Low Urinary Calcium Excretion Research Articles

7297 Clinical Characterization Of FHH Patients With Functional Assessment Of Novel CaSR Variants

Abstract Disclosure: R. Tora: None. L. Canaff: None. J. Welch: None. L. Bliss: None. L.S. Weinstein: None. W.F. Simonds: None. S.K. Agarwal: None. J. Blau: None. D. Goltzman: None. S. Jha: None. Familial Hypocalciuric Hypercalcemia (FHH) is characterized by hypercalcemia and low urinary calcium excretion from birth and its diagnosis is confirmed by the presence of a pathogenic germline heterozygous variant in CaSR, AP2S1, or GNA11. Establishing the diagnosis is important as it prevents unnecessary parathyroidectomies as surgery is not curative. We identified patients with germline variants in CaSR, AP2S1, or GNA11 seen at our institute and performed a retrospective review of available clinical parameters. 43 patients (31 kindreds) with 28 unique CaSR variants (15 pathogenic, 13 variants of uncertain significance [VUS]) and one pathogenic AP2S1 variant were identified. Median age at diagnosis was 35 [IQR: 25 - 39] years, with the youngest patient diagnosed at 4.5 months. Median serum calcium was 10.70 [10.25 - 11.00] mg/dL (ref: 8.4 - 10.2) with a median PTH of 51.7 [35.8 - 74.0] pg/mL (ref: 15 - 65). The median fractional excretion of calcium (FECa) was 1.00 [0.54 - 1.40] while median serum magnesium was 2.30 [2.19 - 2.43] mg/dL (ref: 1.6 - 2.6). 5/43 (12%) patients developed kidney stones and 6/43 (14%) men ≤ 50 years and premenopausal women developed osteoporosis with median age at diagnosis of 38 [28.50 - 45.75] years and no alternate underlying secondary cause. Longitudinal follow-up available on 9/43 patients with median duration of 14.4 ± 4.5 years showed no significant changes in serum and urine biochemistries. 23/43 (53%) patients had undergone failed parathyroidectomy prior to diagnosis, of which 20/23 (87%) had multiple glands removed including one who became hypoparathyroid. Functional characterization was performed for 8/13 CaSR VUS (p.D217G, p.C546Y, p.F563S, p.C694Y, p.A880P, p.E109Q, p.D382N, and p.A880S) in HEK293 cells by examining intracellular Ca2+ and ERK/MAPK responses to extracellular Ca2+ using NFAT-Luc and SRE-Luc constructs, respectively. Compared to the wild type (WT) receptor, p.D217G, p.C546Y, p.F563S, and p.C694Y showed less of the cell-membrane-localized mature glycosylated species in Western blots and a significant rightward shift in their concentration-response curve in both SRE- and NFAT-Luc assays. p.A880P showed a significant rightward shift in its concentration-response curve, while p.E109Q, p.D382N, and p.A880S showed no differences on Western blot or concentration-response curves as compared to WT. Further work to characterize the remaining variants (p.T14A, p.T609M, p.V268_P278del, p.G36R, and p.F589V) is ongoing. We conclude that the diagnosis of FHH is frequently missed and that patients with the disease may rarely develop target organ damage. Presentation: 6/2/2024

Familial hypocalciuric hypercalcemia in an infant: diagnosis and management quandaries.

Familial hypocalciuric hypercalcemia (FHH) is typically a benign condition characterized by elevated serum calcium, low urinary calcium excretion, and non-suppressed circulating levels of parathyroid hormone (PTH), usually requiring no intervention. FHH is inherited in an autosomal-dominant manner. Three subtypes are described, representing variants in genes with critical roles in extracellular calcium-sensing. FHH1, due to heterozygous inactivating variants in the calcium-sensing receptor gene (CASR), accounts for the majority of cases. FHH2, due to variants in GNA11, encoding the α-subunit of the downstream signaling protein, G11, is the rarest form of FHH. FHH3, resulting from variants in AP2S1, may present with a more pronounced phenotype than FHH1 or FHH2. We describe herein a newborn girl presenting with in utero femoral fractures, hypercalcemia, hypophosphatemia, and elevated circulating PTH. She was diagnosed with mild hyperparathyroidism and provided supplemental phosphate upon hospital discharge. However, serum calcium and PTH remained elevated at 5mo of age. The combination of low-calcium formula and cinacalcet improved the biochemical profile. No pathogenic variants in the coding region of CASR were identified; subsequent whole exome sequencing revealed a G- > T transition at c.44 (p.R15L) in AP2S1. Family studies identified this variant in the father and an affected brother. The mother was unexpectedly found to be hypocalcemic and was diagnosed with idiopathic hypoparathyroidism. This case demonstrates successful treatment of FHH3 using a low-calcium formula to limit dietary calcium availability and cinacalcet to modify PTH levels.

Association between Kidney Stones and CKD: A Bidirectional Mendelian Randomization Study.

Kidney stones and CKD are common disorders with a substantial interaction. Although observational studies have suggested a potential for enhanced CKD risk after prior kidney stones, the exact relationship remains ambiguous. Shared comorbidities between two diseases were identified using unbiased screening. Genome-wide association study summary statistics were obtained from the UK Biobank (UKBB), FinnGen, and CKDGen, followed by genetic association analyses across various traits. Bidirectional Mendelian randomization (MR) analyses were performed to define causal links, complemented by multivariable MR that included the shared comorbidities including hypertension, diabetes, and obesity. Observational analyses were undertaken using cohorts from the Mayo Clinic and a UKBB subset. Despite identifying a total of 123 conditions as shared comorbidities, there was no significant genetic correlation between kidney stones and CKD. Unadjusted MR analysis revealed no significant association between kidney stones and CKD risk (UKBB [exposure]/FinnGen [outcome]: OR=0.97, 95% CI: 0.88∼1.06; FinnGen/UKBB: OR=1.17, 95% CI:0.98∼1.39). Kidney stones did significantly associate with a higher urinary albumin-creatinine ratio (UACR) (β=0.014, 95% CI: 0.002∼0.025), but this association disappeared in the multivariable MR model (β=0.009, 95% CI: -0.003∼0.020). Furthermore, in a cross-sectional analysis limited to the UKBB cohort, a robust regression model did not detect an independent association between kidney stones and UACR (β=0.16, 95% CI: -0.04∼0.35) or eGFR (β=0.10, 95% CI: -0.07∼0.28). Conversely, CKD associated with a diminished risk of kidney stones in multivariable MR models (UKBB/FinnGen: OR=0.77, 95% CI: 0.69∼0.87; FinnGen/UKBB: OR=0.73, 95% CI: 0.66∼0.81). Furthermore, in the Mayo Clinic cohort with available urinary biochemistries, lower eGFR was associated with lower urinary calcium excretion and urinary calcium oxalate/phosphate supersaturation. In this study, kidney stones were not independently associated with CKD. Conversely, CKD was associated with a lower risk of calcium kidney stones likely via changes in key urinary traits, including lower calcium excretion.

Longitudinal trends in serum calcium, parathormone and bone mineral density in breast cancer survivors on aromatase inhibitor therapy: A single institutional cohort study.

e12531 Background: Aromatase inhibitors (AIs), when used as adjuvant treatment of hormone receptor positive breast cancer, have been associated with bone density loss. However, any impact on PTH and by extension calcium (Ca) homeostasis is yet to be elucidated as no study till date has investigated longitudinal trends in parathyroid hormone (PTH) while on AI therapy. We explored these to determine if long term AI therapy was associated with hyperparathyroidism. Methods: This is a retrospective study of female breast cancer patients at the Cleveland Clinic Foundation started on AIs (anastrozole, letrozole and exemestane) from January 2015 to April 2022. Included patients had both baseline and subsequent data (after starting AI therapy), on serum Ca, PTH and lumbar spine T-scores (LsTs). These values were compared using paired t-test. Results: We identified n=9657 patients on AI therapy and included n=249 with pre and post AI PTH values. Among these, n= 209 received anastrozole, n=34 received letrozole and n=6 received exemestane. Cohort median age was 68 (IQR 62-72) years and median duration of AI therapy was 3 years. While we observed a significant positive correlation between change in Ca and change in PTH (p=0.02), the differences between baseline and treatment median serum Ca and PTH were not statistically significant. There was no significant association between changes in either serum Ca or PTH and change in LsTs (p-value: 0.69 and p-value: 0.05 respectively). Notably, 4% patients had elevated PTH and Ca after AI therapy was initiated among whom n=4 had markedly elevated PTH with low urinary Ca. Three of these four patients also had a diagnosis of chronic kidney disease (CKD) with normal vitamin D levels. Two patients with elevated PTH were on biotin supplements. There was an overall downtrend in LsTs (p-value: 0.0039) with no significant difference between the three AIs (p-value 0.0613). However, in comparison to other AIs, letrozole had the least depreciation in LsTs. Conclusions: Our findings do not suggest any correlation between AI therapy and hyperparathyroidism among breast cancer survivors. No significant increase in PTH levels occurred during AI therapy and majority of patients with clinically significant PTH elevation also had CKD, which may have contributed also to elevation of PTH level and low urinary calcium excretion.[Table: see text]

Обмен кальция и остеопороз у больных ишемической болезнью сердца в пожилом и старческом возрасте

im. To analyze the relationship between calcium and various clinical and laboratory parameters in old and very old patients with coronary artery disease (CAD) based on the determination of the blood concentration of ionized calcium and daily excretion of calcium in the urine. Materials and methods. This work was a cross-sectional study enrolled 102 patients (84 women and 18 men) with coronary artery disease with a mean age of 83.0±5.9 years. The blood level of ionized calcium (N 1.16–1.32 mmol/l), daily excretion of calcium in the urine (N 2.5– 7.5 mmol/l) and concentration of 25-hydroxycalciferol (25(OH)D) were determined in all patients. Along with this, the bone mineral density of the lumbar spine and proximal femur was analyzed using dual-energy x-ray absorptiometry. Results. The mean daily excretion of calcium in the urine was 1.39±1.1 mmol/l (0.17–7.44 mmol/l). 84.4% of patients had low calcium content in daily urine. The mean blood concentration of ionized calcium was 1.26±0.05 mmol/l (1.16–1.53 mmol/l). Hypercalcemia was observed in 8.3% of patients. Osteoporosis in the proximal femur was registered in 32.9% of patients, in the lumbar spine – in 18.1% of patients. In patients with normal vitamin D level the mean urinary calcium excretion was 1.77±1.2 mmol/l, with vitamin D deficiency – 1.1±0.7 mmol/l (p=0.07). In patients with low calcium excretion in the urine, the mean blood concentration of vitamin D was 18.7±7.7, with normal excretion – 28.5±10.8 ng/ml (p=0.007). Significant inverse relationships were registered between urinary calcium excretion and creatinine (r=-0.36; p<0.0001). In the group of patients with low urinary calcium excretion the mean creatinine level reached 102.3±23.9 µmol/l, with normal excretion – 84.9±11.2 µmol/l (p<0.0001). There was a significant direct correlation between daily urinary calcium excretion and glomerular filtration rate, estimated using the CKD-EPI formula (r=0.39; p<0.0001) and an inverse correlation between blood calcium and GFR (r=-0.26; p=0.01). Significant direct relationship was registered between the blood concentration of ionized calcium and the creatinine (r=0.2; p=0.05) and urea (r=0.32; p=0.04) concentration. Conclusion. The study results demonstrate a low level of calcium excretion in the urine in old and very old patients with coronary artery disease. The urine calcium excretion is primarily due to impaired renal function and low vitamin D concentration.

Gitelman syndrome with normocalciuria – a case report

BackgroundGitelman Syndrome (GS) is a hereditary tubulopathy associated with a biallelic inactivating mutations of the SLC12A3 gene encoding the thiazide-sensitive sodium-chloride cotransporter (NCCT). The typical clinical manifestation is a hypokalemic metabolic alkalosis with significant hypomagnesemia, and low urinary calcium excretion. Hypocalciuria is widely believed to be a hallmark of GS that distinguishes it from Barter’s syndrome, presenting as hypercalciuria. The pathomechanism of hypocalciuria in GS is not fully elucidated. Up to date, a clinical course of GS with normocalciuria has been reported only in men, while women have a milder course of the disease with typical hypocalciuria, which is believed as the result of sex hormone. Additionally, there is a growing evidence that calcium channels of the distal nephron could be regulated by a variety of hormones, including aldosterone (Aldo).Case presentationWe present the case of a 28-year-old Caucasian woman with asymptomatic, chronic hypokalemia, hypomagnesemia, hypochloremic alkalosis and normal urinary calcium excretion. A high renin levels with normal concentration of Aldo in serum have also been found. The values of blood pressure were low. Based on genetic studies, two heterozygous mutations in the trans position were confirmed: c.2186G>T (p.Gly729Val) and c.1247G>C (p.Cys416Ser) in the SLC12A3 gene, which ultimately confirmed the diagnosis of GS.ConclusionsWe report here the first case of genetically confirmed GS manifested as normocalciuria in a Caucasian woman. Thus, our result does not confirm a role of sex hormones on the level of calciuria. Based on the results of normal Aldo concentration despite high renin level in our patient, we hypothesized that Aldo may be connecting with the level of urinary calcium excretion in patients with the GS.

Isolated idiopathic hypoparathyroidism that developed in adulthood: a case report

Hypoparathyroidism is a rare endocrine disease. In most cases in adult patients, the cause of hypoparathyroidism is damage or removal of parathyroid glands during surgical interventions on the neck; other causes are rarely observed.Case report. A 52-year-old man with episodes of seizures, intense muscle pain, progressing for 7 years and resistance to treatment with myorelaxant, anxiolytics and nonsteroidal anti-inflammatory drugs was examined and hypocalcemia associated with low parathyroid hormone and excessive urinary calcium excretion was found. Ultrasound examination didn't reveal any changes in parathyroid glands. The patient was diagnosed with idiopathic hypoparathyroidism. Treatment with calcium supplements and active metabolites of vitamin D led to an improvement in clinical symptoms and laboratory parameters.Discussion. Hypoparathyroidism as part of several genetic syndromes was excluded due to the late- onset of the disease and the absence of concomitant diseases. Ultrasound of the parathyroid glands made it possible to rule out metastasis and storage diseases. It is recommended to perform genetic testing of the chromosomes 22 and 10 to exclude rare variants of syndromic hypoparathyroidism with the late-onset in the form of isolated hypocalcemia.

Determinants and Outcomes Associated With Urinary Calcium Excretion in Chronic Kidney Disease.

Abnormalities in calcium metabolism are common in chronic kidney disease (CKD). Diminished urinary calcium excretion may promote vascular calcification and increased urinary calcium excretion may lead to nephrolithiasis and nephrocalcinosis, conditions associated with CKD. To study predictors of urinary calcium excretion and its association with adverse clinical outcomes in CKD. This study assessed 3768 nondialysis participants in the Chronic Renal Insufficiency Cohort study from April 2003 to September 2008. Participants were followed up to October 2018. Clinically plausible predictors of urinary calcium excretion and 24-h urinary calcium excretion at baseline. Urinary calcium excretion; incident end stage kidney disease (ESKD), CKD progression [50% estimated glomerular filtration rate (eGFR) decline or incident ESKD], all-cause mortality, and atherosclerotic cardiovascular disease events. eGFR was positive correlated with 24-h urinary calcium excretion. The variables most strongly associated with 24-h urinary calcium excretion in males and females were 24-h urinary sodium (β = 0.19 and 0.28, respectively), serum parathyroid hormone (β = -0.22 and -0.20, respectively), loop diuretics (β = 0.36 and 0.26, respectively), thiazide diuretics (β = -0.49 and -0.53, respectively), and self-identified black race (β = -0.23 and -0.27, respectively). Lower urinary calcium excretion was associated with greater risks of adverse outcomes, but these associations were greatly attenuated or nullified after adjustment for baseline eGFR. Urinary calcium excretion is markedly lower in individuals with CKD compared to the general population. Determinants of urinary calcium excretion differed between sexes and levels of CKD. Associations between urinary calcium excretion and adverse clinical events were substantially confounded by eGFR.

Prolonged Hypercalcemia After Recovery From Severe Rhabdomyolysis and Acute Renal Failure

Abstract Introduction: Rhabdomyolysis is characterized by elevated creatine kinase, electrolyte abnormalities and acute renal failure. After the initial hypocalcemic phase, serum calcium levels can normalize or become significantly elevated during the recovery phase. We report a case of persistent hypercalcemia during the recovery phase of rhabdomyolysis-induced acute renal failure. Case Description: A 26-year old woman with Cornelia de Lange syndrome initially presented with septic shock, complicated by severe rhabdomyolysis and acute renal failure requiring hemodialysis. On admission, creatine kinase level was >30,500 U/L [29–168], BUN 95 mg/dL [6–22], Creatinine 3.39 mg/dL [0.57–1.11], and Corrected Calcium 7.1 mg/dL [8.6–10.2]. Approximately 1 month after admission, the patient developed hypercalcemia with corrected calcium values ranging from 10.4 to 14.5 mg/dL with PTH 10.5 pg/mL [8.7–77.1] and 1,25-Dihydroxyvitamin D and 25-OH Vitamin D levels <8.0 pg/dL [18–78] and 21.20 ng/mL [30–80], respectively. Her renal function improved and dialysis was discontinued seven weeks after initial presentation, although her GFR remained impaired (22 mL/min/BSA). She remained hypercalcemic and was treated with intermittent doses of Calcitonin when the level rose above 12.0 mg/dL. Spot urinary calcium to creatinine ratio was low at 0.13. Improvement in her calcium was seen with a level of 10.8 mg/dL eleven weeks after she was initially noted to be hypercalcemic and she was able to be discharged. Two weeks after discharge, an outpatient calcium improved further to 10.4 mg/dL. Discussion: Rhabdomyolysis is a clinical syndrome that results from severe muscle damage with release of the breakdown products from injured muscle cells leading to acute renal failure and electrolyte abnormalities. The initial hypocalcemic phase is due to entry of calcium into damaged myocytes and deposition of calcium salts in damaged muscle. After recovery, most patients have normalization of calcium levels. However, in up to one-third of patients, a rebound hypercalcemia can ensue. The proposed mechanism is massive muscle calcium release during renal recovery. In the cases reported, the hypercalcemia phase resolves by an average of 8 days with the longest reported recovery being 3.5 months. We report a case of prolonged hypercalcemia in the setting of continued renal impairment. It appears that the patient’s low GFR impaired urinary calcium excretion resulting in persistent hypercalcemia which eventually resolved over time with some improvement in renal function. Other case reports have described treatment with IV fluids, Calcitonin, Pamidronate and hemodialysis, although the efficacy of these interventions is unclear. Rhabdomyolysis is an under-recognized cause of hypercalcemia. Our case highlights the potential for prolonged hypercalcemia after recovery from rhabdomyolysis in the setting of renal impairment.

Multi-centre study of the clinical features and gene variant spectrum of Gitelman syndrome in Chinese children.

Based on the Chinese Children Genetic Kidney Disease Database (CCGKDD), we established a pediatric Gitelman syndrome (GS) cohort to explore the phenotype and genotype characteristics. Thirty-two patients with SLC12A3 gene variants were collected. Five cases (16%) were homozygous, 16 (50%) were compound heterozygous, 10 (31%) carried only a single variant, and the other one harbored two de novo variants beyond classification. p.(T60M) was found in eight patients. The average diagnosis age was 7.79 ± 3.54 years. A total of 31% of the patients were asymptomatic. Muscle weakness was the most common symptom, accounting for 50%. Earlier age of onset (4.06 ± 1.17 yr vs. 8.10 ± 3.46 yr vs. 8.61 ± 3.56 yr, p< 0.05) and lower urinary calcium-creatinine ratio (p = 0.024) were found in the homozygous group than those in the heterozygous and compound heterozygous group. Patients with p.(T60M) variant had an earlier age of onset (4.01 ± 2.83 yr vs. 6.92 ± 3.07 yr, p = 0.025) and lower urinary calcium-creatinine ratio (p = 0.056). Thus, more than 30% of GS children have no clinical symptoms. Homozygous variant and the p.(T60M) variant may be associated with earlier onset and lower urinary calcium excretion in Chinese pediatric GS.

A Case of Lithium-Associated Hypocalciuric Hypercalcemia

Lithium is the treatment of choice for acute manic, mixed, and depressive episodes of bipolar disorder, along with long-term prophylaxis. A significant proportion of patients taking lithium develop lithium-associated hypercalcemia. Most cases are due to lithium-associated hyperparathyroidism with underlying parathyroid adenoma or hyperplasia. We present a 67-year-old woman who presented with increasing lethargy and loss of concentration and was found to have slightly raised serum calcium levels with inappropriately low urinary calcium excretion levels characteristic of hypocalciuric hypercalcemia. She had been on lithium therapy for over 15 years for bipolar disease. She had no other cause for these findings and had no family history to suggest familial hypocalciuric hypercalcemia. Neck imaging ruled out any parathyroid adenoma or hyperplasia. A diagnosis of lithium-associated hypocalciuric hypercalcemia was discussed with the patient, and she remains stable under surveillance.

Genetic testing for hereditary hyperparathyroidism and familial hypocalciuric hypercalcaemia in a large UK cohort.

Primary hyperparathyroidism (PHPTH) is a common endocrine disorder and an estimated 10% of cases are hereditary, related to syndromes including; multiple endocrine neoplasia (MEN) type 1, MEN type 4, MEN2A and hereditary hyperparathyroidism-jaw tumour syndrome. Establishing the underlying genetic cause for PHPTH allows for personalized and cost-effective management. Familial hypocalicuric hypercalcaemia (FHH) is a benign disorder of hypercalcaemia associated with an inappropriately low urinary calcium excretion, which is quantified by the calcium creatinine clearance ratio (CCCR). Recent NHS England National Genomic Test Directory testing criteria for familial hyperparathyroidism state testing patients presenting with PHPTH and CCCR>0.02 presenting (i) <35years of age, or (ii) <45y with one of (a) multiglandular disease, or (b) hyperplasia on histology, or (c) ossifying fibroma(s) of the maxilla and/ or mandible, or (d) a family history of unexplained PHPTH. The testing criterion for FHH is a CCCR<0.02. A retrospective review of patients referred for genetic testing over a 4year period for suspected hereditary HPTH was performed. Genetic analysis was performed by next-generation sequencing of the following genes; MEN1, CDC73, CASR, CDKN1A, CDKN1B, CDKN2B, CDKN2C, RET, GCM2, GNA11, and AP2S1 in NHS-accredited Regional Genetic laboratories. Aims of this study were to better define testing criteria for suspected hereditary PHPTH in a UK cohort. A total of 121 patients were included in this study (92 female) with a mean age of 41years (SD 17). A pathogenic germline variant was identified in 16% (n=19). A pathogenic variant was identified in the PHPTH genes CDC73 in a single patient and MEN1 in six patients (6% of total), in the FHH genes, CASR in 11 patients and AP2S1 in a single paediatric case (10% of total). A variant of uncertain significance (VUS) was identified in eight patients (6%) but over the course of this study familial segregation studies and computational analysis enabled re-classification of four of the variants, with two VUS's in the CASR gene being upgraded to likely pathogenic variants. Age at diagnosis and multiglandular disease as sole risk factors were not predictive of a pathogenic germline variant in this cohort but a positive family history was strongly predictive (P=.0002). A significant difference in the mean calcium creatinine clearance ratio (CCCR) in those patients with an identified CASR pathogenic variant versus those without (P=.0001) was demonstrated in this study. Thirty-three patients were aged over 50years and the diagnostic rate of a pathogenic variant was 15.1% in those patients >50years of age compared to 15.9% in those <50years. Five patients >50years and with a CCCR of <0.01, were diagnosed with a pathogenic variant in CASR. Family history was the strongest predictor of hereditary PHPTH in this cohort. This study has highlighted the importance of re-evaluating VUS's in order to inform patient management and enable appropriate genetic counselling. Finally, this study has demonstrated the need to consider genetic testing for PHPTH in patients of any age, particularly those with additional risk factors.

Research process in molecular genetics of Gitelman syndrome

Gitelman syndrome(GS) is an autosomal recessive genetic disease caused by mutations in the SLC12A3 gene located in chromosome 16q13. The incidence of GS is 1-10∶40 000. SLC12A3 encodes thiazide-sensitive sodium-chloride cotransporters(NCC) which play key roles in Na+ and Cl- reabsorption. GS is characterized by hypokalemic metabolic alkalosis in combination with significant hypomagnesaemia and low urinary calcium excretion. There are some correlations between genotypes and phenotypes. In previous studies, more than 500 mutations have been identified and some of them have been functionally analyzed. We review genetic mutations and functional studies related to GS as well as the relationship between genotypes and phenotypes, and summarize the research process in molecular genetics of GS. Key words: Gitelman syndrome; Na+ /Cl- cotransporter; SLC12A3 gene mutation

A Novel Mutation in Calcium-Sensing Receptor Presenting as Familial Hypocalciuric Hypercalcemia in a Young Man

Familial hypocalciuric hypercalcemia (FHH) is considered a relatively benign condition characterized by elevated serum calcium with relatively low urinary calcium excretion. It typically results from an altered set point in calcium homeostasis originating from mutations in the calcium-sensing receptor (CASR), AP2S1, or GNA11 genes, which encode for the calcium-sensing receptor (CaSR), adaptor-related protein complex 2, and G-protein alpha-11 subunit, respectively. Despite numerous reports of novel variants in these genes associated with FHH, new variants continue to be discovered. We describe a 20-year-old man with a family history of hypercalcemia who had clinical findings compatible with FHH and no evidence of multiple endocrine neoplasia who underwent CASR gene sequencing for evaluation of hypercalcemia. Parathyroid gland single-photon emission computerized tomography scan was normal. CASR gene sequencing revealed a previously unreported heterozygous intronic variant at position 1608+3A>G (chromosome 3: 121994892) resulting in a 77-residue deletion. His mother has a history of bipolar disorder and hyperparathyroidism with an adenoma found on imaging, yet our patient had no evidence of adenoma and therefore no surgical intervention was recommended. Given that CaSR plays a role in parathyroid growth, some variants in CASR may ultimately lead to parathyroid hypertrophy and be mistaken for primary hyperparathyroidism. Long-term clinical follow up will be helpful in understanding the ultimate effects of specific CASR mutations on parathyroid growth or progression to significant hypercalcemia.

MON-539 Mice Harboring a Germline Heterozygous AP2S1 Mutation, Arg15Leu, Are a Model for Familial Hypocalciuric Hypercalcemia Type 3 (FHH3)

Familial hypocalciuric hypercalcemia (FHH) is a disorder of calcium homeostasis comprising three reported genetic variants: FHH types 1 and 2 are due to germline loss-of-function mutations of the calcium-sensing receptor (CaSR) and G-protein subunit α-11 (Gα11), respectively, whereas, FHH type 3 (FHH3) is mainly caused by germline heterozygous loss-of-function mutations affecting the Arg15 residue (Arg15Cys, Arg15His or Arg15Leu) of the adaptor-related protein complex 2-sigma subunit (AP2σ), which is encoded by the AP2S1 gene, and regulates CaSR endocytosis and endosomal signaling. FHH is considered to be an asymptomatic disorder characterised by mild-to-moderate hypercalcemia, normal or mildly elevated parathyroid hormone (PTH) concentrations and low urinary calcium excretion. However, some FHH3 patients, especially those harboring the Arg15Leu AP2S1 mutation, have marked and symptomatic hypercalcemia, thereby indicating that FHH3 may represent a more severe clinical disorder. To further evaluate the impact of the Arg15Leu AP2S1 mutation on calcium homeostasis, we utilised CRISPR/Cas9-mediated gene editing to generate C57BL/6J mice harboring the germline Arg15Leu mutation. Plasma and 24-hour urine samples were collected for biochemical analysis, and bone mineral density (BMD) was measured by dual energy X-ray absorptiometry (DEXA). All studies were conducted in mice aged 12-15 weeks (n = 10-12 mice per genotype), and in accordance with institutional welfare guidelines. Wild-type (Ap2s1+/+), heterozygous (Ap2s1+/L15) and homozygous (Ap2s1L15/L15) mice were born as expected for a Mendelian pattern of inheritance, however, Ap2s1L15/L15 mice died within 48 hours of birth. In contrast, male and female Ap2s1+/L15mice were viable and biochemical analysis showed marked hypercalcemia (plasma albumin-adjusted calcium = 2.77±0.02 mmol/L) compared to wild-type (WT) mice (plasma albumin-adjusted calcium = 2.13±0.03 mmol/L, p<0.0001), and this finding was associated with significant hypophosphatemia and hypermagnesemia. Male and female Ap2s1+/L15mice also had significant elevations of plasma PTH (157±14 pmol/L) compared to WT mice (63±10 pmol/L, p<0.0001). Furthermore, urine analysis showed that Ap2s1+/L15mice were significantly hypocalciuric (fractional excretion of calcium (FECa) = 0.016±0.002) compared to WT mice (FECa =0.029±0.002, p<0.01), whilst, DEXA analysis demonstrated that Ap2s1+/L15mice have significantly reduced BMD. Thus, these studies have established a mouse model for FHH3, which highlights the importance of the AP2σ protein in the parathyroid and renal regulation of mineral and bone homeostasis. In addition, these findings demonstrate that the germline heterozygous Arg15Leu AP2S1 mutation causes a severe form of FHH characterized by marked hypercalcemia, hyperparathyroidism and reduced BMD.

Identification of a novel large CASR deletion in a patient with familial hypocalciuric hypercalcemia.

SummaryFamilial hypocalciuric hypercalcemia type I is an autosomal dominant disorder caused by heterozygous loss-of-function mutations in the CASR gene and is characterized by moderately elevated serum calcium concentrations, low urinary calcium excretion and inappropriately normal or mildly elevated parathyroid hormone (PTH) concentrations. We performed a clinical and genetic characterization of one patient suspected of familial hypocalciuric hypercalcemia type I. Patient presented persistent hypercalcemia with normal PTH and 25-hydroxyvitamin D levels. The CASR was screened for mutations by PCR followed by direct Sanger sequencing and, in order to detect large deletions or duplications, multiplex ligation-dependent probe amplification (MLPA) was used. One large deletion of 973 nucleotides in heterozygous state (c.1733-255_2450del) was detected. This is the first large deletion detected by the MLPA technique in the CASR gene.Learning points:Molecular studies are important to confirm the differential diagnosis of FHH from primary hyperparathyroidism.Large deletions or duplications in the CASR gene can be detected by the MLPA technique.Understanding the functional impact of the mutations is critical for leading pharmacological research and could facilitate the therapy of patients.

Cinacalcet corrects hypercalcemia in mice with an inactivating Gα11 mutation.

Loss-of-function mutations of GNA11, which encodes G-protein subunit α11 (Gα11), a signaling partner for the calcium-sensing receptor (CaSR), result in familial hypocalciuric hypercalcemia type 2 (FHH2). FHH2 is characterized by hypercalcemia, inappropriately normal or raised parathyroid hormone (PTH) concentrations, and normal or low urinary calcium excretion. A mouse model for FHH2 that would facilitate investigations of the in vivo role of Gα11 and the evaluation of calcimimetic drugs, which are CaSR allosteric activators, is not available. We therefore screened DNA from > 10,000 mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for GNA11 mutations and identified a Gα11 variant, Asp195Gly (D195G), which downregulated CaSR-mediated intracellular calcium signaling in vitro, consistent with it being a loss-of-function mutation. Treatment with the calcimimetic cinacalcet rectified these signaling responses. In vivo studies showed mutant heterozygous (Gna11+/195G) and homozygous (Gna11195G/195G) mice to be hypercalcemic with normal or increased plasma PTH concentrations and normal urinary calcium excretion. Cinacalcet (30mg/kg orally) significantly reduced plasma albumin–adjusted calcium and PTH concentrations in Gna11+/195G and Gna11195G/195G mice. Thus, our studies have established a mouse model with a germline loss-of-function Gα11 mutation that is representative for FHH2 in humans and demonstrated that cinacalcet can correct the associated abnormalities of plasma calcium and PTH.

Минералогический состав камней, факторы риска и метаболические нарушения у пациентов с кальций-оксалатным уролитиазом

To identify the most likely metabolic disturbances and risk factors for stone formation in a group of patients with calcium oxalate urolithiasis, and to establish the relationship between the mineralogical composition of calculi and impaired excretion of inhibitors and promoters of stone formation. Fifty patients with calcium oxalate urolithiasis were tested using a complex of physicochemical methods. Patients assessment included evaluation of quantitative mineralogical composition of calculi, daily urine pH profile and daily urinary excretion of urates, calcium, magnesium, oxalate, phosphate and citrate ions. The main mineralogical phase of the stones in over 80% of patients was calcium oxalate monohydrate; none of the patients had pure dihydrate stones. The most frequent metabolic disorders were hypocitraturia, hypercalciuria and hyperuricosuria. Predominant risk factors were excessive body weight and insufficient fluid intake. Only one patient had an idiopathic stone formation. It was established for the first time that patients with calcium oxalate stones, containing 10 or more mass percent of apatites had statistically significantly lower daily urinary calcium and oxalate excretion and simultaneously increased phosphate excretion. The study findings showed that patients with calculi based on calcium oxalate dihydrate should undergo testing for daily urinary excretion of calcium and citrate while pa-tients with calcium oxalate stones containing 10 or more mass percent of apatites should also be tested for daily phosphate excretion and urine pH-profile. Daily urinary citrate excretion was reduced in all study patients, and urate excretion was significantly increased, apparently due to an imbalanced diet and excessive intake of animal protein. Menopausal and postmenopausal women are at a particular risk due to low urinary citrate excretion and high urinary calcium excretion regardless of stone composition.

SaRNA-mediated activation of TRPV5 reduces renal calcium oxalate deposition in rat via decreasing urinary calcium excretion.

Hypercalciuria is a main risk factor for kidneystone formation. TRPV5is the gatekeeper protein for mediating calcium transport and reabsorption in the kidney. In the present study, we tested the effect of TRPV5 activation with small activating RNA (saRNA), which could induce gene expression by targeting the promoter of the gene, on ethylene glycol (EG)-induced calcium oxalate (CaOx) crystals formation in rat kidney. Five pairs of RNA activation sequences targeting the promoter of rat TRPV5 were designed and synthesized. The synthesized saRNA with the strongest activating effect was selected, and transcellular calcium transportation was tested by Fura-2 analysis. Subsequently, Sprague-Dawley rats were equally divided into three groups and fed with water, 1% EG for 28days after injecting the negative control saRNA, 1% EG for 28days after injecting the selected TRPV5-saRNA, respectively. The CaOx crystal formation and the 24-h urine components were assessed. In vitro study, saRNA ds-320 could significantly activate the expression of TRPV5 and transcellular calcium transportation. In vivo study, after 28days treatment of EG, rats pre-infected with saRNA ds-320 had lower urinary calcium excretion and renal CaOx crystals formation as compared to that pre-infected with negative control saRNA. Activation of TRVP5 with saRNA ds-320 could inhibit EG-induced calcium oxalate crystals formation via promoting urine calcium reabsorption and decreasing urine calcium excretion in rats.

Stepwise CaSR, AP2S1, and GNA11 sequencing in patients with suspected familial hypocalciuric hypercalcemia.

Patients with familial hyperparathyroidism and low urinary calcium excretion may have familial hypocalciuric hypercalcemia (FHH) with mutations in one of three genes: the calcium-sensing receptor (CaSR) defining FHH-type 1, the adaptor-related protein complex 2 (AP2S1) related to FHH-type 3 or the G-protein subunit alpha11 (GNA11) associated with FHH-type 2. We aimed to evaluate the presence of mutations in these genes and to identify phenotypic specificities and differences in these patients. Selected patients were recruited for genetic evaluation. After informed consent was signed, blood for DNA extraction was obtained and genetic sequencing of CaSR was done. In negative cases, we further performed sequencing of AP2S1 and GNA11. A total of 10 index cases were recruited. CaSR sequencing yielded three missense heterozygous mutations (30%): c.554G > A (p.I32V) previously characterized by our team, c.1394 G > A (p.R465Q) and a novel expected disease-causing mutation c.2479 A > C (p.S827R). We identified 2 additional patients (20%) carrying the deleterious recurrent mutation c.44G > T (p.R15L) in the AP2S1 gene. No GNA11 mutation was found. Clinically, patients with AP2S1 mutations had significant cognitive and behavioral disorders, and higher blood calcium and magnesium levels than patients with FHH1. CaSR and AP2S1 sequencing is worthwhile in patients with familial hyperparathyroidism and phenotype suggesting FHH as it can diagnose up to 50% of cases. GNA11 mutations seem much rarer. Learning disabilities in these patients, associated with higher serum calcium and magnesium levels may suggest the presence of AP2S1 rather than CaSR mutation and may guide the first step in the genetic evaluation.