Bone Density and Trabecular Bone Score Decline Rapidly in the First Year After Bone Marrow Transplantation with a Marked Increase in 10-Year Fracture Risk.

As outcomes from allogeneic bone marrow transplantation (BMT) have improved, prevention of long-term complications, such as fragility fractures, has gained importance. We aimed to assess areal bone mineral density (aBMD) and trabecular bone score (TBS) changes post BMT, and determine their relationship with fracture prevalence. Patients who attended the Royal Melbourne Hospital (RMH) BMT clinic between 2005-2021 were included. Patient characteristics and dual-energy X-ray absorptiometry (DXA) values were collected from the electronic medical record and a survey. TBS iNsight™ was used to calculate TBS for DXA scans performed from 2019 onwards. 337 patients with sequential DXAs were eligible for inclusion. Patients were primarily male (60%) and mean age ± SD was 45.7 ± 13.4 years. The annualised decline in aBMD was greater at the femoral neck (0.066g/cm2 (0.0038-0.17)) and total hip (0.094g/cm2 (0.013-0.19)), compared to the lumbar spine (0.049g/cm2 (- 0.0032-0.16)), p < 0.0001. TBS declined independently of aBMD T-scores at all sites. Eighteen patients (5.3%) sustained 19 fractures over 3884 person-years of follow-up post-transplant (median follow-up 11 years (8.2-15)). This 5.3% fracture prevalence over the median 11-year follow-up period is higher than what would be predicted with FRAX® estimates. Twenty-two patients (6.5%) received antiresorptive therapy, and 9 of 18 (50%) who fractured received or were on antiresorptive therapy. In BMT patients, aBMD and TBS decline rapidly and independently in the first year post BMT. However, FRAX® fracture probability estimates incorporating these values significantly underestimate fracture rates, and antiresorptive treatment rates remain relatively low.

PRECeDe Pilot: Prevention of neonatal respiratory distress with antenatal corticosteroids before elective caesarean section in women with diabetes - a feasibility randomised trial.

OBJECTIVE: The PRECeDe Pilot Trial was designed to determine the feasibility of undertaking a multicentre, randomised controlled trial (RCT) to assess the efficacy of antenatal corticosteroids administration within 7 days before elective caesarean section (CS) in women with pre-gestational diabetes (PGDM) or gestational diabetes (GDM). DESIGN: Triple blind, parallel group, placebo-controlled, pilot RCT. SETTING: Single-centre tertiary maternity hospital in Melbourne, Australia. POPULATION: Pregnant women with PGDM (type 1 or type 2 diabetes) or GDM booked for a planned CS scheduled between 35+0 and 38+6 weeks of gestation. METHODS: Eligible participants were randomised to receive two injections of either betamethasone 11.4 mg or normal saline placebo, 24 hours apart within 7 days before CS scheduled between 35+0 and 38+6 weeks of gestation. MAIN OUTCOME MEASURE: The proportion of eligible women who consented and were randomised. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Registry ACTRN12619001475134. RESULTS: Of 537 women eligible, 182 were approached and 47 (26%) were recruited. Of these, 22 were allocated to the betamethasone group and 25 were allocated to the placebo group. There were no serious adverse events related to participation. CONCLUSION: It is feasible to undertake a triple-blind, placebo-controlled RCT investigating the efficacy of antenatal corticosteroids in preventing respiratory morbidity in infants of women with PGDM or GDM who are undergoing an elective CS between 35+0 and 38+6 weeks.

Ambiguous medical abbreviation study: challenges and opportunities.

BACKGROUND: Healthcare workers often abbreviate for convenience, but ambiguous abbreviations may cause miscommunication, which jeopardises patient care. Robust large-scale research to quantify abbreviation frequency and ambiguity in medical documents is lacking. AIMS: To calculate the frequency of abbreviations used in discharge summaries, the proportion of these abbreviations that are ambiguous and the potential utility of auto-expansion software. METHODS: We designed a software programme to extract all instances of abbreviations from every General Medical Unit discharge summary from the Royal Melbourne Hospital in 2015. We manually expanded abbreviations using published inventories and clinical experience, logging multiple expansions for any abbreviation if identified. Abbreviations were classified based on well defined criteria as standardised and likely to be well understood, or ambiguous. Outcome measures included the range and frequency of standardised and ambiguous abbreviations, and the feasibility of electronic auto-expansion software based on these measures. RESULTS: Of the 1 551 537 words analysed from 2336 documents, 137 997 (8.9%) were abbreviations with 1741 distinct abbreviations identified. Most abbreviations (88.7%) had a single expansion. The most common abbreviation was PO (per os/orally), followed by BD (bis in die/twice daily) and 68.1% of abbreviations were standardised, largely pertaining to pathology/chemicals. This meant, however, that a large proportion (31.9%) of abbreviations (2.8% of all words) were ambiguous. The most common ambiguous abbreviation was Pt (patient/physiotherapy), followed by LFT (liver function test/lung function test). CONCLUSIONS: Close to one-third of abbreviations used in general medical discharge summaries were ambiguous. Electronic auto-expansion of ambiguous abbreviations is likely to reduce miscommunication and improve patient safety.

Acute myeloid leukaemia presenting with diabetes insipidus.

A 41-year-old man was diagnosed with hypogonadotropic hypogonadism managed with gonadotropins after routine fertility review. Eight months later he presented with new polydipsia and polyuria, lethargy and easy bruising. A full blood count showed 28% circulating blasts. A bone marrow biopsy confirmed a diagnosis of acute myeloid leukaemia with inv(3)(q21.3q26.2) with additional monosomy 7. Central diabetes insipidus (DI) was diagnosed following a water deprivation test. Pituitary magnetic resonance imaging showed a slightly thickened pituitary stalk, stable Rathke's cyst, and new absence of the pituitary bright spot. The patient was commenced on desmopressin and induction chemotherapy, subsequently requiring a bone marrow transplant. Bone marrow examination at 100 days post-transplant revealed cytogenetic remission. All symptoms of DI resolved and magnetic resonance imaging showed return of the posterior bright spot and a pituitary stalk of normal thickness. Biochemical hypogonadotropic hypogonadism persisted but was uninterpretable in the context of systemic illness and recent chemotherapy. DI is a rare complication of haematological malignancies, and the prevalence and pathophysiology of DI in this context are poorly understood. Pathogenic mechanisms proposed include leukaemic infiltration of the pituitary, interference with antidiuretic hormone synthesis, and abnormal thrombopoiesis influencing hormone levels. Particular cytogenetic abnormalities such as inv(3)(q21.3q26.2) and monosomy 7 appear to be more commonly associated with DI and also appear to confer worse outcomes. Aetiologies in the literature remain elusive but as DI is a recognised association of haematological malignancies it should be considered in a patient presenting with polydipsia and polyuria.

Quantifying Use of a Health Virtual Community of Practice for General Practitioners' Continuing Professional Development: A Novel Methodology and Pilot Evaluation.

BACKGROUND: Health care practitioners (HPs), in particular general practitioners (GPs), are increasingly adopting Web-based social media platforms for continuing professional development (CPD). As GPs are restricted by time, distance, and demanding workloads, a health virtual community of practice (HVCoP) is an ideal solution to replace face-to-face CPD with Web-based CPD. However, barriers such as time and work schedules may limit participation in an HVCoP. Furthermore, it is difficult to gauge whether GPs engage actively or passively in HVCoP knowledge-acquisition for Web-based CPD, as GPs' competencies are usually measured with pre- and posttests. OBJECTIVE: This study investigated a method for measuring the engagement features needed for an HVCoP (the Community Fracture Capture [CFC] Learning Hub) for learning and knowledge sharing among GPs for their CPD activity. METHODS: A prototype CFC Learning Hub was developed using an Igloo Web-based social media software platform and involved a convenience sample of GPs interested in bone health topics. This Hub, a secure Web-based community site, included 2 key components: an online discussion forum and a knowledge repository (the Knowledge Hub). The discussion forum contained anonymized case studies (contributed by GP participants) and topical discussions (topics that were not case studies). Using 2 complementary tools (Google Analytics and Igloo Statistical Tool), we characterized individual participating GPs' engagement with the Hub. We measured the GP participants' behavior by quantifying the number of online sessions of the participants, activities undertaken within these online sessions, written posts made per learning topic, and their time spent per topic. We calculated time spent in both active and passive engagement for each topic. RESULTS: Seven GPs participated in the CFC Learning Hub HVCoP from September to November 2017. The complementary tools successfully captured the GP participants' engagement in the Hub. GPs were more active in topics in the discussion forum that had direct clinical application as opposed to didactic, evidence-based discussion topics (ie, topical discussions). From our knowledge hub, About Osteoporosis and Prevention were the most engaging topics, whereas shared decision making was the least active topic. CONCLUSIONS: We showcased a novel complementary analysis method that allowed us to quantify the CFC Learning Hub's usage data into (1) sessions, (2) activities, (3) active or passive time spent, and (4) posts made to evaluate the potential engagement features needed for an HVCoP focused on GP participants' CPD process. Our design and evaluation methods for ongoing use and engagement in this Hub may be useful to evaluate future learning and knowledge-sharing projects for GPs and may allow for extension to other HPs' environments. However, owing to the limited number of GP participants in this study, we suggest that further research with a larger cohort should be performed to validate and extend these findings.

Peripheral Quantitative Computed Tomography (pQCT) Measures Contribute to the Understanding of Bone Fragility in Older Patients With Low-trauma Fracture.

Dual-energy X-ray absorptiometry (DXA) as currently used has limitations in identifying patients with osteoporosis and predicting occurrence of fracture. We aimed to express peripheral quantitative computed tomography (pQCT) variables of patients with low-trauma fracture as T-scores by using T-score scales obtained from healthy young women, and to evaluate the potential clinical utility of pQCT for the assessment of bone fragility. Fracture patients were recruited from a fracture liaison service at the Royal Melbourne Hospital. Reference pQCT data were obtained from studies on women's health conducted by our group. A study visit was arranged with fracture patients, during which DXA and pQCT were applied to measure their bone strength. A total of 59 fracture patients were recruited, and reference data were obtained from 78 healthy young females. All DXA variables and most pQCT variables were significantly different between healthy young females and fracture patients (p < 0.05), except polar stress-strain index (p = 0.34) and cortical bone density (p = 0.19). Fracture patients were divided into osteoporosis and non-osteoporosis groups according to their DXA T-scores. Significant differences were observed in most pQCT variables (p < 0.05), except trabecular area and cortical density (p > 0.9 and p = 0.5, respectively). By applying pQCT T-scores, 11 (27%) of patients who were classified as having low or medium risk of osteoporosis on DXA T-scores alone were reclassified as high risk. Results of logistic regression suggested trabecular bone density as an independent predictor of osteoporosis status. More patients can be identified with osteoporosis by applying pQCT T-score variables in older people with low-trauma fracture. Peripheral QCT T-scores contribute to the understanding of bone fragility in this population.

Molecular and thermodynamic mechanisms of the chloride-dependent human angiotensin-I-converting enzyme (ACE).

Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg(522). Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu(403)-Lys(118) salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains.

Bridging the osteoporosis treatment gap: performance and cost-effectiveness of a fracture liaison service.

Individuals who sustain fragility fractures are at high risk of refracture. However, osteoporosis treatment rates remain low for these patients. Therefore, we aimed to assess the performance and cost-effectiveness of introducing a fracture liaison service (FLS) into a tertiary hospital. In "nonhospitalized" ambulatory patients who had sustained fragility fractures, we assessed baseline osteoporosis investigation and treatment rates, and subsequently, the impact of introducing an orthopedic osteoporosis policy and an FLS. Outcomes measured were uptake of osteoporosis intervention, patient satisfaction, and quality-adjusted life years (QALYs) gained. QALYs were calculated over 5 years using predicted fracture risks without intervention and estimated fracture risk reduction with intervention. At baseline (n = 49), 2% of ambulatory patients who had sustained fragility fractures underwent dual-energy X-ray absorptiometry (DXA) and 6% received osteoporosis-specific medication. After introduction of an osteoporosis policy (n = 58), 28% were investigated with DXA (p < 0.0001). However, treatment rates were unchanged. An FLS was introduced, reviewing 203 new patients over the inaugural 2 years (mean age [standard deviation], 67 (11) years; 77% female). All underwent DXA, and criteria for osteoporosis and osteopenia were identified in 44% and 40%, respectively. Osteoporosis medications were prescribed to 61% patients (risedronate: 22%, alendronate: 16%, strontium ranelate: 13%, zoledronic acid: 8%, other: 2%). Eighty-five of 90 questionnaire respondents were very satisfied or satisfied with the FLS. With the treatment prescribed over 5 years, we conservatively estimated that this FLS would reduce nonvertebral refractures from 59 to 50, improving QALYs by 0.054 and costing $1716 per patient (incremental cost-effectiveness ratio: $31749). This FLS model improves uptake of osteoporosis intervention guidelines, is popular among patients, and improves cost-effectiveness. Thus, it has the capacity to substantially improve health in a cost-effective way.

Angiotensin-I converting enzyme (ACE): structure, biological roles, and molecular basis for chloride ion dependence.

Somatic angiotensin-I converting enzyme (sACE) has an essential role in the regulation of blood pressure and electrolyte fluid homeostasis. It is a zinc protease that cleaves angiotensin-I (AngI), bradykinin, and a broad range of other signalling peptides. The enzyme activity is provided by two homologous domains (N- and C-), which display clear differences in substrate specificities and chloride activation. The presence of chloride ions in sACE and its unusual role in activity was identified early on in the characterisation of the enzyme. The molecular mechanisms of chloride activation have been investigated thoroughly through mutagenesis studies and shown to be substrate-dependent. Recent results from X-ray crystallography structural analysis have provided the basis for the intricate interactions between ACE, its substrate and chloride ions. Here we describe the role of chloride ions in human ACE and its physiological consequences. Insights into the chloride activation of the N- and C-domains could impact the design of improved domain-specific ACE inhibitors.

Divided dosing reduces prednisolone-induced hyperglycaemia and glycaemic variability: a randomized trial after kidney transplantation.

BACKGROUND: Prednisolone is a major risk factor for hyperglycaemia and new-onset diabetes after transplantation. Uncontrolled observational data suggest that divided dosing may reduce requirements for hypoglycaemic agents. This study aims to compare the glycaemic effects of divided twice daily (BD) and once daily (QD) prednisolone. METHODS: Twenty-two kidney transplant recipients without diabetes were randomized to BD or QD prednisolone. Three weeks post-transplant, a continuous glucose monitor (iPro2(®) Medtronic) was applied for 5 days with subjects continuing their initial prednisolone regimen (Days 1-2) before crossover to the alternative regimen. Mean glucose, peak glucose, nadir glucose, exposure to hyperglycaemia (glucose ≥7.8 mmol/L) and glycaemic variability were assessed. RESULTS: The mean ± standard deviation (SD) age of subjects was 50 ± 10 years and 77% were male. Median (interquartile range) daily prednisolone dose was 25 (20, 25) mg. BD prednisolone was associated with decreased mean glucose (mean 7.9 ± 1.7 versus 8.1 ± 2.3 mmol/L, P < 0.001), peak glucose [median 10.4 (9.5, 11.4) versus 11.4 (10.3, 13.4) mmol/L, P< 0.001] and exposure to hyperglycaemia [median 25.5 (14.6, 30.3) versus 40.4 (33.2, 51.2) mmol/L/h, P = 0.003]. Median glucose peaked between 14:55-15.05 h with BD and 15:25-15:30 h with QD. Median glycaemic variability scores were decreased with BD: SD (1.1 versus 1.9, P < 0.001), mean amplitude of glycaemic excursion (1.5 versus 2.2, P = 0.001), continuous overlapping net glycaemic action-1 (CONGA-1; 1.0 versus 1.2, P = 0.039), CONGA-2 (1.2 versus 1.4, P = 0.008) and J-index (25 versus 31, P = 0.003). CONCLUSIONS: Split prednisolone dosing reduces glycaemic variability and hyperglycaemia early post-kidney transplant.

A practical limited sampling strategy to predict free prednisolone exposure and glycemia in kidney transplant recipients.

BACKGROUND: Despite significant interindividual variability in prednisolone pharmacokinetics and potentially serious consequences with inadequate or excessive exposure, monitoring of prednisolone levels is not employed to guide therapy. As ultrahigh-performance liquid chromatography-tandem mass spectrometry methods can now measure the active free fraction of prednisolone, this study aimed to evaluate the performance of 15 published limited sampling strategies (LSSs) for predicting free prednisolone exposure in adult kidney transplant recipients and to examine the relationship between free/total prednisolone exposure and plasma glucose. METHODS: The study was performed in 11 subjects without diabetes 3-4 weeks postkidney transplantation. Area under the concentration time curve profiles of total and free prednisolone from 0 to 12 hours postdose (AUC0₋12) were determined and compared with predicted AUC0₋12 values calculated from published LSSs. Venous glucose was measured concurrently with the 13 sampling time points. RESULTS: The mean (±SD) age of subjects was 52 ± 12 years, 5 were men and the median (interquartile range) daily prednisolone dose was 20.0 mg (20.0-22.5). Interindividual variation in dose-adjusted free and total prednisolone exposure was 1.9- and 3.2-fold, respectively. All 15 free prednisolone LSSs exhibited good correlation (r ≥ 0.83), with bias and imprecision less than 15%. An LSS incorporating 1.25- and 3-hour samples had the highest predictive power (r = 0.97, bias 1.2%, imprecision 5.6%). Free prednisolone AUC0₋12 correlated with peak glucose levels (r = 0.65, P = 0.037), as did predicted AUC0₋12 from 14/15 LSSs. CONCLUSIONS: Biologically active free prednisolone exposure can be accurately predicted postkidney transplantation by LSSs incorporating 2-point concentration sampling. Peak plasma glucose concentration correlated well with prednisolone exposure.

Skeletal fluorosis secondary to methoxyflurane use for chronic pain.

Skeletal fluorosis is rare and occurs secondary to chronic high amounts of fluoride consumption, manifesting as diffuse osteosclerosis, skeletal pain, connective tissue calcification, and increased fracture risk. Methoxyflurane is a volatile, fluorinated hydrocarbon-inhaled analgesic, and the maximum recommended dose is 15 mL (99.9 % w/w) per wk. A rodent study found increased skeletal fluoride after methoxyflurane exposure. However, skeletal fluorosis secondary to methoxyflurane use in humans has rarely been reported. We present the case of a 47-yr-old female with diffuse osteosclerosis secondary to fluorosis from methoxyflurane use for chronic pain, presenting with 3 yr of generalized bony pain and multiple fragility fractures. Lumbar spine BMD was elevated. CT and radiographs demonstrated new-onset marked diffuse osteosclerosis, with calcification of interosseous membranes and ligaments, and a bone scan demonstrated a grossly increased uptake throughout the skeleton. Biochemistry revealed an elevated alkaline phosphatase and bone turnover markers, mild secondary hyperparathyroidism with vitamin D deficiency, and mild renal impairment. Zoledronic acid, prescribed for presumed Paget's disease, severely exacerbated bony pain. Urinary fluoride was elevated (7.3 mg/L; reference range < 3.0 mg/L) and the patient revealed using methoxyflurane 9 mL per wk for 8 yr for chronic pain. A decalcified bone biopsy revealed haphazardly arranged cement lines and osteocytes lacunae and canaliculi, which was consistent with an osteosclerotic process. Focal subtle basophilic stippling around osteocyte lacunae was suggestive of fluorosis. Although fluorosis is not a histological diagnosis, the presence of compatible histology features was supportive of the diagnosis in this case with clinical-radiological-pathological correlation. Skeletal fluorosis should be considered as a cause of acquired diffuse osteosclerosis. Methoxyflurane should not be recommended for chronic pain. The risk of repeated low-dose exposure to fluoride from methoxyflurane use as analgesia may be greater than expected, and the maximum recommended dose for methoxyflurane may require re-evaluation to minimize skeletal complications. Abbreviated abstract: Skeletal fluorosis is rare and occurs secondary to chronic high amounts of fluoride consumption, manifesting as diffuse osteosclerosis, skeletal pain, connective tissue calcification, and increased fracture risk. We present the case of a 47-yr-old female with skeletal fluorosis secondary to long-term methoxyflurane for chronic pain. The risk of repeated low-dose exposure to fluoride from methoxyflurane use for analgesia may be greater than expected, and the maximum recommended dose for methoxyflurane may require re-evaluation to minimize skeletal complications.

Simplified gestational diabetes screening with a triaging fasting plasma glucose reduces the burden of oral glucose tolerance tests during pregnancy - A large tertiary comparative cohort study.

AIMS: The study aimed to evaluate the impact of a simplified screeningapproach for gestational diabetes (GDM) compared to conventional screening on OGTT rates, GDM prevalence, and perinatal outcomes. METHOD: A retrospective comparative cohort study included singleton births from 20 weeks' gestation. Pregnancies without diagnostic glucose results from 13 weeks' gestation or incomplete screenings were excluded. Simplified screening consisted of a triaging fasting plasma glucose (FPG), where only those with FPG levels between 4.7 and 5.0 mmol/L proceeded to the 2hr 75 g oral glucose tolerance test (OGTT).The study period was divided into conventional screening (1st January 2019-30th June 2020) and simplified screening (1st January 2021-31st December 2021). RESULTS: Out of 15,138 pregnancies, 12,035 met the inclusion criteria: 7385 underwent conventional and 4650 underwent simplified screening. In the simplified group, 82.9 % avoided an OGTT. The simplified screening group also had a lower GDM prevalence compared to the conventional group ((18.7 % vs. 21.7 %, p < 0.001). Perinatal outcomes, including the rate of large-for-gestational-age infants, were similar between the groups. CONCLUSION: The simplified GDM screening strategy for significantly reduced OGTTs by over 80% without impacting perinatal outcomes. It suggests that prospective studies are necessary to further evaluate this approach.

Increased prevalence of germline pathogenic CHEK2 variants in individuals with pituitary adenomas.

CONTEXT: CHEK2 is a cell cycle checkpoint regulator gene with a long-established role as a clinically relevant, moderate risk breast cancer predisposition gene, with greater risk ascribed to truncating variants than missense variants. METHODS: We assessed 165 individuals with pituitary adenomas for CHEK2 variants. The study consisted of a primary cohort of 29 individuals who underwent germline and tumour whole exome sequencing, and a second, independent cohort of 136 individuals who had a targeted next-generation sequencing panel performed on both germline and tumour DNA (n=52) or germline DNA alone (n=84). RESULTS: We identified rare, coding, non-synonymous germline CHEK2 variants amongst 3/29 (10.3%) patients in our primary cohort and 5/165 (3.0%) patients overall, with affected patients having a range of hormone secretion types (prolactinoma, thyrotrophinoma, somatotrophinoma and non-functioning pituitary adenoma). No somatic variants were identified. Two variants were definitive null variants (c.1100delC, c.444+1G>A), classified as pathogenic. Two variants were missense variants (p.Asn186His, p.Thr476Met), classified as likely pathogenic. Even when considering the null variants only, the rate of CHEK2 variants was higher in our cohort compared to national control data (1.8% vs. 0.5%, P=0.049). CONCLUSIONS: This is the first study to suggest a role for the breast cancer predisposition gene, CHEK2, in pituitary tumorigenesis, with pathogenic/likely pathogenic variants found in 3% of patients with pituitary adenomas. As pituitary adenomas are relatively common and typically lack classical autosomal dominant family histories, risk alleles - such as these variants found in CHEK2 - might be a significant contributor to pituitary adenoma risk in the general population.

PAM variants in patients with thyrotrophinomas, cyclical Cushing's disease and prolactinomas.

Introduction: Germline loss-of-function variants in PAM, encoding peptidylglycine α-amidating monooxygenase (PAM), were recently discovered to be enriched in conditions of pathological pituitary hypersecretion, specifically: somatotrophinoma, corticotrophinoma, and prolactinoma. PAM is the sole enzyme responsible for C-terminal amidation of peptides, and plays a role in the biosynthesis and regulation of multiple hormones, including proopiomelanocortin (POMC). Methods: We performed exome sequencing of germline and tumour DNA from 29 individuals with functioning pituitary adenomas (12 prolactinomas, 10 thyrotrophinomas, 7 cyclical Cushing's disease). An unfiltered analysis was undertaken of all PAM variants with population prevalence <5%. Results: We identified five coding, non-synonymous PAM variants of interest amongst seven individuals (six germline, one somatic). The five variants comprised four missense variants and one truncating variant, all heterozygous. Each variant had some evidence of pathogenicity based on population prevalence, conservation scores, in silico predictions and/or prior functional studies. The yield of predicted deleterious PAM variants was thus 7/29 (24%). The variants predominated in individuals with thyrotrophinomas (4/10, 40%) and cyclical Cushing's disease (2/7, 29%), compared to prolactinomas (1/12, 8%). Conclusion: This is the second study to demonstrate a high yield of suspected loss-of-function, predominantly germline, PAM variants in individuals with pathological pituitary hypersecretion. We have extended the association with corticotrophinoma to include the specific clinical entity of cyclical Cushing's disease and demonstrated a novel association between PAM variants and thyrotrophinoma. PAM variants might act as risk alleles for pituitary adenoma formation, with a possible genotype-phenotype relationship between truncating variants and altered temporal secretion of cortisol.

miR-3156-5p is downregulated in serum of MEN1 patients and regulates expression of MORF4L2.

Multiple endocrine neoplasia type 1 (MEN1), caused by mutations in the MEN1 gene encoding menin, is an autosomal dominant disorder characterised by the combined occurrence of parathyroid, pituitary and pancreatic neuroendocrine tumours (NETs). Development of these tumours is associated with wide variations in their severity, order and ages (from <5 to >80 years), requiring life-long screening. To improve tumour surveillance and quality of life, better circulating biomarkers, particularly for pancreatic NETs that are associated with higher mortality, are required. We, therefore, examined the expression of circulating miRNA in the serum of MEN1 patients. Initial profiling analysis followed by qRT-PCR validation studies identified miR-3156-5p to be significantly downregulated (-1.3 to 5.8-fold, P < 0.05-0.0005) in nine MEN1 patients, compared to matched unaffected relatives. MEN1 knock-down experiments in BON-1 human pancreatic NET cells resulted in reduced MEN1 (49%, P < 0.05), menin (54%, P < 0.05) and miR-3156-5p expression (20%, P < 0.005), compared to control-treated cells, suggesting that miR-3156-5p downregulation is a consequence of loss of MEN1 expression. In silico analysis identified mortality factor 4-like 2 (MOR4FL2) as a potential target of miR-3156-5p, and in vitro functional studies in BON-1 cells transfected with either miR-3156-5p mimic or inhibitors showed that the miR-3156-5p mimic significantly reduced MORF4L2 protein expression (46%, P < 0.005), while miR-3156-5p inhibitor significantly increased MORF4L2 expression (1.5-fold, P < 0.05), compared to control-treated cells, thereby confirming that miR-3156-5p regulates MORF4L2 expression. Thus, the inverse relationship between miR-3156-5p and MORF4L2 expression represents a potential serum biomarker that could facilitate the detection of NET occurrence in MEN1 patients.

Insights into pituitary tumorigenesis: from Sanger sequencing to next-generation sequencing and beyond.

Introduction: This review explores insights provided by next-generation sequencing (NGS) of pituitary tumors and the clinical implications.Areas covered: Although syndromic forms account for just 5% of pituitary tumours, past Sanger sequencing studies pragmatically focused on them. These studies identified mutations in MEN1, CDKN1B, PRKAR1A, GNAS and SDHx causing Multiple Endocrine Neoplasia-1 (MEN1), MEN4, Carney Complex-1, McCune Albright Syndrome and 3P association syndromes, respectively. Furthermore, linkage analysis of single-nucleotide polymorphisms identified AIP mutations in 20% with familial isolated pituitary adenomas (FIPA). NGS has enabled further investigation of sporadic tumours. Thus, mutations of USP8 and CABLES1 were identified in corticotrophinomas, BRAF in papillary craniopharyngiomas and CTNNB1 in adamantinomatous craniopharyngiomas. NGS also revealed that pituitary tumours occur in the DICER1 syndrome, due to DICER1 mutations, and CDH23 mutations occur in FIPA. These discoveries revealed novel therapeutic targets and studies are underway of BRAF inhibitors for papillary craniopharyngiomas, and EGFR and USP8 inhibitors for corticotrophinomas.Expert opinion: It has become apparent that single-nucleotide variants and small insertion/deletion DNA mutations cannot explain all pituitary tumorigenesis. Integrated and improved analyses including whole-genome sequencing, copy number, and structural variation analyses, RNA sequencing and epigenomic analyses, with improved genomic technologies, are likely to further define the genomic landscape.

Predicting experimentally-derived failure load at the distal radius using finite element modelling based on peripheral quantitative computed tomography cross-sections (pQCT-FE): A validation study.

Dual energy X-ray absorptiometry, the current clinical criterion method for osteoporosis diagnosis, has limitations in identifying individuals with increased fracture risk, especially at the distal radius. Peripheral quantitative computed tomography (pQCT) can provide volumetric bone density data, as well as information on bone geometry, which makes it possible to establish finite element (FE) models of the distal radius from which bone strength and stiffness can be calculated. In this study, we compared experimental mechanical failure load data of the forearm with pQCT- based FE (pQCT-FE) modelling properties. Sixteen cadaveric forearm specimens were experimentally loaded until failure. Estimated stiffness and strength variables of compression, shear, bending and torsion were calculated from pQCT-FE modelling of single cross-sections of 0.2 × 0.2 × 2.4 mm of the radius pQCT image. A moderate-to-strong coefficient of determination (r2) was observed between experimental failure load and pQCT-FE variables. The highest r2 was observed for bending stiffness (r2 = 0.83). This study validates the use of pQCT-FE in the assessment of distal radius bone strength for future studies.