The Roles Of CPA1 Gene And SPINK1 Exonic Mutations In Pathogenesis Of Chronic Pancreatitis

Section 1: Identification of Rare Functionally CPA1 Variants in Chinese Han Patients with Idiopathic Chronic PancreatitisObjective Chronic pancreatitis?CP?is a progressive inflammatory pancreatic disease,characterized by long course and recurrent attacks,resulting in poor life quality.At present,it is generally accepted that the genetic factors,environmental factors and their interactions are involved in the occurrence and development of CP.Over the last 20 years,molecular genetics has played an increasingly important role in pathogenesis of chronic pancreatitis.Several studies reported PRSS1?encoding cationic trypsinogen gene?,PRSS2?encoding anionic trypsinogen?,CFTR?encoding cystic fibrosis transmembrane conductance regulator?,SPINK1?encoding pancreatic secretory trypsin inhibitor?and CTRC?encoding chymotrypsin C?,which predisposed to or protected against chronic pancreatitis.In 2013,rare functional CPA1 variants were found to predispose to chronic pancreatitis in a German cohort and then replicated in an European cohort and two Asian?Indian and Japanese?cohorts.As in many other cases,replication in independent populations of different ethnicity is critical in order to confirm a direct disease association.This is particularly the case for the above described association because of the remarkable differences in the occurrence and distribution of these variants among the different populations,the small sizes of the two Asian cohorts and the differences of susceptible genes and loci of chronic pancreatitis between Chinese population and European ancestries.The aim of this study was to investigate the incidence of functional mutations in CPA1 in Chinese Han population and its association with idiopathic chronic pancreatitis,enrich the data of genetic characteristics of chronic pancreatitis in Chinese population,and lay the foundation for the precision medical treatment of chronic pancreatitis.Design We performed targeted next generation sequencing of the entire coding sequence and exon/intron boundaries of the CPA1 gene in 1112 Han Chinese patients with idiopathic chronic pancreatitis?ICP?and 1580 ethically matched controls,using a newly developed multiplex high-throughput method.We used Sanger sequencing to validate all called variants.We measured the CPA1 activity and secretion of all newly found variants.Results A total of 18 rare CPA1 variants were characterized,11 of which have not been previously described.Functionally defective CPA1 variants were found in 3?p.Gly225 Ser × 1,p.His306 Tyr × 1 and p.Glu380 Lys × 1?of Chinese ICP patients and in 2?p.Glu100 Lys × 1,and p.Arg240 Gln × 1?of the 1,580 controls.However,no association was noted with ICP irrespective of whether all rare variants [20/1112?1.8%?in patients vs.24/1580?1.52%?in controls;P=0.573] or functionally impaired variants [3/1112?0.27%?in patients vs.2/1580?0.13%?in controls;P=0.410] were considered.Conclusions This is the first study to attempt to replicate the recently reported association of rare functional CPA1 variants with chronic pancreatitis in the context of a large ICP cohort.This is also the largest ICP cohort to date used for the sequencing of the entire coding region and exon/intron boundaries of the CPA1 gene in a single population.No association with ICP was noted in our Chinese cohort,irrespective of whether all rare variants or functionally-deficient variants were considered.Ethnicity-specific disease risk factors may account for the inconsistent findings between studies.The correlation between CPA1 functional variants and chronic pancreatitis differs in different human ancestry,which proves that it is of great significance to explore the relationship between genetic factors and disease in Chinese population.Section 2: Functional analysis about the effect of SPINK1 exonic mutations on pre-m RNA splicingObjective Pancreatic secretory trypsin inhibitor gene?SPINK1?is one of the susceptible genes of chronic pancreatitis,which is studied widely and early.Loss-of-function mutants of SPINK1 play an significant role in the pathogenesis of chronic pancreatitis.It has been reported that there are 32 mutants in the SPINK1 exonic region,and the exploration of their pathogenic mechanism was focused on the missense mutation affecting the function of protein.Studies have shown that exonic mutations may affect pre-m RNA splicing by generating new splicing sites or changing a variety of cis acting elements related to splicing,such as exonic splicing enhancers and exonic splicing silencers.An increasing number of studies have reported that mutations in the exonregion,including synonymous mutations,can increase the risk of disease by influencing the splicing of pre-m RNA.More studies have estimated that about 20-45% of pathogenic SNPs,which are located at exon ends,may affect the splicing.In addition,the minigene model is commonly used to analyze the effects of mutations on pre-m RNA splicing.However,minigene constructs discard the partial intronic or exonic region of the gene,which may obscure the influence of distal intron and exon on the pre-m RNA splicing,and may change the m RNA secondary structure and reduce its stability.The main purpose of this study was to investigate the effects of SPINK1 exonic variants on pre-m RNA splicing,the pathogenicity of mutations in the exon region of SPINK1,and the pathogenic mechanism of disease susceptible mutations by using a full-length SPINK1 research model.At the meantime we discussed the possible deviation of the minigene model to the research results by comparing the experimental results of the full-length SPINK1 model and minigene model.Design In this study,we investigated the 32 mutations in SPINK1 exons.The wild type or mutant SPINK1 full-length gene plasmids and minigene plasmids were constructed,and then transfected to HEK293 T cell.We studied the effect of mutations on SPINK1 transcript through RT-PCR,the sequence of which was detected by Sanger sequencing.Q-PCR was used to analyse the influence of variants on expression of SPINK1 m RNA.The effect of SPINK1 exonic mutation on splicing was predicted by Alamut software.Results The results of RT-PCR showed that the mutations of SPINK1 exon did not lead to abnormal SPINK1 transcript.We found that the SPINK1 mutations could reduce the relative expression m RNA,including c.27 del C?70.5±9.8,P=0.0092?,c.29G>A?80.0±4.4,P=0.0029?,c.98₉9ins A?71.9±1.9,P=0.0015?,c.190A>G?82.2±4.1,P=0.0170?,c.199C>T?77.1±10.1,P=0.0070?,c.203A>G?87.4±7.0,P=0.0365?.The mutants c.27 del C and c.98₉9ins A produced a premature termination codon.NMD inhibitor cycloheximide could recover the expression of m RNA with c.27 del C?126.5±2.2,P=0.0023?,but had no effect on the c.98₉9ins A?107.2±18.4,P=0.3606?.Alamut software predicted that 7 mutations might have an impact on the canonical splice site,and 10 mutations,such as c.190A>G,might produce new splice sites.It was also predicted that 12 mutations,such as c.199C>T?c.98₉9ins A,might reduce the activity of ESEs,and 8 mutations might enhance the ESEs activity.By comparing the results of SPINK1 full-length gene model and SPINK1 minigene model,we found that the RT-PCR results of the two models were similar.Both results showed that the SPINK1 abnormal transcriptof c.194+2T>C mutation could be detected and exonic mutations of SPINK1 had no effect on SPINK1 transcripts.But the Q-PCR results of these two models were different.The results of minigene model showed that c.110A>G?64.1±2,P=0.001?,c.137T>A?131.1±7.9,P=0.0211?,c.194G>A?44.5±6.4,P=0.0044?can affect the expression of m RNA,but the analysis using full-length gene model did not suggest that these mutations changed the m RNA expression.The result of full-length gene model showed that c.190A>G reduced the relative expression of SPINK1 m RNA,while in the minigene model analysis,c.190A>G increased the relative expression of m RNA?121.4±2,P=0.0030?.Conclusions In this study,the pathogenicity and pathogenic mechanism of SPINK1 exon mutations was further clarified.We also consider that there are some defects in the minigene splicing analysis model.It is suggested that the full-length gene model is used for the analysis.