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Breastfeeding moderates the relationship between fat mass and obesity‐associated gene rs9939609 and body mass index among adolescents

Abstract Objective Breastfeeding, which is important for early growth, is a possible moderator of genetic influence, such as the effect of the fat mass and obesity‐associated gene (FTO) on body mass index (BMI). The aim of this study was to assess the moderating effect of breastfeeding duration on the relationship between FTO rs9939609 and BMI in a Caucasian sample. Methods Adolescents born in 1997 and in 1999, who were living in the Swedish county Västmanland in 2012, were invited to participate in the Survey of Adolescent Life in Västmanland. The adolescents and their parents completed self‐reported questionnaires in 2012, 2015, and 2018. Genotyping of rs9939609 T > A polymorphism was conducted from saliva DNA samples. Interaction effects of parental reported breastfeeding duration in months, including regions of significance, on the relationship between rs9939609 and BMI plus overweight were assessed. Results Considering physical activity levels, parental reported breastfeeding duration was a moderator of the relationship between rs9939609 and BMI for the younger (regions of significance = <1.6 and >28.1 months) and older adolescents (region of significance = >19.9 months), but not for the young adults. Plots of the association between breastfeeding duration and BMI showed higher BMI for AA with short breastfeeding, but lower BMI with longer breastfeeding than AT and TT. Longer breastfeeding lowered the odds for overweight among the younger adolescents, especially among AA individuals. Conclusion Rs9939609 AA individuals were more susceptible than AT and TT individuals to both short and long breastfeeding durations, which is consistent with the differential susceptibility hypothesis. FTO rs9939609 AA might be a plasticity variant with differential susceptibility to environmental influences. Breastfeeding duration may be one of many factors that affect the relationship between rs9939609 and BMI.


| INTRODUCTION
In 1975, <1% of children and adolescents (aged 5-19 years) had obesity (4% if including children and adolescents with overweigh as well) globally. 1 In 2016, approximately 40 years later, the prevalence rate of obesity had increased to 6% for girls and 8% for boys, while 18% of girls and 19% of boys were overweight. 1 Overweight and obesity are major risk factors for cardiovascular diseases, the main cause of death worldwide in 2017, 2 especially for women with insulin resistance. 3 In addition, for adolescents with higher body mass index (BMI) or obesity, the risk for several forms of cancer such as leukemia and colorectal cancer or adult diabetes type II, respectively, is also increased. 4 Even though overweight and obesity are dependent on lifestyle factors, such as diet and physical activity, obesity is a heritable trait with known genetic influence. 5 Genome-wide association studies have identified more than 100 loci that are robustly associated with BMI. [6][7][8] The fat mass and obesity-associated gene (FTO; FTO alphaketoglutarate-dependent dioxygenase) is the gene with the strongest influence on BMI. 9 In 2007, FTO was determined to be one of the contributing factors to early-onset obesity and severe obesity. 10,11 Since then, knowledge regarding the association between FTO and overweight and obesity has increased. [12][13][14][15][16][17] FTO, located on chromosome 16, encodes a 2-oxoglutarate-dependent nucleic acid demethylase, 9 which is expressed in all tissues, but its highest levels are observed in brain areas that control food intake. 18,19 FTO also plays an important role in adipogenesis. 20 Single nucleotide polymorphisms (SNPs) in FTO appear to regulate the expression of other genes in a tissue-specific manner that varies depending on the developmental stage of the tissue. 21 The causal variant rs1421085 disrupts a binding site for a gene repressor, which leads to overexpression of Iroquois homeobox 3 and Iroquois homeobox 5, which in turn changes these cells to energy-storing white adipocytes and increases lipid accumulation. 22 This SNP is in high linkage disequilibrium with many other SNPs in the genetic region, including rs9939609 (distance 19,573 bp, D' = 0.98, and R 2 = 0.92 in the CEU population, which includes Utah residents with Northern and Western European ancestry, according to the LDlink tool 23 ), which, therefore, can act as a proxy SNP.
The A allele of rs9939609 in the intron of FTO has been associated with higher risk for obesity 12,17 and type 2 diabetes. 12 Several studies have found that this association changed throughout the life course. 16,24 Hardy et al. found a stronger association during childhood and adolescence with a peak at 20 years, after which the association declined again during adulthood. 16 Sovio et al. found that the relationship between the A allele and the BMI was inversed for children below 2.5 years of age. 24 However, some did not find any influence by genotype, 25 while others found it only in girls. 26 Because obesity is difficult to reverse once established, 27 it is important to identify early risk factors as well as protective factors for a healthy weight pattern. One factor that occurs during the child's earliest life and may have an impact on obesity is breastfeeding. The World Health Organization recommend exclusive breastfeeding for up to 6 months of age with continued breastfeeding for up to 2 years of age or beyond. 28 The Swedish National Board of Health and Welfare and the National Food Agency have adapted this recommendation to Swedish conditions promoting exclusive breastfeeding for up to 6 months of age and continued breastfeeding for up to 1 year or beyond. 29,30 Despite these breastfeeding promoting recommendations, 29 breastfeeding is declining in Sweden. 31 A dose-response relationship between breastfeeding duration and future overweight risk has been observed. 32 Compared with breastfeeding, formulafeeding is associated with higher levels of protein and energy intake 33 as well as higher plasma insulin levels that promote fat deposition. 34 In contrast, breastfeeding is associated with higher levels of infant control of feeding and self-regulation. 33 Altogether, formulafeeding is associated with a more rapid weight gain 34 through overnutrition. 32 This is considered as one of the main risk factors in the first 1000 days since birth for the development of childhood obesity. 35 Earlier attempts at investigating a possible interaction effect between breastfeeding and FTO rs9939609 variants on future BMI have revealed inconclusive results. In a Chinese study on 1149 children (y = 10.8 years) analyzing BMI or body fat percentage, no significant interaction effect was found. 36 In contrast, a study on 959 children from Australia showed that a longer period of exclusive breastfeeding could reverse the increased BMI otherwise associated with the A allele. 37 Consistent with this, results from a study on British children revealed that at least 5 months of exclusive breastfeeding had a substantial impact on BMI growth trajectories. 38 Dedoussis et al. reported an interaction effect in two Greek samples (FTO rs17817449 was analyzed in one of the samples; N = 1138, mean age = 11.2 years and N = 2374, mean age = 41.4 months). However, their findings could not be replicated in a British sample of 4325 participants (mean age = 11.7 years). 39 Further studies are needed to establish a consensus regarding these relationships. To our knowledge, these associations are yet to be investigated in a Nordic sample.
Dalle Molle et al. suggested that the differential susceptibility to environment model; that individuals vary in their susceptibility to KANDERS ET AL. environmental influences and plasticity, 40 is "highly applicable to the study of genetic and environmental influences on feeding behaviour and obesity risk" as well. 41 In other words, the incorporation of both positive and negative environmental influences in these studies is essential. In this case, shorter or longer breastfeeding duration could be viewed as negative or positive environmental influence, respectively. Due to the lack of studies that apply this theory on obesity research, we consider this a gap in this research field.
This study hypothesized that breastfeeding duration plays a role in weight regulation through links to feeding behavior as well as through the nutritional value and that individuals differ in their susceptibility to this influence due to genetic variants. The aim of this study was to investigate the possible effect of childhood breastfeeding duration on the association between FTO rs9939609 and BMI and overweight in adolescence and young adulthood in a Caucasian sample, taking the levels of physical activity into account.

| Participants
Adolescents born in 1997 and in 1999, who were living in the Swedish county Västmanland in 2012, were invited by regular mail to participate in the Survey of Adolescent Life in Västmanland cohort study. 42 Individuals who had lived in Sweden for <5 years and had language difficulties or any mental disability/severe illness were excluded. In total, 4712 adolescents were eligible for this study. The consenting adolescents and their parents completed self-reported questionnaires during three assessments, first in 2012 (W1), second in 2015 (W2), and third in 2018 (W3). At W1, adolescents were also instructed to provide a saliva sample for deoxyribonucleic acid (DNA) extraction. Response rates were 39.6% for W1, 82.7% of W1 for W2, and 57.8% of W1 for W3 for the questionnaires. Caucasian participants with available data on the variables of interest were included in this study. The study sample is presented in detail in Table 1. All participants signed an informed consent form during the first assessment (W1). For participants aged <15 years, consent was also obtained from their legal guardian. This study was approved by the Ethical Review Board of Uppsala (Dnr 2012/187) and performed in accordance with the Declaration of Helsinki.

| Genotyping
At W1, participants provided a DNA sample using a self-collection kit (Oragene•DNA, DNA Genotek). Of all the eligible study participants, 86.5% was genotyped. DNA was then extracted from 200 μL of saliva using a silica-based extraction method (Kleargene™, LGC, Biosearch Technologies). Genotyping of the FTO SNP rs9939609 T > A polymorphism was performed using KASP™ (LGC, Biosearch Technologies), which is specific to the targeted SNPs and uses allele-specific primers. The allele combinations TT, TA, and AA were analyzed for Hardy-Weinberg equilibrium using the χ 2 test (p = 0.23).

| Assessment of BMI
We calculated the adolescents' BMI from self-reported values of weight and height. Thereafter, we created binary variables for normal weight, overweight, and obesity according to international age-and sex-specific cutoffs for each wave/time point. 43 Due to a small group of adolescents with obesity in the sample, individuals with overweight and obesity were combined into an "overweight including obesity" (hereinafter referred to as "overweight") category for the binary regression model analysis to reduce the risk of overfitting.
This was performed identically for all the three assessments.
Continuous BMI was used as the outcome in PROCESS analyses (see Statistical analyses).

| Assessment of childhood breastfeeding duration
At W2, parents were instructed to answer two questions regarding breastfeeding. The first question was whether the child had been breastfed at all or not (Yes/No) and the second was "If yes, for how long (in months)?" For this study, a "No" response for the first question was regarded as 0 months and included in the continuous variable "breastfeeding duration in months." The breastfeeding duration was categorized into 0-3, 4-6, 7-9, 10-12, and >12 months for descriptive reasons. We also created a categorical variable using the cutoffs from the breastfeeding recommendations (0-6, 7-12, and >12 months) to be used in the binary logistic regression model analysis.

| Confounders
Age was calculated using the date of birth and the date on which the form was sent out. Sex was coded as male (1) or female (2) using information in the participants' personal identity number. For all the three time points, we used self-reported physical activity that lasted >30 min during leisure time and was accompanied by increased breathing/sweating. Responses ranged from "Never" to "Every day" with seven possible responses that were used as a continuous measurement of physical activity.

| Statistical analyses
Using χ 2 tests for categorical variables and the Mann-Whitney U test for continuous variables, we analyzed whether there were any sex differences between rs9939609 allele variants, breastfeeding duration, age, physical activity, and overweight. In addition, we analyzed T A B L E 1 Description of the study sample  -69 rs9939609 allele variants using χ 2 tests for associations with BMI categories (normal weight and overweight) at the three time points.
Rs9939609 allele variants were also analyzed for any effect of breastfeeding duration and physical activity using the Mann- The moderating effect of parental reported breastfeeding duration in months on the relationship between FTO rs9939609 variants and BMI was analyzed using the PROCESS macro tool

| Crude analyses
Female participants had rs9939609 TA and AA more frequently than male participants (Table 1). We found no sex difference regarding parental reported breastfeeding duration or age. Female participants were less likely than male participants to report physical activity levels at the low or high end of the scale (Table 1). Male participants had a higher BMI than female participants at W2 (Table 1). More male participants than can be expected were overweight at W1 in the crude analysis (Table 1). We did not find any sex difference regarding overweight at W2 or W3 (Table 1)

| Breastfeeding as a moderator
Process analyses revealed a significant moderation effect of parental reported breastfeeding on the relationship between rs9939609 and BMI at W1 and W2 but not W3 (Table 2). R 2 for the model summary was 0.054 (p < 0.001) for W1 and 0.026 (p < 0.001) for W2. The R 2 change for the interaction term was 0.004 (p = 0.029) for W1 and W2. Figure 2A,B displays plots for the conditional effect of the focal predictor using data from the PROCESS output at W1 and W2. Figure 2A shows that for TT individuals, the BMI slope starts at a lower level than for AT and AA individuals, and then, it increases slightly with longer breastfeeding duration. For AT individuals, the slope starts at a level between those for TT and AA individuals, and then, it declines with longer breastfeeding duration. The slope for AA individuals starts at the highest level and has the steepest decrease as the breastfeeding duration increases. In Figure 2B, the slopes follow the same pattern as in Figure 2A, but the slopes for AT and AA overlap. The Johnson-Neyman output revealed that values <1.64 and >28.05 for the moderator were statistically significant at W1 ( Figure 3A). For W2, values >19.85 for the moderator were statistically significant ( Figure 3B). We did not find any statistically significant interaction effect for W3 BMI. The binary logistic regression analyses showed that parental reported breastfeeding for 7-12 months was associated with lower odds ratio (OR) for overweight at W1 compared with shorter breastfeeding (Model 1, Table 3). The combination of rs9939609 TA and breastfeeding for >12 months was associated with a lower OR than the reference combination of rs9939609 TT and breastfeeding for 0-6 months (  the analyses. In another sample, with shorter or longer breastfeeding duration, the region of significance will likely be different. In agreement with Horta et al. 45 Wu et al. 38  indicates that AA is more susceptible than AT. If rs9939609 AA indeed is a plasticity variant as suspected, the main effects would vary depending on the non-measured factors among the study participants, as discussed previously regarding serotonin transporter polymorphisms. 46 The interaction effect found in this study was statistically significant for the younger adolescents but declined in the older adolescents and was absent for the young adults. This might in part be caused by lower power for W2 and W3 analyses due to a loss of participants at the follow-up. An addition of factors such as possible injuries that affect mobility and eating habits and that influence BMI throughout a person's life will also dilute the effect of breastfeeding. Some findings suggest an age-dependent relationship between the FTO rs9939609 variants and BMI. 24 Parental reports of longer breastfeeding duration were associated with lower risk for overweight among the younger adolescents.
The findings indicate a protective effect from longer breastfeeding among the AA individuals, but the small sample of participants with overweight and breastfeeding for >12 months has to be considered.
Wu et al. 38 showed that only AA participants with exclusive breastfeeding for <3 months had an upper quartile BMI value above the overweight line. Future studies with a larger sample size of individuals with long breastfeeding duration are warranted.
Only 13% of the participants had a breastfeeding duration >12 months. Data have shown that high-income countries have shorter breastfeeding duration than low-and middle-income countries. 47 Even in the latter, only 37% of the infants aged <6 months are exclusively breastfed as recommended. 47 Previous findings indicate that breastfeeding (partial or exclusive) is associated with a slower growth rate during infancy, 38,48 which is a major determinant of overweight and obesity later. 38 Given the present findings, it is plausible that rs9939609 A is a plasticity allele. Breastfeeding duration might be one small piece in the large puzzle that encompasses the increasing obesity trend. Indeed, there are many other aspects related to obesity, such as built environment, screen time, and agroalimentary environment, 49 that have not been considered in this study.

| Strengths and limitations
One major strength of this study is its longitudinal design with assessment at three time points, which makes it possible to investigate these associations at several time points during the progression from adolescence to adulthood.
One limitation of this study is that due to only few participants with obesity, such individuals were combined into a category together with participants with overweight, which could have masked an actual effect from the rs9939609 risk allele on the risk for obesity.
However, international age-and sex-specific cutoffs for overweight were used, which is a strength of this study. The data were selfreported, which could be subjected to either under-or overestimation of key variables. The response rates were low (approximately 40%) for W1. The parental reported breastfeeding duration can be subjected to recall bias, as with all retrospective research, which has to be considered in the interpretation of the findings.
Rs9939609 is in close proximity to many other SNPs, which means that causality is not possible to state at this point. Information on energy intake was not assessed in the SALVe cohort and could not be incorporated in this study. However, if the hypotheses that early factors such as breastfeeding have an impact on eating behavior are correct, energy intake would be a mediator in the analyses and not have an impact on the total effect. Moreover, the risk that these findings were found by chance, as discussed in depth previously, 50 must be acknowledged.

| CONCLUSION
FTO rs9939609 AA might be a plasticity variant, with differential susceptibility to environmental influences. Breastfeeding duration may act as one of many moderators of the relationship between FTO rs9939609 and BMI among Caucasian adolescents. Future studies on this research topic should consider the differential susceptibility hypothesis.

ACKNOWLEDGMENTS
The authors would like to thank Mattias Rehn for providing help with the data files and syntax. They would also like to thank the