**3. Results**

Table 1 shows the anthropometric and biochemical data of the children according to sex. The mean age was similar in boys and girls. Plasma concentrations of HDL-cholesterol and Apo A-I were significantly higher, and concentrations of TG, LDL-cholesterol, and Apo B were significantly lower, in boys compared to girls. Leptin levels were significantly (*p* < 0.001) higher in girls.

When analyzing the relationship of the *PLIN1* SNPs rs894160, rs2304795, and rs1052700 with the variables under study by sex (Table 3), a significant and opposite association was discovered between *PLIN1* rs1052700 and Apo A-I levels between boys and girls. The TT carriers showed significantly higher Apo A-I levels as compared with CC and CT carriers in boys, while significantly lower Apo A-I levels were observed in girls. When analyzing the SNP of *PLIN2* rs35568725 (Table 3), carriers of the AG and GG genotypes were grouped together due to the small number of children who were homozygous for the less common allele (G). Significant differences between AA carriers and carriers of the G allele were observed for HDL-cholesterol (*p* = 0.005), Apo A-I (*p* = 0.009), and NEFA (*p* = 0.002) concentrations in boys, though no such differences were observed in girls. No significant associations were detected between the PLIN polymorphisms and LDL-cholesterol, Apo B, or leptin levels (data not shown).

The frequencies of the genotypes and alleles for the SNPs studied are shown in Table 2. The genotype distributions were within the Hardy–Weinberg equilibrium. These frequencies were similar to those described in other Caucasian populations.


**Table 1.** Characteristics (means ± SD) of the population studied.

1 *p*-value: Mann–Whitney U test. Abbreviations are as follows: BMI, body mass index; TC, total cholesterol; TG, triglycerides; LDL-C, low-density lipoprotein cholesterol; Apo B, apolipoprotein B; HDL-C, high-density lipoprotein cholesterol; Apo A-I, apolipoprotein A-I; NEFA, non-esterified fatty acids.


**Table 2.** Genotypic and allelic distribution of the SNPs studied in *PLIN1* and *PLIN2*.

To analyze whether the effect of the polymorphisms studied on BMI and lipid levels was mediated by leptin levels, the relationship of the SNPs (rs894160, rs2304795, and rs1052700 in *PLIN1*, and rs35568725 in *PLIN2)* with these parameters was investigated in children classified into two groups according to their plasma leptin levels, i.e., boys and girls with plasma leptin levels above or below their respective median value of leptin. As shown in Figure 1, we observed that the associations of *PLIN1* rs894160 and *PLIN2* rs35568725 with BMI (Figure 1a) and of *PLIN1* rs2304795 and *PLIN2* rs35568725 with NEFA (Figure 1b) were different in boys and girls with lower leptin levels compared to subjects with higher leptin concentrations. Significant associations were observed in boys with leptin levels above the median values, as well as in girls with leptin levels below the median leptin value. Additionally, a different association in boys and girls depending on leptin levels was observed concerning the influence of *PLIN1* rs1052700 on Apo-I levels (Figure 1c). The association of the *PLIN1* rs1052700 SNP with HDL-cholesterol levels (Figure 1d) was evident in girls with high leptin concentrations. Furthermore, the *PLIN1* rs894160 and *PLIN*2 rs35568725 SNPs were also associated with HDL-cholesterol levels depending on leptin concentration and sex (Supplementary Figure S1). No significant results were found when analyzing the association of the SNPs studied with triglyceride concentrations depending on leptin concentration.


**Table 3.** BMI and lipid profile values (means ± SD) by genotype for *PLIN1* and *PLIN2* SNPs in boys and girls.

*p*-value: Mann–Whitney U test; a AA + AT vs. TT; b AA + AG vs. GG; c AA vs. AG + GG. Abbreviations are as follows: ns, not significant; BMI, body mass index; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; Apo A-I, apolipoprotein A-I; NEFA, non-esterified fatty acids.

**Figure 1.** *Cont*.

**Figure 1.** (**a**) BMI values of *PLIN1* rs894160 and *PLIN2* rs35568725 genotypes in boys and girls according to levels of leptin; (**b**) NEFA levels of *PLIN1* rs2304795 and *PLIN2* rs35568725 genotypes in boys and girls by leptin levels; (**c**) Apo A-I levels of *PLIN1* rs1052700 genotypes in boys and girls by levels of leptin. (**d**) HDL-cholesterol levels of *PLIN1* rs1052700 genotypes in boys and girls by levels of leptin. Values are expressed as median and interquartile range. *p*-value: Mann–Whitney U test: \* *p*-value < 0.05; \*\* *p*-value < 0.01.
