Publications

The Melanocortin-4 Receptor (MC4R) plays a pivotal role in energy homeostasis. We used human MC4R mutations associated with an increased or decreased risk of obesity to dissect mechanisms that regulate MC4R function. Most obesity-associated mutations impair trafficking to the plasma membrane (PM), whereas obesity-protecting mutations either accelerate recycling to the PM or decrease internalization, resulting in enhanced signaling. MC4R mutations that do not affect canonical Gαs protein-mediated signaling, previously considered to be non-pathogenic, nonetheless disrupt agonist-induced internalization, β-arrestin recruitment, and/or coupling to Gαs, establishing their causal role in severe obesity. Structural mapping reveals ligand-accessible sites by which MC4R couples to effectors and residues involved in the homodimerization of MC4R, which is disrupted by multiple obesity-associated mutations. Human genetic studies reveal that endocytosis, intracellular trafficking, and homodimerization regulate MC4R function to a level that is physiologically relevant, supporting the development of chaperones, agonists, and allosteric modulators of MC4R for weight loss therapy.

BACKGROUND: GNAS encodes the Gαs (stimulatory G-protein alpha subunit) protein, which mediates G protein-coupled receptor (GPCR) signaling. GNAS mutations cause developmental delay, short stature, and skeletal abnormalities in a syndrome called Albright's hereditary osteodystrophy. Because of imprinting, mutations on the maternal allele also cause obesity and hormone resistance (pseudohypoparathyroidism).

METHODS: We performed exome sequencing and targeted resequencing in 2548 children who presented with severe obesity, and we unexpectedly identified 22 GNAS mutation carriers. We investigated whether the effect of GNAS mutations on melanocortin 4 receptor (MC4R) signaling explains the obesity and whether the variable clinical spectrum in patients might be explained by the results of molecular assays.

RESULTS: Almost all GNAS mutations impaired MC4R signaling. A total of 6 of 11 patients who were 12 to 18 years of age had reduced growth. In these patients, mutations disrupted growth hormone-releasing hormone receptor signaling, but growth was unaffected in carriers of mutations that did not affect this signaling pathway (mean standard-deviation score for height, -0.90 vs. 0.75, respectively; P = 0.02). Only 1 of 10 patients who reached final height before or during the study had short stature. GNAS mutations that impaired thyrotropin receptor signaling were associated with developmental delay and with higher thyrotropin levels (mean [±SD], 8.4±4.7 mIU per liter) than those in 340 severely obese children who did not have GNAS mutations (3.9±2.6 mIU per liter; P = 0.004).

CONCLUSIONS: Because pathogenic mutations may manifest with obesity alone, screening of children with severe obesity for GNAS deficiency may allow early diagnosis, improving clinical outcomes, and melanocortin agonists may aid in weight loss. GNAS mutations that are identified by means of unbiased genetic testing differentially affect GPCR signaling pathways that contribute to clinical heterogeneity. Monogenic diseases are clinically more variable than their classic descriptions suggest. (Funded by Wellcome and others.).

The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. We uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, we identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. We found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength of our approach in predicting novel human obesity genes and signalling pathways and their site of action.

White fat stores excess energy, whereas brown and beige fat are thermogenic and dissipate energy as heat. Thermogenic adipose tissues markedly improve glucose and lipid homeostasis in mouse models, although the extent to which brown adipose tissue (BAT) influences metabolic and cardiovascular disease in humans is unclear1,2. Here we retrospectively categorized 134,529 18F-fluorodeoxyglucose positron emission tomography-computed tomography scans from 52,487 patients, by presence or absence of BAT, and used propensity score matching to assemble a study cohort. Scans in the study population were initially conducted for indications related to cancer diagnosis, treatment or surveillance, without previous stimulation. We report that individuals with BAT had lower prevalences of cardiometabolic diseases, and the presence of BAT was independently correlated with lower odds of type 2 diabetes, dyslipidemia, coronary artery disease, cerebrovascular disease, congestive heart failure and hypertension. These findings were supported by improved blood glucose, triglyceride and high-density lipoprotein values. The beneficial effects of BAT were more pronounced in individuals with overweight or obesity, indicating that BAT might play a role in mitigating the deleterious effects of obesity. Taken together, our findings highlight a potential role for BAT in promoting cardiometabolic health.

The association of brown adipose tissue (BAT) and body fat distribution and their combined effects on metabolic health in humans remains unknown. Here, we retrospectively identify individuals with and without BAT on 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/computed tomography (CT) and assemble a propensity score-matched study cohort to compare body fat distribution and determine its role in mediating the benefits of brown fat. We find that BAT is associated with lower amounts of visceral adipose tissue and higher amounts of subcutaneous adipose tissue, resulting in less central obesity. In addition, BAT is independently associated with lower blood glucose and white blood cell count, improved lipids, lower prevalence of type 2 diabetes mellitus, and decreased liver fat accumulation. These observations are most prominent in individuals with central obesity. Our results support a role of BAT in protection from visceral adiposity and improved metabolic health.

The construction of population-based variomes has contributed substantially to our understanding of the genetic basis of human inherited disease. Here, we investigated the genetic structure of Turkey from 3,362 unrelated subjects whose whole exomes (n = 2,589) or whole genomes (n = 773) were sequenced to generate a Turkish (TR) Variome that should serve to facilitate disease gene discovery in Turkey. Consistent with the history of present-day Turkey as a crossroads between Europe and Asia, we found extensive admixture between Balkan, Caucasus, Middle Eastern, and European populations with a closer genetic relationship of the TR population to Europeans than hitherto appreciated. We determined that 50% of TR individuals had high inbreeding coefficients (≥0.0156) with runs of homozygosity longer than 4 Mb being found exclusively in the TR population when compared to 1000 Genomes Project populations. We also found that 28% of exome and 49% of genome variants in the very rare range (allele frequency < 0.005) are unique to the modern TR population. We annotated these variants based on their functional consequences to establish a TR Variome containing alleles of potential medical relevance, a repository of homozygous loss-of-function variants and a TR reference panel for genotype imputation using high-quality haplotypes, to facilitate genome-wide association studies. In addition to providing information on the genetic structure of the modern TR population, these data provide an invaluable resource for future studies to identify variants that are associated with specific phenotypes as well as establishing the phenotypic consequences of mutations in specific genes.

AIMS: Transient Receptor Potential Melastatin 7 (TRPM7) cation channel is a chanzyme (channel + kinase) that influences cellular Mg2+ homeostasis and vascular signalling. However, the pathophysiological significance of TRPM7 in the cardiovascular system is unclear. The aim of this study was to investigate the role of this chanzyme in the cardiovascular system focusing on inflammation and fibrosis.

METHODS AND RESULTS: TRPM7-deficient mice with deletion of the kinase domain (TRPM7+/Δkinase) were studied and molecular mechanisms investigated in TRPM7+/Δkinase bone marrow-derived macrophages (BMDM) and co-culture systems with cardiac fibroblasts. TRPM7-deficient mice had significant cardiac hypertrophy, fibrosis, and inflammation. Cardiac collagen and fibronectin content, expression of pro-inflammatory mediators (SMAD3, TGFβ) and cytokines [interleukin (IL)-6, IL-10, IL-12, tumour necrosis factor-α] and phosphorylation of the pro-inflammatory signalling molecule Stat1, were increased in TRPM7+/Δkinase mice. These processes were associated with infiltration of inflammatory cells (F4/80+CD206+ cardiac macrophages) and increased galectin-3 expression. Cardiac [Mg2+]i, but not [Ca2+]i, was reduced in TRPM7+/Δkinase mice. Calpain, a downstream TRPM7 target, was upregulated (increased expression and activation) in TRPM7+/Δkinase hearts. Vascular functional and inflammatory responses, assessed in vivo by intra-vital microscopy, demonstrated impaired neutrophil rolling, increased neutrophil: endothelial attachment and transmigration of leucocytes in TRPM7+/Δkinase mice. TRPM7+/Δkinase BMDMs had increased levels of galectin-3, IL-10, and IL-6. In co-culture systems, TRPM7+/Δkinase macrophages increased expression of fibronectin, proliferating cell nuclear antigen, and TGFβ in cardiac fibroblasts from wild-type mice, effects ameliorated by MgCl2 treatment.

CONCLUSIONS: We identify a novel anti-inflammatory and anti-fibrotic role for TRPM7 and suggest that its protective effects are mediated, in part, through Mg2+-sensitive processes.

Attention deficit/hyperactivity disorder (ADHD) is a common and heritable phenotype frequently accompanied by insomnia, anxiety, and depression. Here, using a reverse phenotyping approach, we report heterozygous coding variations in the core circadian clock gene cryptochrome 1 in 15 unrelated multigenerational families with combined ADHD and insomnia. The variants led to functional alterations in the circadian molecular rhythms, providing a mechanistic link to the behavioral symptoms. One variant, CRY1Δ11 c.1657+3A>C, is present in approximately 1% of Europeans, therefore standing out as a diagnostic and therapeutic marker. We showed by exome sequencing in an independent cohort of patients with combined ADHD and insomnia that 8 of 62 patients and 0 of 369 controls carried CRY1Δ11. Also, we identified a variant, CRY1Δ6 c.825+1G>A, that shows reduced affinity for BMAL1/CLOCK and causes an arrhythmic phenotype. Genotype-phenotype correlation analysis revealed that this variant segregated with ADHD and delayed sleep phase disorder (DSPD) in the affected family. Finally, we found in a phenome-wide association study involving 9438 unrelated adult Europeans that CRY1Δ11 was associated with major depressive disorder, insomnia, and anxiety. These results defined a distinctive group of circadian psychiatric phenotypes that we propose to designate as "circiatric" disorders.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) leads to premature stroke and vascular dementia. Mechanism-specific therapies for this aggressive cerebral small vessel disease are lacking. CADASIL is caused by NOTCH3 mutations that influence vascular smooth muscle cell (VSMC) function through unknown processes. We investigated molecular mechanisms underlying the vasculopathy in CADASIL focusing on endoplasmic reticulum (ER) stress and RhoA/Rho kinase (ROCK). Peripheral small arteries and VSMCs were isolated from gluteal biopsies of CADASIL patients and mesentery of TgNotch3R169C mice (CADASIL model). CADASIL vessels exhibited impaired vasorelaxation, blunted vasoconstriction, and hypertrophic remodeling. Expression of NOTCH3 and ER stress target genes was amplified and ER stress response, Rho kinase activity, superoxide production, and cytoskeleton-associated protein phosphorylation were increased in CADASIL, processes associated with Nox5 upregulation. Aberrant vascular responses and signaling in CADASIL were ameliorated by inhibitors of Notch3 (γ-secretase inhibitor), Nox5 (mellitin), ER stress (4-phenylbutyric acid), and ROCK (fasudil). Observations in human CADASIL were recapitulated in TgNotch3R169C mice. These findings indicate that vascular dysfunction in CADASIL involves ER stress/ROCK interplay driven by Notch3-induced Nox5 activation and that NOTCH3 mutation-associated vascular pathology, typical in cerebral vessels, also manifests peripherally. We define Notch3-Nox5/ER stress/ROCK signaling as a putative mechanism-specific target and suggest that peripheral artery responses may be an accessible biomarker in CADASIL.