Screening For PKD1 Mutations In Autosomal Dominant Polycystic Kidney Disease With Intracranial Aneurysms

Background and objectivesIntracranial aneurysm(ICA) is due to some reason of intracranial arterial blood wall outward, thus forming the hemangioma lesions, ruptured intracranial aneurysm is the main cause of subarachnoid hemorrhage(SAH)(about 85%).And intracranial aneurysm rupture mortality and morbidity is high, the threat to the patient’s life and health, also brought a heavy burden for families and society.There are many factors affecting the occurrence of intracranial aneurysm, there are roughly two kinds big, namely: the genetic factors and environmental factors.Its environmental factors mainly include: high blood pressure, smoking, drugs, sex, alcohol, age and so on, the first three are the most important risk factors, also may be two factors work together to promote the happening of the disease.High Resolution Melting curve(HRM) technology is a new developed molecular detection technology at home and abroad in recent years,and is a new type of genetic testing method.HRM technology derives from the melting curve, so it is on the basis of real-time fluorescent quantitative PCR, just increasing a new kind of improved fluorescent dye saturation:HRM is a detection technology which makes use of the fluorescence saturated dye to monitor the change of double-stranded nucleic acid constantly, and then analyzed by the peak shape of melting curve.HRM technology neither need to use a specific sequence tag probe, nor be limited to the mutation of base types and site,It has the advantages of fast speed、easy operating、low cost、high sensitivity and specificity, high accuracy, and realizing the real closed tube operation and so on,so it can be applied to multiple domains rapidly and widely,such as the genetic mutation scanning, genotyping and sequence matching, methylation analysis, short segment repetitive sequence and RNA editing.Autosomal dominant polycystic kidney disease(autosomal dominant polycystic kidney diseases, ADPKD)is the highest incidence of human disease, and is one of the most common genetic disease in clinical.ADPKD is a single-gene genetic disease, its prevalence is about 1/1000 ~ 1/400 [1]and about three out of the five of the patients have a familial heredity history.And this hereditary disease has a high penetrance, as high as over 95%,people who aged over 80 almost all show some signs of the disease.ADPKD has obvious genetic retardance, and comes generally after middle age.Its pathological characteristics mainly reflected in bilateral kidneys, in which can form many kinds of liquid cyst, with the growing of cysts, renal structure will be destroyed, and the function of kidney will be lost, then eventually developing into uremia, that is renal function failure.Through clinical observation found that about 50% of the patients will develop into end-stage kidney failure stage at the age of 60 [2], so in clinical, the kidney function failure is one of the most common cause of death in adult polycystic kidney disease.ADPKD is a kind of systematic genetic disease, its pathological changes mainly affect bilateral kidneys, in addition,it can be spread to the patient’s liver, pancreas, spleen, ovaries and systemic cardiovascular and other multiple systems, such as once occuring in tissues and organs, it can form splenic cyst, liver cyst pancreatic cyst, ovarian cyst and seminal vesicle cystand so on.In systemic cardiovascular systems, such as coronary artery, intracranial artery and other artery place,it can form kinds of aneurysm.Occurring in the groin,it can result in inguinal hernia;occurring in the heart valve, it can lead to abnormal heart valve,such as heart valvular regurgitation.PKD1, PKD2 and PKD3 gene have been reported at home and abroad, and is the three affected genes of ADPKD, but the current study found that PKD1 gene is the main pathogenic genes of ADPKD.To establish a simple and reliable method to detect the mutation of PKD1 gene in patients of autosomal dominant polycystic kidney disease(ADPKD) using the high resolution melting(HRM),and then exploring the technical characteristics of HRM and its feasibility in using of clinical screening and diagnosis of ADPKD patients. MethodsCollected in July 2013 to August 2014, the sixth people’s hospital affiliated to Shanghai jiaotong university, admitted ADPKD patients, all patients by B ultrasonic and CT examination and diagnosis, in the diagnosis of ADPKD patients with skull three dimensional time leap of magnetic resonance angiography(3- dimensiona time- of- flight MR angiography, 3 d TOF MRA) method to screen patients with intracranial aneurysms, altogether collected ADPKD patients with comorbid ICA 32 cases.Collecting blood 4 ml of 32 patients with preoperative early in the morning on an empty stomach peripheral and placing them in the ethylenediamine tetraacetic acid(EDTA) anticoagulant tube, then pouring into the dry gauze,and mading dried blood.Extracting genomic DNA of 32 cases with conventional phenol- chloroform method.Design the primers for PCR reaction and the analysis of HRM, optimize the PCR reaction conditions, and amplified fragment gene.Using HRM method detect ADPKD PKD1 gene mutation of 46 exons,then making direct sequencing test in samples who are suspected of being mutations of genes in the results of HRM and determining the mutation types of mutation samples. ResultsUsing HRM technique testing analysis 56 fragment area in 46 exons of 32 cases of patients with ADPKD,from the knowable melting peak curve charts,knowing that 9 of 28 cases with abnormal melting curve, 9 patients(1、7、8、9、10、16、18、20 �'� 28) specimen confirmed that there are PKD1 gene mutation by sequencing method.There are 62 locus mutations, and the mutation rate is 28.13%, 62 mutations are all point mutations, including 28 species of missense mutation, 1 kind of nonsense mutations, 12 kinds of synonymous mutations. 62 mutations places in 13 exon, among which 11 and 15 exon have the most mutations,and there are 12 cases, accounted for 19.36% of total mutation;there are 9 cases of mutations in exon 10(14.52%);there are 8 cases of mutations in exon13(12.90%); there were 4 cases of mutations in exon 5 and exon 25, respectively accounted for 6.45%;there are 3 cases of mutations in exon29(4.84%);there are 2 cases of mutations in exon7, 16, 23 and 26,respectively accounted for 3.23%;there are 1 case mutation in exon27 and 38, respectively accounted for 1.61%.Direct sequencing method confirms that the test results of two methods are identical. ConclusionHRM method can detect of PKD1 gene mutations fastly, sensitively and accurately in patients with ADPKD, and it can not only detect the known genetic mutations, but can screen out some unknown mutations conveniently and rapidly. The emerging of HRM technology has lots of advantages,such as fast, easy operation and experiment results are accurate, low cost, high sensitivity and high specificity, can be used as a preferred method of screening carriers of the mutation detection of ADPKD patients and ADPKD family, and it is suitable for clinical promotion.