Jeremy Allgrove

A Missense Glial Cells Missing Homolog B (GCMB) Mutation, Asn502His, Causes Autosomal Dominant Hypoparathyroidism

CONTEXT Glial cells missing B (GCMB), the mammalian homolog of the Drosophila GCM gene, encodes a 506-amino-acid parathyroid-specific transcription factor. To date, only two different heterozygous GCMB mutations have been reported in three kindreds with autosomal dominant hypoparathyroidism. OBJECTIVE Our objective was to investigate a family with autosomal dominant hypoparathyroidism for PTH, CaSR, and GCMB mutations. METHODS Leukocyte DNA was used with exon-specific primers for PCR amplification and the DNA sequences of the PCR products determined. Functional analyses using fluorescence microscopy, EMSAs, and luciferase reporter assays were undertaken. Informed consent was obtained using protocols approved by a national ethical committee. RESULTS DNA sequence analysis revealed an A to C transversion at codon 502 of GCMB, which altered the wild-type asparagine (Asn) to histidine (His). Functional studies, using transient transfections of COS7 cells with GCMB wild-type and mutant (Asn502His) tagged constructs, demonstrated that the wild-type and mutant proteins localized to the nucleus and retained the ability to bind the GCM-consensus DNA recognition motif. However, a luciferase reporter assay demonstrated that the Asn502His mutation resulted in a reduction in gene transactivation. Moreover, cotransfection of the wild-type with mutant did not lead to an increase in luciferase activity, thereby demonstrating a dominant-negative effect of the Asn502His mutant that would be consistent with an autosomal dominant inheritance. CONCLUSION Our results, which have identified the first dominant missense GCMB mutation, help to increase our understanding of the mechanism underlying gene transactivation that is a prerequisite for the function of this parathyroid gland-specific transcription factor.

Physiology of Calcium, Phosphate and Magnesium

The physiology of calcium and the other minerals involved in its metabolism is complex and intimately tied in with the physiology of bone. Five principal humoral factors are involved in maintaining plasma levels of calcium, magnesium and phosphate and coordinating the balance between these and their content in bone. The transmembrane transport of these elements is dependent on a series of complex mechanisms that are controlled by these hormones. The plasma concentration of calcium is initially sensed by a calcium-sensing receptor which then sets up a cascade of events that initially determines parathyroid hormone secretion and eventually results in a specific action within the target organs, mainly bone and kidney. This chapter describes the physiology of these humoral factors and relates them to the pathological processes that give rise to disorders of calcium and bone metabolism. It details the stages in the calcium cascade and describes the effects on the various target organs. The pathology of disorders of bone and calcium metabolism is described in detail in the relevant chapters.

Growth and Pubertal Delay in Patients with Epidermolysis Bullosa

Puberty is the acquisition of secondary sexual characteristics, associated with a growth spurt, resulting in the attainment of reproductive function and final adult height. Delayed puberty is defined as the absence of any pubertal development at an age 2 standard deviations (SD) more than the mean, which corresponds to an age of approximately 14 years for boys and 13 years for girls. The degree to which growth and pubertal development are affected in chronic illness depends on the disease itself, as well as factors such as age of onset, duration and severity; the earlier the onset and the more severe the disease, the greater the effect on growth and pubertal development. Most children with severe types of epidermolysis bullosa have abnormal growth and pubertal delay. The possible pathophysiology is discussed.

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.

Calcium deficiency rickets: extending the spectrum of ‘nutritional’ rickets

Rickets is a condition of growing children in which there is failure of normal mineralisation of the epiphyseal growth plate. Normal growth plate development is dependent upon a cascade of events that consists of resting chondrocytes becoming preproliferative, proliferative, hypertrophic and finally apoptotic, which is a signal for the invasion of the growth plate by bone-forming cell precursors that replace the apoptosed cartilage cells with bone cells that mineralise the epiphyseal growth plate into true bone. Eventually, the growth plate is obliterated as the metaphyseal and epiphyseal ends of the bones fuse. It is therefore evident that rickets can only occur in growing children in whom the growth plate is still not fused. The final step of apoptosis is promoted by phosphate which has to be present in sufficient concentration to facilitate apoptosis1. In the absence of enough phosphate, apoptosis does not proceed correctly, the growth plate widens and there is failure of normal invasion of bone-forming cell precursors. This results in the typical radiological appearances of rickets. Thus, it can be seen that any cause of hypophosphataemia may be associated with the development of rickets. Primary hypophosphataemic disorders are usually associated with raised Fibroblast Growth Factor 23 (FGF23) and normal parathyroid hormone (PTH) concentrations. By contrast, secondary hypophosphataemia is caused by relative calcium deficiency resulting from inadequate absorption either because of vitamin D deficiency, or because of an absolute deficiency of calcium in the diet, both of which …

A Practical Approach to Vitamin D Deficiency and Rickets

Rickets is a condition in which there is failure of the normal mineralisation (osteomalacia) of growing bone. Whilst osteomalacia may be present in adults, rickets cannot occur. It is generally caused by a lack of mineral supply, which can either occur as a result of the deficiency of calcium (calciopaenic rickets, now known as parathyroid hormone-dependent rickets) or of phosphate (phosphopaenic rickets, now called FGF23-dependent rickets). Renal disorders may also interfere with the process of mineralisation and cause rickets. Only parathyroid hormone-dependent rickets and distal renal tubular disorders will be discussed in this chapter. The most common cause of rickets is still vitamin D deficiency, which is also responsible for other problems. Disorders of vitamin D metabolism or responsiveness may also cause similar issues. Distal renal tubular acidosis may also be caused by a variety of metabolic errors similar to those of osteoclasts. One form of distal renal tubular acidosis also causes a type of osteopetrosis. This chapter describes these conditions in detail and sets out a logical approach for treatment.

Physiology of Calcium, Phosphate, Magnesium and Vitamin D.

The physiology of calcium and the other minerals involved in its metabolism is complex and intimately linked to the physiology of bone. Five principal humoral factors are involved in maintaining plasma concentrations of calcium, magnesium and phosphate and in coordinating the balance between their content in bone. The transmembrane transport of these elements is dependent on a series of complex mechanisms that are partly controlled by these hormones. The plasma concentration of calcium is initially sensed by a calcium-sensing receptor, which then sets up a cascade of events that initially determines parathyroid hormone secretion and eventually results in a specific action within the target organs, mainly bone and kidney. This chapter describes the physiology of these humoral factors and relates them to the pathological processes that give rise to disorders of calcium, phosphate and magnesium metabolism as well as of bone metabolism. This chapter also details the stages in the calcium cascade, describes the effects of calcium on the various target organs, gives details of the processes by which phosphate and magnesium are controlled and summarises the metabolism of vitamin D. The pathology of disorders of bone and calcium metabolism is described in detail in the relevant chapters.

A new multi-system disorder caused by the Gαs mutation p.F376V.

CONTEXT The α subunit of the stimulatory G protein (Gαs) links numerous receptors to adenylyl cyclase. Gαs, encoded by GNAS, is expressed predominantly from the maternal allele in certain tissues. Thus, maternal heterozygous loss-of-function mutations cause hormonal resistance, as in pseudohypoparathyroidism type Ia, whereas somatic gain-of-function mutations cause hormone-independent endocrine stimulation, as in McCune-Albright syndrome. OBJECTIVE We report two unrelated boys presenting with a new combination of clinical findings that suggest both gain and loss of Gαs function. DESIGN AND SETTING Clinical features were studied and sequencing of GNAS was performed. Signaling capacities of wild-type and mutant Gαs were determined in the presence of different G protein-coupled receptors (GPCRs) under basal and agonist-stimulated conditions. RESULTS Both unrelated patients presented with unexplained hyponatremia in infancy, followed by severe early onset gonadotrophin-independent precocious puberty and skeletal abnormalities. An identical heterozygous de novo variant (c.1136T>G; p.F376V) was found on the maternal GNAS allele in both patients; this resulted in a clinical phenotype that differed from known Gαs-related diseases and suggested gain of function at the vasopressin 2 receptor (V2R) and lutropin/choriogonadotropin receptor (LHCGR), yet increased serum PTH concentrations indicative of impaired proximal tubular PTH1 receptor (PTH1R) function. In vitro studies demonstrated that Gαs-F376V enhanced ligand-independent signaling at the PTH1R, LHCGR, and V2R and, at the same time, blunted ligand-dependent responses. Structural homology modeling suggested mutation-induced modifications at the C-terminal α5 helix of Gαs that are relevant for interaction with GPCRs and signal transduction. CONCLUSIONS The Gαs p.F376V mutation causes a previously unrecognized multisystem disorder.

Early onset cataract in an infant with activating calcium-sensing receptor mutation

CONCLUSION  Hypoparathyroidism is known to be associated with cataract formation  As far as we are aware, this is the first reported case of cataract in infants with activating CaSR mutation, although it has been previously identified in a mouse model  We therefore suggest that evaluation for cataract is required in this subgroup of patients CLINICAL PRESENTATION  Admitted at seven days of life with focal seizures  Hypocalcaemia with inappropriately low PTH levels  Treated with intravenous calcium and magnesium  Discharged on oral calcium, magnesium, alfacalcidol  Re-admitted at five weeks; recurrent focal seizures  Hypocalcaemia persistent despite treatment  Stopped fixing and following over the last week  Nystagmus present and red reflexes absent  Ophthalmology confirmed dense bilateral cataract INVESTIGATIONS  Calcium 1.5 (2.0– 2.7) mmol/l  Magnesium 0.6 (0.7 1.0) mmol/l  Phosphate 3.7(1.3 2.6) mmol/l  PTH <0.3 (1.5 7.6) pmol/l

Anticonvulsant Treatment Associated with Intractable Hypocalcaemia in a Female Child with Hypoparathyroidism

Background: We report the case of a female infant with hypoparathyroidism due to an activating mutation in the calcium-sensing receptor gene. Case Report: The child presented in the neonatal period with clinical seizures associated with severe hypocalcaemia, hyperphosphataemia, low parathyroid hormone levels and elevated urine calcium:creatinine ratios. She required intravenous calcium and phenobarbitone initially, and then oral 1-alfacalcidol (1-AC) and phenobarbitone were started. The patient had intractable hypocalcaemia in the first 5 months of life despite escalating doses of 1-AC. When the phenobarbitone was stopped at 5 months of age she was admitted soon after with symptomatic hypercalcaemia. We postulate that the phenobarbitone increased the metabolism of 1-AC and thus she needed large doses of 1-AC to treat hypocalcaemia until the phenobarbitone was stopped. Her parents had no biochemical abnormalities on testing. Results: Molecular genetic analysis confirmed that our patient had a de novo missense variant, c.682G>A (p.Glu228Lys) in exon 4 of the calcium-sensing receptor. Conclusion: This case report highlights the importance that clinicians caring for children on vitamin D and its analogues are aware of the interaction with phenobarbitone, which can result in symptomatic hypocalcaemia. 1-AC should be stored at 2-8°C, otherwise it will be rendered inactive.