Multiple morbidity across the lifespan in people with Down syndrome or intellectual disabilities: a population-based cohort study using electronic health records.

BACKGROUND: The Down syndrome phenotype is well established, but our understanding of its morbidity patterns is limited. We comprehensively estimated the risk of multiple morbidity across the lifespan in people with Down syndrome compared with the general population and controls with other forms of intellectual disability. METHODS: In this matched population-based cohort-study design, we used electronic health-record data from the UK Clinical Practice Research Datalink (CRPD) from Jan 1, 1990, to June 29, 2020. We aimed to explore the pattern of morbidities throughout the lifespan of people with Down syndrome compared with people with other intellectual disabilities and the general population, to identify syndrome-specific health conditions and their age-related incidence. We estimated incidence rates per 1000 person-years and incidence rate ratios (IRRs) for 32 common morbidities. Hierarchical clustering was used to identify groups of associated conditions using prevalence data. FINDINGS: Between Jan 1, 1990, and June 29, 2020, a total of 10 204 people with Down syndrome, 39 814 controls, and 69 150 people with intellectual disabilities were included. Compared with controls, people with Down syndrome had increased risk of dementia (IRR 94·7, 95% CI 69·9-128·4), hypothyroidism (IRR 10·6, 9·6-11·8), epilepsy (IRR 9·7, 8·5-10·9), and haematological malignancy (IRR 4·7, 3·4-6·3), whereas asthma (IRR 0·88, 0·79-0·98), cancer (solid tumour IRR 0·75, 0·62-0·89), ischaemic heart disease (IRR 0·65, 0·51-0·85), and particularly hypertension (IRR 0·26, 0·22-0·32) were less frequent in people with Down syndrome than in controls. Compared to people with intellectual disabilities, risk of dementia (IRR 16·60, 14·23-19·37), hypothyroidism (IRR 7·22, 6·62-7·88), obstructive sleep apnoea (IRR 4·45, 3·72-5·31), and haematological malignancy (IRR 3·44, 2·58-4·59) were higher in people with Down syndrome, with reduced rates for a third of conditions, including new onset of dental inflammation (IRR 0·88, 0·78-0·99), asthma (IRR 0·82, 0·73-0·91), cancer (solid tumour IRR 0·78, 0·65-0·93), sleep disorder (IRR 0·74, 0·68-0·80), hypercholesterolaemia (IRR 0·69, 0·60-0·80), diabetes (IRR 0·59, 0·52-0·66), mood disorder (IRR 0·55, 0·50-0·60), glaucoma (IRR 0·47, 0·29-0·78), and anxiety disorder (IRR 0·43, 0·38-0·48). Morbidities in Down syndrome could be categorised on age-related incidence trajectories, and their prevalence clustered into typical syndromic conditions, cardiovascular diseases, autoimmune disorders, and mental health conditions. INTERPRETATION: Multiple morbidity in Down syndrome shows distinct patterns of age-related incidence trajectories and clustering that differ from those found in the general population and in people with other intellectual disabilities, with implications for provision and timing of health-care screening, prevention, and treatment for people with Down syndrome. FUNDING: The European Union's Horizon 2020 Research and Innovation Programme, the Jérôme Lejeune Foundation, the Alzheimer's Society, the Medical Research Council, the Academy of Medical Sciences, the Wellcome Trust, and William Harvey Research Limited.

Platelet-derived growth factor D expression in adrenal cells is modulated by corticosteroids: putative role in adrenal suppression.

BACKGROUND: Adrenal suppression is a clinically concerning side effect of inhaled corticosteroid (ICS) treatment in patients with asthma. Increased susceptibility to ICS-induced adrenal suppression has previously been identified in those with the rs591118 polymorphism in platelet-derived growth factor D (PDGFD). The mechanism underpinning this relationship is not known. METHODS: H295R cells were genotyped for rs591118 using a validated Taqman PCR allelic discrimination assay. H295R cell viability was determined after treatment with beclometasone and fluticasone (range 0-330 µM). Cortisol was measured in cell culture medium using competitive enzyme immunoassay. RESULTS: PDGFD protein expression in H295R cells was confirmed using Western blotting. When ACTH and forskolin were added to H295R cells, a reduction in PDGFD expression was seen, which was then restored by incubation with prochloraz, a known inhibitor of steroidogenesis. A dose-dependent, decrease in PDGFD expression was observed with beclometasone (over a 24 h incubation period) but not with beclometasone incubations beyond 24 h nor with fluticasone (at 24 or 48 h). CONCLUSIONS: H295R cells express PDGFD protein, which can be modulated by incubation with steroidogenesis agonists and antagonists and additionally with exogenous beclometasone. IMPACT: PDGFD is expressed in the human adrenal cell line, H295R, and expression can be modulated by beclometasone as well as agonists/antagonists of steroidogenesis. This builds on previous research that identified a SNP in PDGFD (rs591118) as an independent risk factor for adrenal suppression in adults and children with obstructive airway disease treated with inhaled corticosteroids. First in vitro experiments to support a link between the PDGF and cortisol production pathways, supporting the hypothesis that PDGFD variants can affect an individual's sensitivity to corticosteroid-induced adrenal suppression.

Rare disease gene association discovery from burden analysis of the 100,000 Genomes Project data.

To discover rare disease-gene associations, we developed a gene burden analytical framework and applied it to rare, protein-coding variants from whole genome sequencing of 35,008 cases with rare diseases and their family members recruited to the 100,000 Genomes Project (100KGP). Following in silico triaging of the results, 88 novel associations were identified including 38 with existing experimental evidence. We have published the confirmation of one of these associations, hereditary ataxia with UCHL1 , and independent confirmatory evidence has recently been published for four more. We highlight a further seven compelling associations: hypertrophic cardiomyopathy with DYSF and SLC4A3 where both genes show high/specific heart expression and existing associations to skeletal dystrophies or short QT syndrome respectively; monogenic diabetes with UNC13A with a known role in the regulation of ß cells and a mouse model with impaired glucose tolerance; epilepsy with KCNQ1 where a mouse model shows seizures and the existing long QT syndrome association may be linked; early onset Parkinson's disease with RYR1 with existing links to tremor pathophysiology and a mouse model with neurological phenotypes; anterior segment ocular abnormalities associated with POMK showing expression in corneal cells and with a zebrafish model with developmental ocular abnormalities; and cystic kidney disease with COL4A3 showing high renal expression and prior evidence for a digenic or modifying role in renal disease. Confirmation of all 88 associations would lead to potential diagnoses in 456 molecularly undiagnosed cases within the 100KGP, as well as other rare disease patients worldwide, highlighting the clinical impact of a large-scale statistical approach to rare disease gene discovery.

Identification of a novel specific small-molecule melanocortin-2-receptor antagonist.

The overproduction of adrenocorticotropic hormone (ACTH), in conditions such as Cushing's disease and congenital adrenal hyperplasia (CAH), leads to significant morbidity. Current treatment with glucocorticoids does not adequately suppress plasma ACTH, resulting in excess adrenal androgen production. At present, there is no effective medical treatment in clinical use that would directly block the action of ACTH. Such a therapy would be of great clinical value. ACTH acts via a highly selective receptor, the melanocortin-2 receptor (MC2R) associated with its accessory protein MRAP. ACTH is the only known naturally occurring agonist for this receptor. This lack of redundancy and the high degree of ligand specificity suggest that antagonism of this receptor could provide a useful therapeutic strategy in the treatment of conditions of ACTH excess. To this end, we screened an extensive library of low-molecular-weight drug-like compounds for MC2R antagonist activity using a high-throughput homogeneous time-resolved fluorescence cAMP assay in Chinese hamster ovary cells stably co-expressing human MC2R and MRAP. Hits that demonstrated MC2R antagonist properties were counter-screened against the ß2 adrenergic receptor and dose-response analysis undertaken. This led to the identification of a highly specific MC2R antagonist capable of antagonising ACTH-induced progesterone release in murine Y-1 adrenal cells and having selectivity for MC2R amongst the human melanocortin receptors. This work provides a foundation for the clinical investigation of small-molecule ACTH antagonists as therapeutic agents and proof of concept for the screening and discovery of such compounds.

Diabetes and Obesity in Down Syndrome Across the Lifespan: A Retrospective Cohort Study Using U.K. Electronic Health Records.

OBJECTIVE: Down syndrome (DS) is the most common form of chromosomal trisomy. Genetic factors in DS may increase the risk for diabetes. This study aimed to determine whether DS is associated with an increased incidence of diabetes and the relationship with obesity across the life span compared with control patients. RESEARCH DESIGN AND METHODS: This matched population-based cohort study analyzed UK Clinical Practice Research Datalink data from 1990 to 2020. RESULTS: A total of 9,917 patients with DS and 38,266 control patients were analyzed. Diabetes rates were higher in patients with DS (incidence rate ratio 3.67; 95% CI 2.43-5.55; P < 0.0001) and peaked at a younger age (median age at diagnosis 38 [interquartile range 28-49] years vs. 53 [43-61] years in control patients). Incidence rates (per 1,000 person-years) for type 1 diabetes mellitus were 0.44 (95% CI 0.31-0.61) in patients with DS vs. 0.13 (0.09-0.17) in control patients. Type 2 diabetes mellitus (T2DM) rates were higher in patients with DS versus control patients in age-groups from 5 years up to 34 years. In patients with DS, peak mean BMI was higher and at a younger age (males 31.2 kg/m2 at age 31 years; females 32.1 kg/m2 at 43 years) versus control patients (males 29.5 kg/m2 at 54 years; females 29.2 kg/m2 at 51 years). Obesity was associated with an increased incidence of T2DM. CONCLUSIONS: At younger ages, the incidence of diabetes in patients with DS is up to four times that of control patients. Peak mean BMI is higher and established earlier in DS, contributing to T2DM risk. Further investigation into the relationship between obesity and diabetes in DS is required to inform treatment and prevention measures.

Susceptibility to COVID-19 Diagnosis in People with Down Syndrome Compared to the General Population: Matched-Cohort Study Using Primary Care Electronic Records in the UK.

BACKGROUND: During the COVID-19 pandemic, people with Down syndrome (DS) have experienced a more severe disease course and higher mortality rates than the general population. It is not yet known whether people with DS are more susceptible to being diagnosed with COVID-19. OBJECTIVE: To explore whether DS is associated with increased susceptibility to COVID-19. DESIGN: Matched-cohort study design using anonymised primary care electronic health records from the May 2021 release of Clinical Practice Research Datalink (CPRD) Aurum. SETTING: Electronic health records from approximately 1400 general practices (GPs) in England. PARTICIPANTS: 8854 people with DS and 34,724 controls matched for age, gender and GP who were registered on or after the 29th January 2020. MEASUREMENTS: The primary outcome was COVID-19 diagnosis between January 2020 and May 2021. Conditional logistic regression models were fitted to estimate associations between DS and COVID-19 diagnosis, adjusting for comorbidities. RESULTS: Compared to controls, people with DS were more likely to be diagnosed with COVID-19 (7.4% vs 5.6%, p ≤ 0.001, odds ratio (OR) = 1.35; 95% CI = 1.23-1.48). There was a significant interaction between people with DS and a chronic respiratory disease diagnosis excluding asthma and increased odds of a COVID-19 diagnosis (OR = 1.71; 95% CI = 1.20-2.43), whilst adjusting for a number of comorbidities. CONCLUSION: Individuals with DS are at increased risk for contracting COVID-19. Those with underlying lung conditions are particularly vulnerable during viral pandemics and should be prioritised for vaccinations.

C-type natriuretic peptide is a pivotal regulator of metabolic homeostasis.

Thermogenesis and adipogenesis are tightly regulated mechanisms that maintain lipid homeostasis and energy balance; dysfunction of these critical processes underpins obesity and contributes to cardiometabolic disease. C-type natriuretic peptide (CNP) fulfills a multimodal protective role in the cardiovascular system governing local blood flow, angiogenesis, cardiac function, and immune cell reactivity. Herein, we investigated a parallel, preservative function for CNP in coordinating metabolic homeostasis. Global inducible CNP knockout mice exhibited reduced body weight, higher temperature, lower adiposity, and greater energy expenditure in vivo. This thermogenic phenotype was associated with increased expression of uncoupling protein-1 and preferential lipid utilization by mitochondria, a switch corroborated by a corresponding diminution of insulin secretion and glucose clearance. Complementary studies in isolated murine and human adipocytes revealed that CNP exerts these metabolic regulatory actions by inhibiting sympathetic thermogenic programming via Gi-coupled natriuretic peptide receptor (NPR)-C and reducing peroxisome proliferator-activated receptor-γ coactivator-1α expression, while concomitantly driving adipogenesis via NPR-B/protein kinase-G. Finally, we identified an association between CNP/NPR-C expression and obesity in patient samples. These findings establish a pivotal physiological role for CNP as a metabolic switch to balance energy homeostasis. Pharmacological targeting of these receptors may offer therapeutic utility in the metabolic syndrome and related cardiovascular disorders.

Missplicing due to a synonymous, T96= exonic substitution in the T-box transcription factor TBX19 resulting in isolated ACTH deficiency.

SUMMARY: Congenital isolated ACTH deficiency (IAD) is a rare condition characterised by low plasma ACTH and serum cortisol with normal production of other pituitary hormones. TBX19 (also known as TPIT) is a T-box pituitary restricted transcription factor important for POMC gene transcription and terminal differentiation of POMC-expressing cells. TBX19 gene mutations have been shown to cause neonatal-onset congenital IAD. We report a neonate of Romanian origin, who presented at 15 h of life with respiratory arrest and hypoglycaemia which recurred over the following 2 weeks. Biochemical investigations revealed IAD, with undetectable serum cortisol (cortisol < 1 µg/dL; normal range (NR): 7.8-26.2) and plasma ACTH levels within the normal range (22.1 pg/mL; NR: 4.7-48.8). He responded to hydrocortisone treatment. Patient DNA was analysed by a HaloPlex next-generation sequencing array targeting genes for adrenal insufficiency. A novel homozygous synonymous mutation p.Thr96= (Chr1:168260482; c.288G>A; rs376493164; allele frequency 1 × 10-5, no homozygous) was found in exon 2 of the TBX19 gene. The effect of this was assessed by an in vitro splicing assay, which revealed aberrant splicing of exon 2 giving rise to a mutant mRNA transcript whereas the WT vector spliced exon 2 normally. This was identified as the likely cause of IAD in the patient. The predicted protein product would be non-functional in keeping with the complete loss of cortisol production and early presentation in the patient. LEARNING POINTS: Synonymous variants (a nucleotide change that does not alter protein sequence) usually thought to be benign may still have detrimental effects on RNA and protein function causing disease. Hence, they should not be ignored, especially if very rare in public databases. In vitro splicing assays can be employed to characterise the consequence of intronic and exonic nucleotide gene changes that may alter splicing. Establishing a diagnosis due to a TBX19 mutation is important as it defines a condition of isolated ACTH deficiency not associated with additional pituitary deficiencies.

Genetic Analysis of Pediatric Primary Adrenal Insufficiency of Unknown Etiology: 25 Years' Experience in the UK.

CONTEXT: Although primary adrenal insufficiency (PAI) in children and young people is often due to congenital adrenal hyperplasia (CAH) or autoimmunity, other genetic causes occur. The relative prevalence of these conditions is poorly understood. OBJECTIVE: We investigated genetic causes of PAI in children and young people over a 25 year period. DESIGN SETTING AND PARTICIPANTS: Unpublished and published data were reviewed for 155 young people in the United Kingdom who underwent genetic analysis for PAI of unknown etiology in three major research centers between 1993 and 2018. We pre-excluded those with CAH, autoimmune, or metabolic causes. We obtained additional data from NR0B1 (DAX-1) clinical testing centers. INTERVENTION AND OUTCOME MEASUREMENTS: Genetic analysis involved a candidate gene approach (1993 onward) or next generation sequencing (NGS; targeted panels, exomes) (2013-2018). RESULTS: A genetic diagnosis was reached in 103/155 (66.5%) individuals. In 5 children the adrenal insufficiency resolved and no genetic cause was found. Pathogenic variants occurred in 11 genes: MC2R (adrenocorticotropin receptor; 30/155, 19.4%), NR0B1 (DAX-1; 7.7%), CYP11A1 (7.7%), AAAS (7.1%), NNT (6.5%), MRAP (4.5%), TXNRD2 (4.5%), STAR (3.9%), SAMD9 (3.2%), CDKN1C (1.3%), and NR5A1/steroidogenic factor-1 (SF-1; 0.6%). Additionally, 51 boys had NR0B1 variants identified through clinical testing. Although age at presentation, treatment, ancestral background, and birthweight can provide diagnostic clues, genetic testing was often needed to define the cause. CONCLUSIONS: PAI in children and young people often has a genetic basis. Establishing the specific etiology can influence management of this lifelong condition. NGS approaches improve the diagnostic yield when many potential candidate genes are involved.

Structure reveals the activation mechanism of the MC4 receptor to initiate satiation signaling.

Obesity is a global epidemic that causes morbidity and impaired quality of life. The melanocortin receptor 4 (MC4R) is at the crux of appetite, energy homeostasis, and body-weight control in the central nervous system and is a prime target for anti-obesity drugs. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human MC4R-Gs signaling complex bound to the agonist setmelanotide, a cyclic peptide recently approved for the treatment of obesity. The work reveals the mechanism of MC4R activation, highlighting a molecular switch that initiates satiation signaling. In addition, our findings indicate that calcium (Ca2+) is required for agonist, but not antagonist, efficacy. These results fill a gap in the understanding of MC4R activation and could guide the design of future weight-management drugs.

ACTH signalling and adrenal development: lessons from mouse models.

The melanocortin-2-receptor (MC2R), also known as the ACTH receptor, is a critical component of the hypothalamic-pituitary-adrenal axis. The importance of MC2R in adrenal physiology is exemplified by the condition familial glucocorticoid deficiency (FGD), a potentially fatal disease characterised by isolated cortisol deficiency. MC2R mutations cause ~25% of cases. The discovery of a MC2R accessory protein MRAP, mutations of which account for ~20% of FGD, has provided insight into MC2R trafficking and signalling. MRAP is a single transmembrane domain accessory protein highly expressed in the adrenal gland and essential for MC2R expression and function. Mouse models helped elucidate the action of ACTH. The Mc2r-knockout (Mc2r - / - ) mice was the first mouse model developed to have adrenal insufficiency with deficiencies in glucocorticoid, mineralocorticoid and catecholamines. We recently reported the generation of the Mrap - / - mice which better mimics the human FGD phenotype with isolated glucocorticoid deficiency alone. The adrenal glands of adult Mrap - / - mice were grossly dysmorphic with a thickened capsule, deranged zonation and deranged WNT4/beta-catenin and sonic hedgehog (SHH) pathway signalling. Collectively, these mouse models of FGD highlight the importance of ACTH and MRAP in adrenal progenitor cell regulation, cortex maintenance and zonation.

Isolated glucocorticoid deficiency: Genetic causes and animal models.

Hereditary adrenocorticotropin (ACTH) resistance syndromes encompass the genetically heterogeneous isolated or Familial Glucocorticoid Deficiency (FGD) and the distinct clinical entity known as Triple A syndrome. The molecular basis of adrenal resistance to ACTH includes defects in ligand binding, MC2R/MRAP receptor trafficking, cellular redox balance, cholesterol synthesis and sphingolipid metabolism. Biochemically, this manifests as ACTH excess in the setting of hypocortisolaemia. Triple A syndrome is an inherited condition involving a tetrad of adrenal insufficiency, achalasia, alacrima and neuropathy. FGD is an autosomal recessive condition characterized by the presence of isolated glucocorticoid deficiency, classically in the setting of preserved mineralocorticoid secretion. Primarily there are three established subtypes of the disease: FGD 1, FGD2 and FGD3 corresponding to mutations in the Melanocortin 2 receptor MC2R (25%), Melanocortin 2 receptor accessory protein MRAP (20%), and Steroidogenic acute regulatory protein STAR (5-10%) respectively. Together, mutations in these 3 genes account for approximately half of cases. Whole exome sequencing in patients negative for MC2R, MRAP and STAR mutations, identified mutations in minichromosome maintenance 4 MCM4, nicotinamide nucleotide transhydrogenase NNT, thioredoxin reductase 2 TXNRD2, cytochrome p450scc CYP11A1, and sphingosine 1-phosphate lyase SGPL1 accounting for a further 10% of FGD. These novel genes have linked replicative and oxidative stress and altered redox potential as a mechanism of adrenocortical damage. However, a genetic diagnosis is still unclear in about 40% of cases. We describe here an updated list of FGD genes and provide a description of relevant mouse models that, despite some being flawed, have been precious allies in the understanding of FGD pathobiology.

Overexpression of melanocortin 2 receptor accessory protein 2 (MRAP2) in adult paraventricular MC4R neurons regulates energy intake and expenditure.

OBJECTIVE: Melanocortin 2 receptor accessory protein 2 (MRAP2) has a critical role in energy homeostasis. Although MRAP2 has been shown to regulates a number of GPCRs involved in metabolism, the key neurons responsible for the phenotype of gross obesity in MRAP2 deficient animals are unclear. Furthermore, to date, all the murine MRAP2 models involve the prenatal deletion of MRAP2. METHODS: To target Melanocortin 4 receptor (MC4R)-expressing neurons in the hypothalamic paraventricular nucleus (PVN), we performed stereotaxic surgery using AAV to selectively overexpress MRAP2 postnatally in adult Mc4r-cre mice. We assessed energy homeostasis, glucose metabolism, core body temperature, and response to MC3R/MC4R agonist MTII. RESULTS: Mc4r-crePVN-MRAP2 female mice on a standard chow diet had less age-related weight gain and improved glucose/insulin profile compared to control Mc4r-crePVN-GFP mice. These changes were associated with a reduction in food intake and increased energy expenditure. In contrast, Mc4r-crePVN-MRAP2 male mice showed no improvement on a chow diet, but improvement of energy and glucose metabolism was observed following high fat diet (HFD) feeding. In addition, an increase in core body temperature was found in both females fed on standard chow diet and males fed on HFD. Mc4r-crePVN-MRAP2 female and male mice showed increased neuronal activation in the PVN compared to controls, with further increase in neuronal activation post MTII treatment in females. CONCLUSIONS: Our data indicate a site-specific role for MRAP2 in PVN MC4R-expressing neurons in potentiating MC4R neuronal activation at baseline conditions in the regulation of food intake and energy expenditure.

MRAP deficiency impairs adrenal progenitor cell differentiation and gland zonation.

Melanocortin 2 receptor accessory protein (MRAP) is a single transmembrane domain accessory protein and a critical component of the hypothamo-pituitary-adrenal axis. MRAP is highly expressed in the adrenal gland and is essential for adrenocorticotropin hormone (ACTH) receptor expression and function. Human loss-of-function mutations in MRAP cause familial glucocorticoid (GC) deficiency (FGD) type 2 (FGD2), whereby the adrenal gland fails to respond to ACTH and to produce cortisol. In this study, we generated Mrap-null mice to study the function of MRAP in vivo. We found that the vast majority of Mrap-/- mice died at birth but could be rescued by administration of corticosterone to pregnant dams. Surviving Mrap-/- mice developed isolated GC deficiency with normal mineralocorticoid and catecholamine production, recapitulating FGD2. The adrenal glands of adult Mrap-/- mice were small, with grossly impaired adrenal capsular morphology and cortex zonation. Progenitor cell differentiation was significantly impaired, with dysregulation of WNT4/ß-catenin and sonic hedgehog pathways. These data demonstrate the roles of MRAP in both steroidogenesis and the regulation of adrenal cortex zonation. This is the first mouse model of isolated GC deficiency and reveals the role of MRAP in adrenal progenitor cell regulation and cortex zonation.-Novoselova, T. V., Hussain, M., King, P. J., Guasti, L., Metherell, L. A., Charalambous, M., Clark, A. J. L., Chan, L. F. MRAP deficiency impairs adrenal progenitor cell differentiation and gland zonation.

Promiscuity among the MRAPs.

The melanocortin 2 receptor accessory protein (MRAP) was originally discovered to be an essential co-receptor for the ACTH receptor/melanocortin 2 receptor, and it physically interacts with this receptor and is required for receptor trafficking and ligand binding. A related molecule, MRAP2, is mainly expressed in the CNS and appears to have a role with the melanocortin 4 receptor. Consistent with this is the observation that a massively obese phenotype develops when the Mrap2 gene is deleted in mice. However, the characteristics of this phenotype differ from those of Mc4r-deleted mice and suggest that an additional role, possibly resulting from an interaction with other receptors is possible. In support of this, a functional interaction with the prokineticin receptors was recently reported. Evidence for other receptor interactions and aspects of the tissue distribution of MRAP and MRAP2 gene expression may indicate that these accessory proteins have a wider role than with the melanocortin receptors alone.

Central role for melanocortin-4 receptors in offspring hypertension arising from maternal obesity.

Melanocortin-4 receptor (Mc4r)-expressing neurons in the autonomic nervous system, particularly in the paraventricular nucleus of the hypothalamus (PVH), play an essential role in blood pressure (BP) control. Mc4r-deficient (Mc4rKO) mice are severely obese but lack obesity-related hypertension; they also show a reduced pressor response to salt loading. We have previously reported that lean juvenile offspring born to diet-induced obese rats (OffOb) exhibit sympathetic-mediated hypertension, and we proposed a role for postnatally raised leptin in its etiology. Here, we test the hypothesis that neonatal hyperleptinemia due to maternal obesity induces persistent changes in the central melanocortin system, thereby contributing to offspring hypertension. Working on the OffOb paradigm in both sexes and using transgenic technology to restore Mc4r in the PVH of Mc4rKO (Mc4rPVH) mice, we have now shown that these mice develop higher BP than Mc4rKO or WT mice. We have also found that experimental hyperleptinemia induced in the neonatal period in Mc4rPVH and WT mice, but not in the Mc4rKO mice, leads to heightened BP and severe renal dysfunction. Thus, Mc4r in the PVH appears to be required for early-life programming of hypertension arising from either maternal obesity or neonatal hyperleptinemia. Early-life exposure of the PVH to maternal obesity through postnatal elevation of leptin may have long-term consequences for cardiovascular health.

Whole-Exome Sequencing in the Differential Diagnosis of Primary Adrenal Insufficiency in Children.

Adrenal insufficiency is a rare, but potentially fatal medical condition. In children, the cause is most commonly congenital and in recent years a growing number of causative gene mutations have been identified resulting in a myriad of syndromes that share adrenal insufficiency as one of the main characteristics. The evolution of adrenal insufficiency is dependent on the variant and the particular gene affected, meaning that rapid and accurate diagnosis is imperative for effective treatment of the patient. Common practice is for candidate genes to be sequenced individually, which is a time-consuming process and complicated by overlapping clinical phenotypes. However, with the availability, and increasing cost effectiveness of whole-exome sequencing, there is the potential for this to become a powerful diagnostic tool. Here, we report the results of whole-exome sequencing of 43 patients referred to us with a diagnosis of familial glucocorticoid deficiency (FGD) who were mutation negative for MC2R, MRAP, and STAR the most commonly mutated genes in FGD. WES provided a rapid genetic diagnosis in 17/43 sequenced patients, for the remaining 60% the gene defect may be within intronic/regulatory regions not covered by WES or may be in gene(s) representing novel etiologies. The diagnosis of isolated or familial glucocorticoid deficiency was only confirmed in 3 of the 17 patients, other genetic diagnoses were adrenal hypo- and hyperplasia, Triple A, and autoimmune polyendocrinopathy syndrome type I, emphasizing both the difficulty of phenotypically distinguishing between disorders of PAI and the utility of WES as a tool to achieve this.

Melanocortin receptor accessory proteins in adrenal disease and obesity.

Melanocortin receptor accessory proteins (MRAPs) are regulators of the melanocortin receptor family. MRAP is an essential accessory factor for the functional expression of the MC2R/ACTH receptor. The importance of MRAP in adrenal gland physiology is demonstrated by the clinical condition familial glucocorticoid deficiency type 2. The role of its paralog melanocortin-2-receptor accessory protein 2 (MRAP2), which is predominantly expressed in the hypothalamus including the paraventricular nucleus, has recently been linked to mammalian obesity. Whole body deletion and targeted brain specific deletion of the Mrap2 gene result in severe obesity in mice. Interestingly, Mrap2 complete knockout (KO) mice have increased body weight without detectable changes to food intake or energy expenditure. Rare heterozygous variants of MRAP2 have been found in humans with severe, early-onset obesity. In vitro data have shown that Mrap2 interaction with the melanocortin-4-receptor (Mc4r) affects receptor signaling. However, the mechanism by which Mrap2 regulates body weight in vivo is not fully understood and differences between the phenotypes of Mrap2 and Mc4r KO mice may point toward Mc4r independent mechanisms.

NNT pseudoexon activation as a novel mechanism for disease in two siblings with familial glucocorticoid deficiency.

CONTEXT: Intronic DNA frequently encodes potential exonic sequences called pseudoexons. In recent years, mutations resulting in aberrant pseudoexon inclusion have been increasingly recognized to cause disease. OBJECTIVES: To find the genetic cause of familial glucocorticoid deficiency (FGD) in two siblings. PATIENTS: The proband and his affected sibling, from nonconsanguineous parents of East Asian and South African origin, were diagnosed with FGD at the ages of 21 and 8 months, respectively. DESIGN: Whole exome sequencing was performed on genomic DNA (gDNA) of the siblings. Variants in genes known to cause FGD were assessed for causality. Further analysis of gDNA and cDNA was performed by PCR/RT-PCR followed by automated Sanger sequencing. RESULTS: Whole exome sequencing identified a single, novel heterozygous variant (p.Arg71*) in nicotinamide nucleotide transhydrogenase (NNT) in both affected individuals. Follow-up cDNA analysis in the proband identified a 69-bp pseudoexon inclusion event, and Sanger sequencing of his gDNA identified a 4-bp duplication responsible for its activation. The variants segregated with the disease: p.Arg71* was inherited from the mother, the pseudoexon change was inherited from the father, and an unaffected sibling had inherited only the p.Arg71* variant. CONCLUSIONS: FGD in these siblings is caused by compound heterozygous mutations in NNT; one causing pseudoexon inclusion in combination with another leading to Arg71*. Discovery of this pseudoexon activation mutation highlights the importance of identifying sequence changes in introns by cDNA analysis. The clinical implications of these findings include: facilitation of antenatal genetic diagnosis, early institution of potentially lifesaving therapy, and the possibility of preventative or curative intervention.

Thioredoxin Reductase 2 (TXNRD2) mutation associated with familial glucocorticoid deficiency (FGD).

CONTEXT: Classic ACTH resistance, due to disruption of ACTH signaling, accounts for the majority of cases of familial glucocorticoid deficiency (FGD). Recently FGD cases caused by mutations in the mitochondrial antioxidant, nicotinamide nucleotide transhydrogenase, have highlighted the importance of redox regulation in steroidogenesis. OBJECTIVE: We hypothesized that other components of mitochondrial antioxidant systems would be good candidates in the etiology of FGD. DESIGN: Whole-exome sequencing was performed on three related patients, and segregation of putative causal variants confirmed by Sanger sequencing of all family members. A TXNRD2-knockdown H295R cell line was created to investigate redox homeostasis. SETTING: The study was conducted on patients from three pediatric centers in the United Kingdom. PATIENTS: Seven individuals from a consanguineous Kashmiri kindred, six of whom presented with FGD between 0.1 and 10.8 years, participated in the study. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURE: Identification and functional interrogation of a novel homozygous mutation segregating with the disease trait were measured. RESULTS: A stop gain mutation, p.Y447X in TXNRD2, encoding the mitochondrial selenoprotein thioredoxin reductase 2 (TXNRD2) was identified and segregated with disease in this extended kindred. RT-PCR and Western blotting revealed complete absence of TXNRD2 in patients homozygous for the mutation. TXNRD2 deficiency leads to impaired redox homeostasis in a human adrenocortical cell line. CONCLUSION: In contrast to the Txnrd2-knockout mouse model, in which embryonic lethality as a consequence of hematopoietic and cardiac defects is described, absence of TXNRD2 in humans leads to glucocorticoid deficiency. This is the first report of a homozygous mutation in any component of the thioredoxin antioxidant system leading to inherited disease in humans.