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Heritability of body weight: Moving beyond genetics
Nutrition, Metabolism & Cardiovascular Diseases (2010) 20, 691e697
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/nmcd
VIEWPOINT
Heritability of body weight: Moving beyond genetics P. Russo, F. Lauria, A. Siani* Unit of Epidemiology and Population Genetics, Institute of Food Sciences, CNR, Via Roma, 64-83100 Avellino, Italy Received 8 July 2010; received in revised form 7 September 2010; accepted 16 September 2010
KEYWORDS Obesity; Genetics; Genome-wide association studies; Epigenetics
Abstract Obesity is a complex disease, arising from the interaction between several genetic and environmental factors. Until recently, the genetic basis of complex diseases in general, and of obesity in particular, were poorly characterized. While the relatively rare monogenic and syndromic forms of obesity clearly recognize a genetic origin, the actual worldwide epidemics of obesity represent a challenge for the identification of the genetic factors involved, being likely the effect of several loci each having a subtle influence on the phenotypic expression. Progress in DNA analysis techniques and in computational tools, and the increasing level of characterization of the variability of the human genome has recently allowed to study comprehensively the association between genetic variants and obesity. To date, wellconducted and powered genome-wide association studies allowed to consistently identify genomic regions e lying on different chromosomes and affecting different metabolic pathways e influencing the predisposition to the accumulation of body fat, ultimately leading to overweight and obesity. However, the population attributable risk for obesity linked to the most statistically significant loci, like FTO and MC4R, remains discouragingly low, explaining only small fractions of the overall variance of body weight. In the last few years, the role of the complex interaction between genetic determinants and environmental factors in the rapid global increase of obesity has been further challenged by the entry of new players, that is the transcriptional and post-transcriptional regulation, summarized under the emerging discipline of epigenetics. The key challenge now is to move from the identification of causal genes and variants to the integration of different “omics” disciplines, finally allowing the molecular understanding of obesity and related conditions. ª 2010 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: þ39 0825 299353; fax: þ39 0825 299423. E-mail address: [email protected] (A. Siani). 0939-4753/$ - see front matter ª 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.numecd.2010.09.007
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Introduction Obesity is a complex disease, arising from the interaction between several genetic and environmental factors. The current worldwide surge of obesity in both adults and children may be conceivably attributed to the interaction between the ancestral genetic predisposition toward efficient energy storage, which is the legacy of the huntergatherer cultures, and a permissive (obesogenic) contemporary environment of readily available food and sedentary behaviours. This genetic predisposition, often described as “thrifty genotype” [1], appears nowadays maladaptive, increasing the individual predisposition to obesity and related metabolic disorders. This theory, although recently challenged with attractive arguments [2e4], still remains the most accepted, until alternative hypotheses will find confirmation. In the last few years, the role of the complex interaction between genetic determinants and environmental factors in the rapid global increase of obesity has been further challenged by the entry of a new player, that is the epigenetic adaptation [5], fuelling the debate surrounding the aetiology of obesity. From the genetic point of view, obesity is classified into three main categories: monogenic, syndromic, and polygenic obesity [6]. This classification may be applied to both childhood and adult forms of human obesity, although monogenic and syndromic forms are most often evident very early in the life [7]. The study of the extreme Mendelian human obesity phenotypes provided solid evidence in favour of the genetic origin of obesity [8,9]. The discussion of these severe and rare forms is beyond the objectives of this viewpoint, which is mainly devoted to address some questions and challenges about the epidemiologically most relevant form of obesity, that is, polygenic obesity.
Heritability of body weight In 1889, Sir Francis Galton in his book “Natural Inheritance” suggested for the first time the familial aggregation of the individual’s body size [10,11]. This initial observation was corroborated by studies in the first decades of the last century [12], showing a clear association between offspring and parental body weight. However, this observation could not dissect out the relative role of genetic and environmental factors in body weight determination, with parents and their progeny sharing the same environment. Although environmental factors likely play a major role, it is clear that the regulation of body weight has a large underlying genetic component, that usually follows complex patterns of segregation, involving the subtle influence of multiple genes [13]. More than 25 years ago, Stunkard unequivocally reported [14,15] a heritability estimate of 0.78 for body weight, increasing to 0.81 in a 25-year follow-up study. Further twin studies have confirmed heritability estimates of w0.7 for BMI in both adults and children [16,17]. Controlled overfeeding studies in monozygotic twins showed that the tendency towards weight gain within each couple of twins was comparable, confirming the relevant
P. Russo et al. role of genetic factors on body weight regulation, particularly evident when food intake and exercise are controlled to reduce the environmental variance [18e20]. A complementary model to the twin study is the adoption study. In fact, adoptive children share the environment with their adoptive parents while they share their genetic background only with their biological parents. The results of the adoption studies consistently showed that the adoptees present a highly statistically significant association of their BMI with that of their biological parents, but not with that of their adoptive parents [15,21,22]. In summary, quantitative genetics studies clearly demonstrated that genetic factors play a significant role in the causes of individual differences in relative body weight and human body fat accumulation.
The candidate gene approach The characterization of the relative role of geneegene and geneeenvironment interaction in polygenic disorders of body weight regulation, such as obesity, is extremely challenging. It seems conceivable that several gene products, each making a relatively small contribution, interact to build up the genetic background that may explain a variable but significant proportion of the variance of body weight. The candidate-gene strategy has been the traditional approach for identifying the genes involved in complex diseases [23]. The candidate gene approach directly tests the effects of genetic variants of a potentially contributing gene in an association study. This approach involves the assessment of the association between a particular allele (or set of alleles) of a gene that may be involved in the disease (i.e. the candidate gene) and the disease itself. These studies, which may include members of an affected family or unrelated cases and controls, can be performed relatively quickly and inexpensively and may allow identification of genes with small effects. However, this approach is limited by how much is known of the biology of the disease being investigated. In fact, the identification of candidate genes represents a difficult task, given the number of pathways involved and the subsequent huge number of potential candidate genes. In the case of obesity, the potential genetic markers lie indeed on various biochemical and metabolic pathways, such as energy expenditure, appetite regulation, lipid metabolism, endothelial function, adrenergic activation, inflammation etc, highlighting the multiple geneegene, geneenutrient and geneenutrienteenvironment interactions in the pathogenesis of this complex disorder. In the case of rare monogenic diseases, the molecular approach has proven extremely powerful in the identification of the genes responsible and in defining new syndromes [24]. Conversely, in common polygenic disorders like obesity a large number of studies were published, many based on collections as small as only tens of samples, or at best several hundreds, and numerous weak associations were reported in the literature. Some of the identified genes have received confirmation by betterpowered genetic association studies, but most have never been replicated, so raising unwarranted enthusiasm, often followed by scepticism within the scientific community. Among the possible explanations, beside the limited study
Heritability of body weight size and the inadequacy of available bio-computing tools, we also include the failure to detect geneegene interactions and the lack of reporting environmental data of possible importance for geneeenvironment interactions [25,26]. Notwithstanding the above described pitfalls, in recent years sizeable progress has been made toward the understanding of the genetic bases of polygenic obesity. From the first release of the Human Obesity Gene Map [27], the meritorious work of a group of researchers gave us the annual update of the state of art of the research on the genetics of obesity. The last release [28] summarized in 106 pages the scientific evidence available in this field up to October 2005. Their impressive, though in some way discouraging, conclusion was that “The number of studies reporting associations between DNA sequence variation in specific genes and obesity phenotypes has also increased considerably, with 426 findings of positive associations with 127 candidate genes. A promising observation is that 22 genes are each supported by at least five positive studies. The obesity gene map shows putative loci on all chromosomes except Y”. Unfortunately, in publishing the 12th update, the authors announced that it would be the last of the series, due to the lack of economical and human resources to collect and manage such enormous amount of information [29]. From that time on, thousands of researchers worldwide were challenged by the same question: among all the possibilities, what are the inherited genetic variants that really shape the susceptibility to common obesity? Different approaches were adopted to answer this question, including population-based or family-based genetic association studies, linkage disequilibrium mapping, haplotype tagging and the latest gene-finding strategy, that is the whole genome association scans. A discussion of the advantages/disadvantages of each of these techniques is far beyond the objectives of this paper. Indeed, in spite of the difficulties, both the candidate single-nucleotide polymorphisms (SNPs) and the quantitative trait locus (QTL) strategies identified a large number of genetic markers potentially associated with the susceptibility to adult and childhood obesity on various chromosomes [28]. Many excellent reviews summarized progress and delusion on the way of the research of the genetic basis of obesity [30e35]. The scenario rapidly evolved over the last decade. Large international collaborative studies allowed the researcher to access databases of well-defined phenotypes with related DNA and biological sample banks and more precise environmental information.
Genome-wide association studies in obesity: the model of FTO The immense progress in molecular biology tools, like microarrays and bioinformatics, has led to a steeper increase in the identification of the genetic basis of complex, nonMendelian diseases through the genome-wide association (GWA) approach. In short, high-throughput genotyping technologies were used to test hundreds of thousands of SNPs, each of them identified and assigned a unique reference number (rs) in the National Center for Biotechnology
693 Information’s dbSNP database [36]. The GWA approach represented a significant advance in gene-hunting strategies, because it is free from prior hypotheses regarding candidacy of genes, it permits to explore at the highest level of resolution the whole genome and it allows to associate hundreds of thousands of genetic variants with the phenotype (disease) of interest [37]. The GWA studies, driven by the completion of the Human Genome project [38] and, more recently, by the International HapMap [39] updated in 2007 [40] and by the availability of the high-throughput technologies, are able, at least in part, to circumvent the obstacles encountered with both the candidate genes and the linkage approach. In the last years, an avalanche of GWA studies appeared in the scientific literature, linking common genetic variations to obesity and related metabolic traits. Apparently, these studies overcame some of the methodological problems described with the candidate gene approach. Here, we briefly comment on how the information gathered by these studies could help uncovering the genetic basis of obesity. Given that the list of obesity-associated loci is increasing day-by-day, we will only discuss the role of one gene that recently emerged as being robustly and consistently associated to common obesity, that is the Fat Mass and Obesity Associated gene (FTO). Almost simultaneously, three well-powered GWA studies published in 2007 identified a cluster of common SNPs in the first intron of FTO on chromosome 16 as unexpected but strong contributors to both childhood and adult obesity phenotypes [41e43]. Human FTO is a gene of unknown function in an unknown pathway and presents high homology with the murine Fto, located on mouse chromosome 8 [44]. It encodes for a member of the non-heme dioxygenase superfamily and it is ubiquitously expressed in foetal and adult tissues, particularly in the brain. Interestingly, in the first published report by Frayling et al. [41], the FTO locus was strongly associated with type 2 diabetes and secondarily with BMI in 1924 U.K. type 2 diabetes patients and 2938 U.K. population controls. The association with type 2 diabetes disappeared after adjustment for BMI, thus suggesting that BMI mediates the association with the risk of diabetes. In the same report, the association of the rs9939609 SNP of FTO with BMI was confirmed in 13 cohorts of adults and children with 38,759 participants of European origin. The 16% of adults who were homozygous for the risk allele weighed about 3 kg more and had 1.67-fold increased odds of obesity when compared with those not inheriting a risk allele. Effects of comparable magnitude were present in children. The association of a set of SNPs at this locus with BMI and obesity was unequivocally confirmed in the studies by Dina et al. [42] in European adults and children and by Scuteri et al. [43] in Sardinian adults, the latter study extending the analysis also to independent samples of European and Hispanic Americans. To date, several studies confirmed these initial observations, consistently linking SNPs at the FTO locus to other obesity-related phenotypes and markers, including body fat distribution, fat mass, and leptin levels [45]. Of particular interest is the wide distribution of the riskcarrying FTO allele in European populations, with about 63% of the population being at least heterozygous and 16% homozygous for the risk allele. But data are not entirely
694 consistent in some other ethnic groups, notably African American, Chinese and Japanese populations in which the FTO variants are not always associated with BMI or obesity phenotypes [42,46e48]. In all these populations minor allele frequencies and linkage disequilibrium patterns are different form that reported for European populations. Despite the consistency of the association of FTO with BMI in people of European ancestry, the effect size is relatively small, explaining about 1% of the overall phenotypic variation of obesity observed in the general population [49]. The relatively weak association of the FTO gene with metabolic factors and with obesity-associated conditions such as type 2 diabetes is in favour of a non-metabolic pathway of action of this gene. In February 2009, an elegant experimental study was published in Nature, providing clues towards possible mechanisms by which FTO acts on energy homeostasis and in turn on the regulation of body weight [50]. Fischer et al. generated an Fto knock-out mice strain and showed the absence of the FTO protein in the Fto/ mutant. The loss of Fto was associated with post-natal growth retardation and significant reduction in both adipose tissue and lean body mass. The mechanisms advocated for these effects are increased energy expenditure and sympathetic activation. The results of this study provided the first translation of the human epidemiological data into functional evidence of the involvement of Fto/FTO in energy homeostasis and finally into body weight regulation. In summary, the “agnostic” approach of the GWA studies opened a new research avenue to the study of the genetic roots of obesity and body weight regulation [51]. The success stories of GWA studies cannot hide the seemingly paradoxical result that association p-values higher than 105 only result in weak genetic effects, explaining only a small fraction of the phenotypic variation observed in the general population for the diseases under study [52,53]. In fact, the combined effect of common variants in two of the most robustly obesity-associated loci, that is FTO and MC4R, explains not more than 2% of the variance in body weight at the population level. To explain the remaining fraction of the genetic-linked body mass variability, the term “missing heritability” has been coined [49]. As a working hypothesis, this remaining part may reside on a large number of very common genetic variants exerting small effects and/or on rare variants with large effects each. A simulation exercise estimated that for variants with small effects, 50 genes with a genotypic prevalence of 10% would be needed to explain about 50% of the population attributable risk of complex diseases, while for rare variants with large effects as many as hundreds of genes would be needed to explain the same proportion of risk [54]. A plausible biological explanation should indeed consider the possibility of epigenetic effects affecting important regulatory regions/pathways of our genome.
Epigenetic mechanisms in obesity In the previous paragraphs, we sought to document that DNA sequence variants may influence an individual’s susceptibility to obesity and related syndromes. However, a “dark matter” still persists with regard the genetic
P. Russo et al. explanation for the global obesity epidemic of the last five decades, since it is evident that the classical models to describe the geneeenvironment interaction do not provide satisfactory answers. In the past few years, epigenetics emerged as a complementary model to explain the rapid emergence of obesity and metabolic disorders [5]. Epigenetics is defined as “the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence” [55,56]. These non-genetic alterations are under the tight regulation of two major epigenetic mechanisms acting at the transcriptional level: methylation of cytosine residues of DNA and modification of the histone proteins associated with DNA (chromatine remodelling). At the post-transcriptional level, a family of small, non-coding RNAs (MicroRNAs or siRNAs), completes the regulation of gene activity and expression during development or in response to environmental changes. Despite uncertainties about the mechanism(s) involved, epigenetic studies have opened new perspectives to the mechanistic explanation for the genome response under the influence of physiological and environmental factors. The scientific basis and the molecular mechanisms underlying epigenetics inheritance have been extensively reviewed [57]. While the knowledge of the role of epigenetic alterations in cancer is steeply increasing [58], epigenetic studies of obesity, type 2 diabetes and other related metabolic disorders are still in an early stage in humans [5,59e61]. In particular, while there is no doubt that epigenetic modifications are heritable in somatic cells and have an important regulatory action in modifying gene expression in response to environmental stimuli, such as climate, famine, dietary changes etc, the possibility that they may remain stable and heritable from one generation to another is still not confirmed. However, a considerable evidence stems from animal models, showing that the environment can affect stable gene expression through methylation changes at different DNA level [62e65]. As a matter of fact, in certain parts of the genome, notably in the 50 region upstream of genes, there is unusually high frequence of CpG dinucleotides, known as CpG islands, in which the cytosines are unmethylated. About half human genes have CpG islands in their promoter regions and, for genes that are being transcribed, these islands are normally unmethylated. In association with chromatin modifications, promoter methylation prevents access by the transcription factors to specific DNA sequences and results in gene silencing. In effect, the epigenetic marks act as a switch alternatively silencing or inducing the expression of specific genes. Cell differentiation, X chromosome inactivation and genetic imprinting are all examples of epigenetic processes. Two studies [66,67] showed, in a rat model, that a maternal high-fat diet during intrauterine developmental stage may increase body fat accumulation in the offspring, suggesting a role of maternal diet on the health of offspring through epigenetic inheritance mechanisms. These and other observations suggest that some epigenetic modifications of the DNA sequence may persist in the next generations, thus influencing the epigenetic state of the genome and the activity of genes.
Heritability of body weight The discovery of microRNAs (miRNAs), almost 10 years ago, changed dramatically our perspective on eukaryotic gene expression regulation. miRNAs are single-stranded, evolutionarily conserved, small (17e25 ribonucleotides) non-coding RNAs [68]. They are described in the Sanger miRNA database, an international registry for miRNAs’ nomenclature, targets, functions and implications in different diseases (http:// www.mirbase.org/). In the database, each mature miRNA is assigned a unique identifier number for universal standardization. The overall role of miRNAs is therefore to regulate target gene expression to control normal rates of cellular growth, proliferation, differentiation and cell death [69]. Over the past 5 years, it has become increasingly clear that miRNAs are not only important for normal organismal development and physiology, but also in pathologies such as diabetes, cancer, heart disease and inflammation. Due to the strong impact of miRNAs on the biological processes, it is expected that mutations affecting miRNA function have a pathogenic role in human genetic diseases, similar to protein-coding genes. Recent computational and experimental studies have shown that miRNAs play a role in metabolic tissue development, lipid metabolism and glucose homeostasis. In addition, many miRNAs are dysregulated in metabolic tissues from obese animals and humans, which potentially contributes to the pathogenesis of obesity-associated complications [70,71]. Excitingly, the recent discovery of miRNAs in serum and plasma of humans and other animals [72e74] has prompted further exploration of their use as novel minimally invasive translational biomarkers of metabolic processes and nutritional status [71,75,76]. The miRNA levels in blood are stable, reproducible and consistent among individuals of the same species, allowing the identification of specific expression patterns of peripheral miRNA for diseases such as lung and intestinal cancer and diabetes [72]. As a working hypothesis, changes in specific circulating miRNA expression patterns may be used to monitor disease progression and/or to detect differences in the regulation of the gene activity in response to different environmental factors, like dietary behaviours, thus suggesting a potential use of miRNA in the diagnosis and treatment of obesity and related disorders [71]. In summary, understanding the complex interplay between genetic susceptibility and epigenetic modulation will help not only to unravel the molecular causes underlying multifactorial diseases but also to provide a comprehensive explanation of the so called “geneeenvironment” interaction [77].
Future directions The phenotypic expression of polygenic obesity is synthetically the result of a positive energy balance resulting from the changes in energy intake and energy expenditure in the modern environment. Nowadays, we are facing an obesity pandemic with devastating consequences on public health, due to the increased morbidity and mortality associated to obesity and its sequelae. The past decades of research yielded an immense progress in understanding of the
695 genetic factors associated with body weight regulation in humans. In the post-genomic era, the advent of the GWA studies has changed the pace of gene discoveries in the field of complex diseases. We should recognize indeed that most of the recently identified obesity loci may not have a causal role, so that the prediction of the individual’s risk to develop obesity is not straightforward. The strategy of using deep-sequencing techniques will allow to identify a large number of common SNPs with relatively low penetrance and a substantial number of rare SNPs with larger phenotypic effects, up to now discarded because of their very low frequency in the population [78]. The integration of different “omics” disciplines (transcriptomics, proteomics, metabolomics and nutrigenomics) will provide new tools to elucidate the molecular basis of obesity. In addition, epigenetic studies suggested that the DNA sequence alone and its variation do not provide enough information to identify the molecular pathways modulating health and disease at the individual level. The documented involvement of epigenetic factors in metabolic processes, both as transcriptional and post-trascriptional regulators, has provided strong evidence in support of their role as key players in the regulation of complex conditions, like polygenic obesity. The interplay of several environmental factors such as nutrition, lifestyle, social influences, and foetal growth further limits the predictive value of genetic markers for complex diseases. Although the promise that genetic discoveries will have in the foreseeable future potential preventive and clinical application in obesity and related conditions remains a research endeavour, addressing current gaps in knowledge will ultimately lead to the identification of new mechanisms of disease and new options for treatment and prevention.
Conflict of interest None declared.
Acknowledgements FL is the recipient of a Research Grant funded by the IDEFICS Study (Sixth RTD Framework Programme Contract No. 016181 (FOOD), www.idefics.eu).
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VIEWPOINT
Heritability of body weight: Moving beyond genetics P. Russo, F. Lauria, A. Siani* Unit of Epidemiology and Population Genetics, Institute of Food Sciences, CNR, Via Roma, 64-83100 Avellino, Italy Received 8 July 2010; received in revised form 7 September 2010; accepted 16 September 2010
KEYWORDS Obesity; Genetics; Genome-wide association studies; Epigenetics
Abstract Obesity is a complex disease, arising from the interaction between several genetic and environmental factors. Until recently, the genetic basis of complex diseases in general, and of obesity in particular, were poorly characterized. While the relatively rare monogenic and syndromic forms of obesity clearly recognize a genetic origin, the actual worldwide epidemics of obesity represent a challenge for the identification of the genetic factors involved, being likely the effect of several loci each having a subtle influence on the phenotypic expression. Progress in DNA analysis techniques and in computational tools, and the increasing level of characterization of the variability of the human genome has recently allowed to study comprehensively the association between genetic variants and obesity. To date, wellconducted and powered genome-wide association studies allowed to consistently identify genomic regions e lying on different chromosomes and affecting different metabolic pathways e influencing the predisposition to the accumulation of body fat, ultimately leading to overweight and obesity. However, the population attributable risk for obesity linked to the most statistically significant loci, like FTO and MC4R, remains discouragingly low, explaining only small fractions of the overall variance of body weight. In the last few years, the role of the complex interaction between genetic determinants and environmental factors in the rapid global increase of obesity has been further challenged by the entry of new players, that is the transcriptional and post-transcriptional regulation, summarized under the emerging discipline of epigenetics. The key challenge now is to move from the identification of causal genes and variants to the integration of different “omics” disciplines, finally allowing the molecular understanding of obesity and related conditions. ª 2010 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: þ39 0825 299353; fax: þ39 0825 299423. E-mail address: [email protected] (A. Siani). 0939-4753/$ - see front matter ª 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.numecd.2010.09.007
692
Introduction Obesity is a complex disease, arising from the interaction between several genetic and environmental factors. The current worldwide surge of obesity in both adults and children may be conceivably attributed to the interaction between the ancestral genetic predisposition toward efficient energy storage, which is the legacy of the huntergatherer cultures, and a permissive (obesogenic) contemporary environment of readily available food and sedentary behaviours. This genetic predisposition, often described as “thrifty genotype” [1], appears nowadays maladaptive, increasing the individual predisposition to obesity and related metabolic disorders. This theory, although recently challenged with attractive arguments [2e4], still remains the most accepted, until alternative hypotheses will find confirmation. In the last few years, the role of the complex interaction between genetic determinants and environmental factors in the rapid global increase of obesity has been further challenged by the entry of a new player, that is the epigenetic adaptation [5], fuelling the debate surrounding the aetiology of obesity. From the genetic point of view, obesity is classified into three main categories: monogenic, syndromic, and polygenic obesity [6]. This classification may be applied to both childhood and adult forms of human obesity, although monogenic and syndromic forms are most often evident very early in the life [7]. The study of the extreme Mendelian human obesity phenotypes provided solid evidence in favour of the genetic origin of obesity [8,9]. The discussion of these severe and rare forms is beyond the objectives of this viewpoint, which is mainly devoted to address some questions and challenges about the epidemiologically most relevant form of obesity, that is, polygenic obesity.
Heritability of body weight In 1889, Sir Francis Galton in his book “Natural Inheritance” suggested for the first time the familial aggregation of the individual’s body size [10,11]. This initial observation was corroborated by studies in the first decades of the last century [12], showing a clear association between offspring and parental body weight. However, this observation could not dissect out the relative role of genetic and environmental factors in body weight determination, with parents and their progeny sharing the same environment. Although environmental factors likely play a major role, it is clear that the regulation of body weight has a large underlying genetic component, that usually follows complex patterns of segregation, involving the subtle influence of multiple genes [13]. More than 25 years ago, Stunkard unequivocally reported [14,15] a heritability estimate of 0.78 for body weight, increasing to 0.81 in a 25-year follow-up study. Further twin studies have confirmed heritability estimates of w0.7 for BMI in both adults and children [16,17]. Controlled overfeeding studies in monozygotic twins showed that the tendency towards weight gain within each couple of twins was comparable, confirming the relevant
P. Russo et al. role of genetic factors on body weight regulation, particularly evident when food intake and exercise are controlled to reduce the environmental variance [18e20]. A complementary model to the twin study is the adoption study. In fact, adoptive children share the environment with their adoptive parents while they share their genetic background only with their biological parents. The results of the adoption studies consistently showed that the adoptees present a highly statistically significant association of their BMI with that of their biological parents, but not with that of their adoptive parents [15,21,22]. In summary, quantitative genetics studies clearly demonstrated that genetic factors play a significant role in the causes of individual differences in relative body weight and human body fat accumulation.
The candidate gene approach The characterization of the relative role of geneegene and geneeenvironment interaction in polygenic disorders of body weight regulation, such as obesity, is extremely challenging. It seems conceivable that several gene products, each making a relatively small contribution, interact to build up the genetic background that may explain a variable but significant proportion of the variance of body weight. The candidate-gene strategy has been the traditional approach for identifying the genes involved in complex diseases [23]. The candidate gene approach directly tests the effects of genetic variants of a potentially contributing gene in an association study. This approach involves the assessment of the association between a particular allele (or set of alleles) of a gene that may be involved in the disease (i.e. the candidate gene) and the disease itself. These studies, which may include members of an affected family or unrelated cases and controls, can be performed relatively quickly and inexpensively and may allow identification of genes with small effects. However, this approach is limited by how much is known of the biology of the disease being investigated. In fact, the identification of candidate genes represents a difficult task, given the number of pathways involved and the subsequent huge number of potential candidate genes. In the case of obesity, the potential genetic markers lie indeed on various biochemical and metabolic pathways, such as energy expenditure, appetite regulation, lipid metabolism, endothelial function, adrenergic activation, inflammation etc, highlighting the multiple geneegene, geneenutrient and geneenutrienteenvironment interactions in the pathogenesis of this complex disorder. In the case of rare monogenic diseases, the molecular approach has proven extremely powerful in the identification of the genes responsible and in defining new syndromes [24]. Conversely, in common polygenic disorders like obesity a large number of studies were published, many based on collections as small as only tens of samples, or at best several hundreds, and numerous weak associations were reported in the literature. Some of the identified genes have received confirmation by betterpowered genetic association studies, but most have never been replicated, so raising unwarranted enthusiasm, often followed by scepticism within the scientific community. Among the possible explanations, beside the limited study
Heritability of body weight size and the inadequacy of available bio-computing tools, we also include the failure to detect geneegene interactions and the lack of reporting environmental data of possible importance for geneeenvironment interactions [25,26]. Notwithstanding the above described pitfalls, in recent years sizeable progress has been made toward the understanding of the genetic bases of polygenic obesity. From the first release of the Human Obesity Gene Map [27], the meritorious work of a group of researchers gave us the annual update of the state of art of the research on the genetics of obesity. The last release [28] summarized in 106 pages the scientific evidence available in this field up to October 2005. Their impressive, though in some way discouraging, conclusion was that “The number of studies reporting associations between DNA sequence variation in specific genes and obesity phenotypes has also increased considerably, with 426 findings of positive associations with 127 candidate genes. A promising observation is that 22 genes are each supported by at least five positive studies. The obesity gene map shows putative loci on all chromosomes except Y”. Unfortunately, in publishing the 12th update, the authors announced that it would be the last of the series, due to the lack of economical and human resources to collect and manage such enormous amount of information [29]. From that time on, thousands of researchers worldwide were challenged by the same question: among all the possibilities, what are the inherited genetic variants that really shape the susceptibility to common obesity? Different approaches were adopted to answer this question, including population-based or family-based genetic association studies, linkage disequilibrium mapping, haplotype tagging and the latest gene-finding strategy, that is the whole genome association scans. A discussion of the advantages/disadvantages of each of these techniques is far beyond the objectives of this paper. Indeed, in spite of the difficulties, both the candidate single-nucleotide polymorphisms (SNPs) and the quantitative trait locus (QTL) strategies identified a large number of genetic markers potentially associated with the susceptibility to adult and childhood obesity on various chromosomes [28]. Many excellent reviews summarized progress and delusion on the way of the research of the genetic basis of obesity [30e35]. The scenario rapidly evolved over the last decade. Large international collaborative studies allowed the researcher to access databases of well-defined phenotypes with related DNA and biological sample banks and more precise environmental information.
Genome-wide association studies in obesity: the model of FTO The immense progress in molecular biology tools, like microarrays and bioinformatics, has led to a steeper increase in the identification of the genetic basis of complex, nonMendelian diseases through the genome-wide association (GWA) approach. In short, high-throughput genotyping technologies were used to test hundreds of thousands of SNPs, each of them identified and assigned a unique reference number (rs) in the National Center for Biotechnology
693 Information’s dbSNP database [36]. The GWA approach represented a significant advance in gene-hunting strategies, because it is free from prior hypotheses regarding candidacy of genes, it permits to explore at the highest level of resolution the whole genome and it allows to associate hundreds of thousands of genetic variants with the phenotype (disease) of interest [37]. The GWA studies, driven by the completion of the Human Genome project [38] and, more recently, by the International HapMap [39] updated in 2007 [40] and by the availability of the high-throughput technologies, are able, at least in part, to circumvent the obstacles encountered with both the candidate genes and the linkage approach. In the last years, an avalanche of GWA studies appeared in the scientific literature, linking common genetic variations to obesity and related metabolic traits. Apparently, these studies overcame some of the methodological problems described with the candidate gene approach. Here, we briefly comment on how the information gathered by these studies could help uncovering the genetic basis of obesity. Given that the list of obesity-associated loci is increasing day-by-day, we will only discuss the role of one gene that recently emerged as being robustly and consistently associated to common obesity, that is the Fat Mass and Obesity Associated gene (FTO). Almost simultaneously, three well-powered GWA studies published in 2007 identified a cluster of common SNPs in the first intron of FTO on chromosome 16 as unexpected but strong contributors to both childhood and adult obesity phenotypes [41e43]. Human FTO is a gene of unknown function in an unknown pathway and presents high homology with the murine Fto, located on mouse chromosome 8 [44]. It encodes for a member of the non-heme dioxygenase superfamily and it is ubiquitously expressed in foetal and adult tissues, particularly in the brain. Interestingly, in the first published report by Frayling et al. [41], the FTO locus was strongly associated with type 2 diabetes and secondarily with BMI in 1924 U.K. type 2 diabetes patients and 2938 U.K. population controls. The association with type 2 diabetes disappeared after adjustment for BMI, thus suggesting that BMI mediates the association with the risk of diabetes. In the same report, the association of the rs9939609 SNP of FTO with BMI was confirmed in 13 cohorts of adults and children with 38,759 participants of European origin. The 16% of adults who were homozygous for the risk allele weighed about 3 kg more and had 1.67-fold increased odds of obesity when compared with those not inheriting a risk allele. Effects of comparable magnitude were present in children. The association of a set of SNPs at this locus with BMI and obesity was unequivocally confirmed in the studies by Dina et al. [42] in European adults and children and by Scuteri et al. [43] in Sardinian adults, the latter study extending the analysis also to independent samples of European and Hispanic Americans. To date, several studies confirmed these initial observations, consistently linking SNPs at the FTO locus to other obesity-related phenotypes and markers, including body fat distribution, fat mass, and leptin levels [45]. Of particular interest is the wide distribution of the riskcarrying FTO allele in European populations, with about 63% of the population being at least heterozygous and 16% homozygous for the risk allele. But data are not entirely
694 consistent in some other ethnic groups, notably African American, Chinese and Japanese populations in which the FTO variants are not always associated with BMI or obesity phenotypes [42,46e48]. In all these populations minor allele frequencies and linkage disequilibrium patterns are different form that reported for European populations. Despite the consistency of the association of FTO with BMI in people of European ancestry, the effect size is relatively small, explaining about 1% of the overall phenotypic variation of obesity observed in the general population [49]. The relatively weak association of the FTO gene with metabolic factors and with obesity-associated conditions such as type 2 diabetes is in favour of a non-metabolic pathway of action of this gene. In February 2009, an elegant experimental study was published in Nature, providing clues towards possible mechanisms by which FTO acts on energy homeostasis and in turn on the regulation of body weight [50]. Fischer et al. generated an Fto knock-out mice strain and showed the absence of the FTO protein in the Fto/ mutant. The loss of Fto was associated with post-natal growth retardation and significant reduction in both adipose tissue and lean body mass. The mechanisms advocated for these effects are increased energy expenditure and sympathetic activation. The results of this study provided the first translation of the human epidemiological data into functional evidence of the involvement of Fto/FTO in energy homeostasis and finally into body weight regulation. In summary, the “agnostic” approach of the GWA studies opened a new research avenue to the study of the genetic roots of obesity and body weight regulation [51]. The success stories of GWA studies cannot hide the seemingly paradoxical result that association p-values higher than 105 only result in weak genetic effects, explaining only a small fraction of the phenotypic variation observed in the general population for the diseases under study [52,53]. In fact, the combined effect of common variants in two of the most robustly obesity-associated loci, that is FTO and MC4R, explains not more than 2% of the variance in body weight at the population level. To explain the remaining fraction of the genetic-linked body mass variability, the term “missing heritability” has been coined [49]. As a working hypothesis, this remaining part may reside on a large number of very common genetic variants exerting small effects and/or on rare variants with large effects each. A simulation exercise estimated that for variants with small effects, 50 genes with a genotypic prevalence of 10% would be needed to explain about 50% of the population attributable risk of complex diseases, while for rare variants with large effects as many as hundreds of genes would be needed to explain the same proportion of risk [54]. A plausible biological explanation should indeed consider the possibility of epigenetic effects affecting important regulatory regions/pathways of our genome.
Epigenetic mechanisms in obesity In the previous paragraphs, we sought to document that DNA sequence variants may influence an individual’s susceptibility to obesity and related syndromes. However, a “dark matter” still persists with regard the genetic
P. Russo et al. explanation for the global obesity epidemic of the last five decades, since it is evident that the classical models to describe the geneeenvironment interaction do not provide satisfactory answers. In the past few years, epigenetics emerged as a complementary model to explain the rapid emergence of obesity and metabolic disorders [5]. Epigenetics is defined as “the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence” [55,56]. These non-genetic alterations are under the tight regulation of two major epigenetic mechanisms acting at the transcriptional level: methylation of cytosine residues of DNA and modification of the histone proteins associated with DNA (chromatine remodelling). At the post-transcriptional level, a family of small, non-coding RNAs (MicroRNAs or siRNAs), completes the regulation of gene activity and expression during development or in response to environmental changes. Despite uncertainties about the mechanism(s) involved, epigenetic studies have opened new perspectives to the mechanistic explanation for the genome response under the influence of physiological and environmental factors. The scientific basis and the molecular mechanisms underlying epigenetics inheritance have been extensively reviewed [57]. While the knowledge of the role of epigenetic alterations in cancer is steeply increasing [58], epigenetic studies of obesity, type 2 diabetes and other related metabolic disorders are still in an early stage in humans [5,59e61]. In particular, while there is no doubt that epigenetic modifications are heritable in somatic cells and have an important regulatory action in modifying gene expression in response to environmental stimuli, such as climate, famine, dietary changes etc, the possibility that they may remain stable and heritable from one generation to another is still not confirmed. However, a considerable evidence stems from animal models, showing that the environment can affect stable gene expression through methylation changes at different DNA level [62e65]. As a matter of fact, in certain parts of the genome, notably in the 50 region upstream of genes, there is unusually high frequence of CpG dinucleotides, known as CpG islands, in which the cytosines are unmethylated. About half human genes have CpG islands in their promoter regions and, for genes that are being transcribed, these islands are normally unmethylated. In association with chromatin modifications, promoter methylation prevents access by the transcription factors to specific DNA sequences and results in gene silencing. In effect, the epigenetic marks act as a switch alternatively silencing or inducing the expression of specific genes. Cell differentiation, X chromosome inactivation and genetic imprinting are all examples of epigenetic processes. Two studies [66,67] showed, in a rat model, that a maternal high-fat diet during intrauterine developmental stage may increase body fat accumulation in the offspring, suggesting a role of maternal diet on the health of offspring through epigenetic inheritance mechanisms. These and other observations suggest that some epigenetic modifications of the DNA sequence may persist in the next generations, thus influencing the epigenetic state of the genome and the activity of genes.
Heritability of body weight The discovery of microRNAs (miRNAs), almost 10 years ago, changed dramatically our perspective on eukaryotic gene expression regulation. miRNAs are single-stranded, evolutionarily conserved, small (17e25 ribonucleotides) non-coding RNAs [68]. They are described in the Sanger miRNA database, an international registry for miRNAs’ nomenclature, targets, functions and implications in different diseases (http:// www.mirbase.org/). In the database, each mature miRNA is assigned a unique identifier number for universal standardization. The overall role of miRNAs is therefore to regulate target gene expression to control normal rates of cellular growth, proliferation, differentiation and cell death [69]. Over the past 5 years, it has become increasingly clear that miRNAs are not only important for normal organismal development and physiology, but also in pathologies such as diabetes, cancer, heart disease and inflammation. Due to the strong impact of miRNAs on the biological processes, it is expected that mutations affecting miRNA function have a pathogenic role in human genetic diseases, similar to protein-coding genes. Recent computational and experimental studies have shown that miRNAs play a role in metabolic tissue development, lipid metabolism and glucose homeostasis. In addition, many miRNAs are dysregulated in metabolic tissues from obese animals and humans, which potentially contributes to the pathogenesis of obesity-associated complications [70,71]. Excitingly, the recent discovery of miRNAs in serum and plasma of humans and other animals [72e74] has prompted further exploration of their use as novel minimally invasive translational biomarkers of metabolic processes and nutritional status [71,75,76]. The miRNA levels in blood are stable, reproducible and consistent among individuals of the same species, allowing the identification of specific expression patterns of peripheral miRNA for diseases such as lung and intestinal cancer and diabetes [72]. As a working hypothesis, changes in specific circulating miRNA expression patterns may be used to monitor disease progression and/or to detect differences in the regulation of the gene activity in response to different environmental factors, like dietary behaviours, thus suggesting a potential use of miRNA in the diagnosis and treatment of obesity and related disorders [71]. In summary, understanding the complex interplay between genetic susceptibility and epigenetic modulation will help not only to unravel the molecular causes underlying multifactorial diseases but also to provide a comprehensive explanation of the so called “geneeenvironment” interaction [77].
Future directions The phenotypic expression of polygenic obesity is synthetically the result of a positive energy balance resulting from the changes in energy intake and energy expenditure in the modern environment. Nowadays, we are facing an obesity pandemic with devastating consequences on public health, due to the increased morbidity and mortality associated to obesity and its sequelae. The past decades of research yielded an immense progress in understanding of the
695 genetic factors associated with body weight regulation in humans. In the post-genomic era, the advent of the GWA studies has changed the pace of gene discoveries in the field of complex diseases. We should recognize indeed that most of the recently identified obesity loci may not have a causal role, so that the prediction of the individual’s risk to develop obesity is not straightforward. The strategy of using deep-sequencing techniques will allow to identify a large number of common SNPs with relatively low penetrance and a substantial number of rare SNPs with larger phenotypic effects, up to now discarded because of their very low frequency in the population [78]. The integration of different “omics” disciplines (transcriptomics, proteomics, metabolomics and nutrigenomics) will provide new tools to elucidate the molecular basis of obesity. In addition, epigenetic studies suggested that the DNA sequence alone and its variation do not provide enough information to identify the molecular pathways modulating health and disease at the individual level. The documented involvement of epigenetic factors in metabolic processes, both as transcriptional and post-trascriptional regulators, has provided strong evidence in support of their role as key players in the regulation of complex conditions, like polygenic obesity. The interplay of several environmental factors such as nutrition, lifestyle, social influences, and foetal growth further limits the predictive value of genetic markers for complex diseases. Although the promise that genetic discoveries will have in the foreseeable future potential preventive and clinical application in obesity and related conditions remains a research endeavour, addressing current gaps in knowledge will ultimately lead to the identification of new mechanisms of disease and new options for treatment and prevention.
Conflict of interest None declared.
Acknowledgements FL is the recipient of a Research Grant funded by the IDEFICS Study (Sixth RTD Framework Programme Contract No. 016181 (FOOD), www.idefics.eu).
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