Introduction
The epidemic of obesity has major costs on the individual level.The costs encompass excess mortality and morbidity from cardiovascular diseases, type 2 diabetes, and certain forms of cancers, osteoarthritis and sleep apnoea. Moreover, obese subjects tend to suffer from various forms of social stigmatisation and discrimination contributing to low quality of life. The medical-care costs burden of obesity is considerable, and increasing along with the epidemic. Obesity and its related co-morbidities are estimated to account for 5.5–7% of the total health care expenditure in the United States, and 2.0–3.5% in other Western countries (Thompson and Wolf, 2001). Obesity causes a considerable increase in sick leave, and risk of early retirement (Seidell, 1998). Unfortunately, current strategies for prevention and treatment of obesity have failed to reverse the epidemic of obesity, and therefore a continued search for modifiable causes is mandatory.
The development of obesity is determined by both genetic and environmental factors. A considerable proportion of the between-subject variation in body weight is determined by genetic differences, but part of the variation must also be attributed to differences in environment.Whereas changes in the environment must be responsible for the increasing prevalence of obesity, genetic factors together with environmental factors are expected to determine who will become obese, and to which degree obesity will develop.Thus, genetic factors influence the distribution of obesity in a given environment, in a given population, at a given time.
It is generally assumed that the genes and the environment interact in some way, but there is a considerable uncertainty about how this interaction takes place. There must be a tight interaction between the genes and the environment, fully integrated in the biological system that constitutes the organism of any species. This type of interaction does not in itself contribute to the inter-individual differences, and particularly not to the explanation of why some become and stay obese and others do not. If gene–environment interaction contributes to this kind of difference, there must be between-subject variations that cannot be attributed to the genetic differences and/or the environmental differences as such.Thus, this type of gene–environment interaction implies that the response to a certain environmental exposure depends on the particular genotype, and vice versa, that the effect of a particular genotype depends on the environmental exposures.
2.1 Role of genetic factors in obesity
Estimating the role of genetic factors in obesity The estimated quantitative role of genetic factors varies dependent of study type. In family studies, the heritability has been estimated to 20–40% (Maes et al., 1997).The correlations between full siblings are higher than between parents and their offspring, which suggest non-additive genetic influences, possibly due to intra- and inter-locus gene–gene interactions, or higher degree of shared environment between siblings than between parents and their offspring.
However, family studies do not allow separation of genetic effects and effects of shared family environment, which may be achieved in studies of adopted children and their biological and adoptive families and by twin studies. In adoption studies, the resemblance between the adoptee and the biological family members, i.e. parents, full and half-siblings can be ascribed solely to genetic factors. This approach has suggested that genetic factors account for 20–40% of between-subject differences in obesity and associated phenotypes (Maes et al., 1997; Stunkard et al., 1986; Sorensen et al., 1989).
Studies of monozygotic and dizygotic twins have revealed a much greater resemblance in the degree of obesity between monozygotic twins than between dizygotic twins, indicating that the resemblance is related to their similar genetic background rather than to their shared environment.These types of studies indicate a higher heritability, indicating that up to 60–80% of the between-subject differences can be ascribed to genetic factors. It might be argued that monozygotic twins may tend to share more environmental factors than dizygotic twins, which would lead to an overestimation of the heritability. However, twins raised apart show the same resemblance in body weight as twins raised together (Stunkard et al., 1990).
Also, adoption and twin studies suggest that there are non-additive genetic influences, but generally they are difficult to disentangle from shared environmental influences, which naturally make studies of gene–environment interactions in this setting difficult. Both twin studies and adoption studies have indicated that the childhood family environment plays a minor – if any – role in adult obesity and associated phenotypes, whereas the rearing environment may have some influence while the child lives in the parents’ home (Sorensen, 1996). This indicates that the within family resemblance in BMI in adults can be ascribed almost exclusively to genetic background (Sorensen et al., 1992;Vogler et al., 1995).
Selecting the optimal obesity phenotype for genetic research
Obesity represents merely the extreme in continuously distributed phenotypes. Although standardised categorisation of subjects as normal-weight and obese may be relevant in relation to treatment and prevention of obesity, arbitrary classification may hamper the identification of genetic and environmental factors contributing to the between-subject variation in obesity and related traits. Studies aiming to elucidate the role of genetic components and nutrient–gene interactions in obesity should ideally involve detailed characteristics of the obesity state, including a broad range of obesity-related and intermediate phenotypes (Comuzzie and Allison, 1998). Specification such as body fat percentage, or body fat distribution, and the use of intermediate phenotypes such as energy expenditure, fat oxidation and plasma levels of hormones expected to be involved in the regulation of energy balance, has several advantages.
Firstly, assessment of body composition gives a more refined measure of the degree of fat accumulation, as compared to body weight and BMI. Secondly, assessment of parameters related to adipose tissue metabolism, energy expenditure and appetite regulation offers the possibility of studying genetic factors involved in the regulation of energy balance, and exploring the mechanisms of action. Thirdly, it is conceivable that intermediate phenotypes, such as energy expenditure or fat oxidation, may be less influenced by environmental factors than BMI per se. Indeed, when addressing the role of specific candidate genes, the phenotypic profile should include intermediate phenotypes presumed to be closely linked to the function of the candidate genes.
Finally, recognising obesity as a complex heterogeneous phenotype it is of importance to address the common traits, i.e. the high body weight, as well as the heterogeneity with regard to, for example, abdominal fat accumulation, insulin sensitivity, lipid metabolism, etc. Studying the changes in
the phenotype in response to environmental manipulation, e.g. changes in body weight, body composition, or abdominal obesity induced by changes in energy balance, is another potentially profitable approach to study the effect of different genotypes, and it may be particularly suitable to the study of gene–environment interaction.
2.2.2 Candidate genes and the mechanism behind their role of genetic factors
A major aim in obesity research is to identify single gene variants involved in the development of obesity and to explore and clarify the interaction between specific gene variants and specific environmental factors, with the prospect of transforming this knowledge directly into techniques for identification of individuals at risk for developing obesity, and developing strategies for specific prevention and treatment. However, as judged from the phenotypic segregation pattern in the families, the general between-subject variations in body weight and other obesity-related phenotypes undoubtedly involve a complex oligo- or polygenic non-Mendelian pattern of inheritance.
Challenges in identifying obesity genes
There are two strategic approaches for identifying potential candidate genes.The first is to study the association between obesity or obesity-related phenotypes and already identified candidate genes selected on the basis of their known or presumed biological function, and the second one is to search for regions in the genome which appear to be linked to the obese phenotypes (Clement et al., 2002a).
A great number of research groups have contributed to this field by studying candidate genes of interest in cohorts of obese patients. For this purpose they have constituted banks of clinical data and DNA in large cohorts of obese patients and controls. Group of patients and their families have been characterised with regard to clinical and biological parameters related to obesity.
In association studies, the frequency of DNA variations between groups of subjects (i.e. obese vs. non-obese) is compared, or a measurable phenotype (body mass index, fat mass, skin folds, waist/hip ratio) in subjects carrying or not carrying the given polymorphism is compared. Such association studies have been conducted in many populations collected in Europe and in North America.They have provided a huge number of putative susceptibility genes, but with small or uncertain effects (Snyder et al., 2004; Swarbrick and Vaisse, 2003). This strategy has been used for both adults (Clement et al., 2002a) and children (Clement and Ferre, 2003).
For the candidate genes that have shown association to the obese phenotype in one population, the general situation is lack of replication in independent populations. Association studies encounter many pitfalls, including doubtful links between the physiological roles of the candidate genes and body weight regulation. Selecting candidate genes based on rodent models of monogenic forms of obesity have been considered for genetic studies of human obesity. Although previous studies have led mainly to the discovery of rare forms of obesity in humans, it is likely that key regulatory genes discovered in animal forms of monogenic obesity may reveal genetic factors involved in the common forms of obesity in humans.
There are also difficulties related to statistical aspects including too small sample sizes of obese and controls, non-representative control groups, biased population stratification, false-positive results due to multiple testing and suppression of negative results. This state of affairs has led to development of recommendations that should secure more robust results: sufficient sample size, necessity of replication in independent groups, statistical correction for multiple testing and functional assessment of the gene variant (Cardon and Bell, 2001; Tabor et al., 2002). However, only few published studies have met these criteria, such as the study on the recently described new obesity candidate gene, GAD2 (Boutin et al., 2003).
Whereas the candidate gene approach may be successful in addressing the genetic factors influencing mono- or oligogenic traits, this approach seems destined to fail when studying polygenic inheritance where many different genes contribute to the phenotype in interaction with environmental factors and other genes (Comuzzie et al., 2001; Comuzzie and Allison, 1998; Sorensen and Echwald, 2001). One set of problems is related to the identification of importance only in co-existence with other obesity genes, which are present in the selected populations. Another set of problems is related to the study size of relevant candidate genes as described. Further, genes identified as major obesity genes in family-based linkage studies may turn out to be of major importance only in co-existence with other obesity genes which are present in the selected populations, and have only minor influence on the common forms of obesity.
Another set of problems is related to the study size and statistical power, which in these settings is even more demanding. In very large studies it may be possible to study the gene–gene interactions, whereas collection of detailed information regarding the environment, e.g. the habitual lifestyle and thorough phenotypic profile including the response to dietary interventions is feasible only in smaller studies. Moreover, the success of addressing the gene–gene and gene–environment interaction may depend highly on the inclusion of genes and environmental factors with major effects in the model, since even relatively large effects of ‘minor’ genes will only become evident after adjustment for major effect (Williams, 1984). Thus, the ‘major-effect’ factors will need to be clarified before the ‘minor effect’ factors can be addressed.
The other approach for identifying obesity genes does not involve any a priori hypothesis about the genes and their function. Linkage analyses in families offer the possibility of studying the co-segregation of chromosomal markers with obesity or related phenotypes.The technique of genome-wide scan offers a new way of identifying candidate genes, which can then be examined further using the candidate gene approach.
Genome-wide scans have been performed in populations originating from Europe (France, Germany, Finland, Denmark), United States and Canada (Snyder et al., 2004; Swarbrick and Vaisse, 2003; Clement et al., 2002a). The genome-wide scan has been performed mostly in adult populations where the severity of obesity varied (Bell et al., 2004; Adeyemo et al., 2003; Newman et al., 2003; Suviolahti et al., 2003; van Tilburg et al., 2003), but also more recently in families where children sib pairs were collected (Meyre et al., 2004).
In North America, the genome-wide scan has been performed either in Caucasian families, or in selected populations with less admixture such as Pima Indians, Amish, Mexican, Indian or African Americans. Usually, families in which obesity-related traits segregate are analysed using 400 to 600 polymorphic markers regularly spanning the genome, with the goal of finding the genes and pathways underlying these complex traits. The genome-wide scan approach has provided more than 30 genome-wide scans for obesity and related phenotypes.
In general, the validity of chromosomal loci identified in genetic linkage is increased if the association between the loci and the phenotype has been replicated in other studies. Twenty-five regions of the human genome harbour quantitative trait loci (QTL) replicated in two to five studies with high lod score (Snyder et al., 2004; Suviolahti et al., 2003; van Tilburg et al., 2003). Some of the QTLs could explain a significant part of the variance of obesity-related phenotypes. Polymorphisms of candidate genes situated in the regions of linkage to obesity have been identified (Boutin et al., 2003; Durand et al., 2004). Some haplotypes are associated with a higher risk of obesity or diabetes. Genetic maps record annually the genes and polymorphisms implicated in the various European and American populations.
Despite the power of current analyses, it has been difficult to draw conclusions concerning the role of these tested candidate genes in fully explaining links observed on the genome, which include thousands of bases. The risks associated with the development of obesity or diabetes, in subjects with these variants, are generally moderate and should be placed in the context of other, lifestyle-related, risk factors.
In mice, hundreds of QTLs have been linked to body weight or body fat (Snyder et al., 2004). Among them at least six different chromosomal loci (DO1-6) have been identified by genetic mapping studies after crosses of mice strain differentially sensitive to diet-induced obesity (e.g. the AKR/J being the most sensitive strain and the SWR/J, the less sensitive strain to high fat diet) (West and York, 1998). However, the corresponding genes explaining the linkage have not been found in mice or in humans, even in chromosomal regions showing high and replicated statistical linkage. The multi-factorial nature of obesity with a polygenic, non-Mendelian inheritance is probably responsible for the lack of success of gene identification. Several years will probably be needed to clone the genes located in the regions of linkage but the time needed for gene identification will possibly be reduced considerably thanks to the use of strategies combining analysis of genome scans and gene expression.
Monogenic forms of human obesity
Although studying the role of single gene variants may not solve the enigma of obesity, this approach has led to the discovery and classification of a series of rare monogenic types of obesity, which might contribute to the understanding of the molecular basis of a number of well-known rare syndromes in which obesity is a main feature, such as the Bardet–Biedl syndrome, Prader–Willi syndrome, Alstrom and Cohen syndromes. For several of the identified genes, it remains unclear what the role of these genes are in the complex pathogenesis of the disease.
The discovery and characterisation of these rare monogenic forms of obesity provide valuable insight into the complex physiological pathways involved in the control of fat tissue size and energy balance. Such discoveries may pave the way for developing new pharmacological aids for treating obesity, irrespective of the cause. During the last decade, several rare monogenic forms of obesity have been described, involving the genes encoding for the fat cell hormone leptin (LEP) and its receptor (LEPR), pro-opiomelanocortine (POMC) and its converting enzyme, pro-hormone convertase 1, and finally the melanocortin 4 receptor (MC4R) (Clement et al., 2002a). The examples below describe monogenic forms of obesity, in which the discovery of the underlying genetic cause has led to new insight into the pathways involved in the regulation of body weight.
In 1994, the product of the ob-gene, the 16 kd peptide hormone, referred to as ‘leptin’, was described for the first time (Zhang et al., 1994) followed by the description of the rodent leptin receptor (Lee et al., 1996). Mice lacking either functional leptin (ob/ob mice) or leptin receptor (db/db mice) are severely obese. Rare homozygous loss-of-function mutations in the human leptin and leptin receptor genes have been shown to lead to symptoms similar to those seen in ob/ob and db/db animals, including early onset of severe obesity, abnormal eating behaviour, and hypogonadotropic hypogonadism (Clement et al.,1998;Montague et al.,1997;Ozata et al.,1999).Several studies have suggested a possible association between more common polymorphisms in the human leptin and leptin receptor gene and obesity, but it has not been possible to confirm these associations (Cancello et al., 2004).
The role of genetic variants of the Melanocortin 4 receptor (MC4R), the receptor for alpha-melanocyte-stimulating hormone (aMSH), in the regulation of body weight and obesity in humans has been addressed in several studies (Sina et al., 1999;Vaisse et al., 1998, 2000). The frequency of rare heterozygous MC4-R missense and frameshift mutations has been found to be 4% in a population of morbidly obese subjects (Vaisse et al., 2000) but low in normal weight subjects. Altogether, these findings suggest a dominant pattern of inheritance with variable penetrance and reduced expressivity (Vaisse et al., 2000), although also recessive, and dominant negative pattern inheritance have been described (Biebermann et al., 2003; Farooqi et al., 2000).
The molecular mechanisms for the effect of these mutations on body weight regulation are multiple including impaired trafficking of the receptor to the cell surface, impaired binding of aMSH, and impaired ability to generate cAMP (Lubrano-Berthelier et al., 2003;Yeo et al., 2003; Nijenhuis et al., 2003). Among obese subjects, phenotypic characteristics have been shown not to differ between carriers and non-carriers of the mutations, but carriers tended to have a higher prevalence of childhood obesity (Vaisse et al., 2000). Binge eating has been suggested as a major phenotypic trait in obese carriers of MC4R mutations (Branson et al., 2003), but these findings are controversial, and have for instance not been confirmed in a more recent study (Hebebrand et al., 2004).
Where other known forms of monogenetic obesity are recessive, and associated with other endocrine abnormalities, functional polymorphisms of the MC4-R gene have been suggested to be associated with a dominant non-syndromic form of obesity, and it is the most frequent genetic cause of obesity described to date (Sina et al., 1999;Vaisse et al., 2000). Others have, however, identified features of a distinct syndrome, including increased linear growth, hyperphagia and elevated insulin levels (Farooqi et al., 2003). These clinical traits have, however, not been retrieved in all the tested populations.
Different types of obesity mutations
Until recently, mutations in the coding regions have been the major focus in the research addressing obesity genes. Localising the coding region of a gene is far less complicated than localising all of the regulatory elements. In addition, knowing the structure and the function of the gene product, it is possible to predict the potential effect of changes in a specific area of the coding region. However, mutations in the non-coding region have gained increasing attention. Addressing the regulatory regions of putative obesity genes may lead to discovery of gene variants involved in obesity. In addition, combining genotyping with studies of the gene expression in specific tissues and in response to specific exposures, such as changes in fat intake or calorie restriction, will improve the understanding of the specific mechanisms of gene regulation and the mechanism for regulatory gene variants.
During the last years, mutations and polymorphisms in the promoters of several putative obesity genes including 5HT receptor, CART, UCP2, UCP3, TNFalpha, resistin, leptin and more recently adiponectin have been suggested to be associated with obesity-related phenotypes (Cancello et al., 2004; Engert et al., 2002; Esterbauer et al., 2001; Halsall et al., 2001; Hoffstedt et al., 2000; Mammes et al., 2000; Rosmond et al., 2000; Yamada et al., 2002). However, several of these observations are challenged by negative findings, and still need to be replicated in additional studies.
Development of obesity is very slow, ongoing for several years, and is typically considered to be a result of inappropriate adaptation of the systems involved in control of energy balance to either a primarily increased energy intake or reduced energy expenditure, leading to a passive accumulation of surplus energy as body fat.
Studies addressing the heritability of intermediate phenotypes have suggested that 30–50% of between-subject differences in metabolic variables, and 25–50% of between-subject variation in energy intake can be ascribed to genetic factors (Bouchard et al., 1989; de Castro, 1993). These findings suggest that the search for specific genetic effects should encompass both components of the energy balance. However, the paradigm of obesity as a passive storage of the surplus of energy may be insufficient. Active accumulation of fat in the adipose tissue, due to dys-regulations of the adipose tissue balance between release of fat and fat accumulation, followed by a subsequent corresponding regulatory adjustment of the energy balance should be considered (Sorensen, 2003b).
By C. Verdich (Danish Epidemiology Science Centre), K. Clément, (INSERM, France) and T. Sorensen, (Copenhagen University Hospital, Denmark) in the book 'Food, Diet and Obesity', by David J. Mela, Woodhead Publishing Limited, Cambridge UK & CRC Press U.S.A, 2006, p. 17-25. Digitized, adapted and illustrated to be posted by Leopoldo Costa
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