Angela Rogers

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.

Cinacalcet for Symptomatic Hypercalcemia Caused by AP2S1 Mutations

The authors show that cinacalcet, which mediates allosteric modulation of the calcium-sensing receptor, corrects loss of function of AP2S1 mutations that cause familial hypocalciuric hypercalcemia type 3 and ameliorates symptomatic hypercalcemia.

Diagnosis and management of prolactinomas and non-functioning pituitary adenomas.

#### Summary points Pituitary tumours account for a major proportion of intracranial tumours; most are benign adenomas. The tumours present with a range of signs and symptoms. Although evidence suggests that they are diagnosed earlier than previously, some are still missed, often for years, with clues such as headache and visual problems not being picked up.1 Management by a multidisciplinary team improves outcomes and optimises resources. This article focuses on the diagnosis and management of prolactinomas and non-functioning pituitary adenomas, which are the commonest types of pituitary adenoma in clinical practice. #### Sources and selection criteria We searched PubMed to identify peer reviewed original articles and reviews. Search terms were “pituitary tumour”, “pituitary adenoma”, “pituitary neoplasm”, and “prolactinoma”. We only considered those papers that were written in English and published within the past 20 years. Pituitary adenomas are monoclonal benign tumours that arise from differentiated hormone expressing cells in the anterior pituitary. Five major cell types occur in the anterior pituitary: the lactrotroph, producing prolactin; the somatotroph, producing growth hormone; the corticotroph, producing adrenocorticotrophic hormone; the thyrotroph, producing thyroid stimulating hormone; and the gonadotroph, producing luteinising hormone and follicle stimulating hormone. Classification of pituitary adenomas is based in part on the cell type. Tumours that secrete hormones are called functioning adenomas …

Mutational analysis of the adaptor protein 2 sigma subunit (AP2S1) gene: search for autosomal dominant hypocalcemia type 3 (ADH3).

CONTEXT Autosomal dominant hypocalcemia (ADH) types 1 and 2 are due to calcium-sensing receptor (CASR) and G-protein subunit-α11 (GNA11) gain-of-function mutations, respectively, whereas CASR and GNA11 loss-of-function mutations result in familial hypocalciuric hypercalcemia (FHH) types 1 and 2, respectively. Loss-of-function mutations of adaptor protein-2 sigma subunit (AP2σ 2), encoded by AP2S1, cause FHH3, and we therefore sought for gain-of-function AP2S1 mutations that may cause an additional form of ADH, which we designated ADH3. OBJECTIVE The objective of the study was to investigate the hypothesis that gain-of-function AP2S1 mutations may cause ADH3. DESIGN The sample size required for the detection of at least one mutation with a greater than 95% likelihood was determined by binomial probability analysis. Nineteen patients (including six familial cases) with hypocalcemia in association with low or normal serum PTH concentrations, consistent with ADH, but who did not have CASR or GNA11 mutations, were ascertained. Leukocyte DNA was used for sequence and copy number variation analysis of AP2S1. RESULTS Binomial probability analysis, using the assumption that AP2S1 mutations would occur in hypocalcemic patients at a prevalence of 20%, which is observed in FHH patients without CASR or GNA11 mutations, indicated that the likelihood of detecting at least one AP2S1 mutation was greater than 95% and greater than 98% in sample sizes of 14 and 19 hypocalcemic patients, respectively. AP2S1 mutations and copy number variations were not detected in the 19 hypocalcemic patients. CONCLUSION The absence of AP2S1 abnormalities in hypocalcemic patients, suggests that ADH3 may not occur or otherwise represents a rare hypocalcemic disorder.

Transsphenoidal pituitary surgery in the elderly is safe and effective

Abstract Object. With an increasingly ageing population, the number of elderly people diagnosed with pituitary tumours continues to rise. There is a concern that with increasing age and comorbidities, there is higher anaesthetic risk, as well as peri-operative morbidity and mortality from pituitary surgery. This study aimed to audit the benefits and complications of transsphenoidal surgery performed in a large pituitary centre in elderly patients. Methods. Data on all elderly patients (age: ≥ 70 years) undergoing transsphenoidal surgery at a large tertiary referral centre between November 2003 and August 2012 were collected retrospectively. Results. A total of 104 operations were performed on 102 patients during 106 months. Median age was 75.2 years (range: 70–94) and 63 (61%) of the patients were male. Median follow-up was 15.2 months (range: 2.3–84.4). The majority presented with either peripheral visual field defects (26.4%) or pituitary hormone deficits (17.9%). A significant number (21.7%) of tumours were incidental radiological findings while investigating other diagnoses like stroke and dementia. 48.1% of operations were undertaken microscopically and the remaining 51.9% were endoscopic. Median hospital stay was 4 days (range: 3–18). Intra-operative complications included hypotension (1.9%) and blood loss requiring transfusion (2.9%). The 30-day complications included transient diabetes insipidus (9.6%), syndrome of inappropriate anti-diuretic hormone secretion (8.7%), delayed cerebrospinal fluid leak requiring lumbar drainage (0.9%) with no patient requiring formal repair. There were no peri-operative deaths. Long-term assessment suggested 79% had improved or stable endocrine function with 7% achieving biochemical cure and 91% showed improved or stable visual fields. Conclusions. Pituitary surgery in the elderly, whether microscopic or endoscopic, has low morbidity and mortality and is a safe and effective intervention for both symptom control and functional outcomes.

Gα11 mutation in mice causes hypocalcemia rectifiable by calcilytic therapy

Heterozygous germline gain-of-function mutations of G-protein subunit α11 (Gα11), a signaling partner for the calcium-sensing receptor (CaSR), result in autosomal dominant hypocalcemia type 2 (ADH2). ADH2 may cause symptomatic hypocalcemia with low circulating parathyroid hormone (PTH) concentrations. Effective therapies for ADH2 are currently not available, and a mouse model for ADH2 would help in assessment of potential therapies. We hypothesized that a previously reported dark skin mouse mutant (Dsk7) — which has a germline hypermorphic Gα11 mutation, Ile62Val — may be a model for ADH2 and allow evaluation of calcilytics, which are CaSR negative allosteric modulators, as a targeted therapy for this disorder. Mutant Dsk7/+ and Dsk7/Dsk7 mice were shown to have hypocalcemia and reduced plasma PTH concentrations, similar to ADH2 patients. In vitro studies showed the mutant Val62 Gα11 to upregulate CaSR-mediated intracellular calcium and MAPK signaling, consistent with a gain of function. Treatment with NPS-2143, a calcilytic compound, normalized these signaling responses. In vivo, NPS-2143 induced a rapid and marked rise in plasma PTH and calcium concentrations in Dsk7/Dsk7 and Dsk7/+ mice, which became normocalcemic. Thus, these studies have established Dsk7 mice, which harbor a germline gain-of-function Gα11 mutation, as a model for ADH2 and have demonstrated calcilytics as a potential targeted therapy.

AP2σ Mutations Impair Calcium-Sensing Receptor Trafficking and Signaling, and Show an Endosomal Pathway to Spatially Direct G-Protein Selectivity

Summary Spatial control of G-protein-coupled receptor (GPCR) signaling, which is used by cells to translate complex information into distinct downstream responses, is achieved by using plasma membrane (PM) and endocytic-derived signaling pathways. The roles of the endomembrane in regulating such pleiotropic signaling via multiple G-protein pathways remain unknown. Here, we investigated the effects of disease-causing mutations of the adaptor protein-2 σ subunit (AP2σ) on signaling by the class C GPCR calcium-sensing receptor (CaSR). These AP2σ mutations increase CaSR PM expression yet paradoxically reduce CaSR signaling. Hypercalcemia-associated AP2σ mutations reduced CaSR signaling via Gαq/11 and Gαi/o pathways. The mutations also delayed CaSR internalization due to prolonged residency time of CaSR in clathrin structures that impaired or abolished endosomal signaling, which was predominantly mediated by Gαq/11. Thus, compartmental bias for CaSR-mediated Gαq/11 endomembrane signaling provides a mechanistic basis for multidimensional GPCR signaling.

N-ethyl-N-nitrosourea–Induced Adaptor Protein 2 Sigma Subunit 1 (Ap2s1) Mutations Establish Ap2s1 Loss-of-Function Mice

The adaptor protein‐2 sigma subunit (AP2σ), encoded by AP2S1, forms a heterotetrameric complex, with AP2α, AP2β, and AP2μ subunits, that is pivotal for clathrin‐mediated endocytosis, and AP2σ loss‐of‐function mutations impair internalization of the calcium‐sensing receptor (CaSR), a G‐protein–coupled receptor, and cause familial hypocalciuric hypercalcemia type‐3 (FHH3). Mice with AP2σ mutations that would facilitate investigations of the in vivo role of AP2σ, are not available, and we therefore embarked on establishing such mice. We screened >10,000 mice treated with the mutagen N‐ethyl‐N‐nitrosourea (ENU) for Ap2s1 mutations and identified 5 Ap2s1 variants, comprising 2 missense (Tyr20Asn and Ile123Asn) and 3 intronic base substitutions, one of which altered the invariant donor splice site dinucleotide gt to gc. Three‐dimensional modeling and cellular expression of the missense Ap2s1 variants did not reveal them to alter AP2σ structure or CaSR‐mediated signaling, but investigation of the donor splice site variant revealed it to result in an in‐frame deletion of 17 evolutionarily conserved amino acids (del17) that formed part of the AP2σ α1‐helix, α1‐β3 loop, and β3 strand. Heterozygous mutant mice (Ap2s1+/del17) were therefore established, and these had AP2σ haplosufficiency but were viable with normal appearance and growth. Ap2s1+/del17 mice, when compared with Ap2s1+/+ mice, also had normal plasma concentrations of calcium, phosphate, magnesium, creatinine, urea, sodium, potassium, and alkaline phosphatase activity; normal urinary fractional excretion of calcium, phosphate, sodium, and potassium; and normal plasma parathyroid hormone (PTH) and 1,25‐dihydroxyvitamin D (1,25(OH)2) concentrations. However, homozygous Ap2s1del17/del17 mice were non‐viable and died between embryonic days 3.5 and 9.5 (E3.5–9.5), thereby indicating that AP2σ likely has important roles at the embryonic patterning stages and organogenesis of the heart, thyroid, liver, gut, lungs, pancreas, and neural systems. Thus, our studies have established a mutant mouse model that is haplosufficient for AP2σ.

Clinically relevant genetic advances in endocrinology

#### Key points Genetic advances have enabled redefinition of diseases according to their true causes and this molecular-based taxonomy has transformed our understanding of medicine and improved diagnosis and treatments for patients[1][1] Genetic testing is helpful in clinical practice in several