Clinical Observation Of Dent 's Disease Drug Treatment And Construction Of CLCN5 Mutation Expression Vector

Part 1 The analysis of the clinical characteristics and the long-term clinical observation of treatment with potassium citrate and thiazide diuretics and angiotensin-converting enzyme inhibitors in 15 children Dent diseaseObjective To summarize the clinical manifestation of 15 cases of Dent disease patients in our hospital,analyzing the correlation between phenotype and genotype.To investigate the drug effect of patient who received therapy with potassium citrate and thiazide diuretics and angiotensin-converting enzyme(ACE) inhibitors, providing the reference for the clinical treatment.Methods We selected children who were diagnosed with Dent disease during the period of 2006.7 and 2014.3. The patients were administered a therapy of potassium citrate associated with thiazide diuretics and ACE inhibitors according to the level of proteinuria and calciuria and serum potassium.We compared and analysised the underlying changes before and after the treatment. The obvervational index concluded: the level of proteinuria and calciuria, kidney function,the serum levels of muscle enzymes, renal ultrasound, bone mineral density.Results 1.In 15 cases of children with Dent disease, there were all male. Onset age ranged from 3 months to 11 years[median(2.62±3.11)years].The disease duration ranged from 0.5 year to 9.5years[median(2.81±2.34)years].All of them had LWMP and hypercalciuria, three cases had hematuria. In total 15 patients, 4patients had a elevated level of serum muscle enzymes. 10 patients were performed renal biopsy, and the result were 7 cases of minor lesion, 1 case of focal global glomerulosclerosis, 1 case of mesangioproliferative Ig A nephropathy, 1 case of mesangioproliferative Ig M nephropathy. 5 cases had nephrocalcinosis and 1case had renal lithiasis. 3 cases showed growth failure. Osteoporosis existed in 5 cases. 10 patients recived genetic test, six of them carried the mutation in CLCN5 gene, one ofthem carried the mutation in OCRL1 gene, three of them carried no gene mutation.2. The follow-up time ranged from 0.5 to 7.5years[median(3.61±2.26)years].Until the end of follow-up, 15 cases accepted the therapy of potassium citrate which was administered at a average dose of(0.16±0.12)g/kg·d. 11 cases accepted the therapy of thiazide diuretics which was administered at a average dose of(0.75 ±0.41)g/kg·d, 4 of 15 patients gradually stopped using hydrochlorothiazide because of the stable control of hypercalciuria. 13 cases accepted the therapy of ACE inhibitors which was administered at a average dose of(0.17±0.06)g/kg·d, 2 of 15 patients stopped medication of benazepril because of hypotension.3.Therapeutic efficacy: there was significantly statistical difference at calcium/creatinine and daily Ca-creat ratio in contrast to before treatment [(0.41±0.19)mg/mg VS(0.26±0.12)mg/mg, P=0.021;(6.57±2.37)mg/kg Vs(4.60±1.91) mg/kg,P=0.005]. There was no significantly statistical difference at 24-hour urinary protein quantity in contrast to before treatment[(0.96±0.62)g/24 h VS(0.87±0.44)g/24 h,P=0.327]. LWMP was a constant feature no matter before or after treatment. 3 cases still had hematuria and there were not obviously aggravation after treatment. 3 cases had a high level of muscle enzymes, 1 case, muscle enzymes go back to normal level.2 patients repeated renal biopsy, and the finding showed minor lesion and focal glomerulonephritis, respectively. 1 cases with kidney stones and 5 cases with nephrocalcinosis had a negative result of renal ultrasound after treatment. 4 cases had osteoporosis and 1case was cured. The height of 1 caes who recived growth hormone treatment was in the 10 th percentile at the end of follow-up, the other 2 cases still manifested as growth failure, 1 case was diagnosed with short stature in the 1.25 years of follow-up. The 15 cases had a normal kidney function at the beginning of follow-up, and there was no progression of CKD at the end of follow-up period.4.4 cases had hypokalemia after thiazide diuretics treatment and 3 cases had hypotension after ACE inhibitors treatment.Conclusions 1. Patient combined with nephrotic-range proteinuria in our group does not manifest as focal glomeru losclerosis.2. No correlation between genotype and phenotype has been established in Dentdisease.3.Treatment with potassium citrate and thiazide diuretics could significantly decrease urinary calcium excretion, make a disappearance of kidney stone and nephrocalcinosi.4.Treatment with benazepril is not thought to significantly decrease the proteinuria. There is no substantial improvement of LWMP.5.When patient has a therapy of thiazide diuretics, the serum potassium level should be closely monitored and the hypokalemia must be timely corrected.Part 2 Eukaryotic expression vector construction of CLCN5 gene and two mutationObjective We found two new mutations, namely L594 fs X595 and IVS4-2A>G,which had never been reported. To explore the metergasis after mutations, we constructed three CLCN5 eukaryotic expression vector which included a wild type and two mutant type based on the gene synthesis technology.Methods In the study, firstly we manufactured the normal CLCN5 gene and two mutational CLCN5 gene through the gene synthesis technology.The CLCN5-DNA fragment had a correct sequencing and then subcloned into pc DNA3.1(+)/EGFP/IRES vector using Bam HI and Eco RⅠrestriction enzyme. Next,the composition was transformed into competent cells. Finally, the CLCN5 expressing plasmid pc DNA3.1(+)/EGFP/IRES-CLCN5 and pc DNA3.1(+)/EGFP/IRES- c.2073 del GCLCN5 and pc DNA3.1(+)/EGFP/IRES- IVS4-2A>GCLCN5 was constructed.Results The CLCN5 mutation fragment was constructed successfully and direct sequence analyses displayed a deletion mutation of L594 fs X595 and a nonsense mutation of IVS4-2A>G. The plasmid was extracted from the culture-positive colonies, and then the DNA solution was sent to sequence. The sequencing results manifest that the gene sequence alignment was correct, and the recombinant expression plasmid was successfully constructed.Conclusions The successfully construct of three eukaryotic expression vector,named pc DNA3.1(+)/EGFP/IRES-CLCN5 and pc DNA3.1(+)/EGFP/IRES- c. 2073 del G CLCN5 and pc DNA3.1(+)/EGFP/IRES-IVS4-2A>GCLCN5, seems to provide a experimental basis for future research into the function in CLCN5 mutant and the mechanisms of urinary protein reabsorption.