Emma Webb

Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta

Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50–70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.

Current and novel approaches to children and young people with congenital adrenal hyperplasia and adrenal insufficiency

Congenital adrenal hyperplasia (CAH) represents a group of autosomal recessive conditions leading to glucocorticoid deficiency. CAH is the most common cause of adrenal insufficiency (AI) in the paediatric population. The majority of the other forms of primary and secondary adrenal insufficiency are rare conditions. It is critical to establish the underlying aetiology of each specific condition as a wide range of additional health problems specific to the underlying disorder can be found. Following the introduction of life-saving glucocorticoid replacement sixty years ago, steroid hormone replacement regimes have been refined leading to significant reductions in glucocorticoid doses over the last two decades. These adjustments are made with the aim both of improving the current management of children and young persons and of reducing future health problems in adult life. However despite optimisation of existing glucocorticoid replacement regimens fail to mimic the physiologic circadian rhythm of glucocorticoid secretion, current efforts therefore focus on optimising replacement strategies. In addition, in recent years novel experimental therapies have been developed which target adrenal sex steroid synthesis in patients with CAH aiming to reduce co-morbidities associated with sex steroid excess. These developments will hopefully improve the health status and long-term outcomes in patients with congenital adrenal hyperplasia and adrenal insufficiency.

Phenotypic variability in patients with osteogenesis imperfecta caused by BMP1 mutations

Osteogenesis Imperfecta (OI) is an inherited bone fragility disorder most commonly associated with autosomal dominant mutations in the type I collagen genes. Autosomal recessive mutations in a number of genes have also been described, including the BMP1 gene that encodes the mammalian Tolloid (mTLD) and its shorter isoform bone morphogenic protein‐1 (BMP1). To date, less than 20 individuals with OI have been identified with BMP1 mutations, with skeletal phenotypes ranging from mild to severe and progressively deforming. In the majority of patients, bone fragility was associated with increased bone mineral density (BMD); however, the full range of phenotypes associated with BMP1 remains unclear. Here, we describe three children with mutations in BMP1 associated with a highly variable phenotype: a sibship homozygous for the c.2188delC mutation that affects only the shorter BMP1 isoform and a further patient who is compound heterozygous for a c.1293C>G nonsense mutation and a c.1148G>A missense mutation in the CUB1 domain. These individuals had recurrent fractures from early childhood, are hypermobile and have no evidence of dentinogenesis imperfecta. The homozygous siblings with OI had normal areal BMD by dual energy X‐ray absorptiometry whereas the third patient presented with a high bone mass phenotype. Intravenous bisphosphonate therapy was started in all patients, but discontinued in two patients and reduced in another due to concerns about increasing bone stiffness leading to chalk‐stick fractures. Given the association of BMP1‐related OI with very high bone material density, concerns remain whether anti‐resorptive therapy is indicated in this ultra‐rare form of OI.© 2016 Wiley Periodicals, Inc.

Cardiovascular health, growth and gonadal function in children and adolescents with congenital adrenal hyperplasia

After the introduction of replacement therapy with glucocorticoids and mineralocorticoids in the 1950s, congenital adrenal hyperplasia (CAH) is no longer a life-limiting condition. However, due to the successful introduction of medical steroid hormone replacement, CAH has become a chronic condition, with associated comorbidities and long-term health implications. The aim of treatment is the replacement of mineralocorticoids and glucocorticoids and the normalisation of elevated androgen concentrations. Long-term consequences of the condition and current treatment regimens include unfavourable changes in the cardiovascular risk profile, impaired growth, testicular adrenal rest tumours (TART) in male and subfertility in both male and female patients with CAH. Optimising replacement therapy in patients with CAH remains challenging. On one hand, treatment with supraphysiological doses of glucocorticoids might be required to normalise androgen concentrations and decrease size or presence of TARTs. On the other hand, treatment with supraphysiological doses of glucocorticoids is associated with an increased prevalence of unfavourable cardiovascular and metabolic risk profiles as well as impaired longitudinal growth and gonadal function. Therefore, treatment of children and adults with CAH requires an individualised approach. Careful monitoring for early signs of complications is already warranted during paediatric healthcare provision to prevent and reduce the impact of comorbidities in later life.

Keeping the pressure on mineralocorticoid replacement in congenital adrenal hyperplasia

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is an autosomal recessive disorder and represents one of the most common inborn disorders of metabolism. Based on the recent outcome studies, awareness of long-term health problems in CAH has significantly increased among many different medical specialities caring for patients with CAH. One particular issue is the variability of frequency, onset and development of hypertension in patients with CAH. It has been, however, documented that appropriate replacement with fludrocortisone will lower the required amount of glucocorticoids. This finding is significant as cumulative glucocorticoid exposure is associated with several potential long-term adverse effects in patients with CAH. Mineralocorticoid requirement is variable and physiological mineralocorticoid resistance is most pronounced during early life subsequently decreasing with age. During infancy, childhood and adolescence, the relative fludrocortisone dose decreases with improved sensitivity to mineralcorticoids in the kidney. To avoid salt loss in neonates and infants with CAH, the European Society for Paediatric Endocrinology/Lawson Wilkinson Paediatric Endocrine Society (ESPE/LWPES) consensus statement on 21-hydroxylase deficiency from 2002 suggested that sodium supplements are often required. Due the salt-loss continuum in patients with CAH, the most recent Endocrine Society guideline recommends monitoring also for subclinical mineralocorticoid deficiency and treatment with fludrocortisone and sodium chloride supplementation for all patients with classic CAH in the newborn period and early infancy. The majority of studies analysing blood pressure in patients with CAH have studied patients older than 5 years with different studies reporting highly variable outcomes. So far, only one study has analysed the blood pressure in 24 CAH patients during the first year of life. All of these patients received replacement with fludrocortisone and sodium chloride and were found to be normotensive. In this issue of Clinical Endocrinology, Bonfig and Schwarz present an assessment of their practice analysing blood pressure and confounding factors in 33 patients identified with CAH during newborn screening from birth to age 4 years. All patients had a diagnosis of classic CAH and were treated solely with glucocorticoids and mineralocorticoids. None of the patients received sodium replacement. This was based on the assumption that sodium replacement would represent an additional risk factor for the development of hypertension. Plasma renin activity (PRA) was closely monitored on a monthly basis during the first three months of life, followed by three monthly intervals until the age of 2 years. From 2 years of age, PRA was checked every 6 months. The authors aimed for normalization of PRA. The targets for 17-hydroxyprogesterone (<18 nmol/l) were more stringent than the suggested target ranges of 12–30 nmol/l. Interestingly, hydrocortisone doses were relatively low during the study period. The previously suggested glucocorticoid actions of fludrocortisone in early life have not been discussed in this study and might have contributed to the low glucocorticoid requirements. Fludrocortisone doses in the current report were within the range suggested by the most recent Endocrine Society guidelines (absolute doses of 50–200 lg). However, these doses are higher than doses used in our clinical practice where commonly relative fludrocortisone doses do not exceed 150–200 lg/m per day. Patients in this study were on significantly higher doses than this during the first 18 months of life. The authors found a significant rate of hypertension despite titrating fludrocortisone doses according to PRA. This might be partly explained by aiming for PRA targets within the normal range. In concordance with increasing mineralocorticoid sensitivity during life, fludrocortisone requirements decreased during the first 48 months of life. Thirty per cent of patients had an increased blood pressure at three months of age, and 40% of patients were hypertensive at six months of age. This rate further increased to more than half of patients (58%) having elevated blood pressure at 18 months of age. Fludrocortisone doses administered during the first 9 months of life were significantly different between hypertensive and normotensive patients identified during the first 36 months of life. Only at the age of 48 months did the patients with hypertension and normotension receive similar fludrocortisone doses. The highest rate of Correspondence: Nils Krone, Centre for Endocrinology, Diabetes & Metabolism, School of Clinical and Experimental Medicine, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Tel.: +44 121 414 2540; E-mail: n.p.krone@bham.ac.uk

Phenotypic Spectrum in Osteogenesis Imperfecta Due to Mutations in TMEM38B: Unraveling a Complex Cellular Defect

Context Recessive mutations in TMEM38B cause type XIV osteogenesis imperfecta (OI) by dysregulating intracellular calcium flux. Objectives Clinical and bone material phenotype description and osteoblast differentiation studies. Design and Setting Natural history study in pediatric research centers. Patients Eight patients with type XIV OI. Main Outcome Measures Clinical examinations included bone mineral density, radiographs, echocardiography, and muscle biopsy. Bone biopsy samples (n = 3) were analyzed using histomorphometry, quantitative backscattered electron microscopy, and Raman microspectroscopy. Cellular differentiation studies were performed on proband and control osteoblasts and normal murine osteoclasts. Results Type XIV OI clinical phenotype ranges from asymptomatic to severe. Previously unreported features include vertebral fractures, periosteal cloaking, coxa vara, and extraskeletal features (muscular hypotonia, cardiac abnormalities). Proband lumbar spine bone density z score was reduced [median -3.3 (range -4.77 to +0.1; n = 7)] and increased by +1.7 (1.17 to 3.0; n = 3) following bisphosphonate therapy. TMEM38B mutant bone has reduced trabecular bone volume, osteoblast, and particularly osteoclast numbers, with >80% reduction in bone resorption. Bone matrix mineralization is normal and nanoporosity low. We demonstrate a complex osteoblast differentiation defect with decreased expression of early markers and increased expression of late and mineralization-related markers. Predominance of trimeric intracellular cation channel type B over type A expression in murine osteoclasts supports an intrinsic osteoclast defect underlying low bone turnover. Conclusions OI type XIV has a bone histology, matrix mineralization, and osteoblast differentiation pattern that is distinct from OI with collagen defects. Probands are responsive to bisphosphonates and some show muscular and cardiovascular features possibly related to intracellular calcium flux abnormalities.

Quantitative MRI brain in congenital adrenal hyperplasia: in vivo assessment of the cognitive and structural impact of congenital adrenal hyperplasia

Abstract Context Brain white matter hyperintensities are seen on routine clinical imaging in 46% of adults with congenital adrenal hyperplasia (CAH). The extent and functional relevance of these abnormalities have not been studied with quantitative magnetic resonance imaging (MRI) analysis. Objective To examine white matter microstructure, neural volumes, and central nervous system (CNS) metabolites in CAH due to 21-hydroxylase deficiency (21OHD) and to determine whether identified abnormalities are associated with cognition, glucocorticoid, and androgen exposure. Design, Setting, and Participants A cross-sectional study at a tertiary hospital including 19 women (18 to 50 years) with 21OHD and 19 age-matched healthy women. Main Outcome Measure Recruits underwent cognitive assessment and brain imaging, including diffusion weighted imaging of white matter, T1-weighted volumetry, and magnetic resonance spectroscopy for neural metabolites. We evaluated white matter microstructure by using tract-based spatial statistics. We compared cognitive scores, neural volumes, and metabolites between groups and relationships between glucocorticoid exposure, MRI, and neurologic outcomes. Results Patients with 21OHD had widespread reductions in white matter structural integrity, reduced volumes of right hippocampus, bilateral thalami, cerebellum, and brainstem, and reduced mesial temporal lobe total choline content. Working memory, processing speed, and digit span and matrix reasoning scores were reduced in patients with 21OHD, despite similar education and intelligence to controls. Patients with 21OHD exposed to higher glucocorticoid doses had greater abnormalities in white matter microstructure and cognitive performance. Conclusion We demonstrate that 21OHD and current glucocorticoid replacement regimens have a profound impact on brain morphology and function. If reversible, these CNS markers are a potential target for treatment.

How close is the dose? Manipulation of 10 mg hydrocortisone tablets to provide appropriate doses to children

ABSTRACT This study explores the methodology advised by healthcare professionals and the methods used by parents/carers to identify whether there is a best practice method for manipulation of 10mg hydrocortisone tablets to provide an accurate dose to children. Bespoke surveys were used to identify methods recommended and used in manipulation of tablets. Hydrocortisone tablets were manipulated to provide a specified dose by both naïve participants and parents/carers. The accuracy of manipulation was assessed using HPLC analysis. Competed surveys were received from 159 parent/carers reporting doses that ranged from 0.25 to 15mg. Parents/carers most commonly reported splitting the tablet and administering the solid fraction; however more than 30% of those reporting physically splitting tablets were preparing doses that were not simply halving or quartering tablets. In a naïve population the dose accuracy, defined as percent of doses within 20% of the theoretical dose ranged from 57 to 58% depending on the tablet brand and the method of manipulation used. Almost three‐quarters (74.1%) of parent/carers (n=27) were able to produce a dose within 20% of the theoretical value and the most accurate method was to split tablets and administer the solid fraction. This study shows that a lack of age‐appropriate medicines results in children being at risk of sub‐optimal dosing.

G219 Hydrocortisone tablets: human factors in manipulation and their impact on dosing accuracy

Background Exposure to deficient or excess glucocorticoids is associated with increased morbidity in patients with adrenal insufficiency. An age-appropriate low dose hydrocortisone formulation is not available and manipulation of adult medication is required with potential for inaccurate dosing. Licensed pharmaceutical products must contain ±10% of labelled drug content. Aims To assess the variability in manipulation procedures undertaken by parents/carers and to quantify the dose-variability in the manipulated product based on the method of preparation. Methods Parents of children with adrenal insufficiency completed a survey assessing the methods used to manipulate hydrocortisone 10 mg tablets. A sub-group were asked to manipulate a scored 10 mg hydrocortisone tablet (Auden Mackenzie brand) to provide the prescribed dose for their child as they would at home. Hydrocortisone content was analysed according to the current European Pharmacopoeial method. Results One hundred and twenty-nine parents completed the questionnaire. Overall 55% of parents break or cut the tablet and 43% suspend the tablet in water prior to administration. 34% are prescribed a dose indivisible by 2.5 mg of whom 33% break the tablet to acquire the dose. Twenty-seven parents/carers participated in the sub-study and the target doses they prepared ranged from 0.5–7.5 mg. Forty eight percent of the preparations were within 10% of the target dose; 74% were within 20% and 82% were within 30%. Based on this small sample size the most accurate method of tablet manipulation is to split the tablet along the score lines. However, this is only possible for doses divisible by 2.5 mg. Dispersion of the tablet in water and withdrawal of the relevant volume was associated with poor accuracy. Conclusions Children are at risk of suboptimal dosing when parents/carers are required to manipulate adult products to provide the appropriate dose to children. This risk is greatest when doses need to be prepared via dispersion of a tablet and calculation of the volume to withdraw. This may be related to the poor solubility of hydrocortisone which makes formation of a homogenous liquid difficult. There is a need for age-appropriate hydrocortisone products to be available to children. Acknowledgement This study was funded via an unrestricted research grant from Diurnal Ltd.