Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance.

Hypothalamic melanocortin neurons play a pivotal role in weight regulation. Here, we examined the contribution of Semaphorin 3 (SEMA3) signaling to the development of these circuits. In genetic studies, we found 40 rare variants in SEMA3A-G and their receptors (PLXNA1-4; NRP1-2) in 573 severely obese individuals; variants disrupted secretion and/or signaling through multiple molecular mechanisms. Rare variants in this set of genes were significantly enriched in 982 severely obese cases compared to 4,449 controls. In a zebrafish mutagenesis screen, deletion of 7 genes in this pathway led to increased somatic growth and/or adiposity demonstrating that disruption of Semaphorin 3 signaling perturbs energy homeostasis. In mice, deletion of the Neuropilin-2 receptor in Pro-opiomelanocortin neurons disrupted their projections from the arcuate to the paraventricular nucleus, reduced energy expenditure, and caused weight gain. Cumulatively, these studies demonstrate that SEMA3-mediated signaling drives the development of hypothalamic melanocortin circuits involved in energy homeostasis.

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

Diagnostic high-throughput sequencing of 2396 patients with bleeding, thrombotic, and platelet disorders.

A targeted high-throughput sequencing (HTS) panel test for clinical diagnostics requires careful consideration of the inclusion of appropriate diagnostic-grade genes, the ability to detect multiple types of genomic variation with high levels of analytic sensitivity and reproducibility, and variant interpretation by a multidisciplinary team (MDT) in the context of the clinical phenotype. We have sequenced 2396 index patients using the ThromboGenomics HTS panel test of diagnostic-grade genes known to harbor variants associated with rare bleeding, thrombotic, or platelet disorders (BTPDs). The molecular diagnostic rate was determined by the clinical phenotype, with an overall rate of 49.2% for all thrombotic, coagulation, platelet count, and function disorder patients and a rate of 3.2% for patients with unexplained bleeding disorders characterized by normal hemostasis test results. The MDT classified 745 unique variants, including copy number variants (CNVs) and intronic variants, as pathogenic, likely pathogenic, or variants of uncertain significance. Half of these variants (50.9%) are novel and 41 unique variants were identified in 7 genes recently found to be implicated in BTPDs. Inspection of canonical hemostasis pathways identified 29 patients with evidence of oligogenic inheritance. A molecular diagnosis has been reported for 894 index patients providing evidence that introducing an HTS genetic test is a valuable addition to laboratory diagnostics in patients with a high likelihood of having an inherited BTPD.

Germline mutations in the transcription factor IKZF5 cause thrombocytopenia.

To identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an association between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of α granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD4+ T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans.

Expanded repertoire of RASGRP2 variants responsible for platelet dysfunction and severe bleeding.

Heritable platelet function disorders (PFDs) are genetically heterogeneous and poorly characterized. Pathogenic variants in RASGRP2, which encodes calcium and diacylglycerol-regulated guanine exchange factor I (CalDAG-GEFI), have been reported previously in 3 pedigrees with bleeding and reduced platelet aggregation responses. To better define the phenotype associated with pathogenic RASGRP2 variants, we compared high-throughput sequencing and phenotype data from 2042 cases in pedigrees with unexplained bleeding or platelet disorders to data from 5422 controls. Eleven cases harbored 11 different, previously unreported RASGRP2 variants that were biallelic and likely pathogenic. The variants included 5 high-impact variants predicted to prevent CalDAG-GEFI expression and 6 missense variants affecting the CalDAG-GEFI CDC25 domain, which mediates Rap1 activation during platelet inside-out αIIbß3 signaling. Cases with biallelic RASGRP2 variants had abnormal mucocutaneous, surgical, and dental bleeding from childhood, requiring ≥1 blood or platelet transfusion in 78% of cases. Platelets displayed reduced aggregation in response to adenosine 5'-diphosphate and epinephrine, but variable aggregation defects with other agonists. There were no other consistent clinical or laboratory features. These data enable definition of human CalDAG-GEFI deficiency as a nonsyndromic, recessive PFD associated with a moderate or severe bleeding phenotype and complex defects in platelet aggregation.

Rare variants in GP1BB are responsible for autosomal dominant macrothrombocytopenia.

The von Willebrand receptor complex, which is composed of the glycoproteins Ibα, Ibß, GPV, and GPIX, plays an essential role in the earliest steps in hemostasis. During the last 4 decades, it has become apparent that loss of function of any 1 of 3 of the genes encoding these glycoproteins (namely, GP1BA, GP1BB, and GP9) leads to autosomal recessive macrothrombocytopenia complicated by bleeding. A small number of variants in GP1BA have been reported to cause a milder and dominant form of macrothrombocytopenia, but only 2 tentative reports exist of such a variant in GP1BB By analyzing data from a collection of more than 1000 genome-sequenced patients with a rare bleeding and/or platelet disorder, we have identified a significant association between rare monoallelic variants in GP1BB and macrothrombocytopenia. To strengthen our findings, we sought further cases in 2 additional collections in the United Kingdom and Japan. Across 18 families exhibiting phenotypes consistent with autosomal dominant inheritance of macrothrombocytopenia, we report on 27 affected cases carrying 1 of 9 rare variants in GP1BB.

Sphingolipid dysregulation due to lack of functional KDSR impairs proplatelet formation causing thrombocytopenia.

Sphingolipids are fundamental to membrane trafficking, apoptosis, and cell differentiation and proliferation. KDSR or 3-keto-dihydrosphingosine reductase is an essential enzyme for de novo sphingolipid synthesis, and pathogenic mutations in KDSR result in the severe skin disorder erythrokeratodermia variabilis et progressiva-4 Four of the eight reported cases also had thrombocytopenia but the underlying mechanism has remained unexplored. Here we expand upon the phenotypic spectrum of KDSR deficiency with studies in two siblings with novel compound heterozygous variants associated with thrombocytopenia, anemia, and minimal skin involvement. We report a novel phenotype of progressive juvenile myelofibrosis in the propositus, with spontaneous recovery of anemia and thrombocytopenia in the first decade of life. Examination of bone marrow biopsies showed megakaryocyte hyperproliferation and dysplasia. Megakaryocytes obtained by culture of CD34+ stem cells confirmed hyperproliferation and showed reduced proplatelet formation. The effect of KDSR insufficiency on the sphingolipid profile was unknown, and was explored in vivo and in vitro by a broad metabolomics screen that indicated activation of an in vivo compensatory pathway that leads to normalization of downstream metabolites such as ceramide. Differentiation of propositus-derived induced pluripotent stem cells to megakaryocytes followed by expression of functional KDSR showed correction of the aberrant cellular and biochemical phenotypes, corroborating the critical role of KDSR in proplatelet formation. Finally, Kdsr depletion in zebrafish recapitulated the thrombocytopenia and showed biochemical changes similar to those observed in the affected siblings. These studies support an important role for sphingolipids as regulators of cytoskeletal organization during megakaryopoiesis and proplatelet formation.

A gain-of-function variant in DIAPH1 causes dominant macrothrombocytopenia and hearing loss.

Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insight into the autoregulation of DIAPH1 activity.

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

Synaptic NMDA receptor activity boosts intrinsic antioxidant defenses.

Intrinsic antioxidant defenses are important for neuronal longevity. We found that in rat neurons, synaptic activity, acting via NMDA receptor (NMDAR) signaling, boosted antioxidant defenses by making changes to the thioredoxin-peroxiredoxin (Prx) system. Synaptic activity enhanced thioredoxin activity, facilitated the reduction of overoxidized Prxs and promoted resistance to oxidative stress. Resistance was mediated by coordinated transcriptional changes; synaptic NMDAR activity inactivated a previously unknown Forkhead box O target gene, the thioredoxin inhibitor Txnip. Conversely, NMDAR blockade upregulated Txnip in vivo and in vitro, where it bound thioredoxin and promoted vulnerability to oxidative damage. Synaptic activity also upregulated the Prx reactivating genes Sesn2 (sestrin 2) and Srxn1 (sulfiredoxin), via C/EBPbeta and AP-1, respectively. Mimicking these expression changes was sufficient to strengthen antioxidant defenses. Trans-synaptic stimulation of synaptic NMDARs was crucial for boosting antioxidant defenses; chronic bath activation of all (synaptic and extrasynaptic) NMDARs induced no antioxidative effects. Thus, synaptic NMDAR activity may influence the progression of pathological processes associated with oxidative damage.

Coagulation factor V is a T-cell inhibitor expressed by leukocytes in COVID-19.

Clotting Factor V (FV) is primarily synthesized in the liver and when cleaved by thrombin forms pro-coagulant Factor Va (FVa). Using whole blood RNAseq and scRNAseq of peripheral blood mononuclear cells, we find that FV mRNA is expressed in leukocytes, and identify neutrophils, monocytes, and T regulatory cells as sources of increased FV in hospitalized patients with COVID-19. Proteomic analysis confirms increased FV in circulating neutrophils in severe COVID-19, and immunofluorescence microscopy identifies FV in lung-infiltrating leukocytes in COVID-19 lung disease. Increased leukocyte FV expression in severe disease correlates with T-cell lymphopenia. Both plasma-derived and a cleavage resistant recombinant FV, but not thrombin cleaved FVa, suppress T-cell proliferation in vitro. Anticoagulants that reduce FV conversion to FVa, including heparin, may have the unintended consequence of suppressing the adaptive immune system.

A prospective study of risk factors associated with seroprevalence of SARS-CoV-2 antibodies in healthcare workers at a large UK teaching hospital.

OBJECTIVES: To describe the risk factors for SARS-CoV-2 infection in UK healthcare workers (HCWs). METHODS: We conducted a prospective sero-epidemiological study of HCWs at a major UK teaching hospital using a SARS-CoV-2 immunoassay. Risk factors for seropositivity were analysed using multivariate logistic regression. RESULTS: 410/5,698 (7·2%) staff tested positive for SARS-CoV-2 antibodies. Seroprevalence was higher in those working in designated COVID-19 areas compared with other areas (9·47% versus 6·16%) Healthcare assistants (aOR 2·06 [95%CI 1·14-3·71]; p=0·016) and domestic and portering staff (aOR 3·45 [95% CI 1·07-11·42]; p=0·039) had significantly higher seroprevalence than other staff groups after adjusting for age, sex, ethnicity and COVID-19 working location. Staff working in acute medicine and medical sub-specialities were also at higher risk (aOR 2·07 [95% CI 1·31-3·25]; p<0·002). Staff from Black, Asian and minority ethnic (BAME) backgrounds had an aOR of 1·65 (95% CI 1·32 - 2·07; p<0·001) compared to white staff; this increased risk was independent of COVID-19 area working. The only symptoms significantly associated with seropositivity in a multivariable model were loss of sense of taste or smell, fever, and myalgia; 31% of staff testing positive reported no prior symptoms. CONCLUSIONS: Risk of SARS-CoV-2 infection amongst HCWs is highly heterogeneous and influenced by COVID-19 working location, role, age and ethnicity. Increased risk amongst BAME staff cannot be accounted for solely by occupational factors.

Suppression of the intrinsic apoptosis pathway by synaptic activity.

Synaptic activity promotes resistance to diverse apoptotic insults, the mechanism behind which is incompletely understood. We show here that a coordinated downregulation of core components of the intrinsic apoptosis pathway by neuronal activity forms a key part of the underlying mechanism. Activity-dependent protection against apoptotic insults is associated with inhibition of cytochrome c release in most but not all neurons, indicative of anti-apoptotic signaling both upstream and downstream of this step. We find that enhanced firing activity suppresses expression of the proapoptotic BH3-only member gene Puma in a NMDA receptor-dependent, p53-independent manner. Puma expression is sufficient to induce cytochrome c loss and neuronal apoptosis. Puma deficiency protects neurons against apoptosis and also occludes the protective effect of synaptic activity, while blockade of physiological NMDA receptor activity in the developing mouse brain induces neuronal apoptosis that is preceded by upregulation of Puma. However, enhanced activity can also confer resistance to Puma-induced apoptosis, acting downstream of cytochrome c release. This mechanism is mediated by transcriptional suppression of apoptosome components Apaf-1 and procaspase-9, and limiting caspase-9 activity, since overexpression of procaspase-9 accelerates the rate of apoptosis in active neurons back to control levels. Synaptic activity does not exert further significant anti-apoptotic effects downstream of caspase-9 activation, since an inducible form of caspase-9 overrides the protective effect of synaptic activity, despite activity-induced transcriptional suppression of caspase-3. Thus, suppression of apoptotic gene expression may synergize with other activity-dependent events such as enhancement of antioxidant defenses to promote neuronal survival.

Implication of TAp73 in the p53-independent pathway of Puma induction and Puma-dependent apoptosis in primary cortical neurons.

Puma (p53 up-regulated modulator of apoptosis) is a BH3-only protein member of the Bcl-2 family that controls apoptosis by regulating the release of pro-apoptotic factors from mitochondria. Previously, we reported that sodium arsenite (NaAsO(2)) induces Puma-dependent apoptosis in cortical neurons in a p53-independent manner. The following evidence shows that p53-independent Puma activation by NaAsO(2) is mediated by the p53-related protein TAp73: (i) NaAsO(2) causes TAp73alpha accumulation and increases p53-independent expression of p73 target genes; (ii) two p53 response elements in the Puma promoter are required for NaAsO(2)-mediated activation of a Puma reporter construct; (iii) expression of the inhibitory DeltaNp73alpha and DeltaNp73beta isoforms decreases NaAsO(2)-mediated induction of Puma and other p53-family target genes in a p53-null background; (iv) DeltaNp73alpha and DeltaNp73beta expression protects the neurons from NaAsO(2)-dependent apoptosis. Interestingly, although ER stressors also induce p53-independent, Puma-dependent apoptosis, they do not increase TAp73 expression while NaAsO(2) does not induce notable endoplasmic reticulum (ER) stress. In contrast, DNA damaging agents, okadaic acid, and H(2)O(2) all induce apoptosis in a strictly Puma- and p53-dependent manner. Hence, the pivotal position of Puma as mediator of apoptosis in cortical neurons is because of the availability of at least three independent signalling pathways that ensure its activation.

Specific targeting of pro-death NMDA receptor signals with differing reliance on the NR2B PDZ ligand.

NMDA receptors (NMDARs) mediate ischemic brain damage, for which interactions between the C termini of NR2 subunits and PDZ domain proteins within the NMDAR signaling complex (NSC) are emerging therapeutic targets. However, expression of NMDARs in a non-neuronal context, lacking many NSC components, can still induce cell death. Moreover, it is unclear whether targeting the NSC will impair NMDAR-dependent prosurvival and plasticity signaling. We show that the NMDAR can promote death signaling independently of the NR2 PDZ ligand, when expressed in non-neuronal cells lacking PSD-95 and neuronal nitric oxide synthase (nNOS), key PDZ proteins that mediate neuronal NMDAR excitotoxicity. However, in a non-neuronal context, the NMDAR promotes cell death solely via c-Jun N-terminal protein kinase (JNK), whereas NMDAR-dependent cortical neuronal death is promoted by both JNK and p38. NMDAR-dependent pro-death signaling via p38 relies on neuronal context, although death signaling by JNK, triggered by mitochondrial reactive oxygen species production, does not. NMDAR-dependent p38 activation in neurons is triggered by submembranous Ca(2+), and is disrupted by NOS inhibitors and also a peptide mimicking the NR2B PDZ ligand (TAT-NR2B9c). TAT-NR2B9c reduced excitotoxic neuronal death and p38-mediated ischemic damage, without impairing an NMDAR-dependent plasticity model or prosurvival signaling to CREB or Akt. TAT-NR2B9c did not inhibit JNK activation, and synergized with JNK inhibitors to ameliorate severe excitotoxic neuronal loss in vitro and ischemic cortical damage in vivo. Thus, NMDAR-activated signals comprise pro-death pathways with differing requirements for PDZ protein interactions. These signals are amenable to selective inhibition, while sparing synaptic plasticity and prosurvival signaling.

Induction of sulfiredoxin expression and reduction of peroxiredoxin hyperoxidation by the neuroprotective Nrf2 activator 3H-1,2-dithiole-3-thione.

Peroxiredoxins are an important family of cysteine-based antioxidant enzymes that exert a neuroprotective effect in several models of neurodegeneration. However, under oxidative stress they are vulnerable to inactivation through hyperoxidation of their active site cysteine residues. We show that in cortical neurons, the chemopreventive inducer 3H-1,2-dithiole-3-thione (D3T), that activates the transcription factor Nuclear factor erythroid 2-related factor (Nrf2), inhibits the formation of inactivated, hyperoxidized peroxiredoxins following oxidative trauma, and protects neurons against oxidative stress. In both neurons and glia, Nrf2 expression and treatment with chemopreventive Nrf2 activators, including D3T and sulforaphane, up-regulates sulfiredoxin, an enzyme responsible for reducing hyperoxidized peroxiredoxins. Induction of sulfiredoxin expression is mediated by Nrf2, acting via a cis-acting antioxidant response element (ARE) in its promoter. The ARE element in Srxn1 contains an embedded activator protein-1 (AP-1) site which directs induction of Srxn1 by synaptic activity. Thus, raising Nrf2 activity in neurons prevents peroxiredoxin hyperoxidation and induces a new member of the ARE-gene family, whose enzymatic function of reducing hyperoxidized peroxiredoxins may contribute to the neuroprotective effects of Nrf2 activators.

Preconditioning doses of NMDA promote neuroprotection by enhancing neuronal excitability.

Neuroprotection can be induced by low doses of NMDA, which activate both synaptic and extrasynaptic NMDA receptors. This is in apparent contradiction with our recent findings that extrasynaptic NMDA receptor signaling exerts a dominant inhibitory effect on prosurvival signaling from synaptic NMDA receptors. Here we report that exposure to low preconditioning doses of NMDA results in preferential activation of synaptic NMDA receptors because of a dramatic increase in action potential firing. Both acute and long-lasting phases of neuroprotection in the face of apoptotic or excitotoxic insults are dependent on this firing enhancement. Key mediators of synaptic NMDA receptor-dependent neuroprotection, phosphatidylinositol 3 kinase-Akt (PI3 kinase-Akt) signaling to Forkhead box subgroup O (FOXO) export and glycogen synthase kinase 3beta (GSK3beta) inhibition and cAMP response element-binding protein-dependent (CREB-dependent) activation of brain-derived neurotrophic factor (BDNF), can be induced only by low doses of NMDA via this action potential-dependent route. In contrast, NMDA doses on the other side of the toxicity threshold do not favor synaptic NMDA receptor activation because they strongly suppress firing rates below baseline. The classic bell-shaped curve depicting neuronal fate in response to NMDA dose can be viewed as the net effect of two antagonizing (synaptic vs extrasynaptic) curves: via increased firing the synaptic signaling dominates at low doses, whereas firing becomes suppressed and extrasynaptic signaling dominates as the toxicity threshold is crossed.

The dichotomy of NMDA receptor signaling.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors plays a Jekyll and Hyde role in the mammalian central nervous system. In pathological scenarios such as ischemia, Ca2+ influx through the NMDA receptor is a key mediator of cell death. However, physiological levels of NMDA-receptor activity can promote neuronal survival and resistance to trauma and play important roles in synaptic plasticity and transmission. This dichotomy may explain the poor tolerance and efficacy of NMDA-receptor antagonists in clinical trials for excitotoxic trauma. There is a growing understanding of the signaling events that mediate the opposing effects of NMDA-receptor activity and the factors that determine whether an episode of NMDA-receptor activity will promote survival or death. This knowledge may lead to therapeutic strategies that enable the selective blockade of prodeath signaling cassettes while sparing physiological signaling to survival and plasticity.

Rare Variant Analysis of Human and Rodent Obesity Genes in Individuals with Severe Childhood Obesity.

Obesity is a genetically heterogeneous disorder. Using targeted and whole-exome sequencing, we studied 32 human and 87 rodent obesity genes in 2,548 severely obese children and 1,117 controls. We identified 52 variants contributing to obesity in 2% of cases including multiple novel variants in GNAS, which were sometimes found with accelerated growth rather than short stature as described previously. Nominally significant associations were found for rare functional variants in BBS1, BBS9, GNAS, MKKS, CLOCK and ANGPTL6. The p.S284X variant in ANGPTL6 drives the association signal (rs201622589, MAF~0.1%, odds ratio = 10.13, p-value = 0.042) and results in complete loss of secretion in cells. Further analysis including additional case-control studies and population controls (N = 260,642) did not support association of this variant with obesity (odds ratio = 2.34, p-value = 2.59 × 10-3), highlighting the challenges of testing rare variant associations and the need for very large sample sizes. Further validation in cohorts with severe obesity and engineering the variants in model organisms will be needed to explore whether human variants in ANGPTL6 and other genes that lead to obesity when deleted in mice, do contribute to obesity. Such studies may yield druggable targets for weight loss therapies.