The Study Of Pathogenicity Of LPL Gene Mutations D277N,C302R And S474X In A Family With Hypertriglyceridemic Acute Pancreatitis

BACKGROUND:The incidence of hypertriglyceridemic acute pancreatitis(HTG-AP)increased year by year in China,with high incidence of multiple organ dysfunction,significant recurrence trend and dangerous condition.The causes of hypertriglyceridemia(HTG)are mainly divided into two categories,primary causes which are related to gene mutations and secondary causes which are related to diabetes,obesity,high-fat and high-protein diet.Among the primary causes of HTG-AP,lipoprotein lipase(LPL)gene defect/mutation is the most obvious and most significant one.In the previous clinical work,we found a typical gestational HTG-AP patient,who is the third daughter in the family and has a history of HTG for many years.The parents in the family had been both diagnosed with HTG for about 20 years.The eldest daughter was also diagnosed with HTG and had a history of recurrent HTG-AP since adolescence.While the second daughter and fourth son in the family had normal blood lipids levels and no history of acute pancreatitis.The eldest daughter and the third daughter of the family had primary hypertriglyceridemia which is independent of obesity,diabetes and other related diseases,while the level of triglyceride and the recurrence of pancreatitis could not be controlled by drugs or proper diet,which made it critical to study the family's gene background,as well as the pathogenicity of mutation sites.OBJECTIVE:This study focused on LPL and other genes detections in this family,and studied the pathogenicity of the mutations through bioinformatics analysis,in vivo LPL concentration and activity detection,in vitro cell experiments,and structural model analysis.METHODS:This study consists of two parts:(1)Collecting the clinical information of the HTG-AP family,designing LPL and other gene primers,extracting blood samples,extracting DNA,PCR amplification,and Sanger sequencing,then detecting whether the family had LPL and other genes mutations;(2)Comprehensively studying the pathogenicity of the detected mutations.Experiment 1:The frequency of mutations in the population was detected and software prediction,site conservation analysis and other bioinformatics methods were used to study the pathogenicity of the mutations.Experiment 2:The post-heparin blood samples were collected and then in vivo LPL concentration and activity were measured.Experiment 3(in vitro cell experiments):The corresponding plasmids were transiently transfected into HEK-293T cells,and the total protein expression was evaluated by Western blot.The concentration of LPL in the cell media and lysates were measured to evaluate secretion function.LPL activity of media and lysates were detected to evaluate the effect of mutation on LPL activity.Experiment 4:PyMol software structure model prediction was used to study the pathogenicity of the mutation sites based on the newly published real structure of LPL(2019,PNAS Journal).RESULTS:First,we found that there were three compound heterozygous mutation sites of LPL gene:D277N(C.829G>A,GAC>AAC,aspartic acid>asparagine),C302R(C.904T>C,TGC>CGC,cysteine>arginine)and S474X(C.1421C>G,TCA>TGA,serine>stop codon)in the family.C302R was a new mutation that had never been reported,and the family members had no LPL-related gene mutations such as apolipoprotein A-5(APOA5).The father and the fourth son were LPL heterozygous mutation C302R carriers;D277N,S474X occurred in the same DNA chain of the mother and the son of the eldest daughter,and the other DNA chain was normal;Two HTG-AP patients(the eldest daughter and the third daughter)were LPL compound heterozygous D277N,C302R,S474X carriers,without normal DNA chain;Second daughter had no LPL gene mutation;The husband of the third daughter was LPL heterozygous mutation S474X carrier;One chain of the third daughter's son existed C302R and the other chain existed S474X.Next,we conducted a comprehensive pathogenicity study of D277N,C302R and S474X.In the first experiment,population frequency,software prediction pathogenicity,conservative analysis and bioinformatics analysis were done.It was predicted that D227N(rs118204068)and C302R(rs 1064797075)were all highly likely pathogenic mutations,and they have an over 90%possibility of causing the disease.S474X(rs328)had been reported in many related studies previously.It was the only beneficial mutation in LPL gene,which could enhance LPL function,and had been successfully prepared as the first gene therapy drug in Europe.In the second experiment,LPL concentration(55.31%,97.82%of the normal people concentration),and activity(94.14%,93.99%of the normal people activity)were both decreased in mother and the son of the eldest daughter who were D277N&S474X heterozygous mutation carriers;LPL concentration(58.21%,47.32%),and activity(62.72%,91.32%)were both decreased in father and the fourth son who are C302R heterozygous mutation carriers;LPL concentration(112.90%)and activity(140%)of third daughter's husband(S474X heterozygous mutation carrier)were higher than that of normal people.LPL concentration(288.29%,28397.49%)increased,but activity(38.69%,49.40%)decreased in the eldest and third daughters who were compound D277N,C302R,S474X carriers.In the third experiment,we constructed the corresponding plasmids and set eight groups----the WT,D277N,C302R,S474X,D277N&S474X fusion plasmids,D277N&S474X and C302R co-transfection,negative control plasmids,and untreated blank cells.After the transfection of plasmid D277N,the LPL activity of cell media and lysates decreased.C302R decreased the expression of LPL protein in total protein and media,the activity decreased.After transfection of S474X,total protein,media protein,lysates protein,the activity increased.After D277N&S474X fusion plasmids transfection,the expression of LPL protein in total protein and media were decreased,and LPL activity was decreased too.After D277N&S474X and C302R being cotransfected,total protein,media protein and lysate protein were elevated,media and lysate activity decreased.In the fourth experiment,we clarified the pathogenicity of D277N,C302R and S474X from the structural level by constructing a model based on the deciphered GPIHBP1-LPL complex structure published in PNAS in cooperation with the Institute of Biophysics of the Chinese Academy of Sciences.After observing the mutation at position 277,it was speculated that the hydrogen bond and the ionic bond disappeared,which affected protein function.The 302-position Cys formed a pair of disulfide bonds with the 291-position Cys,but the mutant C302R affected the formation of disulfide bonds and destroyed the LPL structure,thereby affecting the function of the enzyme.The C-term tails 472 and 473 of LPL were in the positive charge region,which may participate in the binding to GPIHBP1,while the 474-position mutated into a stop codon,the protein sequence was shortened,the site was exposed more,and the binding of two proteins was enhanced.The positive and negative charge interactions promoted the maintenance of LPL structure and functioned as beneficial mutants.CONCLUSION:We firstly detected LPL gene compound heterozygous mutations D277N,C302R,and S474X in the HTG-AP family,among them C302R was a new mutation.Through bioinformatics,in vivo LPL concentration and activity detection,in vitro cell experiments and structural model simulation analysis,we confirmed that D277N,C302R,D277N&S474X and complex heterozygous mutations D277N,C302R,S474X were harmful mutations,while S474X was a beneficial mutation.This study enriches the understanding of the genetic background of clinical HTG-AP patients and provides new ideas for the diagnosis and treatment of HTG-AP.