Clinical And Genetic Analysis Of Lowe Syndrome In 8 Chinese Children,and Transfection Experiments Of CLCN5 Mutant Genes Into 293 Cells

Part 1 Clinical and genetic analysis of Lowe syndrome in 8Chinese childrenObjective To summarize and analyze the clinical and genetic characteristics of 8Chinese children of Lowe syndrome in our hospital,in order to improve the understanding of the disease.Methods We selected 8 children whose clinical diagnosis were Lowe syndrome during the period of 2011.11 and 2016.6,their clinical and laboratory data were analyzed retrospectively.Genetic testing of 7 cases was carried out by next-generation sequencing technology.Results All of 8 cases were boys.They were all found congenital cataract on set,but the ages of diagnosis were 1 month to 44 months last,average(16.5±17.1)months.Then muscle hypotonia,delayed motor milestones,growth failure,low-molecular-weight proteinuria and hypercalciuria were existed of the 8cases,and 2/8 with glaucoma,7/8 with intellectual disability and 2/8 with behavioral abnormalities,2/8 with seizures,6/8 with cranial MRI abnormalities,6/8 with aminoaciduria,2/8 with renal tubular acidosis,1/8 with hypokalemia,2/8 with rickets,5/8 with osteopenia and 2/8 with facial dysmorphisms.7 cases of them got genetic testing,6 of them with OCRL gene mutations,including R361 G,L423W,Q388 P,N373I,R695 X and I336 N,including 5 missense mutations and a nonsense mutation,the rest one with SLC4A1 gene mutation of c.16-15G>A.Except for R695 X,the rest are novel mutations.Conclusions Lowe syndrome is a multi-system damaged disease,the characteristic clinical manifestations including eye,nervous system and kidney abnormalities,and the most children were observed with eye abnormalities on set,but to the age of diagnosis with a longe interval,so maybe earlier diagnosis has an important significance.Some children with clinical manifestations of Lowe syndrome were not detected with OCRL gene mutations,suggesting that OCRL may not be the only pathogenic gene leading to Lowe syndrome.Part 2 Transfection experiments of CLCN5 mutant genes into 293 cellsObjective In recent years,our hospital received a number of children with Dent disease,we have mastered the diagnosis and treatment of this disease.Now we all know it's a disease with X recessive inheritance,the majority of children with CLCN5 gene mutation,but the exact pathogenesis is still not clear.There're two children with Dent's disease,One had elevated urinary calcium but with low levels of proteinuria,and the other one showed nephrotic proteinuria.The results of these two children's genetic tests are IVS3-2A>G and L594fsX595 mutations of CLCN5,Respectively.these two patients with different phenotypic characteristics and different mutation sites.Therefore,a experiment was designed to explore the gene mutation effect caused by the two mutate genes.We have already synthesized plasmids containing these two mutant genes in previous experiment,in order to finish the next experiment.Methods Two CLCN5 mutant genes of IVS3-2A> G and L594fsX595,as well as CLCN5 wild-type gene,were transfected into 293 cells by the SYBR dye method.The relative expression levels of four target genes including of Megalin,Cubilin,NHE3 and V-H+-ATPase in the 293 cells containing the two mutant genes were tested and compared with those in the 293 cells containing the CLCN5 wild-type gene,respectively.And then the mutation effect of CLCN5 gene was deduced.Results The plasmids containing wild-type CLCN5 gene and IVS3-2A>G,L594fsX595 mutant genes were successfully transfected into 293 cells.Compared to wild-type gene,the expression of Megalin,Cubilin,NHE3,V-H+-ATPase gene was not significantly different from that of 293 cells containing IVS3-2A> G mutant gene.The expression of Megalin and V-H+-ATPase genes in 293 cells with L594fsX595 mutant gene showed a decrease,while the expression of Cubilin and NHE3 genes showed no significant difference when compared to 293 cells with wild-type gene.Conclusions The plasmids containing wild-type CLCN5 gene and IVS3-2A>G,L594fsX595 mutant genes were successfully transfected into 293 cells.It isspeculated that some mutations of ClCN5 such as L594 fs X595 may result in the reduced expression of Megalin and V-H+-ATPase,which may lead to low-molecular-weight proteinuria.