Familial Hypocalciuric Hypercalcemia Type Research Articles

A novel mouse model for familial hypocalciuric hypercalcemia (FHH1) reveals PTH-dependent and independent CaSR defects

The Calcium-sensing receptor (CaSR) senses extracellular calcium, regulates parathyroid hormone (PTH) secretion, and has additional functions in various organs related to systemic and local calcium and mineral homeostasis. Familial hypocalciuric hypercalcemia type I (FHH1) is caused by heterozygous loss-of-function mutations in the CaSR gene, and is characterized by the combination of hypercalcemia, hypocalciuria, normal to elevated PTH, and facultatively hypermagnesemia and mild bone mineralization defects. To date, only heterozygous Casr null mice have been available as model for FHH1. Here we present a novel mouse FHH1 model identified in a large ENU-screen that carries an c.2579 T > A (p.Ile859Asn) variant in the Casr gene (CasrBCH002 mice). In order to dissect direct effects of the genetic variant from PTH-dependent effects, we crossed CasrBCH002 mice with PTH deficient mice. Heterozygous CasrBCH002 mice were fertile, had normal growth and body weight, were hypercalcemic and hypermagnesemic with inappropriately normal PTH levels and urinary calcium excretion replicating some features of FHH1. Hypercalcemia and hypermagnesemia were independent from PTH and correlated with higher expression of claudin 16 and 19 in kidneys. Likewise, reduced expression of the renal TRPM6 channel in CasrBCH002 mice was not dependent on PTH. In bone, mutations in Casr rescued the bone phenotype observed in Pth null mice by increasing osteoclast numbers and improving the columnar pattern of chondrocytes in the growth zone. In summary, CasrBCH002 mice represent a new model to study FHH1 and our results indicate that only a part of the phenotype is driven by PTH.

A large in-frame deletion of the calcium-sensing receptor extracellular domain causes familial hypocalciuric hypercalcaemia type 1 (FHH1) and is partially responsive to cinacalcet

A large in-frame deletion of the calcium-sensing receptor extracellular domain causes familial hypocalciuric hypercalcaemia type 1 (FHH1) and is partially responsive to cinacalcet

OR03-05 Cinacalcet Acts As A Spatially Biased Allosteric Modulator To Enhance CaSR Signaling From Endosomal Membranes

Abstract Disclosure: R. Wyatt: None. C.M. Gorvin: None. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that plays a fundamental role in extracellular calcium homeostasis by activating Gαq/11 and Gαi/o signaling to regulate parathyroid hormone (PTH) release. CaSR can also continue signaling once internalised (known as sustained signaling) to elicit responses that are distinct from those at the plasma membrane. Loss-of-function mutations of CaSR cause familial hypocalciuric hypercalcemia type 1 (FHH1), while Arg15 mutations in the adaptor protein-2 σ-subunit (AP2σ) cause FHH3, by impairing CaSR internalisation and disrupting sustained signaling. However, the sustained signaling pathway has been incompletely defined and the effects of allosteric modulators on the process are unknown. Here we examined CaSR membrane trafficking and G protein activation in different endomembrane compartments using bystander BRET and HILO advanced imaging in HEK293 cells. This showed that internalised CaSR is targeted to Rab5-positive early endosomes and Rab4-positive early-to-recycling membranes, and not to degradation pathways as previously hypothesised. CaSR exclusively recruits active Gq/11 to early endosome membranes to drive sustained signals that produce distinct transcriptional profiles than those from plasma membranes. We next mutated a C-terminal region within CaSR predicted to form a dileucine endocytic motif that interacts with AP2σ. NanoBiT assays confirmed that the CaSR-dileucine mutant disrupted interactions between CaSR and AP2σ to the same magnitude as AP2σ-Arg15 mutations. Moreover, this CaSR-dileucine mutant impaired CaSR internalisation and reduced Gq/11 sustained signals from Rab5 and Rab4 endosomes to similar levels to those observed in cells expressing the FHH3-associated AP2σ-Arg15 mutant. Gi/o and G12/13 responses from plasma membranes were not affected by the CaSR-dileucine mutant. Finally, we assessed whether the CaSR positive allosteric modulator, cinacalcet, affects sustained signalling. When exposed to cinacalcet CaSR significantly increased the recruitment of active Gq/11 to early endosomes, resulting in a significant increase in signaling, while signals from the plasma membrane were not different to vehicle-treated cells. In summary, we have demonstrated that CaSR is targeted to an early-to-recycling endosomal pathway, which could explain how CaSR responds rapidly to fluctutations in serum calcium to maintain calcium homeostasis. Moreover, we have defined the endocytic motif within the CaSR required for sustained signaling and shown that cinacalcet is a spatially-biased allosteric modulator of the CaSR that preferentially enhances signaling from endosomal membranes. Thus, FHH3-associated AP2σ mutations, CaSR dileucine mutants and cinacalcet can be used as tools to better understand CaSR spatiotemporal signaling and define its physiological significance. Presentation: Thursday, June 15, 2023

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.

Neurodevelopmental Abnormalities in Patients with Familial Hypocalciuric Hypercalcemia Type 3

To evaluate the prevalence and degree of any neurodevelopmental abnormalities in children with familial hypocalciuric hypercalcemia type 3 (FHH3). A formal neurodevelopmental assessment was performed in children diagnosed with FHH3. The Vineland Adaptive Behavior Scales, which is a standardized parent report assessment tool for adaptive behavior, was used to assess communication, social skills, and motor function and to generate a composite score. Six patients were diagnosed with hypercalcemia between 0.1 and 8years of age. All had neurodevelopmental abnormalities in childhood consisting of either global developmental delay, motor delay, expressive speech disturbances, learning difficulties, hyperactivity, or autism spectrum disorder. Four out of the 6 probands had a composite Vineland Adaptive Behavior Scales SDS of<-2.0, indicating adaptive malfunctioning. Significant deficits were observed in the domains of communication (mean SDS: -2.0, P<.01), social skills (mean SDS: -1.3, P<.05), and motor skills (mean SDS: 2.6, P<.05). Individuals were equally affected across domains, with no clear genotype-phenotype correlation. All family members affected with FHH3 also described evidence of neurodevelopmental dysfunction, including mild-to-moderate learning difficulties, dyslexia, and hyperactivity. Neurodevelopmental abnormalities appear to be a highly penetrant and common feature of FHH3, and early detection is warranted to provide appropriate educational support. This case series also supports consideration of serum calcium measurement as part of the diagnostic work-up in any child presenting with unexplained neurodevelopmental abnormalities.

Familial hypocalciuric hypercalcaemia type 1 caused by a novel heterozygous missense variant in the CaSR gene, p(His41Arg): two case reports

BackgroundFamilial hypocalciuric hypercalcaemia (FHH) is a rare, inherited disorder of extracellular calcium sensing. It is clinically characterised by mild to moderate parathyroid hormone dependent hypercalcaemia, an autosomal dominant pattern of inheritance, and a normal to reduced urinary calcium excretion in spite of high serum calcium.Case presentationWe report two cases of FHH in a family caused by a novel pathogenic missense variant in the CaSR gene, p. His41Arg. Case 1, describes a 17 year old female with no significant past medical history, admitted with acute appendicitis requiring laparoscopic appendectomy and reporting a six month history of polydipsia. Routine investigations were significant for hypercalcaemia, corrected calcium 3.19 mmol/L (2.21-2.52mmol/L), elevated parathyroid hormone of 84pg/ml (15-65pg/ml) and a low 24-hour urine calcium of 0.75mmol/24 (2.50-7.50mmol/24). She was initially managed with intravenous fluids and Zolendronic acid with temporary normalisation of calcium though ultimately required commencement of Cinacalcet 30 mg daily for persistent symptomatic hypercalcaemia. Genetic analysis was subsequently positive for the above variant. Case 2, a 50-year-old female, was referred to the endocrine outpatient clinic for the management of type 2 diabetes and reported a longstanding history of asymptomatic hypercalcaemia which had not been investigated previously. Investigation revealed hypercalcaemia; corrected calcium of 2.6 mmol/L (reference range: 2.21–2.52 mmol/L); PTH of 53.7ng/L (reference range: 15–65 ng/L) and an elevated 24-hour urine calcium of 10 mmol/24 (2.50–7.50 mmol/24hr) with positive genetic analysis and is managed conservatively. Despite sharing this novel mutation, these cases have different phenotypes and their natural history is yet to be determined. Two further relatives are currently undergoing investigation for hypercalcaemia and the family have been referred for genetic counselling.ConclusionAccurate diagnosis of FHH and differentiation from classic primary hyperparathyroidism can be challenging, however it is essential to avoid unnecessary investigations and parathyroid surgery. Genetic analysis may be helpful in establishing a diagnosis of FHH in light of the biochemical heterogeneity in this population and overlap with other causes of hypercalcaemia.

Reduced affinity of calcium sensing-receptor heterodimers and reduced mutant homodimer trafficking combine to impair function in a model of familial hypocalciuric hypercalcemia type 1.

Heterozygous loss-of-function mutation of the calcium sensing-receptor (CaSR), causes familial hypocalciuric hypercalcemia type 1 (FHH1), a typically benign condition characterized by mild hypercalcemia. In contrast, homozygous mutation of this dimer-forming G-protein coupled receptor manifests as the lethal neonatal severe hyperparathyroidism (NSHPT). To investigate the mechanisms by which CaSR mutations lead to these distinct disease states, we engineered wild-type (WT) and an exon 5-deficient disease-causing mutation, and transfected expression constructs into human embryonic kidney (HEK) cells. WT protein was mainly membrane-expressed whereas the mutant CaSR protein (mCaSR) was confined to the cytoplasm. Co-expression of WT CaSR directed mCaSR to the cell membrane. In assays of CaSR function, increases in extracellular [Ca2+] ([Ca2+]o) increased intracellular [Ca2+] ([Ca2+]i) in cells expressing WT CaSR while the response was reduced in cells co-expressing mutant and WT receptor. Untransfected cells or those expressing mCaSR alone, showed minimal, equivalent responses to increased [Ca2+]o. Immunoprecipitation experiments confirmed an association between mutant and wild-type CaSR. The affinity of the WT CaSR for calcium was three times greater than that of the heterodimer. The maximal functional response to [Ca]o was dependent on localization of CaSR to the membrane level and independent of homo- or heterodimerizations. In summary, these results suggest that heterodimerization of WT and mCaSR receptors, rescues the trafficking defect of the mutant receptors and also reduces the affinity of the WT-mutant heterodimer for [Ca]o. In contrast, the homozygous mutants do not produce functional receptors on cell membrane. These data indicate how substantial differences between signaling of hetero- and homodimeric mutants may lead to profound differences in the severity of disease in heterozygous and homozygous carriers of these mutations.

Familial hypocalciuric hypercalcaemia (FHH) type 3: A rare cause of parathyroid (PTH) dependent hypercalcaemia with associated learning disabilities and behavioural problems

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Clinical Presentation and Management Approach in a Case of Familial Hypocalciuric Hypercalcemia Type 3 Due to APS21 Gene Mutation

Familial hypocalciuric hypercalcemia (FHH) is a genetic disorder caused by dysfunctional calcium homeostasis. Thus far, three types of FHH are known to be caused by mutations inCASR (FHH1), GNA11 (FHH2), and AP2S1 (FHH3). The patient in this case report is a 36-year old male that initially presented for a second opinion after being diagnosed with Primary Hyperparathyroidism(PHPT) with subsequent parathyroidectomy done at another institute, and developed recurrent symptomatic hypercalcemia. Prior to considering this patient for further surgical options, he underwent genetic testing, which revealed he had c.43C>T (p.Arg15Cys) mutation in the AP2S1 gene diagnostic of Familial Hypocalciuric Hypercalcemia Type 3 (FHH3). The patient’s father and sister also have hypercalcemia, and have been offered genetic testing. There have been cases reported of patients with FHH3 that have symptomatic hypercalcemia and that have associated cognitive issues. Many patients with FHH can be misdiagnosed and may undergo unnecessary parathyroidectomy. This case report further elucidates the need to raise awareness of FHH.

Performance of the Pro-FHH Score in a Delayed Diagnosis of Familial Hypocalciuric Hypercalcemia Type-1

We present a case of a 50-year-old woman with familial hypocalciuric hypercalcemia type 1 (FHH-1) that was missed during an initial evaluation of hypercalcemia in the setting of normal serum parathyroid hormone (PTH), leading to unnecessary parathyroidectomy. She subsequently reported years later with an inappropriately elevated PTH in the setting of hypercalcemia. Sequencing of the calcium-sensing receptor (CASR) gene revealed a known inactivating variant associated with FHH-1. We assessed the clinical utility of the recently reported pro-FHH (probability of having familial hypocalciuric hypercalcemia) score at the time of her surgery and subsequent re-evaluation years later. This case highlights the diagnostic challenges in differentiating familial hypocalciuric hypercalcemia (FHH) from primary hyperparathyroidism (PHPT).

GNA11 Variants Identified in Patients with Hypercalcemia or Hypocalcemia.

Familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2) are due to loss- and gain-of-function mutations, respectively, of the GNA11 gene that encodes the G protein subunit Gα11, a signaling partner of the calcium-sensing receptor (CaSR). To date, four probands with FHH2-associated Gα11 mutations and eight probands with ADH2-associated Gα11 mutations have been reported. In a 10-year period, we identified 37 different germline GNA11 variants in >1200 probands referred for investigation of genetic causes for hypercalcemia or hypocalcemia, comprising 14 synonymous, 12 noncoding, and 11 nonsynonymous variants. The synonymous and noncoding variants were predicted to be benign or likely benign by in silico analysis, with 5 and 3, respectively, occurring in both hypercalcemic and hypocalcemic individuals. Nine of the nonsynonymous variants (Thr54Met, Arg60His, Arg60Leu, Gly66Ser, Arg149His, Arg181Gln, Phe220Ser, Val340Met, Phe341Leu) identified in 13 probands have been reported to be FHH2- or ADH2-causing. Of the remaining nonsynonymous variants, Ala65Thr was predicted to be benign, and Met87Val, identified in a hypercalcemic individual, was predicted to be of uncertain significance. Three-dimensional homology modeling of the Val87 variant suggested it was likely benign, and expression of Val87 variant and wild-type Met87 Gα11 in CaSR-expressing HEK293 cells revealed no differences in intracellular calcium responses to alterations in extracellular calcium concentrations, consistent with Val87 being a benign polymorphism. Two noncoding region variants, a 40bp-5'UTR deletion and a 15bp-intronic deletion, identified only in hypercalcemic individuals, were associated with decreased luciferase expression in vitro but no alterations in GNA11 mRNA or Gα11 protein levels in cells from the patient and no abnormality in splicing of the GNA11 mRNA, respectively, confirming them to be benign polymorphisms. Thus, this study identified likely disease-causing GNA11 variants in <1% of probands with hypercalcemia or hypocalcemia and highlights the occurrence of GNA11 rare variants that are benign polymorphisms. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Cell Surface Calcium-Sensing Receptor Heterodimers: Mutant Gene Dosage Affects Ca2+ Sensing but Not G Protein Interaction.

ABSTRACTThe calcium‐sensing receptor is a homodimeric class C G protein‐coupled receptor (GPCR) that senses extracellular Ca2+ (Ca2+ o) via a dimeric extracellular Venus flytrap (VFT) unit that activates G protein‐dependent signaling via twin Cysteine‐rich domains linked to transmembrane heptahelical (HH) bundles. It plays a key role in the regulation of human calcium and thus mineral metabolism. However, the nature of interactions between VFT units and HH bundles, and the impacts of heterozygous or homozygous inactivating mutations, which have implications for disorders of calcium metabolism are not yet clearly defined. Herein we generated CaSR‐GABAB1 and CaSR‐GABAB2 chimeras subject to GABAB‐dependent endoplasmic reticulum sorting to traffic mutant heterodimers to the cell surface. Transfected HEK‐293 cells were assessed for Ca2+ o‐stimulated Ca2+ i mobilization using mutations in either the VFT domains and/or HH bundle intraloop‐2 or intraloop‐3. When the same mutation was present in both VFT domains of receptor dimers, analogous to homozygous neonatal severe hyperparathyroidism (NSHPT), receptor function was markedly impaired. Mutant heterodimers containing one wild‐type (WT) and one mutant VFT domain, however, corresponding to heterozygous familial hypocalciuric hypercalcemia type‐1 (FHH‐1), supported maximal signaling with reduced Ca2+ o potency. Thus two WT VFT domains were required for normal Ca2+ o potency and there was a pronounced gene‐dosage effect. In contrast, a single WT HH bundle was insufficient for maximal signaling and there was no functional difference between heterodimers in which the mutation was present in one or both intraloops; ie, no gene‐dosage effect. Finally, we observed that the Ca2+ o‐stimulated CaSR operated exclusively via signaling in‐trans and not via combined in‐trans and in‐cis signaling. We consider how receptor asymmetry may support the underlying mechanisms. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Clinical characteristics of familial hypocalciuric hypercalcaemia type 1: A multicentre study of 77 adult patients.

Familial hypocalciuric hypercalcaemia type 1 (FHH1), related to heterozygous loss-of-function mutations of the calcium-sensing receptor gene, is the main differential diagnosis for primary hyperparathyroidism. The aim of our study was to describe clinical characteristics of adult patients living in France with a genetically confirmed FHH1. This observational, retrospective, multicentre study included 77 adults, followed up in 32 clinical departments in France, with a genetic FHH1 diagnosis between 2001 and 2012. Hypercalcaemia was diagnosed at a median age of 53years [IQR: 38-61]. The diagnosis was made after clinical manifestations, routine analysis or familial screening in 56, 34 and 10% of cases, respectively, (n=58; data not available for 19 patients). Chondrocalcinosis was present in 11/51 patients (22%), bone fractures in 8/56 (14%) and renal colic in 6/55 (11%). The median serum calcium was 2.74mmol/L [IQR: 2.63-2.86mmol/L], the median plasma parathyroid hormone level was 4.9pmol/L [3.1-7.1], and the median 24-hour urinary calcium excretion was 2.8mmol/24hours [IQR: 1.9-4.0]. Osteoporosis (dual X-ray absorptiometry) or kidney stones (renal ultrasonography) were found in 6/38 patients (16%) and 9/32 patients (28%), respectively. Fourteen patients (18%) underwent parathyroid surgery; parathyroid adenoma was found in three patients (21%) and parathyroid hyperplasia in nine patients (64%). No correlation between genotype and phenotype was established. This large cohort study demonstrates that FHH1 clinical characteristics can be atypical in 33 patients (43%). Clinicians should be aware of this rare differential diagnosis in order to adopt an appropriate treatment strategy.

SAT-404 Neonatal Hypocalcemic Seizures in Offspring of a Mother with Familial Hypocalciuric Hypercalcemia Type 1 (FHH1)

Background: Familial hypocalciuric hypercalcemia type 1 (FHH1) is caused by loss-of-function mutations of the calcium-sensing receptor (CaSR), and considered to be a benign condition associated with mild-to-moderate hypercalcemia (1). However, the children of parents with FHH1 can develop a variety of disorders of calcium homeostasis in infancy. Objective: To further characterise the range of calcitropic phenotypes in the children of a mother with FHH1. Methods: We assessed a three generation FHH kindred by clinical, biochemical and mutational analysis following informed consent. Results: The kindred comprised a hypercalcemic male, his daughter who had hypercalcemia and hypocalciuria, and her four children, of whom two had asymptomatic hypercalcemia, one was normocalcemic, and one suffered from transient hypocalcemic seizures during infancy. The hypocalcemic infant had a serum calcium of 1.57 mmol/L (normal, 2.0-2.8) and PTH of 2.2 pmol/L (normal, 1.0-9.3) as a consequence of maternal hypercalcemia, and required treatment with I-V calcium gluconate infusions. Mutational analysis identified a novel heterozygous p.Ser448Pro CaSR variant in the hypercalcemic family members, but not in the children with hypocalcemia or normocalcemia. Three-dimensional modelling using a reported crystal structure of the dimeric CaSR showed the mutated Ser448 residue to be located in the CaSR extracellular domain, and predicted the p.Ser448Pro variant to disrupt a hydrogen bond interaction across the extracellular CaSR dimer interface. The variant Pro448 CaSR, when expressed in HEK293 cells, was shown to significantly impair CaSR-mediated intracellular calcium mobilisation and mitogen-activated protein kinase (MAPK) responses following stimulation with extracellular calcium, thereby demonstrating it to represent a loss-of-function mutation. Conclusion: These studies have identified a novel loss-of-function CaSR mutation which caused asymptomatic hypercalcemia in a mother and her children who had inherited the mutation. However, one child who did not inherit the mutation developed transient neonatal hypocalcemic seizures as a consequence of maternal hypercalcemia. These findings highlight the importance of assessing serum calcium and undertaking CaSR mutational analysis in the newborn offspring of a mother with FHH1. Reference: (1) Hannan FM, Kallay E, Chang W, Brandi ML, Thakker RV. The calcium-sensing receptor in physiology and in calcitropic and noncalcitropic diseases. Nat Rev Endocrinol. 2018; 15(1): 33-51.

SAT-361 Familial Hypocalciuric Hypercalcemia Type 3: AP2S1 Mutation

Background: Familial hypocalciuric hypercalcemia (FHH) type 3 can appear similar to primary hyperparathyroidism and make the diagnosis of etiology of hypercalcemia challenging.Clinical Case: A 45-year-old man with hypertension and glaucoma was evaluated in clinic for hypercalcemia. His calcium was 12.3 mg/dL (8.4–10.2), PTH 41.9 pg/mL (15–65), Vitamin D 14.8 ng/mL (6.6–49) and phosphorus 1.7 mg/dL (2.7–4.5). He denied history of thiazide diuretic use, fragility fracture, nephrolithiasis and family history of calcium disorders. Further workup revealed normal kidney function, undetectable PTH related peptide and dual-energy x-ray absorptiometry (DEXA) scan with a T-score of -3.3 at spine L1-L4, -2.7 at femoral neck and -2.1 at distal one-third forearm. A 24-hour urine collection revealed a urinary calcium of 42.4 mg/24-hour (100–300) and calcium: creatinine clearance ratio of 0.003. Diagnosis of primary hyperparathyroidism was made despite low urinary calcium as this was thought to be due to vitamin D deficiency. Sestamibi scintigraphy and four-dimensional computed tomography did not localize a parathyroid adenoma, however, the patient was sent to surgery for four gland parathyroid exploration for primary hyperparathyroidism in setting of high calcium and young age with evidence of end-organ failure of osteoporosis. During surgery, three large abnormal parathyroid glands were identified and one normal parathyroid gland. Patient had a three-gland parathyroidectomy with intraoperative drop in PTH by 26.5%. Pathology returned as benign parathyroid tissues. After surgery, patient had persistently elevated calcium level of 12.6 mg/dL and an inappropriately non-suppressed PTH. He was then started on bisphosphonate and cinacalcet for osteoporosis and hypercalcemia, respectively and sent for genetic testing of FHH. His CASR gene was negative but his AP2S1 gene was positive which confirmed the diagnosis of FHH type 3. His calcium responded well to cinacalcet and repeat DEXA scan showed stability of bone mineral density in spine and hip after two years of treatment with bisphosphonate therapy.Conclusion: Familial hypocalciuric hypercalcemia type 3 is caused by an inactivating mutation in the AP2S1 gene. This gene encodes the adaptor-related protein complex 2, sigma 1 subunit which is located downstream from calcium-sensing receptor. This genetic mutation can appear similar to primary hyperparathyroidism in that it produces high levels of calcium and PTH and low phosphorus. Hypercalcemia, however, persists despite removal of parathyroid gland. This genetic mutation can be treated with cinacalcet in patients with high levels of calcium (>1 upper limit of normal) or symptoms of hypercalcemia.

SAT-340 Familial Hypocalciuric Hypercalcemia Due to a C.571G&amp;gt;A (p.Glu191Lys) Variant in the GNA11 Gene

Background: Familial hypocalciuric hypercalcemia (FHH) type 1 is caused by inactivating pathogenic variants in the calcium sensing receptor (CASR) gene and clinically presents with serum hypercalcemia, low urine calcium excretion, and inappropriately normal PTH response. In a minority of patients with CASR-negative FHH (up to 26%), variants in AP2S1 are linked to FHH type 3. Similarly, inactivating pathogenic variants in the GNA11 gene have been shown to cause FHH type 2 in up to 10% of CASR and AP2S1 negative cases.1 Clinical case: A four-month old male presented to the ED for symptoms of polyphagia and polyuria. Biochemical evaluation found a serum calcium of 11.9 mg/dL (8.7-11.0 mg/dL), magnesium 2.4 mg/dL (1.6-2.3 mg/dL), 25-OH Vit D 38 ng/mL (30 -100 mg/dL) and intact PTH 27.8 pg/mL (15-65 pg/mL). His further labwork fit clinically with FHH with a urine calcium-creatinine ratio < 0.09 (<= 0.20), accompanied by a serum calcium level of 11.9 mg/dL and iCal of 1.48 mmol/L (1.13 – 1.37 mmol/L). Subsequent monitoring of serum calcium levels showed improvement with time, along with improvement in polyphagia and polyuria.Of note, mother also has a history of hypercalcemia that was diagnosed at age fifteen as part of a rheumatology evaluation. At diagnosis, her calcium was 12.1 mg/dL (9.3-10.6 mg/dL), Mg 2.3 mg/dL, phosphorous 3.3 mg/dL (2.3-4.8 mg/dL), intact PTH 39 pg/mL, and urine calcium-creatinine ratio of 0.04 (<= 0.20). She was treated with calcitonin, during which she developed hypercalciuria with elevated PTH of 88 pg/mL. A parathyroid uptake scan was concerning for adenomatous changes in the right upper parathyroid gland, but she was subsequently lost to follow-up without additional evaluation.Sequencing of the AP2S1, CASR, CDC73, CYP24A1, GCM2, GNA11, MEN1, PTH1R, or SLC34A1 genes by Next-generation sequencing methodology was completed in the patient and only revealed a maternally inherited missense variant of unknown significance of c.571G>A (p.Glu191Lys) in GNA11. This G to A transition in exon 4 substitutes a glutamic acid for a lysine at codon 191 of a highly conserved amino acid. This variant has not been previously reported in affected individuals nor has it been observed in the healthy population per the gnomAD population database. It is predicted to be deleterious/probably damaging by in silico tools (SIFT, PolyPhen2). Conclusion: To our knowledge, this is the first case demonstrating the presence of a c.571G>A (p.Glu191Lys) variant in the GNA11 gene, in a child with familial hypocalciuric hypercalcemia as well as in his affected mother. In cases with strong suspicion of FHH despite negative CASR and AP2S1 analyses, genetic analysis of GNA11 should also be considered.Reference:(1) Hovden, S., Rejnmark, L., Ladefoged, S., & Nissen, P. (2017). AP2S1 and GNA11 mutations – not a common cause of familial hypocalciuric hypercalcemia, European Journal of Endocrinology, 176(2), 177-185.

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.

Mice harbouring a germline heterozygous AP2S1 mutation, Arg15Leu, are a model for familial hypocalciuric hypercalcaemia type 3 (FHH3)

Mice harbouring a germline heterozygous AP2S1 mutation, Arg15Leu, are a model for familial hypocalciuric hypercalcaemia type 3 (FHH3)

About a case of familial hypocalciuric hypercalcemia (FHH) type 3 with neurological involvement

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

SAT-036 Extracellular Domain Calcium-Sensing Receptor (CaSR) Mutations Leading to Hypercalcemic and Hypocalcemic Disorders Cluster at the Homodimeric Interface

The calcium-sensing receptor (CaSR) is a homodimeric G-protein coupled receptor that plays a pivotal role in extracellular Ca2+ (Ca2+e) homeostasis, and loss- and gain-of-function mutations of the CaSR cause familial hypocalciuric hypercalcemia type 1 (FHH1) and autosomal dominant hypocalcemia type 1 (ADH1), respectively. Crystal structure analysis has shown that the CaSR has a large extracellular domain (ECD) comprising lobes 1 and 2, and a cysteine-rich domain (CRD), which connects the ECD to the transmembrane domain. The CaSR ECD binds Ca2+e at multiple sites, which induces conformational changes at the extracellular dimer interface that lead to intracellular signaling via the inositol trisphosphate (IP3) and mitogen-activated protein kinase (MAPK) pathways. To further elucidate the structure-function relationships of the ECD, we examined the location of all CaSR ECD mutations reported to-date in FHH1 and ADH1 probands using available CaSR crystal structures. These studies identified that 127 FHH1 and 66 ADH1 mutations affected ECD residues, with >50% of these mutations being located at the dimer interface. Mutations predicted to disrupt key CaSR dimer-dimer interactions included: a lobe 1 Tyr161Cys mutation, which impaired an interprotomer interaction with the lobe 1 Pro55 residue; a Ser171Asn mutation predicted to disrupt a lobe 2 interprotomer salt bridge, which forms upon agonist binding; and a Gly553Arg mutation, which altered interprotomer hydrophobic interactions within the CRD. We investigated the effect of the Tyr161Cys, Ser171Asn and Gly553Arg mutations on receptor dimerization using western blotting and showed that these mutant proteins all form dimers similar to the wild-type (WT) CaSR. Furthermore, we evaluated the effect of these mutations on CaSR function in HEK293 cells following stimulation with Ca2+e, by measuring accumulation of inositol monophosphate (IP1), which is an IP3 metabolite, and also by measuring serum response element (SRE)-containing luciferase reporter fold-change responses, which is an indicator of MAPK activation. The Tyr161Cys, Ser171Asn and Gly553Arg dimer interface mutations were shown to significantly impair CaSR-mediated signaling. Indeed, cells expressing the Tyr161Cys, Ser171Asn or Gly553Arg mutant CaSRs all showed a >30% reduction in IP1 accumulation and >50% reduction in SRE-reporter responses, respectively, compared to WT-expressing cells (p<0.05, results are from 3 independent experiments). Thus, these studies demonstrate that the majority of FHH1- and ADH1-causing CaSR ECD mutations are located at the dimer interface, but likely do not disrupt dimer formation. Instead, these mutations may potentially influence conformational changes that occur at the dimer interface upon CaSR activation.