Familial Hypocalciuric Hypercalcemia Type Research Articles

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.

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.

SAT-506 Identification of a Mutation in AP2S1 Causing Familial Hypocalciuric Hypercalcemia Type 3 in a Patient with Unknown Family History of the Disease

Background: Calcium homeostasis is mediated by the CaSR on the parathyroid gland and kidney. Mutations in the CaSR or downstream signaling proteins can lead to dysregulation in serum calcium levels. It is estimated that AP2S1 missense mutations may be the underlying cause of 20% of cases of hypocalciuric hypercalcemia that are negative for mutations in the CaSR (1).Case: The patient is a 29 yo female with a past medical history of IBD who presented for evaluation of hypercalcemia. She had symptoms of joint and abdominal pain concerning for symptomatic hypercalcemia. Labs revealed a normal vitamin D level of 48 ng/ml (ref: 25 to 80), a calcium of 12.0 mg/dL (ref: 8.5-10.5), albumin of 4.2 g/dL (ref: 3.4-5.0), PTH of 54 pg/ml (ref: 16-77), and urinary calcium of < 28 mg/24h (ref: 100-300). Given her symptomatology, the patient had a thyroid US and sestamibi scan that were unremarkable for parathyroid adenoma. In lieu of these negative findings and a low 24h urinary calcium suggestive of FHH, the Patient was sent for genetic testing for over 130 mutations in the CaSR, a common cause of FHH. No mutation in the CaSR was identified. Initial lab results, imaging, and genetic analysis were unrevealing as to the cause of her hypercalcemia. Further consideration of neck exploration was considered given the severity of symptoms that were attributed to elevated serum calcium at the time. Additional genetic evaluation was undertaken prior to exposing the patient to the risk of surgery. The patient was found to have a rare cause of FHH, a heterozygous c.43C>T, Arg15Cys missense mutation in AP2S1 (rs.39751448). Surprisingly, the patient had never been evaluated for hypercalcemia nor has a known family history.Discussion: FHH is comprised of 3 genetically distinct causes: mutations in the CaSR (FHH1), mutations in G α11 that couples with the CaSR (FHH2), and mutations in AP2S1 involved in CaSR-mediated endocytosis (FHH3). To date, at least 3 kindreds have been identified with the AP2S1 mutation (1). The calcimimetic drug, cinacalcet, has been trialed for treatment of FHH3 with more than 20% reduction in serum calcium levels and improvement of symptoms (2).Conclusion: FHH3 is a rare cause of hypercalcemia. To avoid unnecessary neck exploration, it may be beneficial to broaden the initial gene panels to include all 3 genetic causes of FHH to identify Patients with FHH3 foremost. Moreover, screening of family members is also important to avoid unnecessary surgery in kin. Identifying overlapping symptomatology of hypercalcemia with comorbid conditions will prevent extraneous treatment and side effect exposure. When the patient’s IBD was controlled, her symptoms also improved abrogating the need for cinacalcet. 1. Nesbit et al. Nat. Genet. 2013 Jan;45(1):93-72. 2. Howles et al. N Engl J Med. 2016 Apr 7; 374(14):1

OR30-5 Germline Ablation of G-Protein Subunit Alpha-11 in Mice Causes Hypercalcemia That Is Rectified by Treatment with Cinacalcet

G-protein subunit α-11 (Gα11) is a signalling partner of the calcium-sensing receptor (CaSR), which is a G-protein coupled receptor that plays a major role in calcium homeostasis by regulating parathyroid hormone (PTH) secretion, urinary calcium excretion and skeletal development. Although loss-of-function point mutations of the GNA11 gene, which encodes Gα11, have been shown to increase plasma calcium concentrations and cause familial hypocalciuric hypercalcemia type 2 (FHH2), the overall contribution of Gα11 to the parathyroid, bone and renal regulation of calcium homeostasis remains to be established. The aim of this study was to characterise the impact of selective germline ablation of Gα11 on plasma concentrations of albumin-adjusted calcium (adj-calcium) and PTH, 24-hour urinary calcium excretion, and bone mineral density (BMD) in adult male and female heterozygous and homozygous Gna11 null C57BL/6N mice (n=13-16 mice per genotype), which had been previously generated by the International Mouse Phenotyping Consortium (IMPC). These mice were also treated with cinacalcet to assess whether this CaSR positive allosteric modulator can rectify any alterations in calcium homeostasis. All studies were conducted in accordance with institutional welfare guidelines. Male and female heterozygous Gna11 null (Gna11+/-) mice were viable and significantly hypercalcemic (plasma adj-calcium = 2.47±0.02 mM) compared to wild-type (WT) mice (plasma adj-calcium = 2.33±0.01 mM, p<0.0001). Gna11+/- mice showed no alterations in plasma PTH or 24-hour urinary calcium. In contrast, homozygous Gna11 null (Gna11-/-) mice showed reduced viability and the proportion of viable Gna11-/- mice was >25% less than would be expected from a Mendelian pattern of inheritance (p=0.01, Chi-squared analysis). Moreover, male and female Gna11-/- mice had significantly reduced body weight and were significantly more hypercalcemic (plasma adj-calcium = 2.70±0.02 mM) than Gna11+/- or WT mice (p<0.0001). Female Gna11-/- mice also showed significant increases in plasma PTH and 24-hour urinary calcium compared to female Gna11+/- and WT mice. However, no alterations in BMD were observed in male and female Gna11+/- or Gna11-/- mice. Administration of a single oral 30mg/kg cinacalcet dose significantly reduced plasma adj-calcium and PTH in Gna11+/- and Gna11-/- mice, and normalised plasma adj-calcium concentrations in Gna11-/- mice at 1-hour post-dose. Thus, these findings demonstrate that Gα11 plays a major role in regulating plasma calcium concentrations, but does not influence BMD, nor represent a key mediator of urinary calcium excretion. Furthermore, cinacalcet rectifies the hypercalcemia of Gna11 null mice, thus indicating that this CaSR-targeted compound may modulate parathyroid gland function in a Gα11-independent manner.

MON-335 Phenocopy of Multiple Endocrine Neoplasia Type 1 (MEN1) Due to a Germline Cell Division Cycle 73 (CDC73) Variant

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the combined occurrence of parathyroid tumors, and neuroendocrine tumors (NETs) of the pituitary and pancreas. MEN1 is caused by germline mutations of the tumor suppressor gene MEN1, which are found in >75% of MEN1 patients. The remaining patients may have mutations involving: as yet unidentified genes; or other genes with known involvement in endocrine tumors, e.g. cyclin dependent kinase inhibitor 1B (CDKN1B) which is associated with MEN4. Here, we report a patient who had primary hyperparathyroidism, acromegaly, and a pancreatic NET that showed immuno-staining for glucagon and chromogranin A, which are consistent with MEN1. However, mutational analysis did not identify a MEN1 mutation, and analysis of other genes was undertaken, after informed consent was obtained from the patient. These genes include: CDKN1B; CDKN1A; CDKN2B; CDKN2C;cell cycle division 73 (CDC73), causative for hyperparathyroidism-jaw tumour (HPT-JT) syndrome, an autosomal dominant disorder characterised by occurrence of parathyroid tumors, ossifying fibromas of the jaw, renal tumours and uterine tumors; calcium sensing receptor (CASR), causative for familial hypocalciuric hypercalcemia type 1 (FHH1); and aryl-hydrocarbon receptor-interacting protein (AIP), causative for familial pituitary tumors. This revealed a heterozygous c.1138C>T (p.Leu380Phe) missense mutation of CDC73. This is likely a disease-causing mutation as: 1) the p.Leu380Phe substitution is not present in >120,000 alleles of The Exome Aggregation Consortium (ExAc) database; 2) involves an evolutionary conserved Leu380 residue; and 3) is situated within the parafibromin domain predicted to interact with the polymerase associated factor 1 (PAF1) and RNA polymerase II, (POLR2A) complex. In addition, RNA-Scope analysis, to detect specific CDC73 mRNA transcripts in paraffin embedded sections, of the patient’s pancreatic NET revealed that CDC73 mRNA expression was significantly decreased by >13% (p<0.005) in the tumor cells, compared to normal adjacent islets. Thus, our results suggest the spectrum of tumours associated with CDC73 mutations also includes pancreatic NETs, and demonstrate that CDC73 mutations may result in a phenocopy of MEN1.

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.

Large-scale exome datasets reveal a new class of adaptor-related protein complex 2 sigma subunit (AP2σ) mutations, located at the interface with the AP2 alpha subunit, that impair calcium-sensing receptor signalling.

Mutations of the sigma subunit of the heterotetrameric adaptor-related protein complex 2 (AP2σ) impair signalling of the calcium-sensing receptor (CaSR), and cause familial hypocalciuric hypercalcaemia type 3 (FHH3). To date, FHH3-associated AP2σ mutations have only been identified at one residue, Arg15. We hypothesized that additional rare AP2σ variants may also be associated with altered CaSR function and hypercalcaemia, and sought for these by analysing >111 995 exomes (>60 706 from ExAc and dbSNP, and 51 289 from the Geisinger Health System-Regeneron DiscovEHR dataset, which also contains clinical data). This identified 11 individuals to have 9 non-synonymous AP2σ variants (Arg3His, Arg15His (x3), Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) with 3 of the 4 individuals who had Arg15His and Met117Ile AP2σ variants having mild hypercalcaemia, thereby indicating a prevalence of FHH3-associated AP2σ mutations of ∼7.8 per 100 000 individuals. Structural modelling of the novel eight AP2σ variants (Arg3His, Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) predicted that the Arg3His, Thr112Met, Glu122Gly and Glu142Lys AP2σ variants would disrupt polar contacts within the AP2σ subunit or affect the interface between the AP2σ and AP2α subunits. Functional analyses of all eight AP2σ variants in CaSR-expressing cells demonstrated that the Thr112Met, Met117Ile and Glu142Lys variants, located in the AP2σ α4-α5 helical region that forms an interface with AP2α, impaired CaSR-mediated intracellular calcium () signalling, consistent with a loss of function, and this was rectified by treatment with the CaSR positive allosteric modulator cinacalcet. Thus, our studies demonstrate another potential class of FHH3-causing AP2σ mutations located at the AP2σ-AP2α interface.

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.

Variable learning disability and behavioural difficulties in children with familial hypocalciuric hypercalcaemia type 3

Variable learning disability and behavioural difficulties in children with familial hypocalciuric hypercalcaemia type 3

Disorders of the calcium-sensing receptor and partner proteins: insights into the molecular basis of calcium homeostasis.

The extracellular calcium (Ca2+o)-sensing receptor (CaSR) is a family C G protein-coupled receptor, which detects alterations in Ca2+o concentrations and modulates parathyroid hormone secretion and urinary calcium excretion. The central role of the CaSR in Ca2+o homeostasis has been highlighted by the identification of mutations affecting the CASR gene on chromosome 3q21.1. Loss-of-function CASR mutations cause familial hypocalciuric hypercalcaemia (FHH), whereas gain-of-function mutations lead to autosomal dominant hypocalcaemia (ADH). However, CASR mutations are only detected in ≤70% of FHH and ADH cases, referred to as FHH type 1 and ADH type 1, respectively, and studies in other FHH and ADH kindreds have revealed these disorders to be genetically heterogeneous. Thus, loss- and gain-of-function mutations of the GNA11 gene on chromosome 19p13.3, which encodes the G-protein α-11 (Gα11) subunit, lead to FHH type 2 and ADH type 2, respectively; whilst loss-of-function mutations of AP2S1 on chromosome 19q13.3, which encodes the adaptor-related protein complex 2 sigma (AP2σ) subunit, cause FHH type 3. These studies have demonstrated Gα11 to be a key mediator of downstream CaSR signal transduction, and also revealed a role for AP2σ, which is involved in clathrin-mediated endocytosis, in CaSR signalling and trafficking. Moreover, FHH type 3 has been demonstrated to represent a more severe FHH variant that may lead to symptomatic hypercalcaemia, low bone mineral density and cognitive dysfunction. In addition, calcimimetic and calcilytic drugs, which are positive and negative CaSR allosteric modulators, respectively, have been shown to be of potential benefit for these FHH and ADH disorders.

Allosteric Modulation of the Calcium-sensing Receptor Rectifies Signaling Abnormalities Associated with G-protein α-11 Mutations Causing Hypercalcemic and Hypocalcemic Disorders

Germline loss- and gain-of-function mutations of G-protein α-11 (Gα11), which couples the calcium-sensing receptor (CaSR) to intracellular calcium (Ca2+i) signaling, lead to familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2), respectively, whereas somatic Gα11 mutations mediate uveal melanoma development by constitutively up-regulating MAPK signaling. Cinacalcet and NPS-2143 are allosteric CaSR activators and inactivators, respectively, that ameliorate signaling disturbances associated with CaSR mutations, but their potential to modulate abnormalities of the downstream Gα11 protein is unknown. This study investigated whether cinacalcet and NPS-2143 may rectify Ca2+i alterations associated with FHH2- and ADH2-causing Gα11 mutations, and evaluated the influence of germline gain-of-function Gα11 mutations on MAPK signaling by measuring ERK phosphorylation, and assessed the effect of NPS-2143 on a uveal melanoma Gα11 mutant. WT and mutant Gα11 proteins causing FHH2, ADH2 or uveal melanoma were transfected in CaSR-expressing HEK293 cells, and Ca2+i and ERK phosphorylation responses measured by flow-cytometry and Alphascreen immunoassay following exposure to extracellular Ca2+ (Ca2+o) and allosteric modulators. Cinacalcet and NPS-2143 rectified the Ca2+i responses of FHH2- and ADH2-associated Gα11 loss- and gain-of-function mutations, respectively. ADH2-causing Gα11 mutations were demonstrated not to be constitutively activating and induced ERK phosphorylation following Ca2+o stimulation only. The increased ERK phosphorylation associated with ADH2 and uveal melanoma mutants was rectified by NPS-2143. These findings demonstrate that CaSR-targeted compounds can rectify signaling disturbances caused by germline and somatic Gα11 mutations, which respectively lead to calcium disorders and tumorigenesis; and that ADH2-causing Gα11 mutations induce non-constitutive alterations in MAPK signaling.

Familial Hypocalciuric Hypercalcemia Types 1 and 3 and Primary Hyperparathyroidism: Similarities and Differences.

Familial hypocalciuric hypercalcemia (FHH) is a genetically heterogeneous condition resembling primary hyperparathyroidism (PHPT) but not curable by surgery; FHH types 1, 2, and 3 are due to loss-of-function mutations of the CASR, GNA11, or AP2S1 genes, respectively. This study aimed to compare the phenotypes of patients with genetically proven FHH types 1 or 3 or PHPT. This was a mutation analysis in a large cohort, a cross-sectional comparison of 52 patients with FHH type 1, 22 patients with FHH type 3, 60 with PHPT, and 24 normal adults. There were no interventions. Abnormalities of the CASR, GNA11, and AP2S1 genes, blood calcium, phosphate, and PTH concentrations, urinary calcium excretion were measured. In 133 families, we detected 101 mutations in the CASR gene, 68 of which were previously unknown, and in 19 families, the three recurrent AP2S1 mutations. No mutation was detected in the GNA11 gene. Patients with FHH type 3 had higher plasma calcium concentrations than patients with FHH type 1, despite having similar PTH concentrations and urinary calcium excretion. Renal tubular calcium reabsorption levels were higher in patients with FHH type 3 than in those with FHH type 1. Plasma calcium concentration was higher whereas PTH concentration and urinary calcium excretion were lower in FHH patients than in PHPT patients. In patients with FHH or PHPT, all data groups partially overlapped. In our population, AP2S1 mutations affect calcium homeostasis more severely than CASR mutations. Due to overlap, the risk of confusion between FHH and PHPT is high.

Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype–phenotype correlations, codon bias and dominant-negative effects

The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca2+o) homeostasis. To elucidate the role of AP2σ2 in Ca2+o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype–phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype–phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Increased trabecular volumetric bone mass density in Familial Hypocalciuric Hypercalcemia (FHH) type 1: a cross-sectional study.

Familial Hypocalciuric Hypercalcaemia (FHH) Type 1 is caused by an inactivating mutation in the calcium-sensing receptor (CASR) gene resulting in elevated plasma calcium levels. We investigated whether FHH is associated with change in bone density and structure. We compared 50 FHH patients with age- and gender-matched population-based controls (mean age 56years, 69% females). We assessed areal BMD (aBMD) by DXA-scans and total, cortical, and trabecular volumetric BMD (vBMD) as well as bone geometry by quantitative computed tomography (QCT) and High-Resolution peripheral-QCT (HR-pQCT). Compared with controls, FHH females had a higher total and trabecular hip vBMD and a lower cortical vBMD and hip bone volume. Areal BMD and HRpQCT indices did not differ except an increased trabecular thickness and an increased vBMD at the transition zone between cancellous and cortical bone in of the tibia in FHH. Finite element analyses showed no differences in bone strength. Multiple regression analyses revealed correlations between vBMD and P-Ca(2+) levels but not with P-PTH. Overall, bone health does not seem to be impaired in patients with FHH. In FHH females, bone volume is decreased, with a lower trabecular volume but a higher vBMD, whereas cortical vBMD is decreased in the hip. This may be due to either an impaired endosteal resorption or corticalization of trabecular bone. The smaller total bone volume suggests an impaired periosteal accrual, but bone strength is not impaired. The findings of more pronounced changes in females may suggest an interaction between sex hormones and the activity of the CaSR on bone.

A mutation in the 5'-UTR of GNA11 causes familial hypocalciuric hypercalcemia type 2 due to reduced translational efficiency

A mutation in the 5'-UTR of GNA11 causes familial hypocalciuric hypercalcemia type 2 due to reduced translational efficiency

Clinical studies of adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 reveal genotype-phenotype correlations and effectiveness of cinacalcet

Clinical studies of adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 reveal genotype-phenotype correlations and effectiveness of cinacalcet

Identification of 12 adaptor protein-2 sigma 2 subunit mutations in familial hypocalciuric hypercalcaemia type 3 and expansion of phenotypic spectrum.

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

Ruling in a Suspect: The Role ofAP2S1Mutations in Familial Hypocalciuric Hypercalcemia Type 3

Ruling in a Suspect: The Role of<i>AP2S1</i>Mutations in Familial Hypocalciuric Hypercalcemia Type 3

Identification ofAP2S1Mutation and Effects of Low Calcium Formula in an Infant With Hypercalcemia and Hypercalciuria

Although AP2S1 has recently been shown to be a causative gene for familial hypocalciuric hypercalcemia type 3 (FHH3), knowledge about FHH3 remains poor. Our objective was to report AP2S1 mutation and effects of low calcium formula in a patient with hypercalcemia and hypercalciuria. This Japanese female infant was found to have hypercalcemia by a routine laboratory test for poor weight gain on breast feeding. At 49 days of age, serum calcium (adjusted by Payne's formula) was 13.1 mg/dL, intact PTH 27 pg/mL, and urinary calcium-to-creatinine ratio 1.29 mg/mg. There was no evidence for hyperparathyroidism, PTHrP-producing neoplasm, and vitamin D excess. These data, except for hypercalciuria, appeared to be consistent with defective calcium-sensing receptor-mediated signaling. With use of low calcium formula containing 2.6 mg/dL of calcium, she showed catch-up growth, and serum calcium was decreased, as was urinary calcium-to-creatinine ratio. Furthermore, feeding with a mixture of low calcium formula and standard formula with a 2:1 ratio maintained serum calcium ∼12 mg/dL without markedly increasing serum PTH. Although no pathologic mutation was detected in CASR or GNA11, a presumably de novo heterozygous mutation (p.Arg15Leu), a previously reported causative mutation for FHH3, was identified in AP2S1 of this patient. The results imply that lack of hypocalciuria does not necessarily argue against the presence of AP2S1 mutations. The early infantile age of this patient would have played a certain role in the occurrence of hypercalciuria, and low calcium formula is worth attempting in infants with FHH.

Mutations Affecting G-Protein Subunit α11 in Hypercalcemia and Hypocalcemia

BackgroundFamilial hypocalciuric hypercalcemia is a genetically heterogeneous disorder with three variants: types 1, 2, and 3. Type 1 is due to loss-of-function mutations of the calcium-sensing receptor, a guanine nucleotide–binding protein (G-protein)–coupled receptor that signals through the G-protein subunit α11 (Gα11). Type 3 is associated with adaptor-related protein complex 2, sigma 1 subunit (AP2S1) mutations, which result in altered calcium-sensing receptor endocytosis. We hypothesized that type 2 is due to mutations effecting Gα11 loss of function, since Gα11 is involved in calcium-sensing receptor signaling, and its gene (GNA11) and the type 2 locus are colocalized on chromosome 19p13.3. We also postulated that mutations effecting Gα11 gain of function, like the mutations effecting calcium-sensing receptor gain of function that cause autosomal dominant hypocalcemia type 1, may lead to hypocalcemia.MethodsWe performed GNA11 mutational analysis in a kindred with familial hypocalciuric hypercalcemia type 2 and in nine unrelated patients with familial hypocalciuric hypercalcemia who did not have mutations in the gene encoding the calcium-sensing receptor (CASR) or AP2S1. We also performed this analysis in eight unrelated patients with hypocalcemia who did not have CASR mutations. In addition, we studied the effects of GNA11 mutations on Gα11 protein structure and calcium-sensing receptor signaling in human embryonic kidney 293 (HEK293) cells.ResultsThe kindred with familial hypocalciuric hypercalcemia type 2 had an in-frame deletion of a conserved Gα11 isoleucine (Ile200del), and one of the nine unrelated patients with familial hypocalciuric hypercalcemia had a missense GNA11 mutation (Leu135Gln). Missense GNA11 mutations (Arg181Gln and Phe341Leu) were detected in two unrelated patients with hypocalcemia; they were therefore identified as having autosomal dominant hypocalcemia type 2. All four GNA11 mutations predicted disrupted protein structures, and assessment on the basis of in vitro expression showed that familial hypocalciuric hypercalcemia type 2–associated mutations decreased the sensitivity of cells expressing calcium-sensing receptors to changes in extracellular calcium concentrations, whereas autosomal dominant hypocalcemia type 2–associated mutations increased cell sensitivity.ConclusionsGα11 mutants with loss of function cause familial hypocalciuric hypercalcemia type 2, and Gα11 mutants with gain of function cause a clinical disorder designated as autosomal dominant hypocalcemia type 2. (Funded by the United Kingdom Medical Research Council and others.)