*2.1. Subjects*

Our study comprised a population of 1264 children, aged 6–8 years (633 boys and 631 girls), who participated of the Four Provinces Study (4P Study), a cross-sectional study aiming to examine cardiovascular risk factors in Spanish children [30]. All children with a parent-reported chronic disease were excluded. Parents or legal guardians were required to provide written informed consent for their children to participate in the study. The study protocol complied with the Helsinki Declaration guidelines and was approved by the Clinical Research Ethics Committee of the IIS-Fundación Jiménez Díaz (PIC016-2019 FJD).

### *2.2. Anthropometric Data*

Measurements were taken with the children lightly dressed and barefoot as previously described [30]. Height was measured to the nearest millimeter using a portable stadiometer, weight was recorded to the nearest 0.1 kg using a standardized electronic digital scale, and body mass index (BMI, weight in kilograms divided by height in meters squared, kg/m2) was calculated from these parameters.

### *2.3. Biochemical Data*

Fasting (12 h) blood samples were obtained by venipuncture and centrifuge. Serum and plasma samples were separated and stored at −70 ◦C. Biochemical determinations were performed as previously described [30]. Cholesterol and triglyceride (TG) concentrations were measured enzymatically (Menarini Diagnostics, Florence, Italy) in a RA-1000 Autoanalyzer (Technicon Ltd., Dublin, Ireland). High-density lipoprotein cholesterol (HDL-cholesterol) was measured after precipitation of apolipoprotein B-containing lipopro-

teins with phosphotungstic acid and Mg (Roche Diagnostics, Madrid, Spain). Plasma apolipoprotein A-I (Apo A-I) and apolipoprotein B (Apo B) concentrations were measured by immunonephelometry (Dade Berhing, Frankfurt, Germany). Low-density lipoprotein cholesterol (LDL-cholesterol) was calculated according to the Friedewald formula. Non-esterified fatty acids (NEFA)were measured by using the Wako NEFA-C kit (Wako Industries, Osaka, Japan). Leptin concentrations were determined by ELISA using a commercial kit (Leptin EIA-2395, DRG, Marburg, Germany), as described elsewhere [30].

#### *2.4. Genotyping Assays of SNPs in PLIN1 and PLIN2*

All DNA was isolated from 10-mL EDTA-blood samples according to standard procedures. To determine the polymorphism in the perilipin genes, the following predesigned TaqMan SNP Genotyping Assays from Applied Biosystems (Waltham, MA, USA) were used: C\_8722593\_10, C\_8722587\_10, and C\_9304320\_20 for the SNPs in *PLIN1* rs894160, rs1052700, and rs2304795, respectively, and C\_25764255\_10 for the SNPrs35568725 in *PLIN2*. A StepOnePlus ™ Real-Time PCR System (Applied Biosystems) was used for allelic discrimination. Additionally, PCR was performed with a mixture of 10 ng of genomic DNA, TaqMan® SNP Genotyping Assay (20X), and TaqPath ™ ProAmp ™ Master Mix (Applied Biosystems). Samples were cycled under the following recommended conditions: 95 ◦C for 10 min, 95 ◦C for 15 sec, and 60 ◦C for 1 min, repeated over 40 cycles.

### *2.5. Statistical Analysis*

Statistical analyses were performed using the SPSS software package, version 26.0 (IBM, New York, NY, USA) and the GraphPad Prism statistical software (San Diego, CA, USA, Version 8). The normality of quantitative variables was analyzed by the Kolmogorov– Smirnov test, revealing a non-parametric distribution. The Mann–Whitney U test was used to perform sex-based comparisons of median BMI values, lipid profile variables (TC, TG, LDL-cholesterol, Apo B, HDL-cholesterol, Apo A-I, NEFA), and leptin. Differences in median values for the variables under study according to the different genotypes of the SNPs studied were tested using the Mann–Whitney or Kruskal–Wallis tests in our population, divided by sex and grouped according to median plasma leptin levels in each sex (2.26 ng/mL in boys and 5.25 ng/mL in girls).
