Research Analysis of SLC12A3 Gene Mutation and Molecular Mechanism in Gitelman Syndrome / 罕见病研究

ObjectiveThis study aimed at conducting retrospective analysis of the clinical symptoms and genetic mutations in 20 children with Gitelman syndrome treated at the Affiliated Children′s Hospital of Nanjing Medical University from August 2015 to November 2022 and also explored the molecular mechanism of the pathogenic high-frequency mutation D486N in the Chinese population.MethodsWe collected the clinical manifestations, growth and development status, laboratory examination results, and SLC12A3 gene variations of the patients. We distinguished the wild-type and mutant SLC12A3 genes overexpressed in human embryonic kidney 293T cells (HEK293T). We used protein immunoblotting to detect the expression level of NCC, and used immunofluorescence techniques to examine the subcellular localization of NCC. In addition, we investigated the impact of the high-frequency SLC12A3 gene mutation D486N on NCC protein expression and localization.ResultsIn the 20 patients with Gitelman syndrome, all of them had hypokalemia. We indemnified twenty-six SLC12A3 gene mutations, 13 of which are missense mutation, 1 of which synonymous mutation, 1 nonsense mutation, 4 frameshift mutation, and 7 splicing site mutation. Among them, four mutations (p.T235K, c.1096-1G > A, p.A464A, and c.2660+1_2660+2insT) were novel mutations.ConclusionsWe found the preliminary evidence that the high-frequency mutation D486N in the Chinese population affected the expression of total and membrane-bound NCC protein and influenced the membrane localization of NCC protein. The findings of this study provides experimental evidence for genetic counseling, diagnosis, and treatment of Gitelman syndrome.

Adolescent Gitelman syndrome:a case report and literature review / 中国综合临床

Objective:To explore the clinical characteristics and genetics of a Chinese patient with Gitelman syndrome (GS) and improve the awareness and diagnosis of GS among clinicians.Methods:Retrospectively analyzed the GS patient's clinical feature, laboratory examination, diagnosis, treatment and literature review admitted to Hebei General Hospital in September 2022.Results:A twelve-year-old boy was admitted to our department due to weakness of lower limbs. Laboratory tests after admission showed hypokalemia, hypomagnesemia, hypocalcemia and metabolic alkalosis. Genetic testing showed tow compound heterozygous mutations in the SLC12A3 gene (c.1456G>A and c.634G>A), which ultimately diagnosed as GS. The patient is on the mend and allowed to leave the hospital after treated by potassium supplement.Conclusion:The rate of leak diagnosis is high. Genetic testing should be undergo earlier if the patients suspected GS.

Whole-exome sequencing in diagnosing 2 cases of Gitelman syndrome / 中南大学学报(医学版)

Two patients with Gitelman syndrome were admitted to the Department of Endocrinology, Third Xiangya Hospital of Central South University. The genomic DNA from the patients' peripheral blood was extracted and the whole-exome sequencing was performed to detect the possible mutations. The function of the mutation sites was analyzed by bioinformatics software. Through whole-exome sequencing and Sanger sequencing, we have found that 2 patients with Gitelman syndrome carried compound heterozygous mutations of SLC12A3 gene, which were c.486_490delTACGGinsA, p.R943W, p.D486N, and p.R928C. Among them, c.486_490delTACGGinsA insertion deletion mutation causes frame shift and protein truncation. The p.R943W, p.D486N, and p.R928C of SLC12A3 gene were predicted to be pathogenic mutations by SIFT, PolyPhen2, and Mutation Taster. These 4 mutations were all reported, but p.R943W was first reported in Chinese population. Gitelman syndrome is rare in clinic and the rate of missed diagnosis is high. Early genetic analysis in patients with Gitelman syndrome is helpful to determine the etiology and guide the treatment.

A novel heterozygous duplication of the SLC12A3 gene in two Gitelman syndrome pedigrees: indicating a founder effect

Gitelman syndrome is an autosomal recessive salt-wasting tubulopathy caused by mutations in the SLC12A3 gene. A female and a male sibling from two unrelated Greek-Cypriot families presenting with a severe salt-wasting tubulopathy dueto compound heterozygous mutations of a novel duplication and a previously reported missense mutation in the SLC12A gene are described. Sanger sequencing was used to identify possible mutations in the SLC12A3 gene. For the detection of duplications/conversions and deletions in the same gene, Multiplex ligation probe amplification (MLPA) analysis was performed. Direct sequencing and MLPA analysis of the SLC12A3 gene identified two compound heterozygous mutations in both unrelated probands. Both probands were identified to carry in compound heterozygosity the known p.Met581Lys and a novelheterozygous duplication of exons 9-14 (E9_E14dup). The diagnosis of Gitelman syndrome was made through clinical assessment, biochemical screening and genetic analysis. The identification of the novel SLC12A3 duplication seems to be characteristic of Greek-Cypriot patients and suggests a possible ancestral mutational event that has spread in Cyprus due to a possible founder effect. Testing for Gitelman syndrome probable variants can be performed before proceeding to a full gene sequencing dropping the diagnostic cost. In addition, this report adds to the mutational spectrum observed.

Gitelman syndrome caused by SLC12A3 gene mutations: a report of pedigree analysis / 临床儿科杂志

Objective To explore the gene diagnosis of Gitelman syndrome.Methods The clinical data of a child with Gitelman syndrome were retrospectively analyzed along with gene detection results of his elder sister and parents.Results A 6-year-old boy was hospitalized for fever and hypokalemia.Gene detection of SLC12A found a new locus mutation of EXON21 c.2522A＞G p.(Asp841Gly) and a heterozygosis of EXON16 c.1946C＞T p.(Thr649Met).The diagnosis of Gitelman syndrome was confirmed.His mother carried a heterozygosis mutation of EXON21 c.2522A＞G p.(Asp841Gly),while his father and elder sister carried a heterozygosis mutation of EXON16 c.1946C＞T p.(Thr649Met).Conclusion Gene detection of SLC12A is helpful in the diagnosis of Gitelman syndrome.The newly discovered mutation of SLC12A3 gene has enriched the mutation spectrum of Gitelman syndrome.

Genotype, phenotype, and follow-up of Chinese patients with Gitelman's syndrome / 中华内分泌代谢杂志

Objective To analyze the characteristics of the genotype, phenotype, and follow-up of Gitelman's syndrome (GS) in the largest group of Chinese patients. Methods Sixty seven patients with GS underwent SLC12A3 gene analysis. Clinical characteristics and biochemical findings at the first presentation as well as follow-up were reviewed. Additionally, the associations of genotypes and phenotypes were explored. Results Forty-one different SLC12A3 mutations were identified in 67 patients with GS, including 11 novel ones, and 5 recurrent ones. 3 families (5. 7% ) had triple SLC12A3 mutations. Typical hypocalciuria and hypomagnesemia were not found in 6(9% ) and 8 (11. 9% )patients, respectively. In addition, male patients had an earlier age of onset and a higher urinary fraction excretion of electrolytes. 2 patients presented with chronic kidney disease, 13 (19. 4% ) with type 2 diabetes, 14 (20. 9% )with impaired glucose tolerance, and 5(7. 5% ) with impaired fasting glucose. Conclusion This study revealed 41 mutations in 67 Chinese patients with GS, including 11 novel variants and 5 high-frequency ones. Fraction excretion of electrolyte in urine may be more sensitive in the evaluation of phenotype compared with those of blood. It is difficult to correct hypokalemia and hypomagnesemia in GS. Patients with GS are at higher risk of the development of diabetes than ordinary people.

Effects of interaction between SLC12A3 polymorphism, salt-sensitive gene, and sodium intake on risk of child obesity / 한국영양학회지

PURPOSE: Obesogenic environments in children, in particular excessive intake of sodium, generate hypertension, which is a major risk factor for chronic diseases. METHODS: In all, 725 children, 379 boys and 373 girls, aged 8∼9 years were recruited from seven elementary schools in Kuro-ku, Seoul. To evaluate whether or not obesity risk was modulated by salt-sensitive genes, Solute Carrier Familiy 12 member 3 (SLC12A3) was used as the target. After children were assigned into obese (BMI > 85 percentile) or non-obese groups, anthropometry, blood biochemistry, and dietary intakes were measured according to the genotypes GG (wild) or GA+AA (hetero+mutant). RESULTS: Without gender differences, high TG and low HDLc were detected in the obese group compared to the non-obese group. Regardless of obesity, weight gain and blood pressure (BP) increased in the SLC12A3 GA+AA genotype rather than in the GG type. HDLc was associated with obesity risk without genotype difference. Odd ratios for risk of obesity were 15.57 (95% CI 2.192∼110.654), 22.84 (95% CI 1.565∼333.469), and 9.32 (95%CI 1.262∼68.817) in boys and girls with GA+AA genotypes as sodium intake increased above 4,000 mg/day. Dietary calcium, sodium, folate, and vit C were associated with obesity risk according to gender or genotype differences. Since high folate intake reduced obesity risk in only boys with GG type. Risk for overweight and obesity increased in boys with GA+AA genotypes and dietary habits with high sodium and cholesterol and low folate. CONCLUSION: The A allele of SLC12A3 rs11643718 was sensitive to development of obesity in children as sodium intake increased.

Analysis of mutations of two Gitelman syndrome family SLC12A3 genes and proposed treatments using Chinese medicine / 中国结合医学杂志

<p><b>OBJECTIVE</b>To determine the gene location of two Gitelman syndrome (GS) family SLC12A3 genes and explore treatments using Chinese medicine (CM) prescriptions.</p><p><b>METHODS</b>In order to locate the two GS mutations, samples were collected from 11 people from two different pedigrees for direct genetic sequencing and comparison of the 26 exons of SLC12A3. Furthermore, the change of serum potassium was monitored throughout the therapy and those two probands undertook a sequential superposition of Western medicine (including potassium, Panangin and potassium-sparing diuretics) with CM prescription based on Buyang Huanwu Decoction () and Sijunzi Decoction (). The treatment included three stages, oral potassium chloride for the first 2 weeks (stage 1), potassium-sparing diuretic and Panangin with potassium chloride for the next 2 weeks (stage 2), CM along with the medicine in stage 2 for the final 2 weeks (stage 3).</p><p><b>RESULTS</b>The three mutations occurring in proband 1 from pedigree I were Thr60Met, 965-1_976del13ins12 (small indels mutation) and Ala122Ala (homozygous silent mutation). Likewise, three mutations, Asn359Lys, Thr382Met and Arg913Gln, appeared in the proband 2 from pedigree II. The serum potassium levels increasing from baseline to sequential stages were 1.63 mmol/L (baseline), 2.5 mmol/L (stage 1), 3.1 mmol/L (stage 2) and 3.9 mmol/L (stage 3) in the proband 1, and 2.8 mmol/L (baseline), 3.1 mmol/L (stage 1), 3.5 mmol/L (stage 2) and 4.3 mmol/L (stage 3) in the proband 2, respectively. The symptoms (numbness of limbs, weakness, palpitations, etc.) of both probands were all alleviated.</p><p><b>CONCLUSIONS</b>The mutations of both GS pedigrees can be defined as compound heterozygous mutations, most of which are known as missense mutations. Applying CM could be an appropriate choice for future intervention of GS.</p>

A novel homozygous mutation in the solute carrier family 12 member 3 gene in a Chinese family with Gitelman syndrome

Loss of function of mutated solute carrier family 12 member 3 (SLC12A3) gene is the most frequent etiology for Gitelman syndrome (GS), which is mainly manifested by hypokalemia, hypomagnesemia and hypocalciuria. We report the genetic characteristics of one suspicious Chinese GS pedigree by gene sequencing. Complete sequencing analysis of the SLC12A3 gene revealed that both the proband and his elder sister had a novel homozygous SLC12A3 mutation: c.2099T>C and p.Leu700Pro. Moreover, the SLC12A3 genes of his mother and daughter encoded the same mutated heterozygote. It was noted that in this pedigree, only the proband complained about recurrent episodes of bilateral lower limb weakness over 8 years, while his elder sister, mother and daughter did not present symptoms. The inconsistent clinical features of this pedigree implied that besides diverse phenotypes possibly originated from the same genotype, gender difference may also dominate the variant GS phenotypes. Further genetic and proteomic research are needed to investigate the precise mechanisms of GS, including the study of specific ethnicities.

A Pedigree with c.179 Cytosine to Threonine Missense Mutation of SLC12A3 Gene Presenting Gitelman's Syndrome

A 42-year-old man came to the hospital presenting chest discomfort and general weakness. He had come to the hospital with the same symptoms 3 months ago and 12 years prior. His laboratory test showed hypokalemia, hypomagnesemia and hypocalciuria. The arterial blood gas analysis showed hypochloremic metabolic alkalosis. He had an ultrasonography guided renal biopsy, the result was normal at light microscopy and immunofluorescence microscopy. However, a special stain for Na-Cl cotransporter was weakly expressed compared with the control. The patient and his family underwent genetic sequencing about the SLC12A3 gene. He had a homozygous mutation in the 179th nucleotide of Exon 1 on the SLC12A3 gene (p.Thr60Met) and his parents and sisters were diagnosed as carrier state of Gitelman's syndrome (GS). GS is an inherited tubular disorder which presents mild hypokalemia, hypomagnesemia and hypocalciuria. Since the symptoms and laboratory results are not severe, it can go unnoticed by physicians. Herein we present a family with GS, diagnosed by genetic sequencing.

Clinical features and genetic analysis of three patients with Gitelman syndrome / 中华内分泌代谢杂志

[Summary] The clinical data of 3 inpatients clinically diagnosed as Gitelman syndrome ( GS ) were collected. The genomic DNA was isolated from the peripheral blood and the primers were designed to amplify all the exons and flanking introns in the SLC12A3 and CLCNKB genes by PCR. Direct sequencing of PCR products in the two genes was performed in all patients. Three patients manifested with recurrent hypokalemia, hypomagnesemia, hypocalciuria, hypochloremic metabolic alkalosis, but normal blood pressure. Gene sequencing results showed that one novel mutation p. L891V was identified in SLC12A3 gene in case 2. Seven and 12 types of polymorphic loci in the CLCNKB gene were found in case 1 and case 3, respectively. However, mutations were not found in the SLC12A3 and CLCNKB gene.

Mutations in SLC12A3 and CLCNKB and Their Correlation with Clinical Phenotype in Patients with Gitelman and Gitelman-like Syndrome

Gitelman's syndrome (GS) is caused by loss-of-function mutations in SLC12A3 and characterized by hypokalemic metabolic alkalosis, hypocalciuria, and hypomagnesemia. Long-term prognosis and the role of gene diagnosis in GS are still unclear. To investigate genotype-phenotype correlation in GS and Gitelman-like syndrome, we enrolled 34 patients who showed hypokalemic metabolic alkalosis without secondary causes. Mutation analysis of SLC12A3 and CLCNKB was performed. Thirty-one patients had mutations in SLC12A3, 5 patients in CLCNKB, and 2 patients in both genes. There was no significant difference between male and female in clinical manifestations at the time of presentation, except for early onset of symptoms in males and more profound hypokalemia in females. We identified 10 novel mutations in SLC12A3 and 4 in CLCNKB. Compared with those with CLCNKB mutations, patients with SLC12A3 mutations were characterized by more consistent hypocalciuria and hypomagnesemia. Patients with 2 mutant SLC12A3 alleles, compared with those with 1 mutant allele, did not have more severe clinical and laboratory findings except for lower plasma magnesium concentrations. Male and female patients did not differ in their requirement for electrolyte replacements. Two patients with concomitant SLC12A3 and CLCNKB mutations had early-onset severe symptoms and showed different response to treatment. Hypocalciuria and hypomagnesemia are useful markers in differentiation of GS and classical Bartter's syndrome. Gender, genotypes or the number of SLC12A3 mutant alleles cannot predict the severity of disease or response to treatment.

Clinical characteristics and SLC12A3 gene mutation profile of Gitelman syndrome: One case report / 中华内分泌代谢杂志

Objective To describe the clinical characteristics of a patient with Gitelman syndrome,and to identify the associated SLC12A3 gene mutations.Methods A suspected case of teenager-onset Gitelman syndrome was observed in our hospital.It was further confirmed by clinical manifestations and auxiliary examination.In addition,direct sequencing for the exons of SLC12A3 gene and CLCNKB gene region was conducted to identify the probable disease-associated mutations.Results The case showed characteristics of hypokalemia,hypomagnesemia,and low level of urinary calcium and onset by age of 18.By excluding the possibilities of long-term use of thiazide diuretics,laxatives,chronic vomiting and diarrhea,he was finally diagnosed as a case of Gitelman syndrome.Furthermore,by Sanger direct sequencing,2 coding variations were identified in SLC12A3 gene region,including T304M and L488P.L488P was a new heterozygous mutation.Conclusion Detection of SLC12A3 gene mutation could facilitate the diagnosis of Gitelman syndrome and improve prognosis.

The study of SLC12A3 complicated heterozygotic mutation in Children Gitelman syndrome / 医学研究生学报

Objective Gitelman Syndrome is a disease caused by the mutation of Na-Cl cotransporter gene(SLC12A3).The article studied the significance of diagnosis and identification by genetic mutation. Methods We collected the clinical data, then we sequenced the SLC12A3 gene by the first sequencing technology and MLPA. Results SLC12A3 complicated heterozygotic mutation was observed.One of them showed c.1964G>A, p.(Arg655His) and exon 8 deletion mutation, the other showed c.2543A>T, p.(Asp848Val) and c.976delG, p.(Val326fs) mutation of SLC12A3 gene in children. Conclusion The final diagnosis depended on gene diagnosis. Pediatrician must recognize the manifestations to advoid misdiagnosis.

Gitelman Syndrome with Normal Serum Magnesium

Gitelman syndrome is an autosomal recessive renal tubular disorder characterized by hypokalemic metabolic alkalosis, and it is distinguished from Batter syndrome by hypomagnesemia and hypocalciuria. This disorder is caused by mutation in SLC12A3 gene which encodes thiazide-sensitive Na(+)-Cl(-)cotransporter (NCCT) which is expressed in the apical membrane of cells, lining distal convoluted tubule. A 8-year old boy who presented with Rolandic epilepsy, and horseshoe kidney accidentally showed clinical features of metabolic alkalosis, hypokalemia, hypocalciuria without hypomagnesemia. So we identified a heterozygote mutation and an abnormal splicing in the SLC12A3 gene, encoding NCCT. The mutation was detected in the exon 15 and 22 of SLC12A3 gene.

Coexistence of Gitelman's syndrome and thyroid disease: SLC12A3 gene analysis in two patients / 中华内分泌代谢杂志

Two patients with coexistence of thyroid disease and suspected Gitelman's syndrome underwent SLC12A3 gene analysis. The results confirmed that both patients were compound heterozygotes of SLC12A3 gene mutation. Three novel variants of SLC12A3 were found in this study. This report suggests that Gitelman's syndrome may coexist with other disorders associated with hypokalemia, such as Graves' disease.

Relationship between SLC12A3 gene Arg913Gln polymorphism and type 2 diabetic nephropathy in Han population of Shanghai / 上海第二医科大学学报

Objective To explore the relationship between Arg913Gln(G→A) polymorphism of solute carrier family 12 member 3 (SLC12A3) gene and diabetic nephropathy (DN) in type 2 diabetes mellitus (T2DM) in Han population of Shanghai. Methods Two hundred and fifty-eight Han ethnic people in Shanghai with T2DM (T2DM group) were divided into non-DN group (DN0 group, n=95) and DN group (n=163) according to 24 h urine albumin excretion rate (AER), and those in DN group were subdivided into microalbuminuria group (DN1 group, n=95) and macroalbuminuria group (DN2 group, n=68). Besides, 82 people with normal results of oral glucose tolerance test (OGTT), without diabetes mellitus and nephropathy were served as controls. PCR-sequencing was used to detect the genotypes of Arg913Gln polymorphism of SLC12A3 gene. Genotypic and allelic frequencies and clinical characteristics were compared among groups. Results Three genotypes (GG, GA and AA) were detected. The frequencies of GA+AA genotype and A allele in T2DM group were higher than those in control group, while there was no significant difference between groups (P>0.05). There was no significant difference in genotypic or allelic frequencies among subgroups of T2DM group (P>0.05). The level of triglyeeride (TG), AER, level of fasting insulin (FINS) and HOMA-IR in patients with GA+AA genotype were significantly higher than those in patients with GG genotype in T2DM group (P<0.05). Conclusion Arg913Gln(G→A) polymorphism of SLC12A3 gene is not significantly associated with T2DM and DN in Han population of Shanghai. The AER of people with GA+AA genotype is significantly higher than that with GG genotype. Arg913Gln (G→A) polymorphism of SLC12A3 gene may predict the risk of increase of albuminuria in patients with T2DM in Han population of Shanghai.

A Case of Gitelman Syndrome Presented with Epileptic Seizure

Both Gitelman syndrome and Bartter syndrome are autosomal recessively inherited renal tubular disorders characterized by hypokalemic metabolic alkalosis, salt wasting and normal to low blood pressure. Gitelman syndrome is caused by mutations in the thiazide-sensitive Na- Cl cotransporter (NCCT) and distinguished from Bartter syndrome, which is associated with mutations of several genes, by the presence of hypomagnesemia and hypocalciuria. In most of the patients with Gitelman syndrome, the disease manifests with transient episodes of muscular weakness and tetany in the adult period, but, often, is asymptomatic. We report here an 11 years-old female with Gitelman syndrome who presented with aggravation of epileptic seizure. The diagnostic work-up showed typical clinical features of metabolic alkalosis, hypokalemia, hypomagnesemia and hypocalciuria. We also identified a heterozygote mutation(642CGC(Arg)>TGC(Cys)) and an abnormal splicing in the SLC12A3 gene encoding NCCT.