CASR Variants Research Articles

Heterogenous Origins of Calcium Homeostasis Disorders Arising from Five Heterozygous Calcium-Sensing Receptor Variants.

The human calcium-sensing receptor (CaSR) plays a key role in calcium homeostasis, and most identified CASR variants are associated with hypercalcemic and hypocalcemic disorders. Here we characterized the pharmacological implications of five heterozygous CASR variants from individuals with familial hypocalciuric hypercalcemia 1 [FHH1: Y63C, I81T, Q459R, W818stop] or autosomal dominant hypocalcemia 1 [ADH1: R955stop]. Total and cell surface expression levels of wild-type (WT) and variant CaSRs expressed in human embryonic kidney 293T (HEK293T) cells were determined using ELISA, and the pharmacological properties of the receptors were delineated in two functional assays. The Y63C and I81T variations in the extracellular domain (ECD) of CaSR yielded markedly reduced cell surface expression and Ca2+ responsiveness, while Q459R displayed WT-like expression and functional properties. Truncation of the 7-transmembrane domain (7TMD) in W818stop eliminated cell surface expression, whereas R955stop in the intracellular carboxy-terminal yielded modestly increased surface expression and Ca2+ potency compared with WT CaSR. Interestingly, the effectiveness of positive allosteric modulators (PAMs) at the variants varied. Ca2+-mediated signaling through Y63C and I81T was significantly augmented by 7TMD-binding PAMs (NPS R-568 and Evocalcet) but not by ECD-binding PAMs (Etelcalcetide and Nb4), whereas signaling through Q459R and R955stop were robustly potentiated by all four PAMs. While the molecular phenotypes exhibited by the five CaSR variants concord with the clinical phenotypes in individuals harboring them, CASR variant-induced calcium homeostasis disorders clearly arise from diverse molecular origins, and the effectiveness of calcimimetics in these disorders could differ depending on the specific variants.

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

Cinacalcet Reverses Short QT Interval in Familial Hypocalciuric Hypercalcemia Type 1.

Familial hypocalciuric hypercalcemia type 1 (FHH-1) defines an autosomal dominant disease, related to mutations in the CASR gene, with mild hypercalcemia in most cases. Cases of FHH-1 with a short QT interval have not been reported to date. Three family members presented with FHH-1 and short QT interval (<360 ms), a condition that could lead to cardiac arrhythmias, and the effects of cinacalcet, an allosteric modulator of the CaSR, in rectifying the abnormal sensitivity of the mutant CaSR and in correcting the short QT interval were determined. CASR mutational analysis was performed by next-generation sequencing and functional consequences of the identified CaSR variant (p.Ile555Thr), and effects of cinacalcet were assessed in HEK293 cells expressing wild-type and variant CaSRs. A cinacalcet test consisting of administration of 30 mg cinacalcet (8 Am) followed by hourly measurement of serum calcium, phosphate, and parathyroid hormone during 8 hours and an electrocardiogram was performed. The CaSR variant (p.Ile555Thr) was confirmed in all 3 FHH-1 patients and was shown to be associated with a loss of function that was ameliorated by cinacalcet. Cinacalcet decreased parathyroid hormone by >50% within two hours, and decreases in serum calcium and increases in serum phosphate occurred within 8 hours, with rectification of the QT interval, which remained normal after 3 months of cinacalcet treatment. Our results indicate that FHH-1 patients should be assessed for a short QT interval and a cinacalcet test used to select patients who are likely to benefit from this treatment.

OR21-2 Encaleret (CLTX-305) Restored Mineral Homeostasis in a Phase 2 Study in Autosomal Dominant Hypocalcemia Type 1 (ADH1)

Abstract Autosomal dominant hypocalcemia type 1 (ADH1), caused by gain-of-function variants in the gene encoding the calcium-sensing receptor (CaSR), is characterized by hypocalcemia, hyperphosphatemia, hypomagnesemia, low parathyroid hormone (PTH), and hypercalciuria. Conventional therapy (calcium and activated Vitamin D) worsens hypercalciuria and can lead to renal morbidity. Calcilytics (negative allosteric modulators of the CaSR) decrease the sensitivity of hyperactive receptors to extracellular calcium and normalize blood and urine abnormalities in ADH1 rodent models. Encaleret is an oral calcilytic under investigation as a potential treatment for ADH1. Thirteen adults (22-60y) with ADH1 due to 9 unique CASR variants participated in a 3-period, Phase 2b, open-label, dose-ranging study. Conventional therapy was discontinued prior to encaleret initiation. Period 1 (P1) was a 5-day inpatient dose-escalation course (n=6). Period 2 (P2) was a 5-day inpatient course (n=13) in which doses were individually titrated to normalize albumin-corrected blood calcium (cCa) and minimize hypercalciuria and hypophosphatemia. All 13 participants continued into Period 3 (P3), a 24-week outpatient maintenance period. Patients underwent serial 24hr blood and urine sampling in P1, P2, and in P3 at Weeks 8, 16, and 24, with additional outpatient laboratory timepoints. The mean±SD encaleret dose at the end of P2, Day 5 (P2D5) was 94±64mg BID (range: 10-180 BID); by P3, Week 24 (P3W24), the mean was 78±67mg BID (5-190 BID). Encaleret was well-tolerated with no serious adverse events reported; there were no treatment discontinuations or study withdrawals. Twenty-four hour mean±SD values from P2D5 (n=13) and P3W24 (n=12) compared to baseline are presented. Baseline PTH was low at 6.3±7.8 pg/mL (nl 10-65) and had normalized by P2D5 (40.5±37.5, p&amp;lt;0.01); this was sustained through P3W24 (31.3±20.8, p&amp;lt;0.01). Likewise, baseline hypocalcemia (cCa=7.1±0.4 mg/dL [nl 8.4-10.2]) corrected to 8.6±0.7 by P2D5 (p&amp;lt;0.01) and remained normal through P3W24 (9.0±0.6, p&amp;lt;0.01). Baseline urinary calcium was 395±216 mg/d (nl &amp;lt;250-300) and decreased to 179±108 by P2D5 (p&amp;lt;0.01) and 189±72 at P3W24 (p&amp;lt;0.05); urinary calcium excretion was normal in 10/12 patients at P3W24. Baseline phosphate decreased from 4.5±1.1 mg/dL (nl 2.3-4.7) to 3.2±0.7 on P2D5 (p&amp;lt;0.01) and was maintained through P3W24 (3.5±0.6, p&amp;lt;0.05). Magnesium increased from low- to mid-normal (baseline 1.7±0.2 mg/dL [nl 1.6-2.6]; P2D5 1.9±0.2 [p&amp;lt;0.01]; P3W24 2.0±0.2, [p&amp;lt;0.01]). 1,25-dihydroxy-Vitamin D increased from 19.5±4.4 pg/mL (nl 20-70) to 32.0±16.7 on P2D5 (p&amp;lt;0.05) and 30.2±14.0 on P3W24 (p&amp;lt;0.05). Bone turnover markers were not different between baseline (n=7, CTX=253±111 pg/mL; P1NP=34±10 mcg/L) and P2D5 (CTX=241±181, p=NS; P1NP=26±12, p=NS) but had increased by P3W24 (CTX=784±686, p&amp;lt;0.01; P1NP=102±87, p&amp;lt;0.01). In conclusion, this study represents a molecularly targeted, precision medicine approach to the treatment of ADH1. The consistent and sustained results from all periods of this Phase 2 study establish a clinically meaningful efficacy, tolerability, and safety profile for encaleret as a potential treatment of adults with ADH1. Presentation: Monday, June 13, 2022 11:15 a.m. - 11:30 a.m.

Autosomal dominant hypocalcemia with a novel CASR mutation: a case study and literature review.

Autosomal dominant hypocalcemia type 1 (ADH1) is a rare inherited disorder characterized by hypocalcemia with low parathyroid hormone (PTH) levels and high urinary calcium. Its clinical presentation varies from mild asymptomatic to severe hypocalcemia. It is caused by gain-of-function mutations in the calcium-sensing receptor gene (CASR) which affect PTH secretion from the parathyroid gland and calcium resorption in the kidney. Here, we describe a case who presented with symptoms of recurrent seizure caused by hypocalcemia with a novel CASR variant. We comprehensively analyzed the phenotypic features of this presentation and reviewed the current literature to better understand clinical manifestations and the genetic spectrum.

Sequencing the CaSR locus in Pakistani stone formers reveals a novel loss-of-function variant atypically associated with nephrolithiasis

BackgroundNephrolithiasis (NL) affects 1 in 11 individuals worldwide and causes significant morbidity and cost. Common variants in the calcium sensing receptor gene (CaSR) have been associated with NL. Rare inactivating CaSR variants classically cause hyperparathyroidism, hypercalcemia and hypocalciuria. However, NL and familial hypercalciuria have been paradoxically associated with select inactivating CaSR variants in three kindreds from Europe and Australia.MethodsTo discover novel NL-associated CaSR variants from a geographically distinct cohort, 57 Pakistani families presenting with pediatric onset NL were recruited. The CaSR locus was analyzed by directed or exome sequencing.ResultsWe detected a heterozygous and likely pathogenic splice variant (GRCh37 Chr3:122000958A>G; GRCh38 Chr3:12228211A>G; NM_000388:c.1609-2A>G) in CaSR in one family with recurrent calcium oxalate stones. This variant would be predicted to cause exon skipping and premature termination (p.Val537Metfs*49). Moreover, a splice variant of unknown significance in an alternative CaSR transcript (GRCh37 Chr3:122000929G>C; GRCh38 Chr3:122282082G >C NM_000388:c.1609-31G >C NM_001178065:c.1609-1G >C) was identified in two additional families.ConclusionsSequencing of the CaSR locus in Pakistani stone formers reveals a novel loss-of-function variant, expanding the connection between the CaSR locus and nephrolithiasis.

Is routine 24-hour urine calcium measurement useful during the evaluation of primary hyperparathyroidism?

BackgroundPrimary hyperparathyroidism and familial hypocalciuric hypercalcemia have similar biochemical profiles, and calcium-to-creatinine-clearance ratio helps distinguish the two. Additionally, 24-hour urine calcium >400 mg/day indicates surgery and guidelines recommend obtaining 24-hour urine calcium preoperatively. Our aim was to assess how 24-hour urine calcium altered care in the evaluation of suspected primary hyperparathyroidism. MethodsConsecutive patients assessed for primary hyperparathyroidism from 2018 to 2020 were reviewed. Primary hyperparathyroidism was diagnosed by 2016 American Association of Endocrine Surgeons Parathyroidectomy Guidelines criteria. 24-hour urine calcium-directed change in care was defined as familial hypocalciuric hypercalcemia diagnosis, surgical deferment for additional testing, or 24-hour urine calcium >400 mg/day as the sole surgical indication. ResultsOf 613 patients, 565 (92%) completed 24-hour urine calcium and 477 (84%) had concurrent biochemical testing to calculate calcium-to-creatinine-clearance ratio. 24-hour urine calcium was <100 mg/day in 9% (49/565) and calcium-to-creatinine-clearance ratio was <0.01 in 17% (82/477). No patient had confirmed familial hypocalciuric hypercalcemia, although 1 had a CASR variant of undetermined significance. When calcium-to-creatinine-clearance ratio was <0.01, familial hypocalciuric hypercalcemia was excluded by 24-hour urine calcium >100 mg/day (56%), prior normal calcium (16%), renal insufficiency (11%), absence of familial hypercalcemia (3%), normal repeat 24-hour urine calcium (10%), or interfering diuretic (1%). 24-hour urine calcium-directed change in care occurred in 25 (4%), including 4 (1%) who had genetic testing. Four-gland hyperplasia was more common with calcium-to-creatinine-clearance ratio <0.01 (17% vs calcium-to-creatinine-clearance ratio ≥ 0.01, 4%, P < .001), but surgical failure rates were equivalent (P = .24). Conclusion24-hour urine calcium compliance was high, and results affected management in 4%, including productive identification of hypercalciuria as the sole surgical indication in 2 patients. When calcium-to-creatinine-clearance ratio <0.01, clinical assessment was sufficient to exclude familial hypocalciuric hypercalcemia and only 1% required genetic testing. 24-hour urine calcium should be ordered judiciously during primary hyperparathyroidism assessment.

Calcium-Sensing Receptor Polymorphisms at rs1801725 Are Associated with Increased Risk of Secondary Malignancies.

Dysregulation of systemic calcium homeostasis during malignancy is common in most patients with high-grade tumors. However, it remains unclear whether single nucleotide polymorphisms (SNPs) that alter the sensitivity of the calcium-sensing receptor (CaSR) to circulating calcium are associated with primary and/or secondary neoplasms at specific pathological sites in patients of European and African ancestry. Multivariable logistic regression models were used to analyze the association of CASR SNPs with circulating calcium, parathyroid hormone, vitamin D, and primary and secondary neoplasms. Circulating calcium is associated with an increased risk for breast, prostate, and skin cancers. In patients of European descent, the rs1801725 CASR SNP is associated with bone-related cancer phenotypes, deficiency of humoral immunity, and a higher risk of secondary neoplasms in the lungs and bone. Interestingly, circulating calcium levels are higher in homozygous patients for the inactivating CASR variant at rs1801725 (TT genotype), and this is associated with a higher risk of secondary malignancies. Our data suggest that expression of CaSR variants at rs1801725 is associated with a higher risk of developing secondary neoplastic lesions in the lungs and bone, due in part to cancer-induced hypercalcemia and/or tumor immune suppression. Screening of patients for CASR variants at this locus may lead to improved management of high calcium associated tumor progression.

Common CASR variants in Hungarian chronic pancreatitis patients

Common CASR variants in Hungarian chronic pancreatitis patients

Outcome of Clinical Genetic Testing in Patients with Features Suggestive for Hereditary Predisposition to PTH-Mediated Hypercalcemia.

Primary hyperparathyroidism (pHPT) is associated with familial syndromes such as multiple endocrine neoplasia type 1 (MEN1), 2A (MEN2A), MEN-like syndromes (CDKN1B), and CDC73-related disorder (hyperparathyroidism - jaw tumor syndrome (HPJT)). Familial hypocalciuric hypercalcemia (FHH) caused by CASR variants is an important differential diagnosis for pHPT. In order to evaluate the contribution of hereditary causes to pHPT in patients encountered in a specialized clinic, we conducted a retrospective study on patients with pHPT that underwent germline genetic testing. We evaluated 46 patients referred to a Cancer Genetics Clinic. Reasons for referral were young age (age < 40) for 29 patients (63%), multi-gland disease for 23 patients (50%), and a positive family history of pHPT for 11 patients (24%). All 46 patients underwent genetic evaluation. A total of 11 rare variants were found (CASR (4), CDC73 (2), MEN1 (2) CDKN1B (1), and RET (2)). One MEN1 variant was classified as pathogenic, and all others were variants of uncertain significance (VUS). All patients with CASR variants had clinical features of FHH and were counselled against parathyroidectomy. Both patients with CDC73 variants were counselled about recurrence of pHPT and parathyroid cancer. Neither of the RET variants were MEN2-associated. The CDKN1B variant was regarded as a true VUS and no action was taken. In this study, genetic testing impacted clinical care in 7 (15%) patients. We suggest that all patients < 40years of age, with multi-gland disease, single gland disease refractory to treatment, and a positive family history for pHPT or associated tumors should be considered for genetic evaluation.

A cell function study on calcium regulation of a novel calcium-sensing receptor mutation (p.Tyr825Phe).

PurposeAutosomal dominant hypocalcemia with hypercalciuria is a genetic disease characterized by hypoparathyroidism with hypercalciuria. We discovered a novel variant (p.Tyr825Phe[Y825F]) of the <i>CASR</i> gene in a neonate with congenital hypoparathyroidism and hypercalciuria and conducted a cell function study to determine whether the CASR-Y825F variant was pathogenic.MethodsTo perform a functional study on CaSR-Y825F, we constructed expression vectors expressing wild-type (WT) CASR and CASR-Y825F. After transfection of each expression vector into HEK293 cells, we examined alterations in intracellular signaling. Mitogen-activated protein kinase (MAPK) signaling activity of HEK293 cells expressing CASR-WT or CASR-Y825F was determined. Changes in intracellular calcium ions ([Ca2+]i) by extracellular calcium ion ([Ca2+]e) stimulation were quantitatively compared and analyzed.ResultsCells expressing CASR-Y825F showed elevated of MAPK signaling (phospho-ERK [pERK], phospho-JNK [pJNK], phospho-p38 [pp38]) and increased [Ca2+]i levels at low [Ca2+]e stimulation compared with cells expressing CASR-WT. Additionally, [Ca2+]i levels in HEK293 cells expression CASR-WT and CASR-Y825F were determined at 340 nm/380 nm wavelength ratios using Fura-2 AM. At [Ca2+]e concentrations of 2.5 mM and 3 mM, the ratios of CASR-Y825F cells were higher (2.6 and 3.5, respectively) than those of CASR-WT cells (1.04 and 1.40, respectively).ConclusionsThis cell function study proved that the CASR-Y825F expressed in HEK293 cells elevated MAPK signaling (pERK, pJNK, pp38) and increased [Ca2+]i to induce hypocalcemia.

Functional Analysis of Calcium-Sensing Receptor Variants Identified in Families Provisionally Diagnosed with Familial Hypocalciuric Hypercalcaemia.

Identification of variants in the calcium-sensing receptor (CASR) gene is an important means of distinguishing between familial hypocalciuric hypercalcaemia (FHH) and primary hyperparathyroidism. However, identification and bioinformatics analysis of genetic variants alone is now considered insufficient as definitive proof; additional functional assessment is required to diagnose FHH with certainty. We identified two novel variants, D433Y and C739Y, and one previously reported variant G509R in the CASR of four kindreds provisionally diagnosed with FHH and aimed to functionally characterise these variants to confirm the diagnosis. Variant receptors were cloned as FLAG-tagged constructs into the mammalian expression vector, pcDNA3.1. Wild type and variant receptor constructs were expressed in HEK293 cells and their expression assessed by Western blot analysis and their functionality analysed using an IP-One assay which measures myo-inositol 1-phosphate accumulation following CaSR activation. Western blot analysis showed that the D433Y receptor had diminished mature glycosylated receptor compared with wild type CaSR whereas the G509R receptor had a complete lack of mature receptor. The C739Y receptor was consistently overexpressed. Functional assessment showed the D433Y receptor to be mildly inactivating at physiological calcium concentrations whereas the G509R receptor was inactive at all calcium concentrations. By contrast, the C739Y variant was activating compared to wild type receptor which is inconsistent with it causing FHH. We conclude that functional assessment of CaSR variants using the IP-One assay was useful in the investigation of suspected FHH probands, confirming the D433Y and G509R variants as likely pathogenic/pathogenic, but dismissing the C739Y variant as causing FHH.

SUN-366 Neonatal Severe Hyperparathyroidism: Extreme Hypercalcemia as a Robust Marker for Homozygous Dosage of Pathogenic CASR Variants

Context: Neonatal severe hyperparathyroidism (NSHPT) is a rare and life-threatening emergency. It includes generalized hyperparathyroid bone disease and respiratory distress from combinations among a narrowed thorax, rib fractures, hypotonia, and biochemical disturbances. Successful therapy is compatible with long life and a healthy prognosis. However, neuromotor retardation may persist after otherwise successful therapy. The time and amplitude of hypercalcemia likely correlate with irreversible neuromotor retardation; thus, early intervention seems critical in many cases. NSHPT is usually caused by homozygous or heterozygous pathogenic variant(s) of the CASR; a heterozygous variant of this gene is also the usual cause of familial hypocalciuric hypercalcemia (FHH or FHH1). Homozygotes and heterozygotes with NSHPT are often not distinguished in the current literature. In theory, their management should differ. Optimum treatment in homozygotes is early total parathyroidectomy with induction of postoperative hypoparathyroidism. Optimal management of heterozygotes is more complex. It consists in temporizing measures and varies from careful observation without surgery, to bisphosphonates and/or calcimimetics, and to subtotal parathyroidectomy. The heterozygotes can then develop into healthy babies with asymptomatic FHH1.Evidence Acquisition: Each case met strict criteria for “severe” and neonatal disease. We analyzed the core biochemical parameters of the maximal serum calcium and maximal PTH. To compare different immunoassays, PTH was analyzed as a ratio (thus without units) of the raw data divided by the upper limit of its normal range. Each case also required information about the allelic dosage of the pathogenic or likely pathogenic CASR variant(s).Evidence Synthesis: There were 36 cases with homozygous pathogenic CASR variants and 21 cases with heterozygous pathogenic or likely pathogenic variants. Maximal serum calcium was far higher in homozygotes 5.8 +/- 1.5 versus 3.2 +/- 0.2 (P<.001) (normal 2.2–2.6 mM). Maximal serum PTH as a ratio was also far higher in homozygotes 17 +/- 12 versus 8 +/- 9 (P=.003) (normal ratio 0.3–1.0). We defined extreme hypercalcemia empirically as any calcium value above 4.5 mM. No heterozygote had a maximal calcium above 3.7 mM; however, 30 of 36 (81%) of the homozygotes had maximal calcium above 4.5 mM. Whenever tested early, this extreme hypercalcemia was usually recognized in the first week of life.Conclusions: Extreme hypercalcemia greater than 4.5 mM was newly identified as a conservative cutoff that was 100% predictive of homozygosity for pathogenic CASR variants. Extreme hypercalcemia in NSHPT is an early, facile, rapid, and inexpensive determinant of homozygosity for pathogenic variants of the CASR. It should be sought and used to promote early and definitive total parathyroidectomy, whenever it is identified in NSHPT.

Familial Hypocalciuric Hypercalcemia Type 1 and Autosomal-Dominant Hypocalcemia Type 1: Prevalence in a Large Healthcare Population

The calcium-sensing receptor (CaSR) regulates serum calcium concentrations. CASR loss- or gain-of-function mutations cause familial hypocalciuric hypercalcemia type 1 (FHH1) or autosomal-dominant hypocalcemia type 1 (ADH1), respectively, but the population prevalence of FHH1 or ADH1 is unknown. Rare CASR variants were identified in whole-exome sequences from 51,289 de-identified individuals in the DiscovEHR cohort derived from a single US healthcare system. We integrated bioinformatics pathogenicity triage, mean serum Ca concentrations, and mode of inheritance to identify potential FHH1 or ADH1 variants, and we used a Sequence Kernel Association Test (SKAT) to identify rare variant-associated diseases. We identified predicted heterozygous loss-of-function CASR variants (6 different nonsense/frameshift variants and 12 different missense variants) in 38 unrelated individuals, 21 of whom were hypercalcemic. Missense CASR variants were identified in two unrelated hypocalcemic individuals. Functional studies showed that all hypercalcemia-associated missense variants impaired heterologous expression, plasma membrane targeting, and/or signaling, whereas hypocalcemia-associated missense variants increased expression, plasma membrane targeting, and/or signaling. Thus, 38 individuals with a genetic diagnosis of FHH1 and two individuals with a genetic diagnosis of ADH1 were identified in the 51,289 cohort, giving a prevalence in this population of 74.1 per 100,000 for FHH1 and 3.9 per 100,000 for ADH1. SKAT combining all nonsense, frameshift, and missense loss-of-function variants revealed associations with cardiovascular, neurological, and other diseases. In conclusion, FHH1 is a common cause of hypercalcemia, with prevalence similar to that of primary hyperparathyroidism, and is associated with altered disease risks, whereas ADH1 is a major cause of non-surgical hypoparathyroidism.

Neonatal Severe Hyperparathyroidism: Novel Insights From Calcium, PTH, and the CASR Gene.

Neonatal severe hyperparathyroidism (NSHPT) is rare and potentially lethal. It is usually from homozygous or heterozygous germline-inactivating CASR variant(s). NSHPT shows a puzzling range of serum calcium and parathyroid hormone (PTH) levels. Optimal therapy is unclear. We categorized genotype/phenotype pairings related to CASRs. For the 2 pairings in NSHPT, each of 57 cases of neonatal severe hyperparathyroidism required calcium, PTH, upper normal PTH, and dosage of a germline pathogenic CASR variant. Homozygous and heterozygous NSHPT are 2 among a spectrum of 9 genotype/phenotype pairings relating to CASRs and NSHPT. For the 2 NSHPT pairings, expressions differ in CASR allelic dosage, CASR variant severity, and sufficiency of maternofetal calcium fluxes. Homozygous dosage of CASR variants was generally more aggressive than heterozygous. Among heterozygotes, high-grade CASR variants in vitro were more pathogenic in vivo than low-grade variants. Fetal calcium insufficiency as from maternal hypoparathyroidism caused fetal secondary hyperparathyroidism, which persisted and was reversible in neonates. Among NSHPT pairings, calcium and PTH were higher in CASR homozygotes than in heterozygotes. Extreme hypercalcemia (above 4.5 mM; normal 2.2-2.6 mM) is a robust biomarker, occurring only in homozygotes (83% of that pairing). It could occur during the first week. In NSHPT pairings, the homozygotes for pathogenic CASR variants show higher calcium and PTH levels than heterozygotes. Calcium levels above 4.5 mM among NSHPT are frequent and unique only to most homozygotes. This cutoff supports early and robust diagnosis of CASR dosage. Thereby, it promotes definitive total parathyroidectomy in most homozygotes.

Role of common CASR variants in chronic pancreatitis

Role of common CASR variants in chronic pancreatitis

The role of the calcium-sensing receptor in disorders of abnormal calcium handling and cardiovascular disease.

The calcium-sensing receptor (CaSR) has a central role in parathyroid gland function. Genetic alterations in CaSR are well known to cause inherited forms of abnormal calcium homeostasis. This review focuses on studies investigating the role of CaSR in common disorders of abnormal calcium handling and in cardiovascular calcification. Genetic population studies tested the association of common allelic CASR variants with serum and urine calcium levels, kidney stone disease, primary hyperparathyroidism and bone mineral density. The results of these association studies suggested either minor or no effects of CASR variants in these phenotypes. Decreased expression of CaSR was associated with the etiology of cardiovascular calcification in individuals with advanced chronic kidney disease. Ionized calcium plays a central role in the physiology of many organ systems and disease states, but the roles of CaSR other than as illustrated by Mendelian forms of CaSR dysfunction remain unclear. The contributions of CaSR to bone mineral homeostasis, vascular calcification and other forms of cardiovascular disease need further investigation.

Identification of rare and frequent variants of the CASR gene by high-resolution melting

BackgroundCalcium metabolic disorders like familial hypocalciuric hypercalcemia (FHH) and autosomal dominant familial isolated hypoparathyroidism (FIH) can be caused by rare variants of the calcium sensing receptor gene (CASR). Molecular genetic screening of the CASR is often based on DNA sequencing. MethodsWe sought to develop a pre-screening method in the diagnostic procedure and pursued variant scanning by high-resolution melting analysis (HRM) on a LightScanner instrument. We used 50 samples, representing 45 different rare variants, to validate the HRM method. In addition, we implemented small amplicon genotyping of three frequent CASR variants (c.1732+16T/C, c.2956G>T and c.2968A>G). ResultsUsing HRM, we identified 43 of 45 variants confidently (~96%) while two variants escaped immediate detection. Implementing this method in clinical use further resulted in the identification of seven new CASR variants and nine recurrent. HRM variant scanning, in combination with small amplicon genotyping, provides a simple workflow with reduced sequencing burden. Bioinformatics analyses using two freely available prediction tools (PolyPhen2 and SIFT) for evaluating amino acid substitutions were compared and indicated discrepancies in the prediction for 25% of the variants. ConclusionThis study demonstrates the utility of HRM as a pre-screening method, adds 24 novel rare CASR variants, and further emphasizes the importance of clinical decision making based on all available information rather than bioinformatics alone.