Identification Of Pathogenic Genes Of Gitelman Syndrome And Functional Study Of Novel Mutations Of SLC12A3

BackgroundGitelman syndrome(GS,OMIM:263800)is an autosomal recessive disorder which is characterized by hypokalemia,hypomagnesemia,and hypocalciuria,accompanied by metabolic alkalosis and activation of the renin-angiotensin-aldosterone system(RAAS).GS patients have low or normal blood pressure.GS is the most common inherited renal tubular disease.Mutations in solute carrier family 12,member 3(SLC12A3)gene on chromosome 16q13 which encodes thiazide-sensitive sodiumchloride cotransporters(NCC)lead to GS.NCC is responsible for reabsorption of Na+and Cl-in distal convoluted tubule.Dysfunction of NCC caused by mutations in SLC12A3 leads to complex clinical symptoms of GS.The prevalence is about 1:40000 with a higher prevalence of about 1:1000 in Asian populations.Criteria for the diagnosis of GS is identification of biallelic inactivating mutations in SLC12A3.The association between genotype and phenotype needs to be further explored.The main treatment is oral potassium and magnesium supplementation,and the long-term prognosis is generally good.To date,more than 500 mutations in SLC12A3 have been reported.A small number of mutations have been confirmed by functional studies.Most of the functional studies were carried out on Xenopus oocytes and several on mammalian polar cells.Meanwhile,two mutations have been studied in vivo.The two sites were T60M,as the classic phosphorylation site of NCC which has a high mutation frequency in Asian populations,and S707X,a nonsense mutation.Both homozygous mice showed typical symptoms of Gitelman syndrome which indicated pathogenicity of two mutations.However,the vast majority of Gitelman syndrome patients were compound heterozygotes.Therefore,a whole functional study of mutant NCC and a better GS mouse model remains to be resolved.In this study,we identified of pathogenic gene of Gitelman syndrome and conducted functional study of novel mutations of SLC12A3.We performed wholeexome sequencing in unexplained hypokalemic population.screened out Gitelman syndrome patients,and analyzed their genotypes and phenotypes.We made definitive diagnosis of Gitelman syndrome patients and performed genotype and phenotype of GS patients.Meanwhile,bioinformatics analysis was performed for R158Q and G212S,novel mutations of SLC12A3.Functional studies were performed on MDCT cell.We constructed a compound heterozygous mouse model,NccR156Q/G210S mice,to evaluate the pathogenicity of two novel mutations.Effects of the mutations on protein expression,localization and function were evaluated in vivo to confirm the pathogenicity of R158Q and G212S.Our new findings confirm the pathogenicity of R158Q and G212S and provide an optimal mouse model for further research of GS.Objectives1.To clarify the diagnosis of people with unexplained hypokalemia.2.To analyse mutation profiles of SLC12A3 and the correlation between genotype and phenotype of patients with Gitelman syndrome.3.Bioinformatics analysis on R158Q and G212S was conducted to analyze the pathogenicity of novel mutations.Three-dimensional structure of the protein was predicted to analyze the potential influence of mutations on NCC.4.In vitro,we used MDCT cells transfected with wild-type and mutant NCC plasmids to study the direct effects of mutations in SLC12A3 on expression and localization of NCC.5.1n vivo,Ncc-R156Q and Ncc-G210S knock in mice were conducted.The phenotype of heterozygous,homozygous and compound heterozygous mice was evaluated.Part 1.Identification of pathogenic genes and genotype and phenotype analysis of Gitelman syndromeMethods1.SubjectsThe research subjects were from patients with unkown hypokalemia in Shandong Provincial Hospital.According to the inclusion and exclusion criteria,73 patients were included in our study.The peripheral blood of patients with hypokalemia was subjected to genetic testing.2.Genetic testingGenetic testing of patients with hypokalemia was performed by Whole-Exome Sequencing(WES,Whole-Exome Sequencing).3.Data collectionMutations were analyzed by sequence alignment.Profiles of SLC12A3 were analyzed.Basic information of patients was obtained through consultation,physical examination and laboratory examination including blood pressure,serum Na+,K+,Mg2+,serum creatinine,blood pH,HCO3-,24h Urine volume,24h Na+excretion,24h K+excretion,24h Ca2+excretion,etc.4.Statistical analysisPositive rate of genetic testing in patients with unknown hypokalemia and the characteristics of mutations in SLC12A3 were performed.The effects of gender and genotype on the phenotype of Gitelman syndrome were analyzed.Values were shown as the mean ± standard deviation(SD).All data were analyzed using SPSS 20.0.Means were compared using Student’t t-test for comparisons between two groups.P<0.05 was considered a statistically significant difference.Results1.The positive rate of genetic testing in patients with unknown hypokalemia was 58.8%.Gitelman syndrome caused by SLC12A3 mutation was the most common cause of hereditary hypokalemia.2.SLC12A3 mutations were distributed in the whole exons.Most of Gitelman syndrome patients were compound heterozygote.In all mutations in SLC12A3,missense mutations accounted for 69.4%.A total of 46 mutations in SLC12A3 were identified including 12 novel mutations.3.Most of GS patients presented with hypomagnesemia.Some patients have hypocalciuria.The average age of onset in male was earlier than in female.4.Genotype-phenotype analysis.Patients with frameshift mutation had lower blood magnesium levels(P<0.05)than those without frameshift mutation.Conclusions1.Gitelman syndrome was the most common cause of hereditary hypokalemia.2.Most of Gitelman syndrome patients were compound heterozygote.Most of the SLC12A3 mutations were missense mutations.3.Gender and genotype were important factors affecting the phenotype of Gitelman syndrome.Part 2.Functional study of novel mutations of SLC12A3Methods1.Bioinformatics analysis of novel mutations(c.473G>A/p.R158Q and c.634G>A/p.G212S).The pathogenicity of novel mutations was predicted by online prediction software.Conservation analysis and three-dimensional protein structure prediction were conducted.2.In vitro functional study(1)Expression detection of mutant protein.The wild-type and mutant NCC plasmids were transfected in MDCT cell lines.Membrane proteins and cytoplasmic proteins were extracted.The expression levels of mutant NCC on membrane were analyzed by Western blot.(2)Localization of mutant protein.The wild-type and mutant NCC plasmids were transfected in MDCT cell lines.The localization of mutant NCC and wild-type NCC were observed by immunofluorescence.3.The NCCR156Q/+and NCCG210S/+knock in mice were constructed by Crispr Cas9.4.Homozygous NccR156Q/R156Q/NccG210S/G210S mice(F2)were produced by mating NccR156Q/+or NccG210S/+mice with each other.Compound heterozygous NccR156Q/G210S mice were produced by mating NccR156Q/+and NccG210S/+mice with each other.5.The body weight and blood pressure of the mice were measured when the mice were 12-16 weeks old.6.Blood was drawn from the angular venous plexus when the mice were 12-16 weeks old.Serum Na+,K+,Mg2+,and creatinine were detected.24h urine was collected from the mice in the metabolic cage.The volume,Na+,K+,Mg2+,Ca2+and creatinine of urine were detected.7.Expression level of Ncc in wild-type mice,heterozygous mice(NccR156Q/+and NccG210S/+),homozygous mice(NccR156Q/R156Q and NccG210S/G210S)and compound heterozygous mice(NccR156Q/G210S)were detected by RT-PCT and Western blot.8.Localization of Ncc in all groups of mice was observed by immunofluorescence and immunohistochemistry.9.Sex effect on phenotype in NCCR156Q/G210S mice was performed on female and male compound heterozygous mice.Serum K+ and Mg2+ levels,excretion fraction of K+and Mg2+,and 24h Ca2+ excretion were analyzed.Difference of Ncc localization between female and male compound heterozygous mice was observed by immunohistochemical staining.10.The mRNA and protein levels of renal Na+ channel(Ncc),Mg2+channel(Trpm6)and Ca2+ channels(Trpv5 and Trpv6)of female and male compound heterozygous mice were detected by RT-PCR and Western blot.11.HCTZ test was conducted to detect the function of Ncc in compound heterozygous mice.12.Statistical analysis.Values were shown as mean ± standard deviation.All data were statistically analyzed by SPSS 20.0 software.P<0.05 indicated a statistical difference.Results1.Two novel mutations was pathogenic mutations by bioinformatic analysis.R158 and G212 were highly conserved in multiple species and were predicted to be pathogenic by Mutation Taster,PolyPhen-2,and PROVEN.Both mutations were predicted to alter protein’s three-dimensional structure by SWISS-MODEL.2.The mutations resulted in decreased membrane expression and abnormal localization of NCC.The surface expression of R158Q-NCC and G212S-NCC decreased compared with WT-NCC detected by Western Blotting.Immunofluorescence showed that wildtype NCC was expressed on the surface of the cell membrane and mutant NCC was expressed in the cytoplasm.3.Compared with wild-type mice,homozygous mice and compound heterozygous mice exhibited a GS-like phenotype including hypokalemia,hypomagnesemia,normal blood pressure,and the same body weight as wild-type mice.Heterozygous mice were the same as WT mice.4.The levels of mRNA and protein expression of renal Ncc were significantly decreased in homozygous mice and compound heterozygous mice compared to wildtype mice.5.The localization of renal Ncc in homozygous mice and compound heterozygous mice was abnormal compared to wild-type mice.Mutant Ncc was mainly localized in the cytoplasm of DCT cells.6.Increased excretion fraction of Mg2+ and decreased 24h Ca2+ excretion were observed in male compound heterozygous mice compared to female mice.There were no significant differences in serum K+and Mg2+ levels,excretion fraction of K+and localization of Ncc between male and female compound heterozygous mice.7.The expression levels of mRNA and protein of Trpv5 and Trpv6 were increased in male compound heterozygous mice compared to female mice.8.Ncc function was reduced in NccR156Q/G210S mice confirmed by HCTZ test.Conclusions1.c.473G>A/p.R158Q and c.634G>A/p.G212S,were predicted to be pathogenic mutations.2.R158Q and G212S lead to decreased membrane expression and abnormal localization of NCC protein in vitro experiments.3.Homozygous and compound heterozygous mice exhibited typical Gitelman syndrome-like phenotype including hypokalemia and hypomagnesemia.4.Dysfunction of Ncc could be caused by decreased Ncc expression and abnormal localization in homozygous and compound heterozygous mice.5.Female and male mice exhibited different phenotypes in compound heterozygous mice.Increased excretion fraction of Mg2+ and decreased 24h Ca2+excretion in males were observed.Meanwhile,Ca2+ channel(Trpv5 and Trpv6)in kidney was up-regulation in male mice.