Sex-dependent relationship of polymorphisms in CLOCK and REV-ERBα genes with body mass index and lipid levels in children

Circadian rhythms, which are governed by a circadian clock, regulate important biological processes associated with obesity. SNPs in circadian clock genes have been linked to energy and lipid homeostasis. The aim of our study was to evaluate the associations of CLOCK and REV-ERBα SNPs with BMI and plasma lipid levels in pre-pubertal boys and girls. The study sample population comprised 1268 children aged 6–8 years. Information regarding anthropometric parameters and plasma lipid concentrations was available. Genotyping of CLOCK SNPs rs1801260, rs4580704, rs3749474, rs3736544 and rs4864548 and REV-ERBα SNPs rs2017427, rs20711570 and rs2314339 was performed by RT-PCR. The CLOCK SNPs rs3749474 and rs4864548 were significantly associated with BMI in girls but no in boys. Female carriers of the minor alleles for these SNPs presented lower BMI compared to non-carriers. A significant association of the REV-ERBα SNP rs2071570 with plasma total cholesterol, LDL-cholesterol and Apo B in males was also observed. Male AA carriers showed lower plasma levels of total cholesterol, LDL-cholesterol and Apo B levels as compared with carriers of the C allele. No significant associations between any of the studied REV-ERBα SNPs and plasma lipid levels were observed in females. In summary, CLOCK and REV-ERBα SNPs were associated with BMI and plasma lipid levels respectively in a sex-dependent manner. Our findings suggest that sex-related factors may interact with Clock genes SNPs conditioning the effects of these polymorphisms on circadian alterations.

Circadian rhythms regulate multiple aspects of physiology and metabolism 1 .These rhythms are generated by an endogenous mechanism involved in endocrine signaling 2 , which is comprised of circadian clocks 3 .This circadian clock system has been related to metabolic processes 4 and metabolic and cardiovascular disease [5][6][7][8] .In this regard, the role of the circadian clock system regulating adipose tissue physiology 9 and therefore energy balance and glucose and lipid metabolism has been established [10][11][12][13] .
The control of the circadian rhythms in mammals is carried out by the Clock genes that encode proteins implicated in positive and negative regulatory pathways 14 .Among them, CLOCK is a key component of the molecular circadian clock, regulating the expression of an important number of transcription factors 15 .CLOCK has been linked to metabolism homeostasis, including lipid metabolism 16 .Several studies in different racial populations have associated single nucleotide polymorphisms (SNPs) in the CLOCK gene with obesity and body mass index (BMI) [17][18][19][20][21] as well as with metabolic syndrome 18,22 and type-2 diabetes 23,24 .
Other of the components of the circadian clock that has a key role in the machinery of circadian rhythms is REV-ERBα, that regulates the circadian rhythms in a negative way.REV-ERBα suppresses the expression of the main components of this machinery ARTNL/BMAL1, CLOCK and CRY1 14 .REV-ERBα is considered to be essential regulating circadian behavior and metabolism 25,26 .Thus, SNPs in the REV-ERBα gene have been investigated in relationship with metabolic alterations and have been associated with obesity and body mass www.nature.com/scientificreports/index in adults [27][28][29] as well as in children population 27,30 .However, a sex-dependent association of the SNPs in the REV-ERBα gene with anthropometric variables has been described 28,30 .
The association of polymorphisms in CLOCK and REV-ERBα with blood lipid levels has been less studied.Studies analyzing CLOCK SNPs failed to find any association 24,31,32 .A study analyzing the implication of CLOCK SNPs in weight reduction reported their association with differences in plasma cholesterol in response to dietary treatment 19 .Studies analyzing the association of plasma lipid levels with REV-ERBα SNPs failed to find any significant association 27 .However, no differences between sexes were examined.
Table 2 shows the location, minor allele frequencies and HWE p values for the studied SNPs.The minor allele frequencies for the analyzed SNPs ranged from 27.1 to 38.5% for CLOCK SNPs and from 12.3 to 28.7% for REV-ERBα SNPS.Frequencies for the studied SNPs were consistent with Hardy-Weinberg equilibrium (p > 0.05).

Associations of the studied SNPs with BMI
The analysis of the association between the studied CLOCK gene SNPs and BMI is shown in Table 3.The variants rs3749474 and rs4864548 were found to be significantly associated with BMI, in girls but no in boys.Carriers of the minor allele T of the SNP rs3749474 and the minor allele A of the SNP rs4864548 presented significantly lower BMI compared to non-carriers (p value 0.032 and 0.022, respectively).No association between BMI and the CLOCK SNPs rs1801260 and rs4580704 was found in any sex.
The analysis of the association between the analyzed REV-ERBα SNPs and BMI revealed no significant associations.www.nature.com/scientificreports/

Associations of the studied SNPs with plasma lipid levels
When analyzing the association of these SNPs in CLOCK and in REV-ERBα with plasma lipid levels, we observed, in males, a significant association of the SNP in REV-ERBα rs2071570 with total cholesterol (Fig. 1a), LDLcholesterol (Fig. 1b) and Apo B (Fig. 1c) that is not observed in females (Fig. 1d,e,f).Boys homozygous for the minor allele A showed lower total cholesterol levels compared to carriers of the major allele C (AA vs. CA p = 0.053) (Fig. 1a).Significantly lower plasma levels of LDL-cholesterol were observed in male carriers of the AA allele when comparing with carriers of the C allele (AA vs. CC p = 0.021; AA vs. CA p = 0.027) (Fig. 1b).Similar results were found for Apo B levels (Fig. 1c).AA males showed lower plasma levels of Apo B as compared with homozygous for the C allele (p = 0.009) and as compared with CA carriers (p = 0.061).No associations with plasma lipid levels were found for the rest of SNPs analyzed in CLOCK and REV-ERBα genes in any sex.

Discussion
In this study we have analyzed the potential associations of SNPs of the CLOCK and REV-ERBα genes with body mass index (BMI) and lipid levels in a cohort of boys and girls aged 6-8 years.We found sex-dependent associations of the SNPs rs3749474 and rs4864548 of CLOCK with BMI and of the SNP rs20711570 of REV-ERBα with plasma total cholesterol, LDL-cholesterol and Apo-B.The association between SNPs of the CLOCK gene and anthropometric variables has been widely investigated.In our cohort, we found that carriers of the less common alleles for both, the rs3749474 and rs4864548 CLOCK SNPs, presented lower BMI compared to non-carriers.These SNPs, alone or combined in haplotypes, have been linked to the individual susceptibility to obesity in adults [17][18][19][20][21] .However, we found no association with rs1801260, one of the CLOCK SNPs most frequently studied in relationship with anthropometric variables in adult populations.Studies conducted in Caucasian populations have been able to find consistent associations of the SNP rs1801260 with obesity and BMI 19,20,22,24,33,34 that are not evident in our population.Furthermore, the associations between the CLOCK SNPs and BMI found in our study are presented in girls but no in boys.A sex-dependent association between CLOCK SNPs and overweight and obesity has been previously described in adults 35 .We hypothesized that age-related factors may be affecting the relationship of these SNPs with anthropometric variables.It has been described that aging may result in gain or loss of rhythmic circadian expression 36 .In this sense, a different regulation of circadian gene expression between middle/older aged individuals and younger adults has been reported 37 which may contribute to explain the described discrepancies in findings depending on age.
Regarding the relationship of CLOCK SNPs with plasma lipid levels, no consistent associations have been found in our cohort.The relationship of CLOCK SNPs with plasma lipid levels remains imprecise.No association was found in the PREDIMED trial analyzing the association of the SNP of CLOCK rs4580704 with total cholesterol, LDL-cholesterol or HDL-cholesterol 24 .However, a study analyzing the implication of Clock genes in weight reduction in obese patients found that CLOCK SNPs were associated with serum cholesterol changes in response to a dietary intervention 19 .Unlike this lack of association with lipid levels observed for CLOCK SNPs, we found an association of plasma total cholesterol, LDL-cholesterol and Apo B levels with the SNP rs20711570 in the promoter region of the REV-ERBα, which is another important regulator of metabolism.Again, we described a sex-dependent association of this SNP with lipid parameters, as we observed that male carriers of the AA genotype showed significantly lower plasma levels of total cholesterol, LDL-cholesterol and Apo B concentrations as compared with C allele carriers.However no significant differences in lipid levels among genotypes were observed in females.REV-ERBα polymorphisms have been studied in relationship with www.nature.com/scientificreports/body weight regulation, but its association with lipid levels has been less explored.An association of genetic variants in the REV-ERBα gene, including the REV-ERBα SNP rs2071570, with obesity has been reported 27,28,30 .However, as happened with CLOCK SNPs, this association appears to be sex-dependent 28 .In a study including adolescents, Nascimento Ferreira et al. described a significant association between REV-ERBα SNPs and BMI only in boys 30 .In our study, although we failed to find any association of the REV-ERBα SNPs with BMI, we observed a sex-dependent association of the SNP rs20711570 of REV-ERBα with lipid levels, which reinforces the hypothesis of the existence of sex-related factor interacting with these Clock genes polymorphisms affecting metabolic alterations.In this sense, a sexual dimorphism in Clock genes expression in human adipose tissue has been described 38 .The role of sex in the different influence of circadian rhythms on cardiovascular disease in males and females has been extensively discussed 39 .In this regard, the sex-related differences in Clock controlled processes have been associated with suprachiasmatic nucleus (SCN) morphology and signaling 40 , as sex differences in SCN morphology and neuropeptide expression are known 41 .SCN receives both estrogenic and androgenic inputs 42 .These aspects may be related to the different association of the studied SNPs with anthropometric and lipid parameters observed in males and females.More studies are needed taking into account how the possible interactions between the Clock genes polymorphisms and sex, as a biological variable, influence the development of obesity and metabolic alterations, including variations in lipid levels 43 .
In conclusion, in our study we report sex-dependent associations between the CLOCK SNPs rs3749474 and rs4864548 and BMI as well as between the REV-ERBα SNP rs20711570 and plasma LDL-cholesterol and Apo-B concentrations.Our findings suggest the role of the interaction between sex-related factors and Clock genes polymorphisms in obesity and plasma lipid levels.

Table 1 .
Characteristics (means ± SD) of the study participants by sex.BMI, body mass index; LDL-Cholesterol, low-density lipoprotein cholesterol; HDL-Cholesterol, high-density lipoprotein cholesterol; Apo B, apolipoprotein B; Apo A-I, apolipoprotein A-I.

Table 3 .
BMI (kg/m 2 ), expressed by means and standard deviation, according to the genotypes of the studied CLOCK SNPs by sex. a p value < 0.05, comparing BMI in carriers of the minor allele T of the SNP rs3749474 versus BMI in non-carriers.b p value < 0.05, comparing BMI in carriers of the minor allele A of the SNP rs4864548 versus BMI in non-carriers.