Genetic Basis Of Obesity Research Articles

The Genetic Basis of Obesity and Related Metabolic Diseases in Humans and Companion Animals.

Obesity is one of the most prevalent health conditions in humans and companion animals globally. It is associated with premature mortality, metabolic dysfunction, and multiple health conditions across species. Obesity is, therefore, of importance in the fields of medicine and veterinary medicine. The regulation of adiposity is a homeostatic process vulnerable to disruption by a multitude of genetic and environmental factors. It is well established that the heritability of obesity is high in humans and laboratory animals, with ample evidence that the same is true in companion animals. In this review, we provide an overview of how genes link to obesity in humans, drawing on a wealth of information from laboratory animal models, and summarise the mechanisms by which obesity causes related disease. Throughout, we focus on how large-scale human studies and niche investigations of rare mutations in severely affected patients have improved our understanding of obesity biology and can inform our ability to interpret results of animal studies. For dogs, cats, and horses, we compare the similarities in obesity pathophysiology to humans and review the genetic studies that have been previously reported in those species. Finally, we discuss how veterinary genetics may learn from humans about studying precise, nuanced phenotypes and implementing large-scale studies, but also how veterinary studies may be able to look past clinical findings to mechanistic ones and demonstrate translational benefits to human research.

Genetic Basis of Obesity and Type 2 Diabetes in Africans: Impact on Precision Medicine.

Recent advances in genomics provide opportunities for novel understanding of the biology of human traits with the goal of improving human health. Here, we review recent obesity and type 2 diabetes (T2D)-related genomic studies in African populations and discuss the implications of limited genomics studies on health disparity and precision medicine. Genome-wide association studies in Africans have yielded genetic discovery that would otherwise not be possible; these include identification of novel loci associated with obesity (SEMA-4D, PRKCA, WARS2), metabolic syndrome (CA-10, CTNNA3), and T2D (AGMO, ZRANB3). ZRANB3 was recently demonstrated to influence beta cell mass and insulin response. Despite these promising results, genomic studies in African populations are still limited and thus genomics tools and approaches such as polygenic risk scores and precision medicine are likely to have limited utility in Africans with the unacceptable possibility of exacerbating prevailing health disparities. African populations provide unique opportunities for increasing our understanding of the genetic basis of cardiometabolic disorders. We highlight the need for more coordinated and sustained efforts to increase the representation of Africans in genomic studies both as participants and scientists.

Why are individuals with autism spectrum disorder at risk group for unhealthy weight?

Several studies have indicated that the prevalence of unhealthy body weight in individuals with autism spectrum disorder (ASD) is high. It is unclear whether factors related to the weight status of children and adolescents with ASD are the same or different from factors associated with the weight status of typically developing children (TDC). The objective of this review was to identify key factors associated with the higher rates of unhealthy weight observed in individuals with ASD and create a structural model, which could be used for future autism intervention research. This review summarizes the current state of knowledge on the genetic basis of obesity in ASD, special treatment (behavioral and medical, including diet and psychotropic medications), psychological (depression and anxiety) as well as somatic comorbid disorders such as feeding problems, sensory processing disorders, gastrointestinal problems and sleep disorders, physical activity and sedentary behaviors, loneliness and social isolation, and family functioning (maternal depression and stress).

Gene-Environment Interactions on Body Fat Distribution.

The prevalence of obesity has been increasing markedly in the U.S. and worldwide in the past decades; and notably, the obese populations are signified by not only the overall elevated adiposity but also particularly harmful accumulation of body fat in the central region of the body, namely, abdominal obesity. The profound shift from “traditional” to “obesogenic” environments, principally featured by the abundance of palatable, energy-dense diet, reduced physical activity, and prolonged sedentary time, promotes the obesity epidemics and detrimental body fat distribution. Recent advances in genomics studies shed light on the genetic basis of obesity and body fat distribution. In addition, growing evidence from investigations in large cohorts and clinical trials has lent support to interactions between genetic variations and environmental factors, e.g., diet and lifestyle factors, in relation to obesity and body fat distribution. This review summarizes the recent discoveries from observational studies and randomized clinical trials on the gene–environment interactions on obesity and body fat distribution.

Whole exome sequencing reveals novel LEPR frameshift mutation in severely obese children from Western India.

BackgroundObesity, especially early onset of obesity is a serious health concern in both developed and developing countries. This is further associated with serious comorbidities like a fatty liver disease, cardiovascular diseases, type‐2 diabetes, obstructive sleep apnea, renal complications and respiratory problems. Many times early onset of obesity is linked with heritable monogenic, polygenic and syndromic forms. Globally, studies on roles of genes involved in early onset of obesity are limited.MethodsHere in this study, a consanguineous family of Western Indian origin having four siblings, one unaffected and three affected with severe early onset of obesity was enrolled. Affected siblings also displayed comorbidities like mild to moderate obstructive sleep apnea, raised Renal Resistance Index, oliguria, and severe anemia. Whole Exome Sequencing (WES) of Trio with one affected and unaffected sibling was done. Data analysis was performed to check pathogenic mutation segregation in unaffected parents with affected and unaffected sibling.ResultsWES of trio identified novel frameshift mutation in the LEPR gene resulting in truncated leptin receptor (LEPR). The same mutation was confirmed in other affected siblings and two siblings of distant relatives by Sanger sequencing. The possible effects of truncating mutation in LEPR function by in silico analysis were also studied.ConclusionUnderstanding genetic basis of obesity might provide a clue for better management and treatment in times to come. This work demonstrates identification of novel mutation in LEPR gene resulting into early onset of obesity. Discovery of novel, population‐specific genomics markers will help population screening programs in creating base for possible therapeutic applications and prevention of this disease for next generations.

The case of GWAS of obesity: does body weight control play by the rules?

As yet, genome-wide association studies (GWAS) have not added much to our understanding of the mechanisms of body weight control and of the etiology of obesity. This shortcoming is widely attributed to the complexity of the issues. The appeal of this explanation notwithstanding, we surmise that (i) an oversimplification of the phenotype (namely by the use of crude anthropometric traits) and (ii) a lack of sound concepts of body weight control and, thus, a lack of a clear research focus have impeded better insights most. The idea of searching for polygenetic mechanisms underlying common forms of obesity was born out of the impressive findings made for monogenetic forms of extreme obesity. In the case of common obesity, however, observational studies on normal weight and overweight subjects never provided any strong evidence for a tight internal control of body weight. In addition, empirical studies of weight changes in normal weight and overweight subjects revealed an intra-individual variance that was similar to inter-individual variance suggesting the absence of tight control of body weight. Not least, this lack of coerciveness is reflected by the present obesity epidemic. Finally, data on detailed body composition highlight that body weight is too heterogeneous a phenotype to be controlled as a single entity. In summary GWAS of obesity using crude anthropometric traits have likely been misled by popular heritability estimates that may have been inflated in the first place. To facilitate more robust and useful insights into the mechanisms of internal control of human body weight and, consequently, the genetic basis of obesity, we argue in favor of a broad discussion between scientists from the areas of integrative physiologic and of genomics. This discussion should aim at better conceived studies employing biologically more meaningful phenotypes based on in depth body composition analysis. To advance the scientific community-including the editors of our top journals-needs a re-launch of future GWAS of obesity.

Genetic basis of obesity: a review

Obesity is a state of “positive” energy balance when energy intake in is much greater than energy expenditure. The familial aggregation of body size was first published by Sir Francis Galton in 1889, which become a well-established risk factor for childhood obesity. Obesity is often allied with insulin resistance, dyslipidemia and cardiovascular disease. Monogenic obesity includes the involvements of Leptin (LEP) gene, leptin receptor(LEPR), Proopiomelanocortin (POMC), Melanocortin-4 receptor (MC4R), single-minded gene 1 (SIM 1), Proprotein convertase subtilisin/ kexin type 1 (PCSK1), Neuropeptide Y (NPY). Polygenic obesity is due to Adrenoceptor beta 1 (ADRB1), ADRB2, ADRB3, Uncoupling protein 1(UCP1), UCP2, UCP3. Monogenic and polygenic obesity; pleiotropic syndromes, chromosomal rearrangement are different types of obesity with genetic cause. Surprisingly all “obesity genes” carriers do not become overweight, and exercise, maintaining healthy routine is a significant contributing factor to nullify genetic prediposition.Journal of Biomedical Sciences, Vol. 3, No.2, 2016, Page: 24-28

Future management of human obesity: understanding the meaning of genetic susceptibility

Gene-environment interactions are central to the expression of obesity. The condition is strongly heritable (ie, genetic), and most of the variation in obesity levels between countries and between individuals can be explained by the effects of obesogenic environments on individual genetic susceptibilities. The nature of the obesogenic environmental influences is not clear in detail, but they correlate closely with measures of affluence. The causes of variation in genetic susceptibility are also not clearly defined, but their general nature has become clearer. The failure of genome-wide association studies or large linkage studies to identify or replicate causative genetic variants, together with the segregation of obesity-related traits in families, implicates a heterogenetic mechanism in which rare, dominantly or additively expressed genetic variants are responsible for most of common obesity. The search for rare causative variants continues with some successes, but those identified contribute very little to the overall burden and, assuming heterogenetics, there are many more to find. The time when genomic risk factors provide more information than do currently available markers, such as family history, is a long way off. Genomic studies to date have contributed little, if anything, to the prevention and treatment of common obesity and its associated disorders. This contrasts with the obvious and immediate potential implications of the well-established overall genetic basis of obesity, which have not yet been exploited in the clinical or public health arenas. Genomic studies, which have helped to define the genetic basis of common obesity mainly by exclusion, will in the future play an increasingly important role in understanding and managing obesity, but only with parallel studies of the physiological, behavioral, and economic influences. Disciplines Medicine and Health Sciences | Social and Behavioral Sciences Publication Details Jenkins, A. & Campbell, L. (2014). Future management of human obesity: understanding the meaning of genetic susceptibility. Advances in Genomics and Genetics, 4 219-232. This journal article is available at Research Online: http://ro.uow.edu.au/smhpapers/2772

EJE Prize 2012: Obesity: from genes to behaviour.

An increase in the consumption of highly palatable foods coupled with a reduction in the amount of voluntary exercise undertaken has contributed to the rising prevalence of obesity. However, despite the obvious environmental influences, there is considerable evidence to support a genetic component to weight gain. In some people, particularly those who are severely obese, genetic factors play a major role in the development of their obesity and associated complications. Studies into the genetic basis of obesity have yielded insights into the mechanisms involved in the regulation of weight. We now understand that weight is regulated by neural mechanisms that regulate appetite and energy expenditure and that disruption of these pathways can result in severe obesity in some patients. These studies provide a starting point for investigating patients with severe obesity and may ultimately guide the development of more rational targeted therapies.

Association study of common polymorphisms in MSRA, TFAP2B, MC4R, NRXN3, PPARGC1A, TMEM18, SEC16B, HOXB5 and OLFM4 genes with obesity-related traits among Portuguese children

At least 52 genetic loci were associated with obesity-related traits. However, little is known about the genetic basis of obesity among children. This study aims to test whether 10 polymorphisms in obesity-related genes methionine sulfoxide reductase A (MSRA), transcription factor AP-2 beta (TFAP2B), melanocortin 4 receptor (MC4R), neurexin 3 (NRXN3), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A), transmembrane protein 18 (TMEM18), homolog of S. cerevisiae Sec16 (SEC16B), homeobox B5 (HOXB5) and olfactomedin 4 (OLFM4) are associated with the risk of obesity in Portuguese children. A total of 730 children aging from 6 to 12 years old, recruited randomly from public schools in Portugal, were analysed. Anthropometric measurements were obtained and children were classified into three phenotypic groups, normal weight (n=256), overweight (n=320) and obese (n=154), according to the International Obesity Task Force cutoffs. Polymorphisms were genotyped by allelic discrimination TaqMan assays. The MC4R rs12970134 polymorphism was nominally associated with body mass index (BMI) (P=0.035), BMI Z-score (P=0.043) and waist circumference (P=0.020), and borderline associated with weight (P=0.053). Near nominal associations were also found for the PPARGC1A rs8192678 polymorphism with weight (P=0.061), and for the MSRA rs545854 polymorphism with BMI (P=0.055) and BMI Z-score (P=0.056). Furthermore, logistic regression showed that MC4R rs12970134 and TFAP2B rs987237 were nominally, respectively, associated (P=0.029) and borderline associated (P=0.056) with the obese phenotype. This study highlighted the possible association of MC4R, PPARGC1A, MSRA and TFAP2B polymorphisms with several obesity-related traits in a sample of Portuguese children. The two significant associated TFAP2B rs987237 and MC4R rs12970134 polymorphisms showed an opposite direction of effect to that in the original reports.

On the Evolutionary Origins of Obesity: A New Hypothesis

Obesity is an escalating threat of pandemic proportions, currently affecting billions of people worldwide and exerting a devastating socioeconomic influence in industrialized countries. Despite intensive efforts to curtail obesity, results have proved disappointing. Although it is well recognized that obesity is a result of gene-environment interactions and that predisposition to obesity lies predominantly in our evolutionary past, there is much debate as to the precise nature of how our evolutionary past contributed to obesity. The "thrifty genotype" hypothesis suggests that obesity in industrialized countries is a throwback to our ancestors having undergone positive selection for genes that favored energy storage as a consequence of the cyclical episodes of famine and surplus after the advent of farming 10 000 years ago. Conversely, the "drifty genotype" hypothesis contends that the prevalence of thrifty genes is not a result of positive selection for energy-storage genes but attributable to genetic drift resulting from the removal of predative selection pressures. Both theories, however, assume that selection pressures the ancestors of modern humans living in western societies faced were the same. Moreover, neither theory adequately explains the impact of globalization and changing population demographics on the genetic basis for obesity in developed countries, despite clear evidence for ethnic variation in obesity susceptibility and related metabolic disorders. In this article, we propose that the modern obesity pandemic in industrialized countries is a result of the differential exposure of the ancestors of modern humans to environmental factors that began when modern humans left Africa around 70 000 years ago and migrated through the globe, reaching the Americas around 20 000 years ago. This article serves to elucidate how an understanding of ethnic differences in genetic susceptibility to obesity and the metabolic syndrome, in the context of historic human population redistribution, could be used in the treatment of obesity in industrialized countries.

Altered lipid homeostasis inDrosophilaInsP3 receptor mutants leads to obesity and hyperphagia

SUMMARYObesity is a complex metabolic disorder that often manifests with a strong genetic component in humans. However, the genetic basis for obesity and the accompanying metabolic syndrome is poorly defined. At a metabolic level, obesity arises from an imbalance between the nutritional intake and energy utilization of an organism. Mechanisms that sense the metabolic state of the individual and convey this information to satiety centers help achieve this balance. Mutations in genes that alter or modify such signaling mechanisms are likely to lead to either obese individuals, who in mammals are at high risk for diabetes and cardiovascular disease, or excessively thin individuals with accompanying health problems. Here we show that Drosophila mutants for an intracellular calcium signaling channel, the inositol 1,4,5-trisphosphate receptor (InsP3R) store excess triglycerides in their fat bodies and become unnaturally obese on a normal diet. Although excess insulin signaling can rescue obesity in InsP3R mutants to some extent, we show that it is not the only cause of the defect. Through mass spectrometric analysis of lipids we find that homeostasis of storage and membrane lipids are altered in InsP3R mutants. Possibly as a compensatory mechanism, InsP3R mutant adults also feed excessively. Thus, reduced InsP3R function alters lipid metabolism and causes hyperphagia in adults. Together, the metabolic and behavioral changes lead to obesity. Our results implicate altered InsP3 signaling as a previously unknown causative factor for metabolic syndrome in humans. Importantly, our studies also suggest preventive dietary interventions.

Genetic studies of common types of obesity: a critique of the current use of phenotypes

Recent research into the genetic basis of obesity has focused upon the study of candidate genes, both functional and positional, of genes underlying weight-related Mendelian disorders and of susceptibility loci identified in genome-wide association studies. Three large genome-wide association studies on obesity, together involving more than 150,000 individuals, were published in Nature Genetics last year. The results suggested the involvement of a large number of genetic variants in disease susceptibility. Most genetic effects upon body weight are likely to become obscured by the use of inappropriate phenotypes. In particular, clinical categories such as the body mass index and Metabolic Syndrome do not provide sufficient etiological information for them to be used sensibly in genetic studies on obesity or obesity-related disease. Alleviation of this situation will not come from new genomic research tools, sophisticated statistical algorithms or ever larger sample sizes. Instead, the above notions argue in favour of so-called 'deep phenotyping'.

The New Obesity Genes

Individual susceptibility to obesity is recognized to be influenced significantly by genetic inheritance. Recently, candidate obesity genes have been identified that may contribute to the inheritance of body fat mass and the partitioning of fat between central and peripheral fat depots. In studies of animal models of obesity, the genetic basis for obesity in the obese (Ob/Ob) mouse, the Fat mouse, and the Yellow (Vvy) mouse has been identified. Further research is needed to determine whether abnormalities in these genes contribute to human obesity as well. In studies of humans, sequence variation in at least six genes has been linked to increased body fatness and/or susceptibility to obesity. In addition, five other encoding genes have been linked to a disproportionate storage of fat in the abdominal region. These genes identified in studies of humans are currently thought to be modifying or background genes, each separately conferring a modest increase in susceptibility to fatness. Further research is needed to identify additional candidate genes that confer susceptibility to obesity and to determine the relative importance of each one in a range of human populations with distinct environments.

The contribution of animal models to the study of obesity

Obesity results from prolonged imbalance of energy intake and energy expenditure. Animal models have provided a fundamental contribution to the historical development of understanding the basic parameters that regulate the components of our energy balance. Five different types of animal model have been employed in the study of the physiological and genetic basis of obesity. The first models reflect single gene mutations that have arisen spontaneously in rodent colonies and have subsequently been characterized. The second approach is to speed up the random mutation rate artificially by treating rodents with mutagens or exposing them to radiation. The third type of models are mice and rats where a specific gene has been disrupted or over-expressed as a deliberate act. Such genetically-engineered disruptions may be generated through the entire body for the entire life (global transgenic manipulations) or restricted in both time and to certain tissue or cell types. In all these genetically-engineered scenarios, there are two types of situation that lead to insights: where a specific gene hypothesized to play a role in the regulation of energy balance is targeted, and where a gene is disrupted for a different purpose, but the consequence is an unexpected obese or lean phenotype. A fourth group of animal models concern experiments where selective breeding has been utilized to derive strains of rodents that differ in their degree of fatness. Finally, studies have been made of other species including non-human primates and dogs. In addition to studies of the physiological and genetic basis of obesity, studies of animal models have also informed us about the environmental aspects of the condition. Studies in this context include exploring the responses of animals to high fat or high fat/high sugar (Cafeteria) diets, investigations of the effects of dietary restriction on body mass and fat loss, and studies of the impact of candidate pharmaceuticals on components of energy balance. Despite all this work, there are many gaps in our understanding of how body composition and energy storage are regulated, and a continuing need for the development of pharmaceuticals to treat obesity. Accordingly, reductions in the use of animal models, while ethically desirable, will not be feasible in the short to medium term, and indeed an expansion in activity using animal models is anticipated as the epidemic continues and spreads geographically.

돼지 melanocortin-4 receptor (MC4R) 유전자의 경제형질과의 연관성에 관한 연구

본 연구는 Duroc, Landrace, Berkshire, Yorkshire를 기초 축으로 이용한 1003두에 대해 MC4R유전자의 PCR-RFLP를 이용하여 그 다형성을 조사하고 돼지의 일당증체량, 등지방 두께, 사료 요구율, 정육율과 그 유전자형 간의 연관성을 규명하고자 실시하였다. MC4R유전자에 대해 PCR-RFLP를 이용하여 226bp산물을 증폭한후 Taq I 체한효소로 사용하였다. 얻어진 MC4R gene의 유전자 빈도는 품종별로 다르게 나타났다. 통계적 분석을 통하여 각 유전자형에 대한 경제형질과 관련성을 분석한 결과 일당 증체량과 사료요구량은 NN 유전자형을 가진 개체들이 DN이나 DD유전자형을 가진 개체들에 비해 유의적으로 우수한 능력을 보였다(P < 0.05). 하지만 D 대립유전자는 높은 정육율과 낮은 등지방두께에 연관성이 있음을 관찰하였다. 따라서 돼지의 성장과 정육율과 관련된 선발력을 높이기 위해서 MC4R유전자의 다형성분석에서 검증된 PCR marker를 우량돼지육종 계획에 있어 분자생물학적 선발 marker로 사용할 수 있을 것으로 사료된다. The Melanocortin-4 receptor gene (MC4R) regulates the energy balance and the genetic basis of obesity. A polymorphism in the porcine melanocortin-4 receptor has previously shown to be associated with growth, fat deposition and feed intake. In this study, the polymorphism of the gene was studied in several pig breeds of Duroc, Landrace, Berkshire, and Yorkshire. The results showed that the frequencies of MC4R genotype varied among those breeds. Association analyses were also performed between the MC4R polymorphism and average daily gain, feed conversion ratio, backfat thickness and lean percentage phenotypes. The results strongly support that the MC4R polymorphism can be used DNA marker selection indicator for economically important traits for pig breeding program in Korea.

The epidemic of obesity and changes in food intake: the Fluoride Hypothesis

The epidemic of obesity is worldwide. It will be followed by an epidemic of diabetes. Although there is a genetic basis for obesity and diabetes, the current epidemic reflects the failure of our ancient genes to cope with a modern toxic environment. To put it another way, the genetic background loads the gun, but the environment pulls the trigger. Diet, lifestyle and exercise are the cornerstones of current approaches to treating obesity. However, these approaches that depend on individuals making lifestyle changes have been ineffective in preventing the epidemic. An alternative model views obesity as an epidemiological disease with food(s) and other environmental agents acting on the host to produce disease. The consumption patterns for many foods have changed over the past 30 years, but the increase in the consumption of high-fructose corn syrup (HFCS) for soft drinks is far and away the largest. Moreover, the rise in HFCS intake is an environmental insult that has occurred at exactly the same time as obesity began to increase in prevalence. Rising soft drink consumption is associated with a decrease in milk consumption and a decrease in calcium intake, which has an inverse relationship to body mass index (BMI). To combat the epidemic of obesity, we need new strategies that flow from the epidemiological model. The Fluoride Hypothesis for obesity proposes that we can make environmental changes that when made, will reduce the epidemic of obesity, in much the same way as fluoride reduced the incidence of dental disease. Fluoride-like strategies can work without the personal effort required by changes in lifestyle. In this context, fluoride is also an acronym for treatment and prevention of obesity: For Lowering Universal Obesity Rates are Implement ideas that Don't demand Effort (FLUORIDE).

Etiology of massive obesity: role of genetic factors.

It is well established that there is a significant familial aggregation of obesity, although most of the evidence regarding the genetic basis of obesity has been derived from overweight and moderately obese cases. Less is known about the contribution of genetic factors in severely obese individuals. This paper reviews the available evidence regarding the extent of familial aggregation of morbid obesity and the contribution of specific genes. The results of available studies suggest a stronger degree of familial resemblance for morbid obesity [body mass index (BMI > 40 kg/m2)] than for more moderate levels of obesity (BMI < 40 kg/m2). Evidence from human association and linkage studies, performed with markers surrounding human homologs of the genes involved in mouse models of obesity, revealed that these genes tend to be linked more often to severe obesity than to moderate levels of obesity.

The genetic basis of obesity in rodents

The genetic basis of obesity in rodents

Regulated expression of the obese gene product (leptin) in white adipose tissue and 3T3-L1 adipocytes.

A mutation within the obese gene was recently identified as the genetic basis for obesity in the ob/ob mouse. The obese gene product, leptin, is a 16-kDa protein expressed predominantly in adipose tissue. Consistent with leptin's postulated role as an extracellular signaling protein, human embryonic kidney 293 cells transfected with the obese gene secreted leptin with minimal intracellular accumulation. Upon differentiation of 3T3-L1 preadipocytes into adipocytes, the leptin mRNA was expressed concomitant with mRNAs encoding adipocyte marker proteins. A factor(s) present in calf serum markedly activated expression of leptin by fully differentiated 3T3-L1 adipocytes. A 16-hr fast decreased (by approximately 85%) the leptin mRNA level of adipose tissue of lean (ob/+ or +/+) mice but had no effect on the approximately 4-fold higher level in obese (ob/ob) littermates. Since the mutation at the ob locus fails to produce the functional protein, yet its cognate mRNA is overproduced, it appears that leptin is necessary for its own downregulation. Leptin mRNA was also suppressed in adipose tissue of rats during a 16-hr fast and was rapidly induced during a 4-hr refeeding period. Insulin deficiency provoked by streptozotocin also markedly down-regulated leptin mRNA and this suppression was rapidly reversed by insulin. These results suggest that insulin may regulate the expression of leptin.