Association between polymorphisms in the Clock gene, obesity and ...

International Journal of Obesity (2008) 32, 658–662 & 2008 Nature Publishing Group All rights reserved 0307-0565/08 $30.00 www.nature.com/ijo

ORIGINAL ARTICLE Association between polymorphisms in the Clock gene, obesity and the metabolic syndrome in man EM Scott, AM Carter and PJ Grant LIGHT Laboratories, Academic Unit of Molecular Vascular Medicine, Leeds Institute of Genetics Health and Therapeutics, University of Leeds, Leeds, UK Objective: Accumulating evidence raises the hypothesis that dysregulation of intrinsic clock mechanisms are involved in the development of the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease. The aim of the present study was to investigate the relationship between three known common polymorphisms in the Clock gene and features of the metabolic syndrome in man. Methods: Genotype and haplotype analysis was carried out in a cohort of 537 individuals from 89 families characterized for inflammatory, atherothrombotic and metabolic risk associated with insulin resistance. Results: Heritability of the metabolic syndrome, defined according to International Diabetes Federation criteria, was 0.40. Haplotype analysis indicated three common haplotypes: CAT, TGT and CGC (rs4864548-rs3736544-rs1801260) with frequencies of 31, 33 and 28%, respectively. The CGC haplotype was less prevalent in subjects with the metabolic syndrome (P ¼ 0.0015) and was associated with lower waist circumference (P ¼ 0.007), lower hip circumference (P ¼ 0.023), lower body mass index (P ¼ 0.043) and lower leptin levels (P ¼ 0.028). The CAT haplotype was significantly associated with the presence of the metabolic syndrome (P ¼ 0.020). Conclusions: These findings suggest that the Clock gene CGC haplotype may be protective for the development of obesity and support the hypothesis that genetic variation in the Clock gene may play a role in the development of the metabolic syndrome, type 2 diabetes and cardiovascular disease. International Journal of Obesity (2008) 32, 658–662; doi:10.1038/sj.ijo.0803778; published online 11 December 2007 Keywords: Clock gene; haplotypes; metabolic syndrome; waist circumference; BMI

Introduction In recent years understanding of the importance of circadian rhythmicity and its role in normal physiological and pathological processes has increased tremendously.1–5 Glucose, lipids, adiponectin, insulin, leptin, plasminogen activator inhibitor-1 (PAI-1), and blood pressure (BP) in humans are all known to exhibit circadian variation,3 and it is of interest and concern that disturbing circadian rhythms in humans by shift work, or repeated sleep deprivation is associated with an increased prevalence of features of the metabolic syndrome6–10 and an increased risk of cardiovascular disease.11

Correspondence: Professor PJ Grant, LIGHT Laboratories, Academic Unit of Molecular Vascular Medicine, Leeds Institute of Genetics, Health and Therapeutics, Clarendon Way, University of Leeds, Leeds LS2 9JT, UK. E-mail: [email protected] Received 29 August 2007; revised 22 October 2007; accepted 6 November 2007; published online 11 December 2007

Circadian and seasonal rhythms in mammals, including man, are predominantly organized by light exposure regulating a central clock in the hypothalamic suprachiasmatic nucleus.12,13 This in turn appears to entrain clocks in peripheral cells, through neural or hormonal signals, to keep the organism functioning in time with itself and the environment.12,13 Central to the timing and accuracy of these rhythms is a complex system of positive and negative feedback loops based on transcriptional activation and repression of Clock genes and degradation of clock proteins.12,13 There are several core clock proteins (CLOCK, BMAL1, Per1, Per2, Per3, Cry1 and Cry2), which drive the feedback loops leading to the development of an oscillating system that regulates the cyclical phenotype in response to day length and the seasons.12,13 Their expression plays an important role regulating the physiological function of the cell and organism, particularly regarding metabolism and energy homeostasis,1,4,5,14,15 indeed 3–10% of transcripts from all tissues are under circadian control.1 Recent evidence in animals links disruption of synchronous clock activity with the pathogenesis of features of the

Clock polymorphisms and obesity EM Scott et al

659 metabolic syndrome.1,16–19 Homozygous Clock mutant mice become obese and develop hepatic steatosis associated with hyperglycaemia, hypertriglyceridaemia, hypercholesterolaemia and hyperleptinaemia.18 Mutations in Bmal and Clock modify circadian variation in glucose and triglycerides17 and are associated with impaired glucose tolerance.19 In the mouse adipocyte, Clock gene expression is attenuated in the presence of obesity with loss of the circadian rhythmicity of expression of adipocytokines and attenuated adiponectin levels.16 These findings support the hypothesis that a functional relationship exists between the circadian clock and development of the metabolic syndrome. The aim of the present study was to investigate the relationship between three known common polymorphisms in the Clock gene and features of the metabolic syndrome in man. This was carried out by genotype and haplotype analysis in a cohort of 537 individuals from 89 families characterized for inflammatory, atherothrombotic and metabolic risk associated with insulin resistance.

Subjects, materials and methods Subjects Details of the subject recruitment and characteristics have been previously published20,21 and are shown in Tables 1 and 2. In brief, potential subjects were selected at random from the Family Health Authority register in Leeds, and included in the study if healthy and if X4 first-, second- or thirddegree relatives were able to take part. Subjects were X16 years, of white European origin, and gave informed consent according to a protocol approved by the Leeds Teaching Hospitals Trust Research Ethics Committee. The study was performed in accordance with the Declaration of Helsinki 1989. Past medical history, smoking and alcohol consumption were recorded. BP and waist circumference were measured. Presence of the metabolic syndrome and discrete

Table 1

Table 2

Breakdown of relationships

Relationship Parent–offspring Sibling Grandparental Avuncular Grand-avuncular First cousins Second cousins Second cousins once removed Unrelated Total

Total (n) 413 422 102 561 60 305 96 2 110 2071

traits were defined according to International Diabetes Federation (IDF) criteria.22 A total of 16 subjects were taking b-blockers, 12 calcium antagonists, 14 angiotensin converting enzyme inhibitors, 25 diuretics and 23 lipid lowering therapies.

Sampling methods To minimize the influence of circadian rhythm on plasma measurements, subjects were studied between 0700 and 1100 after a 10 h overnight fast. Blood samples were drawn from an antecubital vein with a 19-gauge needle without venous stasis. Aliquots of plasma were snap-frozen in liquid nitrogen and stored at 40 1C until assay. Genomic DNA was extracted from 10 ml of whole blood using a Nucleon DNA extraction kit (Tepnel Life Sciences PLC, Manchester, UK).

Plasma measurements Measures of insulin, glucose, homeostasis model of assessmentFinsulin resistance (HOMA-IR), PAI-1, cholesterol and triglycerides were assayed as previously described.20 Adiponectin was assayed by an in-house enzyme-linked immunosorbant assay (ELISA) using antibodies from R&D systems (Oxon, UK). Leptin was assayed using a commercial ELISA from ADI (Autogen Bioclear, Wilts, UK).

Clinical characteristics of the 537 subjects studied

Total (n) Age (y) BMI (kg m2) Waist–hip ratio Systolic BP (mm Hg) Fasting glucose (mmol l1) Triglycerides (mmol l1) HDL cholesterol (mmol l1) Insulin (mU l1) HOMA-IR

Male

Female

217 41.9±16.1 26.0±4.6 0.91 (0.08) 133.9±15.3 5.2 (4.8–5.5) 1.3 (1.1–2.1) 1.2 (1–1.5) 7.13 (5.2–9.8) 1.56 (1.2–2.3)

320 44.1±16.8 25.8±5.2 0.83 (0.09) 130.1±20.1 5.0 (4.7–5.3) 1.1 (0.8–1.6) 1.5 (1.3–1.8) 6.32 (5.1–8,6) 1.41 (1.1–2.0)

Abbreviations: BMI, body mass index; BP, blood pressure; HDL, high-density lipoprotein cholesterol; HOMA-IR, homeostasis model of assessmentFinsulin resistance. Normally distributed variables expressed as mean (s.d.). Skewed variables expressed as median (interquartile range).

Genotyping Rs4864548, a C/T polymorphism in the Clock gene promoter, was genotyped by restriction fragment length polymorphism (RFLP) using Mbo II, which recognizes the T allele but not the C allele, to digest a 190 bp PCR product amplified with forward primer 50 -CTTTTCCAGTAGAAGCACTGAA-30 and a mismatch reverse primer designed to destroy a common Mbo II site two bases downstream of the polymorphic site 50 -AATCAAGTGACAGTGTAATGCAA-30 (mismatch base underlined). Rs3736544 (2121G/A), a synonymous coding region polymorphism (Asn588Asn), was genotyped by RFLP using Fok I, which recognizes the G allele but not the A allele, to digest a International Journal of Obesity


660 330 bp product amplified with forward primer 50 -TCCAAC CACTGATCACTCCA-30 and reverse primer 50 -TTTGCCTTCT TTTTAATGCTCA-30 . Rs1801260 (3111T/C), a polymorphism in the 30 -UTR, was genotyped by RFLP using Bsp1286I, which recognizes the C allele but not the T allele, to digest a 387 bp product amplified with forward primer 50 -TTGACATCAAGGGAG GAAGG-30 and reverse primer 50 -CCCTGACATGGCAGTG GTAT-30 . An evaluation of these three polymorphisms using Tagger (http://www.broad.mit.edu/mpg/tagger/) indicated that of the 196 polymorphisms in the chromosomal region of Clock (ch4: 56 200 000–56 350 000) genotyping of these three polymorphisms alone would capture 70.9% of alleles with r240.8.

Sample size Little of a formal nature is known about sample size requirements for studies of traits based on pedigrees, partly because pedigrees vary in their information content as a function of both their size and structure. However, we have previously shown that this study has sufficient power to detect minor contributions (B2%) of polymorphisms to variance in quantitative traits in the Leeds Family Study23 and many analyses of this type to date have used sample sizes of around 500 or fewer individuals.

Statistical analysis SPSS v12 (SPSS Inc.) was used for data preparation and initial analysis of the data. Linkage disequilibrium between polymorphisms was analysed using Haploview.24 The heritability of the metabolic syndrome and composite discrete traits were analysed using a liability threshold model in Southwest Foundation for Biomedical Research. PBAT v3.525 was used to determine the association of haplotype with the metabolic syndrome, composite discrete traits and other quantitative traits associated with the metabolic syndrome, using the population mean for the traits as the offset value. Three genetic models were tested additive, dominant and recessive. A P-value of 0.05 was taken as significant. Mendelian errors were indicated in two pedigrees and the two individuals with inconsistent genotyping data were excluded from analysis.

Results The clinical characteristics of the subjects and the breakdown of their relationships are shown in Tables 1 and 2, respectively. Utilizing the IDF definition,22 132 of the 537 family members had the metabolic syndrome. The heritability of the metabolic syndrome and its component discrete traits are shown in Table 3. The heritability of the metabolic syndrome was 0.40 and that of the component traits ranged between 0.18 and 0.50. International Journal of Obesity

Table 3 The heritability of discrete traits related to the IDF definition of metabolic syndrome, adjusted for age and gender h2 (s.e.)

Metabolic syndrome Raised waist circumference Raised triglyceride Reduced HDL cholesterol Previously diagnosed type 2 diabetes/ raised fasting plasma glucose Raised blood pressure

0.40 0.34 0.15 0.50 0.49

(0.14) (0.12) (0.15) (0.14) (0.18)

0.18 (0.12)

Proportion of variance explained by age and sex 0.096 0.098 0.074 0.003 0.078 0.19

Abbreviation: HDL, high-density lipoprotein cholesterol. Proportion of variance explained by age and sex as indicated by Kullback–Leibler R2.

The genotype distributions of the three Clock gene polymorphisms were in Hardy–Weinberg equilibrium. The T-allele frequency of rs4864548 was 0.32, A-allele frequency of rs3736544 was 0.35 and C-allele frequency of rs1801260 was 0.34. There were no significant associations between any of the polymorphisms and the metabolic syndrome or its subcomponents (data not shown). The three polymorphisms were in significant linkage disequilibrium, with the rarer allele of each polymorphism occurring more frequently with the more common allele of the other. Haplotype analysis indicated three common haplotypes: CAT, TGT and CGC (rs4864548-rs3736544-rs1801260) with frequencies of 31, 33 and 28%, respectively. In recessive model analyses of the metabolic syndrome and its components, the CGC haplotype was less prevalent in those with the metabolic syndrome (P ¼ 0.0015) and in those with increased waist circumference (P ¼ 0.036), whereas the CAT haplotype was more prevalent in those with the metabolic syndrome (P ¼ 0.020). For analyses of quantitative traits, the CGC haplotype was associated with lower waist circumference (P ¼ 0.007), lower hip circumference (P ¼ 0.023), lower body mass index (BMI; P ¼ 0.043) and lower leptin levels than the other haplotypes (P ¼ 0.028). There were no significant associations between Clock haplotypes and lipids, insulin, glucose, PAI-1 or adiponectin (data not shown).

Discussion This is the first study to report an association between Clock gene polymorphisms and the metabolic syndrome in man. It is also the first to report the heritability of the metabolic syndrome and its component traits in a White European population using the IDF criteria, which places waist circumference as a primary determinant of the definition. After adjusting for age and sex the heritability of the metabolic syndrome was 0.40 in our White European population. This is similar to a heritability of 0.38 recently reported in an Arab population also using the IDF definition26 and a previous study using National Cholesterol Education


661 Program/Adult Treatment Panel III criteria showed the heritability of the metabolic syndrome to be 0.24 in a Caribbean-Hispanic population.27 The present study further supports a role for genetic factors in the familial clustering of the metabolic syndrome and its component and related traits. Haplotype analysis demonstrated that the Clock CGC haplotype was less prevalent in subjects with the metabolic syndrome and in those with increased waist circumference defined according to the IDF metabolic syndrome criteria. Furthermore the CGC haplotype was associated with decreased waist circumference, hip circumference, BMI and leptin levels, suggesting that this haplotype may be protective for the development of obesity. This is the only haplotype to include the C allele of rs1801260, suggesting that this polymorphism may be functional or in linkage disequilibrium with a functional polymorphism elsewhere in the Clock gene locus and warrants further investigation. In support of these findings a recent study has demonstrated an association of the C allele of rs1801260 with lifetime lower body weight in a group of subjects with eating disorders,28 supporting a protective role for this allele and associated haplotype with obesity. The CAT haplotype showed an association with the presence of the metabolic syndrome. This is the only haplotype to include the A allele of the rs3736544 polymorphism. It was not however, associated with any of the component traits of the metabolic syndrome. Taken together, these findings support the evolving view that the circadian oscillator has an important role in the development of obesity and the metabolic syndrome,1,29,30 which is largely based on animal studies:17–19 the Clock mutant mouse has altered feeding patterns accompanied by the development of obesity, elevated triglycerides, glucose and leptin18 and mutations in Clock and Bmal alter diurnal variation in triglycerides and glucose.17 Although we did not perform any assessment of sleep patterns in our subjects, it is of interest that the rs1801260 polymorphism has previously been associated with sleep dysregulation in humans in many31–38 but not all studies.39 These findings, together with the present results may provide genetic evidence to support epidemiological studies associating sleep disturbance with the development of diabetes and obesity.9,10 In addition, linkage studies in several populations have reported some indication of linkage with obesity, type 2 diabetes mellitus and insulin resistance in a region on chromosome 4q that encompasses the Clock gene.40–45 It has also recently been reported that the expression of several components of the molecular clock (Per2,Cry1, Bmal1) in adipose tissue are associated with features of the metabolic syndrome in man.46

Study limitations When interpreting these findings it should be borne in mind that this is a small family study performed in largely healthy

individuals. It will be important to confirm these findings in future studies with larger populations. A further limitation of the study is that only three polymorphisms were examined and they may not be functional. In the light of our findings it will be important to further establish the effects of rs1801260 on gene regulation.

Conclusions It has been proposed that loss of circadian and seasonal rhythms of light exposure and food intake with disruption of clock synchronicity may be responsible for the development of obesity and the metabolic syndrome.1,29,30 The finding that the Clock CGC haplotype is associated with decreased waist circumference and other measures of obesity and is less prevalent in those with the metabolic syndrome defined according to the IDF criteria, in which increased waist circumference is a requirement, support the Clock gene as a candidate gene for obesity. Understanding the mechanisms underpinning the relationship between the environment, our circadian rhythms and obesity in the development of type 2 diabetes and cardiovascular disease will provide important pathways for both prevention and management of these conditions.

References 1 Staels B. When the Clock stops ticking, metabolic syndrome explodes. Nat Med 2006; 12: 54–55. 2 Rajaratnam SM, Arendt J. Health in a 24-h society. Lancet 2001; 358: 999–1005. 3 Walters J, Skene D, Hampton SM, Ferns GA. Biological rhythms, endothelial health and cardiovascular disease. Med Sci Monit 2003; 9: RA1–RA8. 4 Liu C, Li S, Liu T, Borjigin J, Lin JD. Transcriptional coactivator PGC-1alpha integrates the mammalian clock and energy metabolism. Nature 2007; 447: 477–481. 5 Yang X, Downes M, Yu RT, Bookout AL, He W, Straume M et al. Nuclear receptor expression links the circadian clock to metabolism. Cell 2006; 126: 801–810. 6 Lund J, Arendt J, Hampton SM, English J, Morgan LM. Postprandial hormone and metabolic responses amongst shift workers in Antarctica. J Endocrinol 2001; 171: 557–564. 7 Nagaya T, Yoshida H, Takahashi H, Kawai M. Markers of insulin resistance in day and shift workers aged 30–59 years. Int Arch Occup Environ Health 2002; 75: 562–568. 8 Karlsson BH, Knutsson AK, Lindahl BO, Alfredsson LS. Metabolic disturbances in male workers with rotating three-shift work. Results of the WOLF study. Int Arch Occup Environ Health 2003; 76: 424–430. 9 Spiegel K, Knutson K, Leproult R, Tasali E, Van Cauter E. Sleep loss: a novel risk factor for insulin resistance and type 2 diabetes. J Appl Physiol 2005; 99: 2008–2019. 10 Reilly JJ, Armstrong J, Dorosty AR, Emmett PM, Ness A, Rogers I et al. Early life risk factors for obesity in childhood: cohort study. BMJ 2005; 330: 1357. 11 Knutsson A. Health disorders of shift workers. Occup Med (Lond) 2003; 53: 103–108. 12 Dunlap JC. Molecular bases for circadian clocks. Cell 1999; 96: 271–290.

International Journal of Obesity


662 13 Lincoln GA, Andersson H, Loudon A. Clock genes in calendar cells as the basis of annual timekeeping in mammalsFa unifying hypothesis. J Endocrinol 2003; 179: 1–13. 14 Holzberg D, Albrecht U. The circadian clock: a manager of biochemical processes within the organism. J Neuroendocrinol 2003; 15: 339–343. 15 Fontaine C, Dubois G, Duguay Y, Helledie T, Vu-Dac N, Gervois P et al. The orphan nuclear receptor Rev-Erbalpha is a peroxisome proliferator-activated receptor (PPAR) gamma target gene and promotes PPARgamma-induced adipocyte differentiation. J Biol Chem 2003; 278: 37672–37680. 16 Ando H, Yanagihara H, Hayashi Y, Obi Y, Tsuruoka S, Takamura T et al. Rhythmic mRNA Expression of Clock Genes and Adipocytokines in Mouse Visceral Adipose Tissue. Endocrinology 2005; 146: 5631–5636. 17 Rudic RD, McNamara P, Curtis AM, Boston RC, Panda S, Hogenesch JB et al. BMAL1 and CLOCK, two essential components of the circadian clock, are involved in glucose homeostasis. PLoS Biol 2004; 2: e377. 18 Turek FW, Joshu C, Kohsaka A, Lin E, Ivanova G, McDearmon E et al. Obesity and metabolic syndrome in circadian Clock mutant mice. Science 2005; 308: 1043–1045. 19 Kennaway DJ, Owens JA, Voultsios A, Boden MJ, Varcoe TJ. Metabolic homeostasis in mice with disrupted Clock gene expression in peripheral tissues. Am J Physiol Regul Integr Comp Physiol 2007; 293: R1528–R1537. 20 Freeman MS, Mansfield MW, Barrett JH, Grant PJ. Insulin resistance: an atherothrombotic syndrome. The Leeds family study. Thromb Haemost 2003; 89: 161–168. 21 Freeman MS, Mansfield MW, Barrett JH, Grant PJ. Heritability of features of the insulin resistance syndrome in a communitybased study of healthy families. Diabet Med 2002; 19: 994–999. 22 Alberti KG, Zimmet P, Shaw J. The metabolic syndromeFa new worldwide definition. Lancet 2005; 366: 1059–1062. 23 Freeman MS, Mansfield MW, Barrett JH, Grant PJ. Genetic contribution to circulating levels of hemostatic factors in healthy families with effects of known genetic polymorphisms on heritability. Arterioscler Thromb Vasc Biol 2002; 22: 506–510. 24 Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps 1. Bioinformatics 2005; 21: 263–265. 25 Lange C, DeMeo D, Silverman EK, Weiss ST, Laird NM. PBAT: tools for family-based association studies. Am J Hum Genet 2004; 74: 367–369. 26 Bayoumi RA, Al Yahyaee SA, Albarwani SA, Rizvi SG, Al Hadabi S, Al Ubaidi FF et al. Heritability of determinants of the metabolic syndrome among healthy Arabs of the Oman family study. Obesity (Silver Spring) 2007; 15: 551–556. 27 Lin HF, Boden-Albala B, Juo SH, Park N, Rundek T, Sacco RL. Heritabilities of the metabolic syndrome and its components in the Northern Manhattan Family Study. Diabetologia 2005; 48: 2006–2012. 28 Tortorella A, Monteleone P, Martiadis V, Perris F, Maj M. The 3111T/C polymorphism of the CLOCK gene confers a predisposition to a lifetime lower body weight in patients with anorexia nervosa and bulimia nervosa: a preliminary study. Am J Med Genet B Neuropsychiatr Genet 2007; 144: 992–995. 29 Bray MS, Young ME. Circadian rhythms in the development of obesity: potential role for the circadian clock within the adipocyte. Obes Rev 2007; 8: 169–181.

International Journal of Obesity

30 Scott EM, Grant PJ. Neel revisited: the adipocyte, seasonality and type 2 diabetes. Diabetologia 2006; 49: 1462–1466. 31 Benedetti F, Dallaspezia S, Fulgosi MC, Lorenzi C, Serretti A, Barbini B et al. Actimetric evidence that CLOCK 3111 T/C SNP influences sleep and activity patterns in patients affected by bipolar depression. Am J Med Genet B Neuropsychiatr Genet 2007; 144: 631–635. 32 Mishima K, Tozawa T, Satoh K, Saitoh H, Mishima Y. The 3111T/C polymorphism of hClock is associated with evening preference and delayed sleep timing in a Japanese population sample. Am J Med Genet B Neuropsychiatr Genet 2005; 133: 101–104. 33 Katzenberg D, Young T, Finn L, Lin L, King DP, Takahashi JS et al. A CLOCK polymorphism associated with human diurnal preference. Sleep 1998; 21: 569–576. 34 Iwase T, Kajimura N, Uchiyama M, Ebisawa T, Yoshimura K, Kamei Y et al. Mutation screening of the human Clock gene in circadian rhythm sleep disorders. Psychiatry Res 2002; 109: 121–128. 35 Benedetti F, Serretti A, Colombo C, Barbini B, Lorenzi C, Campori E et al. Influence of CLOCK gene polymorphism on circadian mood fluctuation and illness recurrence in bipolar depression. Am J Med Genet B Neuropsychiatr Genet 2003; 123: 23–26. 36 Pirovano A, Lorenzi C, Serretti A, Ploia C, Landoni S, Catalano M et al. Two new rare variants in the circadian ‘clock’ gene may influence sleep pattern. Genet Med 2005; 7: 455–457. 37 Serretti A, Cusin C, Benedetti F, Mandelli L, Pirovano A, Zanardi R et al. Insomnia improvement during antidepressant treatment and CLOCK gene polymorphism. Am J Med Genet B Neuropsychiatr Genet 2005; 137: 36–39. 38 Serretti A, Benedetti F, Mandelli L, Lorenzi C, Pirovano A, Colombo C et al. Genetic dissection of psychopathological symptoms: insomnia in mood disorders and CLOCK gene polymorphism. Am J Med Genet B Neuropsychiatr Genet 2003; 121: 35–38. 39 Pedrazzoli M, Louzada FM, Pereira DS, Benedito-Silva AA, Lopez AR, Martynhak BJ et al. Clock polymorphisms and circadian rhythms phenotypes in a sample of the Brazilian population. Chronobiol Int 2007; 24: 1–8. 40 Gorlova OY, Amos CI, Wang NW, Shete S, Turner ST, Boerwinkle E. Genetic linkage and imprinting effects on body mass index in children and young adults. Eur J Hum Genet 2003; 11: 425–432. 41 Almasy L, Goring HH, Diego V, Cole S, Laston S, Dyke B et al. A novel obesity locus on chromosome 4q: the Strong Heart Family Study. Obesity (Silver Spring) 2007; 15: 1741–1748. 42 King DP, Zhao Y, Sangoram AM, Wilsbacher LD, Tanaka M, Antoch MP et al. Positional cloning of the mouse circadian clock gene. Cell 1997; 89: 641–653. 43 Humphreys P, McCarthy M, Tuomilehto J, Tuomilehto-Wolf E, Stratton I, Morgan R et al. Chromosome 4q locus associated with insulin resistance in Pima Indians. Studies in three European NIDDM populations. Diabetes 1994; 43: 800–804. 44 Permutt MA, Wasson JC, Suarez BK, Lin J, Thomas J, Meyer J et al. A genome scan for type 2 diabetes susceptibility loci in a genetically isolated population. Diabetes 2001; 50: 681–685. 45 Prochazka M, Lillioja S, Tait JF, Knowler WC, Mott DM, Spraul M et al. Linkage of chromosomal markers on 4q with a putative gene determining maximal insulin action in Pima Indians. Diabetes 1993; 42: 514–519. 46 Gomez-Abellan P, Hernandez-Morante JJ, Lujan JA, Madrid JA, Garaulet M. Clock genes are implicated in the human metabolic syndrome. Int J Obes (Lond) 2007 [e-pub ahead of print].