SLC12A3 Mutations Research Articles

7293 An Unusual Case Of Adult-Onset Heterozygous HHRH In A Male With Normal Phosphorus And Severe Osteoporosis

Abstract Disclosure: M. El Najjar: None. A. Sabet: None. D. Willard: None. Introduction: Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare genetic disorder characterized by renal phosphate wasting, which can affect bone metabolism. Although onset is typically in childhood, adult-onset forms have been described, and the manifestation depends on the zygosity of each patient. Most commonly, adults with HHRH demonstrate markedly reduced bone density and multiple fractures. Heterozygotes have milder presentations with mild hypophosphatemia, hypercalciuria, and nephrolithiasis, usually without bone disease; however, it is less well characterized. Case report: A 73-year-old man suffered an L1 vertebral compression fracture while playing golf. Bone mineral density testing revealed T-scores of -0.9 at the lumbar spine and -3.4 at the femoral neck (Z-score -2.6). He started alendronate 70 mg weekly for osteoporosis treatment. A year later, the patient sustained an L3 vertebral compression fracture while on treatment with no improvement in his bone density. His family history was remarkable for osteoporosis in his mother but no nephrolithiasis or hypercalcemia. Physical examination was unremarkable. Laboratory evaluation was notable for a 1,25-dihydroxyvitamin D of 87 pg/mL (18-72 pg/mL) , a 24-hour urine calcium of 240 mg/g Cr (30-210 mg/g),a serum PTH of 9 pg/mL (16-77 pg/ml) , but otherwise a normal serum calcium level of 9.4 mg/dL (8.6-10.3 mg/dL) and serum phosphate level of 2.2 mg/dl (2.1-4.3 mg/dl). Genetic testing revealed heterozygosity for SLC34A3 mutation, which is associated with HHRH. The patient discontinued alendronate and started 250 mg phosphorus supplementation 4 times daily. At 6 months follow up, he reported improvement in his body aches, and biochemical testing showed normalization of his 24-hour urine calcium to 171 mg/g Cr and serum PTH to 21 pg/mL. Repeat bone density scan after 1 year of phosphorus supplementation showed T-scores of -0.3 at the lumbar spine (5.8% increase) and -2.6 at the femoral neck (15.5% increase). Clinical Lesson: HHRH is an autosomal recessive hypophosphatemic rickets associated with mutations of the SLC34A3 sodium-phosphate cotransporter, where it is believed that selective phosphate wasting leads to increased calcitriol, in contrast to other forms of hypophosphatemic rickets, causing increased intestinal calcium absorption and resulting hypercalciuria. Adult-onset forms of HHRH have been described in heterozygous carriers of the SLC34A3 mutation and usually present with low to low-normal phosphate levels, hypercalciuria, and nephrolithiasis but without bone disease. However, clinical variations of heterozygous carriers have also been recognized, and therefore are likely underdiagnosed. Our patient’s serum phosphorus level was within the normal range, and hypophosphatemic rickets could have been missed if a more extensive laboratory evaluation had not been performed. Presentation: 6/1/2024

SLC5A2 Mutation in a Patient Presenting with Volume Depletion

SLC5A2 Mutation in a Patient Presenting with Volume Depletion

Novel Compound Heterozygous Mutations of the SLC12A3 Gene in Gitelman Syndrome with Growth Hormone Deficiency and Hypothyroidism

Introduction: Gitelman syndrome (GS) is an inherited kidney disease that causes an imbalance of charged ions in the body. SLC12A3 mutation is the predominant cause of GS. There are different known and unknown pathogenic mutations in SLC12A3. Case Presentation: In the present case report, an 8-year-old girl was referred to our pediatric endocrinology clinic for evaluation of short stature. Her height was 113 cm (-2.94 SD). Her growth hormone peak was 5.81 ng/mL. IGF-1 was lower than -2SD. Thyroid stimulating hormone was high. The blood potassium was 3.37 mmol/L. After 3 months of GH treatment, her blood potassium dropped further (3.01 mmol/L). The gene test results showed that there were two heterozygous mutations of the SLC12A3 gene: C.1456G>A (p.D486N) and c.1065_1072 delGCAGGG (p.A356Qfs*5), which her parents verified. Conclusions: Gitelman syndrome can be associated with growth hormone deficiency and hypothyroidism in addition to short stature, hypokalemia, and hypomagnesemia, and the underlying molecular mechanisms need to be explored in the coexistence of these three diseases. The experience, in this case, is that blood electrolytes should be checked monthly for the first three months after growth hormone treatment for short stature. Once the blood potassium level is low, much attention should be paid to further diagnosis to avoid misdiagnosis.

Declaration: Novel SLC3A1 mutation in a cystinuria patient with xanthine stones: a case report

BackgroundCystinuria and xanthinuria are both rare genetic diseases involving urinary calculi. However, cases combining these two disorders have not yet been reported.Case PresentationIn this study, we report a case of cystinuria with xanthine stones and hyperuricemia. The 23-year-old male patient was diagnosed with kidney and ureteral stones, solitary functioning kidney and hyperuricemia after admission to the hospital. The stones were removed by surgery and found to be composed of xanthine.ConclusionGenetic testing by next-generation sequencing technology showed that the patient carried the homozygous nonsense mutation c.1113 C> A (p.Tyr371*) in the SLC3A1 gene, which was judged to be a functionally pathogenic variant. Sanger sequencing revealed that the patient’s parents carried this heterozygous mutation, which is a pathogenic variant that can cause cystinuria. The 24-h urine metabolism analysis showed that the cystine content was 644 mg (<320 mg/24 h), indicating that the patient had cystinuria, consistent with the genetic test results. This case shows that cystinuria and xanthine stones can occur simultaneously, and provides evidence of a possible connection between the two conditions. Furthermore, our findings demonstrate the potential value of genetic testing using next-generation sequencing to effectively assist in the clinical diagnosis and treatment of patients with urinary calculi.

#4942 GENOTYPE-PHENOTYPE ANALYSIS IN FAMILIAL RENAL GLUCOSURIA PATIENTS: THE CRUCIAL ROLE OF CONSERVED RESIDUES

Abstract Background and Aims Familial renal glucosuria (FRG) is a hereditary disorder caused by mutations in SLC5A2, the gene encoding sodium-glucose cotransporter 2 (SGLT2) in proximal tubule cells. In this study, we aimed to characterize the genotype-phenotype relationship in FRG patients using data from our cohort and literature review. Method We sequenced SLC5A2 in a cohort of 21 FRG patients and measured the renal threshold of glucose (RTG) in 15 patients using urinary glucose excretion in a 4-hour oral glucose tolerance test (OGTT). We built an open-source online calculator to facilitate the calculation of RTG. Besides, we reviewed published literature and obtained information on SLC5A2 variants and 24-hour urinary glucose (24hUG) in FRG patients. Results We identified 27 rare SLC5A2 variants, including 13 novel variants (G484D, R564W, A212S, c.574+1G&amp;gt;C, W649*, S592Cfs*6, Q579*, A89T, Y339*, V39F, G491E, A464E, and G360D), in our cohort and yielded 107 SLC5A2 variants from literature review. RTG in our cohort ranged from 1.0 to 9.2mmol/L. Patients carrying two SLC5A2 variants had lower RTG (3.9 vs. 6.2 mmol/L, p = 0.057) and larger amounts of 24-hour urinary glucose excretion (24hUG) (52.39 vs. 7.20 g/1.73m2, p = 2.7*10−8) than those carrying a single variant. Patients with homozygous missense or in-frame indels had mean 24hUG of 82.30g/1.73m2, comparable to those with homozygous truncating variants (81.66g/1.73m2) and significantly more than those with homozygous splicing variants (35.38g/1.73m2, p = 0.026). Patients with homozygous missense variants involving conservative residues had more 24hUG (104.76g/1.73m2) than those with variants at non-conservative residues (46.37g/1.73m2, p = 0.0031). Conclusion We built a comprehensive map showing the impact of SLC5A2 variant type, variant location, and zygosity on glucosuria severity. Our results highlighted that conserved residues play an essential role in maintaining the transport function of SGLT2.

Identification of mutations in 15 nephrolithiasis-related genes leading to a molecular diagnosis in 85 Chinese pediatric patients.

The aim of this study was to describe the genotypic and phenotypic characteristics of Chinese pediatric patients with hereditary nephrolithiasis. Whole-exome sequencing (WES) was performed on 218 Chinese pediatric patients with kidney stones, and genetic and clinical data were collected and analyzed retrospectively. The median age at onset in our cohort was 2.5years (age range, 0.3-13years). We detected 79 causative mutations in 15 genes, leading to a molecular diagnosis in 38.99% (85/218) of all cases. Monogenic mutations were present in 80 cases, and digenic mutations were present in 5 cases; 34.18% (27/79) of mutations were not included in the databases. Six common mutant genes, i.e., HOGA1, AGXT, GRHPR, SLC3A1, SLC7A9, and SLC4A1, were found in 84.71% of the patients overall. Furthermore, three mutations (A278A, c.834_834 + 1GG > TT, and C257G) in HOGA1, two mutations (K12QfX156 and S275RfX28) in AGXT, and one mutation (C289DfX22) in GRHPR represented hotspot mutations. The patients with HOGA1 mutations had the earliest onset age (0.8years), followed by those with SLC7A9 (1.8years), SLC4A1 (2.7years), AGXT (4.3years), SLC3A1 (4.8years), and GRHPR (8years) mutations (p = 0.002). Nephrocalcinosis was most commonly observed in patients with AGXT gene mutations. Fifteen causative genes were detected in 85 Chinese pediatric patients with kidney stone diseases. The most common mutant genes, novel mutations, hotspot mutations, and genotype-phenotype correlations were also found. This study contributes to the understanding of genetic profiles and clinical courses in pediatric patients with hereditary nephrolithiasis. A higher resolution version of the Graphical abstract is available as Supplementary information.

A Case Series of Cystinuric Stone Formers in Western Cape, South Africa: SLC3A1 or SLC7A9 Mutations and Phenotype

Objective To describe the genetic mutations and phenotype in the first African series of patients with cystinuria. Methods Patients with cystinuria were recruited from a specialist metabolic renal stone clinic in Cape Town, South Africa, for DNA sequencing to detect mutations in SLC3A1 and SLC7A9. Chart reviews and patient interviews were conducted to record demographics, previous medical history, family history, stone-specific history, age at first presentation, cystinuria complications, urine cystine:creatinine ratio, stone analysis, and serum creatinine. Results Nine patients were included: 3 male patients and 6 female patients. The mean age (± SD) of patients was 33.43 ± 19.96 years. The median age (± IQR) at initial diagnosis of cystinuria was 16 ± 18 years, but the age ranged from 2 to 66 years. Three of 9 patients included (33.3%) had chronic kidney disease (CKD); however, none were receiving dialysis. Most patients initially presented with a staghorn calculus (4/9; 44.4%). The mean serum creatinine (± SD) was 84 ± 38 μmol/L. The mean urine cystine (± SD) was 2083 ± 1249 nmoL/mg creatinine. Eight patients had mutations in the SLC3A1 gene; 1 had mutations in both SLC3A1 and SLC7A9. Of the patients with only SLC3A1 mutations, 1 patient was homozygous and the rest were compound heterozygotes (two different mutations identified in the same gene). Four patients had a pathogenic variant in addition to an “uncertain significance” variant in SLC3A1. There were 9 mutations (5 pathogenic and 4 “unknown significance”) in SLC3A1 and 1 mutation in SLC7A9. Two of these were novel mutations. Conclusion This “first in Africa” series of cystinuria patients showed marked heterogeneity in both phenotype and genotype, with a predominance of SLC3A1 mutations. This heterogeneity is similar to that reported in international cohorts.

Resolving the thiazide-induced hypocalciuria conundrum using single cell transcriptomics

Thiazide diuretics lower urinary calcium excretion and are used to prevent calcium stone recurrence. Thiazides inhibit the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Hypocalciuria also occurs in Gitelman syndrome, caused by mutations in SLC12A3, encoding NCC. The mechanism by which loss of NCC function affects calcium handling remains very controversial. The two prevailing hypotheses invoked are 1) hypocalciuria results from volume-dependent increases in proximal tubule reabsorption; 2) thiazides activate calcium reabsorption along the DCT. Here we combined single nucleus RNA sequencing with physiologic analysis to unravel the causes of hypocalciuria.Eight to 10-week old male mice were given 50 mg/kg/day metolazone (MTZ) orally in food for 4 days. Littermates receiving only vehicle were used as controls. As DCT cells comprise a small percentage of the kidney mass, we used the NCC-Cre +/- - INTACT +/- mouse line to enrich for DCT via Cre-dependent GFP expression in DCT nuclei. After nuclei isolation, GFP+ events (DCT nuclei) were enriched by fluorescence assisted nuclei sorting. The purified DCT nuclei were sequenced using 10X Chromium controller. Reads were then analyzed using a standard transcriptomic bioinformatics pipeline with Seurat.The MTZ-treated mice showed lower urinary calcium excretion compared to controls recapitulating thiazide-induced hypocalciuria. Our snRNAseq integrated dataset contained 51,876 mapped genes across 45,953 samples from 3 MTZ-treated and 3 control mice. Roughly 99% of the nuclei were singlets expressing Slc12a3 showing successful enrichment for the DCT. Dimensional reduction analysis and projection confirmed two clusters, DCT1 and DCT2, based on canonical segment markers consistent with previously published transcriptomic atlases. Gene expression analysis showed the DCT2 contained a calcium cassette consisting of the known calciotropic genes Trpv5, Calb1, S100g, Slc8a1, Ryr2, Vdr, Casr, and Atp2b1. These genes were also expressed in the connecting tubule (CNT), as documented using the Calb1-Cre-INTACT system. From this set of genes, we defined a calcium score computed from the pooled average expression of all these genes in a single cell. Although all calcium cassette genes, and the average calcium score, were lower in the MTZ-treated group, the proportion of DCT2/DCT1 cells increased (0.27 ± 0.03 vs 0.35 ± 0.03; p = 0.027). These data suggest that lower calcium delivery to the DCT reduces calcium cassette expression per cell, but that distal fractional reabsorption is nonetheless increased owing to hypertrophy and elongation of the calcium transporting segments, DCT2 and CNT.We propose a modified mechanism to account for thiazide -induced hypocalciuria: 1) volume-dependent increase in proximal tubule reabsorption; 2) increased fractional (not absolute) DCT2 reabsorption owing to structural remodeling of DCT2 and CNT providing more surface area for calcium reabsorption. Funding: NIH DK51496, NIH DK054983, VA 1I01BX002228, LeDucq Foundation, DOST-PCHRD-ASTHRDP This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Evaluation of the value of genetic testing for cystinuria in the Danish population of English bulldogs.

Cystinuria is a genetic disease that can lead to cystine urolith formation. The English bulldog is the dog breed most frequently affected. In this breed, three missense mutations have been suggested to be associated with cystinuria: c.568A>G and c.2086A>G in SLC3A1 and c.649G>A in SLC7A9. In this study, the occurrence of these three mutations in the Danish population of English bulldogs was investigated. Seventy-one English bulldogs were genotyped using TaqMan assays. The dogs' owners were given questionnaires concerning the medical histories of their dogs. Allele frequencies of 0.40, 0.40, and 0.52 were found for the mutant alleles in the three loci: c.568A>G, c.2086A>G, and c.649G>A, respectively. For both mutations in SLC3A1, a statistically significant association was found between cystinuria and homozygosity for the G allele among male, English bulldogs. For the mutation in SLC7A9, there was no statistically significant association between homozygosity for the mutant allele and cystinuria. Due to high allele frequencies, limited genetic diversity, continued uncertainty about the genetic background of cystinuria, and more severe health problems in the breed, selection based on genetic testing for the mutations in SLC3A1 cannot be recommended in the Danish population of English bulldogs. However, results of the genetic test may be used as a guide to recommend prophylactic treatment.

102 Isolated Glycosuria in a Patient With Confirmed SLC5A2 Mutation

102 Isolated Glycosuria in a Patient With Confirmed SLC5A2 Mutation

CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman's Disease Phenotype in a Patient-Derived Kidney Organoid System.

The aim of this study is to explore the possibility of modeling Gitelman's disease (GIT) with human-induced pluripotent stem cell (hiPSC)-derived kidney organoids and to test whether gene correction using CRISPR/Cas9 can rescue the disease phenotype of GIT. To model GIT, we used the hiPSC line CMCi002 (CMC-GIT-001), generated using PBMCs from GIT patients with SLC12A3 gene mutation. Using the CRISPR-Cas9 system, we corrected CMC-GIT-001 mutations and hence generated CMC-GIT-001corr. Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001corr group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group (12.2 ± 0.7/cm2 vs. 3.7 ± 0.2/cm2, p < 0.05). In qRT-PCR, performed using harvested kidney organoids, relative sodium chloride cotransporter (NCCT) mRNA levels (normalized to each iPSC) were increased in the CMC-GIT-001corr group compared with the CMC-GIT-001 group (4.1 ± 0.8 vs. 2.5 ± 0.2, p < 0.05). Consistently, immunoblot analysis revealed increased levels of NCCT protein, in addition to other tubular proteins markers, such as LTL and ECAD, in the CMC-GIT-001corr group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001corr group was colocalized with ECAD (a distal tubule marker) using confocal microscopy. Kidney organoids from GIT patient-derived iPSC recapitulated the Gitelman's disease phenotype, and correction of SLC12A3 mutation utilizing CRISPR-Cas9 technology provided therapeutic insight.

Genetic and clinical analysis of Chinese pediatric patients with cystinuria.

This study aimed to investigate the genotypic and phenotypic characteristics of Chinese pediatric patients with cystinuria. This was a retrospective study of 14 Chinese pediatric patients with cystine stones. All published studies of the Chinese pediatric cystinuria population were searched and enrolled based on the inclusive standard. Among the 14 pediatric patients with cystinuria, 8 were males and 6 were females. The mean age of first stone onset was 4.0 ± 3.3years (4months-9years). All of the patients had multiple stones, and 57.1% (8/14) had bilateral stones. The mean maximum stone diameter was 1.7 ± 0.6 (range 0.5-2.6) cm. A total of 13 SLC3A1 gene mutations and 9 SLC7A9 gene mutations were detected, of which 41% (9/22) of mutations were novel. Patients with SLC7A9 mutations were more likely to develop bilateral stones than those with SLC3A1 mutations (100% vs. 33.3%, p = 0.03). Thirty-four SLC3A1 gene mutations and twenty-eight SLC7A9 gene mutations were found in a total of fifty-five Chinese children with cystinuria. The SLC7A9 gene mutation distribution was more dispersed, while the SLC3A1 mutation was clustered in exons 6-8. The c.647C > T (p. T216M) (4/53) and c.1113C > A (p. Y371Ter) (4/53) mutations in the SLC3A1 gene and the c.1399 + 2_3insT (3/36) mutation in the SLC7A9 gene represent potential hotspots in cystinuria. Our results present a comprehensive genetic spectrum for pediatric cystinuria patients in China. Patients with SLC7A9 mutations were more likely to develop bilateral stones than those with SLC3A1 mutations. A wide mutation spectrum and the potential mutation hotspots associated with cystinuria were also identified.

Prevalence and Phenotypic Spectrum of Heterozygous SLC34A3 Mutations in a Large Genetic Database

Prevalence and Phenotypic Spectrum of Heterozygous SLC34A3 Mutations in a Large Genetic Database

A case of Gitelman syndrome with membranous nephropathy

BackgroundGitelman syndrome (GS) is a rare autosomal recessive inherited salt-losing tubulopathy (SLT). Here, we report, for the first time, a case of GS overlapping nephrotic syndrome (NS) related to PLA2R-associated membranous nephropathy (MN).Case presentationWe described a male patient had a 4-year history of recurrent fatigue. Serum biochemistry revealed hypokalemia with renal potassium wasting, hypomagnesemia, metabolic alkalosis, hyperreninemia, hypocalciuria, as well as nephrotic-range proteinuria, hypoalbuminemia, and elevated serum anti-phospholipase A2 receptor (PLA2R) antibody. Gene sequencing identified compound heterozygous mutations in SLC12A3 [c.536T > A(p.V179D) and c.1456G > A(p.D486N)]. The unusual association of SLTs and nephrotic-range glomerular proteinuria prompted us to perform a renal biopsy. Renal biopsy showed idiopathic MN. Due to the potential to activate the sodium-chloride co-transporter (NCC) and cause hyperkalemia, tacrolimus was selected to treat NS. Following treatment with potassium chloride, magnesium oxide, low-dose glucocorticoid combined with tacrolimus, the fatigue significantly improved, and concurrently hypokalemia, hypomagnesemia were corrected and NS was remitted.ConclusionsRenal biopsy should be warranted for GS patients with moderate to nephrotic-range proteinuria. Tacrolimus was preferred to the management of GS patients with NS.

The genetic spectrum of Gitelman(-like) syndromes.

Purpose of reviewGitelman syndrome is a recessive salt-wasting disorder characterized by hypomagnesemia, hypokalemia, metabolic alkalosis and hypocalciuria. The majority of patients are explained by mutations and deletions in the SLC12A3 gene, encoding the Na+-Cl−-co-transporter (NCC). Recently, additional genetic causes of Gitelman-like syndromes have been identified that should be considered in genetic screening. This review aims to provide a comprehensive overview of the clinical, genetic and mechanistic aspects of Gitelman(-like) syndromes.Recent findingsDisturbed Na+ reabsorption in the distal convoluted tubule (DCT) is associated with hypomagnesemia and hypokalemic alkalosis. In Gitelman syndrome, loss-of-function mutations in SLC12A3 cause impaired NCC-mediated Na+ reabsorption. In addition, patients with mutations in CLCKNB, KCNJ10, FXYD2 or HNF1B may present with a similar phenotype, as these mutations indirectly reduce NCC activity. Furthermore, genetic investigations of patients with Na+-wasting tubulopathy have resulted in the identification of pathogenic variants in MT-TI, MT-TF, KCNJ16 and ATP1A1. These novel findings highlight the importance of cell metabolism and basolateral membrane potential for Na+ reabsorption in the DCT.SummaryAltogether, these findings extend the genetic spectrum of Gitelman-like electrolyte alterations. Genetic testing of patients with hypomagnesemia and hypokalemia should cover a panel of genes involved in Gitelman-like syndromes, including the mitochondrial genome.

Fluconazole in hypercalciuric patients with increased 1,25(OH)2D levels: the prospective, randomized, placebo-controlled, double-blind FLUCOLITH trial

BackgroundHypercalciuria is one of the most frequent metabolic disorders associated with nephrolithiasis and/or nephrocalcinosis possibly leading to chronic kidney disease (CKD) and bone complications in adults. Orphan diseases with different underlying primary pathophysiology share inappropriately increased 1,25(OH)2D levels and hypercalciuria, e.g., hypersensitivity to vitamin D and renal phosphate wasting. Their management is challenging, typically based on hyperhydration and dietary advice.The antifungal azoles are known to inhibit the 1α-hydroxylase and therefore decrease 1,25(OH)2D levels; they are commonly used, with well described pharmacokinetic and tolerability data. Fluconazole has been successfully reported to reduce calciuria in patients with CYP24A1 or SLC34A3 mutations, with no safety warnings. Thus, based on these case reports, we hypothesize that fluconazole is effective to decrease and normalize calciuria in patients with hypercalciuria and increased 1,25(OH)2D levels.MethodsThe FLUCOLITH trial is a prospective, interventional, randomized in parallel groups (1:1), placebo-controlled, double-blind trial. A total of 60 patients (10–60 years) with nephrolithiasis and/or nephrocalcinosis history, hypercalciuria (> 0.1 mmol/kg/day), increased 1,25(OH)2D levels (> 150 pmol/L), and 25-OH-D levels >20 nmol/L will be included. Inclusions will be performed only from mid-September to the beginning of February to avoid bias due to sunlight-induced vitamin D synthesis. The primary endpoint will be the proportion of patients with normalization of 24-h calciuria between baseline and 16 weeks, or with a relative decrease of at least 30% of 24-h calciuria in patients who still display at W16 a 24-h hypercalciuria.DiscussionThe current challenge is to propose an efficient treatment to patients with hypercalciuria and increased 1,25(OH)2D levels in order to prevent later complications and notably CKD that can ultimately lead to end-stage renal disease. Based on improvement of knowledge in phosphate/calcium metabolism, pathophysiology and genetics, the “off-label” use of fluconazole was recently reported to be useful in hypercalciuric patients with increased 1,25(OH)2D levels. Thus, the FLUCOLITH study is a unique opportunity to develop a new indication of a well-known and not expensive drug in orphan renal diseases, the ultimate objective being the secondary prevention of CKD worsening in these patients.Trial registrationClinicalTrials.gov NCT04495608. Registered on July 23, 2020.

MULTIPLE ENDOCRINE NEOPLASIA TYPE 2A IN A FAMILY WITH RET C.1901G&gt;A AND NOVEL SLC12A3 MUTATIONS

Objective: In recent years, there has been a lot of controversy about the factors affecting the penetrance of multiple endocrine neoplasia type 2A(MEN2A). Some studies considered it may be related to gene mutations. In the present study, we aimed to support the hypothesis that RET gene mutation affect the penetrance of MEN2A and study the effects of novel SLC12A3 variants in MEN2A. Design and method: Peripheral blood samples were collected from the proband and his relatives. Whole-exome sequencing and Sanger sequencing were performed to identify the gene mutations. The protein structure prediction with comparative modelling was used to predict the effects of novel gene variants on protein function. We also used the clinical examinations to evaluate the phenotypes of patients comprehensively. Results: Genetic analysis identified RET c.1901G&gt;A and novel variants in SLC12A3 c.3070_3079delinsCAG in the proband and his mother. The patients carrying the novel SLC12A3 variants didn’t show the typical clinical manifestations of Gitelman syndrome because of heterozygosity. But it is theoretically explained that the novel variants of SLC12A3 may affect the function of protein. We also found SLC12A3 gene was not expressed in typical target organs of MEN2A.We are the first to describe compound RET c.1901G&gt;A and SLC12A3 mutations in one family with MEN2A. Our study supports the hypothesis that RET gene mutations affect the penetrance of MEN2A. The novel variants in SLC12A3 may be disease causing, but it is not significantly related to the clinical phenotype of MEN2A in our study. Conclusions: RET gene mutation affect the penetrance of MEN2A. The novel variants in SLC12A3 may affect the function of protein, but it is not significantly related to the clinical phenotype of MEN2A.We need more studies about the phenotypes and related mechanisms of gene mutations to guide individual treatment.

RET c.1901G>A and Novel SLC12A3 Mutations in Familial Pheochromocytomas

Familial PHEOs (pheochromocytomas) are inherited as an autosomal dominant trait, and inherited PHEOs can be one clinical phenotype of clinical syndromes, such as multiple endocrine neoplasia type 2A (MEN2A). In recent years, there has been a lot of controversy about the factors affecting the penetrance of PHEOs in MEN2A, of which the effects of RET (rearranged during transfection) proto-oncogene mutations are the primary concern. In this report, we performed genetic screening of patients in one family presenting with PHEOs and found they carried a RET c.1901G>A mutation. They were ultimately diagnosed with familial MEN2A. We found that MEN2A patients with the RET c.1901G>A mutation tended to have bilateral PHEOs that appeared earlier than medullary thyroid carcinoma. Genetic analysis showed that the patients also carried novel SLC12A3 (solute carrier family 12 member 3) variants, which are highly associated with Giteman syndrome. The results of protein structure prediction models suggest this SLC12A3 mutant has altered both the protein structure and the interaction with surrounding amino acids. Further studies of the phenotypes and related mechanisms of the gene mutations are required to guide individual assessment and treatment.

Gitelman Syndrome: a Сlinical and Molecular Overview

Gitelman syndrome (OMIM #263800) is an autosomal recessive renal tubular disorder due to loss of function mutations of SLC12A3 gene, encoding the thiazide-inhibitable, electroneutral Na+-Cl– cotransporter (NCC) of the distal convoluted tubule. Clinical consequences include chronic normotensive hypokalemic alkalosis, hypomagnesemia, hypocalciuria, polyuria/nocturia, chronic asthenia, muscular cramps, chondrocalcinosis and rarely cardiac arrhythmias.Impaired reabsorption of glomerular filtrate through NCC drives compensatory reabsorption of Na+ in more distal tubular segments (connecting and cortical collecting tubules) via both the «electrogenic» channel ENa (which also enhances tubular secretion of potassium and protons, explaining the hypokalemic alkalosis), and pendrin-dependent electroneutral NaCl reabsorption. Thus volume depletion is seldom severe in these patients.There exists wide variability and severity of clinical symptoms between subjects, ranging from an almost asymptomatic disease to a severely disabling one. More than 400 SLC12A3 mutations have been so far described, evenly distributed along the protein sequence and without any hot spot. Mutation detection rate by gene sequencing actually is about 80 %. There are no genotype-phenotype correlations.Commonly considered a benign condition, Gitelman syndrome may be associated with reduced quality of life, increased medicalization and high hospitalization rate.

Frequent SLC12A3 mutations in Chinese Gitelman syndrome patients: structure and function disorder

PurposesThis study was conducted to identify the frequent mutations from reported Chinese Gitelman syndrome (GS) patients, to predict the three-dimensional structure change of human Na–Cl co-transporter (hNCC), and to test the activity of these mutations and some novel mutations in vitro and in vivo.MethodsSLC12A3 gene mutations in Chinese GS patients previously reported in the PubMed, China National Knowledge Infrastructure, and Wanfang database were summarized. Predicted configurations of wild type (WT) and mutant proteins were achieved using the I-TASSER workplace. Six missense mutations (T60M, L215F, D486N, N534K, Q617R, and R928C) were generated by site-directed mutagenesis. 22Na+ uptake experiment was carried out in the Xenopus laevisoocyte expression system. In the study, 35 GS patients and 20 healthy volunteers underwent the thiazide test.ResultsT60M, T163M, D486N, R913Q, R928C, and R959frameshift were frequent SLC12A3 gene mutations (mutated frequency >3%) in 310 Chinese GS families. The protein’s three-dimensional structure was predicted to be altered in all mutations. Compared with WT hNCC, the thiazide-sensitive 22Na+ uptake was significantly diminished for all six mutations: T60M 22 ± 9.2%, R928C 29 ± 12%, L215F 38 ± 14%, N534K 41 ± 15.5%, Q617R 63 ± 22.1%, and D486N 77 ± 20.4%. In thiazide test, the net increase in chloride fractional excretion in 20 healthy controls was significantly higher than GS patients with or without T60M or D486N mutations.ConclusionsFrequent mutations (T60M, D486N, and R928C) and novel mutations (L215F, N534K, and Q617R) lead to protein structure alternation and protein dysfunction verified by 22Na+ uptake experiment in vitro and thiazide test on the patients.