Study And Analysis Of PKD1 Gene Mutation In Patients With ADPKD Associated With IA

Background and purposeAutosomal Dominant Polycystic Kidney Disease(ADPKD)is a genetically susceptible autosomal monogenic disease with a fatal risk.ADPKD patients are usually diagnosed in the youth.About 1 / 1000-1 / 400 people suffer from the disease around the world,and more than 1200 million people achieving the diagnostic criteria for the diagnosis of the disease.Studies have shown that ADPKD is usually caused by PKD1 and PKD2 mutations.PKD1 is located in the zone 3 sub-band 3of the short arm of 16 th chromosome.PKD2 is located in the 4q21 area.Polycystin-1(PC1)is obtained by transcriptional translation of PKD1,and polycystin-2(PC2)is obtained by transcriptional translation of PKD2.Polycystin-1 is a plasma membrane-like receptor protein,mainly distributed in the cell membrane,to maintain cells the interaction among cells or between cells and the extracellular fluid.Polycystin-2 is a non-selective cation channel.A complex of PC2 and PC1,which has a strong permeability for calcium ions,play a role in the gated channel of calcium ions.Mutation of PKHD1 in the short arm region 2 of chromosome 6 leads to the formation of recessive polycystic kidney disease,in which the product FPC protein is obtained by transcriptional translation of mutated PKHD1.A study of the pathogenic sites of the discovered polycystic kidney disease gene found that large deletions,duplication or rearrangement mutations were found to be minimal(about 3% to 4%),and ADPKD was predominantly spot mutations.Therefore,the direct sequencing of the disease gene sequence of the patient is still the main technique for gene diagnosis of polycystic kidney disease.ADPKD is a clear locus heterogeneous disease,its genetic diagnosis is quite complex.PKD1 or PKD2 each has a mutation can cause ADPKD mutation.About 85% of polycystic kidney disease is caused by the PKD1 gene mutation,and the clinical phenotype is also the most serious,leading to the development of patients with end-stage renal disease 20 years earlier than PKD2.Second,from the existing research,ADPKD gene mutation does not have a fixed siteas other diseases.Its mutation may occur in PKD1 and PKD2 gene at any site,more which makes it difficult to detect.ADPKD is characterized by multiple renal cysts and early onset chronic renal failure,eventually leading to end-stage renal disease(ESRD).The only treatment to sustain life is renal replacement therapy or kidney transplantation.In addition to renal cysts,ADPKD is usually accompanied by renal cysts,spleen cysts,pancreatic cysts,ovarian cysts and seminal vesicle and other renal manifestations.It is also an important risk factor for the development of intracranial aneurysms.The probability of polycystic kidney disease patient with aneurysms is 4% to 41%.Because ADPKD has a tendency to gather to the family,and the disease generally late onset,especially at adult,it is detrimental to the patient's own treatment.Most patients were diagnosed with polycystic kidney disease after marriage,with a great probability to have children with pathogenic mutations,which also caused a heavy burden on patients and their families and health care system.Intracranial aneurysm(IA)is the pathological localized dilatation of intracranial artery wall forming hemangiomatous lesions,more likely to cause spontaneous subarachnoid hemorrhage.Intracranial aneurysms in the event of rupture,with high lethality and disability,and there is no specific drugs to stabilize or prevent the rupture.So it is a great threat to the patient's life and health,and it also bring a heavy burden to the family and society.The pathogenesis of IA is not clear,and it is generally thought that its onset is caused by a variety of factors.The current study has confirmed that age,sex,smoking,alcoholism,hypertension,drug use,seasonal,family history are high risk factors for intracranial aneurysms,and is likely to be the result of a combination of changes in hemodynamics and acquired degeneration.However,the relevant research reports that genetic factors for the initiation of IA also play a very important role.Cardiovascular complications are the leading cause of death in ADPKD,and IA-induced subarachnoid hemorrhage is one of the most important complications.ADPKD IA in the diagnosis and treatment strategy has not been fully established.IA is the most serious of cardiovascular complications in all ADPKD patients because of the 50% high mortality rate following the rupture of the hemangioma.Despite theexpression of polycystin in arterial smooth muscle,the formation mechanism of this PKD mutant-induced aneurysm remains unclear,and PKD1 and PKD2 mutations are equally risky for IAs.Mutations near the 5' end of the PKD1 site are more likely to develop into IAs.Mathematical modeling shows that the formation of multiple fluid-filled renal interstitial cysts begins in childhood,leading to the early detection of intracranial aneurysms.Therefore,we hope to establish a highly efficient and specific detection of mutations in the system,to study ADPKD associated with patients with PKD1 mutation and general ADPKD mutations in patients with the genetic level to identify the auxiliary diagnosis of the method,so that ADPKD and IA in the formation and incubation period can be detected for early treatment,thereby relieving the pain of the patient.MethodADPKD patients were collected and sorted from July 2013 to August 2014 in the Sixth People's Hospital Affiliated to Shanghai Jiaotong University.All patients were screened by B ultrasound,CT and magnetic resonance angiography to enrolled aneurysm patients,a total of 23 patients with ADPKD associated with ICA.We used ethylenediaminetetraacetic acid(EDTA)anticoagulant to get 4 mL peripheral blood of twenty-three patients in the morning without eating anything and placed in a refrigerator,and extracted genomic DNA from 23 patients by the kit.The nested PCR reaction was designed and the specific primers needed were analyzed.Six pairs of long fragment primers were selected to amplify exon 1,exon 2 to 12,exon 13 to 15 D,exon 15 E 22,exons 22 to 32,36 to 46 exon of PKD1,and selected the specific PCR reaction conditions for amplification.The amplified fragment products were sequenced and the results were analyzed.Results1 The PCR products were confirmed by electrophoresis.The bands were bright and the products were unique with a good specificity.2 A total of 85 mutations were detected in 23 samples after by sequencing,.All these mutations were point mutations,including 47 missense mutations,35 synonymous mutations,1 nonsense mutation,and 1 frameshift mutation.And all samples were detected the same mutation in exon 7.3 A total of 19 unknown mutation species were found,including 14 missense mutation species,3 synonymous mutation species,1 nonsense mutation specie,and 1frameshift mutation specie.Conclusion1 All the samples were detected the same mutation in the PKD1 exon 7,which is different from the normal PKD patients or normal people.It can be the guidance for the polycystic kidney disease associated with aneurysm.2 A total of 72 mutations were found in the PKD1 near the 5 'end,accounting for84.70% of the total number of mutations.It proved that patients with mutations near the 5' end of the PKD1 were more likely to develop into intracranial aneurysms.