Lifetime body mass index and later atherosclerosis risk in young adults: examining causal links using Mendelian randomization in the Cardiovascular Risk in Young Finns study

Study population

The Young Finns study is a multicentre follow-up study of cardiovascular risk factors in Finnish children and adolescents.^7,13 The original sample was 4320 Caucasian children and adolescents aged 3, 6, 9, 12, 15, and 18 years who were randomly sampled from five areas of Finland using the national register. The baseline examination was conducted in 1980, with response being 83% (3596 of those invited). Follow-ups were conducted in 1983, 1986, and, the latest, 2001 when the participants had reached 24–39 years of age. The 2230 individuals (1012 men and 1218 women) with full data on FTO polymorphism rs9939609 and BMI in childhood and adulthood formed the present analytical sample (Figure 2). Differences in the baseline characteristics of participants and non-participants were small but achieved statistical significance owing to the large numbers: BMI (17.9 vs. 17.8 kg/m², P = 0.22); age (10.7 vs. 10.0 years, P < 0.0001); and sex (55% vs. 45% female, P < 0.0001). The study was conducted according to the guidelines of the Helsinki declaration, and the study protocol was approved by local ethics committees. All participants gave their full informed consent.

FTO genotyping

Genomic DNA was extracted from peripheral blood leucocytes using a commercially available kit and Qiagen BioRobot M48 Workstation according to the manufacturer’s instructions (Qiagen Inc., Hilden, Germany). We genotyped FTO single nucleotide polymorphism (SNP) rs9939609 by employing the 5′ nuclease assay and fluorogenic allele-specific TaqMan MGB probes,¹⁴ using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The samples were pipetted using an automated TECAN Freedom EVO-100 instrument (Tecan Group Ltd, Männedorf, Switzerland). The nucleotide sequences of the primers and probes used in the polymerase chain reaction (PCR) were deduced from published sequences deposited in the GenBank and Celera databases and synthesized by Applied Biosystems. The PCR containing genomic DNA, 1× Universal PCR Master Mix, 900 nM of each primer and 200 nM of each probe were performed in 384-well plates using the standard protocol in a total volume of 5 µL. Endpoint fluorescence was measured post-PCR and genotype calling carried out by the allelic discrimination analysis module (ABI Prism SDS software, ABI, Foster City, CA, USA). Random duplicates were used as quality control. All genotypes were analysed in the Department of Clinical Chemistry at Tampere University Hospital and University of Tampere.

Body mass index and other life course atherosclerotic risk factors

Life course measures were based on repeated assessments in 1980 (participant age range 3–18 years), 1983 (6–21 years), 1986 (9–24 years), and 2001 (24–39 years), except C-reactive protein, which was assessed in 1980 and 2001 only. Measurements of weight (kg) and height (mm) were obtained using standard research protocols and were used to calculate BMI (weight in kg/height² in m).¹⁵ Measurements of blood pressure were taken with the participant in the sitting position after 5 min rest with a mercury sphygmomanometer (for the 3 year olds with ultrasound device, Arteriosonde 1020, Roche) in 1980, 1983, and 1986 and with a random zero sphygmomanometer (Hawksley & Sons Ltd, West Sussex, UK) in 2001.^16,17 Readings to the nearest even number of millimetres of mercury were performed at least three times on each subject; the average of the systolic blood pressure measurements was used in the analysis.

In 1980, 1983, 1986, and 2001, all blood samples were taken after an overnight fast and analysed in duplicate in the same laboratory on each time occasion.⁷ Standard enzymatic methods were used for serum total cholesterol, HDL-cholesterol and triglycerides, and plasma glucose concentrations. LDL-cholesterol concentration was calculated using the Friedewald formula.¹⁸ In 2001, we assessed serum high-sensitive C-reactive protein using an automated analyser (Olympus AU400, Olympus, USA) and a highly sensitive turbidimetric immunoassay kit (CRP-UL-assay, Wako Chemicals, Neuss, Germany). Serum C-reactive protein samples taken in 1980 were stored at −20°C and analysed in 2005 using the same method as in 2001. During the storage, the samples were not thawed or refrozen.

Carotid intima-media thickness and adulthood atherosclerotic risk factors

In 2001–2002, ultrasound studies were performed using Sequoia 512 ultrasound mainframes (Acuson, Mountain View, CA, USA) to measure CIMT. The measurement has previously been described in detail.^7,19 In brief, the image was focused on the posterior (far) wall of the left carotid artery. A minimum of four measurements of the common carotid far wall were taken approximately 10 mm proximal to the carotid bifurcation to derive mean carotid IMT. The digitally stored scans were manually analysed by one reader blinded to subjects’ details (M.J.). The between-visit coefficient of variation of IMT measurements was 6.4%.

Several additional assessments were carried out in 2001.²⁰ Waist circumference (mm, measured in duplicate at the level of the twelfth rib or level with the navel in thin subjects) and hip circumference (mm) were obtained to calculate waist–hip ratio. Standard enzymatic methods were used for plasma glucose concentrations. Serum insulin was measured by microparticle enzyme immunoassay kit (Abbott Laboratories, Diagnostic Division, Dainabot). Insulin resistance was estimated according to the homeostasis model as the product of fasting glucose and insulin divided by the constant 22.5, i.e. the HOMA index.²¹ Serum apolipoproteins A-1 and B were analysed immunoturbidometrically (Orion Diagnostica, Espoo, Finland).

Parental factors and adult lifestyle factors

Parental socioeconomic position was measured in 1980 using parental occupational status as classified by Statistics Finland and categorized as manual vs. non-manual.²² Where socioeconomic position differed between parents, data on the parent with the higher occupational status were used. Parental BMI in 1980 was calculated from self-reported height and weight.

The participant’s own adult socioeconomic position was measured in 2001 by occupational status and categorized as for parental socioeconomic position. Educational attainment was categorized as academic; secondary education but not academic; and comprehensive school as the highest level of education. Information on adult smoking (status and pack-years of tobacco smoking) and alcohol consumption (units per week) was obtained by questionnaire in 2001. One unit of alcohol (12 g) was equal to a glass of wine, a single 4 cL measure of spirits or a 33 cL bottle of beer.²⁰

Statistical analysis

Statistical significance in all tests was inferred at a two-tailed P < 0.05. The following tests were performed.

Gene-phenotype associations

FTO polymorphism explained 0.4% of the variation in lifetime BMI. Each additional copy of the A allele in the FTO gene was associated with higher lifetime BMI, with higher maternal BMI and, more weakly, paternal BMI (Table 3). FTO polymorphism was also associated with higher lifetime systolic blood pressure, but no association was found with lipids or C-reactive protein. The association between FTO polymorphism and lifetime systolic blood pressure attenuated after adjustment for lifetime BMI (adjusted change per A allele, 0.05; 95% CI, −0.01, 0.10; P = 0.11), suggesting that adiposity as indexed by BMI at least partially mediates this association.

Of the adulthood outcomes, a greater number of A alleles was associated with higher BMI (although less strongly than with lifetime BMI), larger waist circumference, and higher fasting glucose levels. Adjustment for BMI and waist circumference had little effect on the association between FTO polymorphism and fasting glucose (adjusted change per A allele 0.05; 95% CI, 0.01, 0.10; P = 0.03). A greater number of A alleles was weakly associated with greater CIMT and higher insulin resistance. Progression of atherosclerosis is more rapid in men than in pre-menopausal women. In sex-specific analysis, each additional copy of A allele was associated with a CIMT increase of 0.009 mm (95% CI, 0.000, 0.017; P = 0.049) in men. This figure was 0.000 mm (95% CI, −0.006, 0.007; P = 0.95) in women (P for sex interaction = 0.11; for all other genotype associations, P for sex interaction exceeded 0.2). Adjustment for BMI and waist circumference slightly reduced the effect of FTO genotype on CIMT in men (per A allele change, 0.008; 95% CI, −0.001, 0.016; P = 0.08).

Standard regression analysis and instrumental variables regression analysis

The standard regression analysis suggests positive associations of lifetime BMI with adult CIMT, lifetime systolic blood pressure, adult glucose, and adult insulin resistance (Table 4, upper panel). In the instrumental variables regression analysis with FTO polymorphism as the instrument for unconfounded lifetime BMI levels (path a in Figure 1), all the effects of BMI on CIMT and other phenotypes are in the same direction but less precisely estimated (P-values range between 0.02 and 0.15). In general, the effects appeared larger in the instrumental analysis than in the standard regression analysis, but only for adult fasting glucose does the difference in effect sizes between the two analyses reach conventional statistical significance. The wide confidence intervals in the instrumental variables regression analysis suggest that our data, despite the relatively large sample size, had limited power for the instrumental variable analyses (i.e. path a in Figure 1 was relatively weak). Indeed, F-values in the first-stage regression testing the strength of instruments were <10 (range, 7.5–9.2). Running these analyses with FTO haplotypes as instruments did not solve this problem as the strength of instrument was not improved (see Supplementary material online, Appendix 1).

The lower panel of Table 4 shows the results from the standard and instrumental variables regression analyses using simulated data corresponding to ours, but the sample size increased two-fold to that we had at the baseline (i.e. n = 2 × 3596). In such data, F-value in the first-stage regression is 31.6; all of the associations of lifetime BMI with CIMT and atherosclerotic risk factors were now robust in instrumental variables regression analysis and for all outcomes except HOMA index these associations were of greater magnitude compared with those in the standard regression analyses. We repeated these analyses with five independently simulated data sets. The associations in instrumental variables regression analysis and standard regression analysis remained across all of the simulations repeated five times, whereas the difference in the magnitude of the associations between the two analyses was consistently replicated only for adult glucose (see Supplementary material online, Appendix 2).

Utility of the Mendelian randomization approach

At least three issues suggest that Mendelian randomization approach may add to the evidence from conventional cohort studies in testing the contribution of lifetime BMI to atherosclerosis risk. First, confirming previous studies, FTO polymorphism was associated with BMI (path a in Figure 1).^8–11 This association was stronger for lifetime BMI than adult BMI, which is, as would be expected, given that genotypes should have life-long influence on risk factor levels.³⁵ Our analysis using the FTO genotype as an instrument for life-long BMI levels is therefore likely to be less biased by attenuation by errors than those based on a single measurement of BMI. In addition, FTO polymorphism was associated with parental BMI, as would be expected, given that offspring share all genes with their parents (50% with each parent). The slightly stronger association with maternal than paternal BMI may reflect intrauterine influences of maternal obesity programming increased life-long obesity risk in the offspring, non-paternity generating attenuated paternal associations, or random variation in associations.^36–38

Secondly, FTO polymorphism was not associated with factors such as sex, age, early or adult socioeconomic circumstances, educational attainment, smoking, or alcohol consumption. Thus, these common confounding factors are unlikely to distort the instrumental variables analysis of the effect of BMI on atherosclerosis risk (path b in Figure 1), as has been demonstrated previously.³⁹ Thirdly, as the FTO genotype determines the increased risk of higher BMI levels from early life onwards, reverse causality where subclinical atheroma affects risk factor levels was largely eliminated.

A full understanding of the function of the FTO gene is undergoing further exploration. However, both high BMI and type 2 diabetes contribute to atherosclerotic vascular changes,^40–43 and an additive effect of the FTO variant rs9939609 on BMI has been replicated in at least 13 unselected independent population studies with 38 759 participants.⁸ In the Avon Longitudinal Study of Parents and Children (ALSPAC) offspring, the variant was shown to have a specific association with fat mass.⁸ In genome wide studies, the FTO gene has been identified to predispose to type 2 diabetes via an effect on BMI.⁸ Furthermore, it has been shown that the FTO gene is associated with insulin resistance, hyperglycaemia, dyslipidaemia, and hypertension, as one would predict based on observational evidence of the association of BMI with these outcomes,⁴⁴ and of randomized trials of the effect of weight change interventions on these outcomes.⁴⁴ These findings suggest that greater adiposity is indeed causally related to these outcomes and that the observational studies for these associations provide valid estimates of effect. Furthermore, these findings provide some support that FTO polymorphism is a valid instrument for adiposity. If it were not a valid instrument of adiposity, then one would not expect such accurate prediction of the causal effect of adiposity on outcomes that are generally widely accepted as being causally related to greater adiposity,⁴⁵ and that alter in relation to changes in BMI in randomized controlled trials of drug and surgery treatments that reduce BMI.^46–48

Study limitations

Limitations of Mendelian randomization testing have been discussed in detail previously.⁵ It is possible that FTO has other direct phenotypic effects in addition to those on BMI, and it is these that influence the outcomes. Furthermore, the expression of FTO during early-life development could trigger a canalizing response that would mitigate the effects of increased adiposity. Studies utilizing additional independent genetic variants related to BMI, and investigations of when, during development, the variants begin to influence adiposity, could help resolve these issues. It could be argued that, as adjustment for BMI did not abolish the FTO-outcome associations, this suggests that there are indeed alternative pathways linking FTO to the outcomes. However, it must be remembered that even the repeat measures of BMI utilized in our lifetime BMI score will not be an accurate indicator of true lifetime adiposity, and a residual effect of FTO after adjustment for our lifetime BMI measure would be expected even if the entire effect of FTO was mediated through adiposity.

In this study, the wider confidence intervals for effect estimates from instrumental variables analysis compared with those obtained from the standard regression models demonstrate a drawback. Body mass index is a complex trait, and the small proportion of variation in BMI explained by the genetic instrument based on a single gene leads to the much lower precision of the instrumental variables analysis estimates compared with the estimates from standard regression models. Thus, the instrumental variable estimates are typically less efficient than the standard regression estimates if there is no confounding, reverse causality, or measurement error, but unlike the standard regression estimators the instrumental variables estimates should remain consistent when these are present. Besides supporting the role of BMI as a contributing factor to CIMT and various atherosclerotic risk factors, our findings suggest that the true influence of BMI on these factors might be underestimated in cohort studies. However, larger samples and/or stronger genetic instruments are needed before this conclusion can be convincingly supported by statistically robust results after correction for multiple testing. Furthermore, as the function of FTO is not fully known, we cannot be certain that the FTO polymorphism conferred effect on atherosclerosis risk factors only through adiposity.