efficiently has been lacking.Herein,we present just such a pipeline and explore its efficacy and potential utility in potentiating the classification of newly described SPINK1 intronic variants.We confirm a close correlation between in silico splicing prediction and results from the cell culture-based full-length gene assay in the context of three recently reported pathogenic SPINK1 intronic variants.We then integrated in silico splicing prediction and the full-length gene assay into a stepwise approach and tested its utility in the classification of two novel datasets of SPINK1 intronic variants.The first dataset comprised 16 deep intronic variants identified in 52 genetically unexplained Chinese CP patients by sequencing the entire intronic sequence of the SPINK1 gene.The second dataset comprised five novel rare proximal intronic variants identified through the routine analysis of the SPINK1 gene in French pancreatitis patients.Employing a minor allele frequency of >?5% as a population frequency filter,6 of the 16 deep intronic variants were immediately classified as benign.In silico prediction of the remaining ten deep intronic variants and the five rare proximal intronic variants with respect to their likely impact on splice site selection suggested that only one proximal intronic variant,c.194?+?5G?>?A,was likely to be of functional significance.Employing the cell culture-based full-length gene assay,we functionally analyzed c.194+5G?>A,together with seven predicted non-functional variants,thereby validating their predicted effects on splicing in all cases.In conclusion,we demonstrated the accuracy and efficiency of in silico prediction in combination with the cell culture-based full-length gene assay for the classification of SPINK1 intronic variants.Based upon these findings,we propose an operational pipeline for classifying SPINK1 intronic variants in the clinical diagnostic setting.