The influence of the rs1137101 genotypes of leptin receptor gene on the demographic and metabolic profile of normal Saudi females and those suffering from polycystic ovarian syndrome

Leptin receptor (LEP-R; OB-R; CD295), which is encoded by the LEPR gene, is a single-trans-membrane domain protein receptor of the cytokine receptor family [1] that binds the hormone leptin [2]. Cell membranes of a variety of cells in different organs and tissues harbor the LEP-R, signifying the role played by leptin in these tissues [3‐5]. Even in the hypothalamus, the LEP-R is located in the cell membrane and is actively involved in the control of hunger and thirst and influences sleep, moods, and body temperature [6, 7]. Leptin, a polypeptide hormone, is a major player in regulating adipose tissue mass through hypothalamus effects on hunger and energy usage [6, 8]. Leptin binding to its receptor activates a series of chemical signals that influence hunger and helps in the generation of a feeling of fullness (satiety). Lean people generally have lower levels of leptin compared to their obese counterparts, and female gender, produce more leptin than their male counterpart [9]. Interest in the role of leptin in the weight control developed in the early ‘90s, and for over two decades leptin is closely linked to body weight fluctuations [10‐13]. More recently it has become evident that such weight-related alterations may be a consequence of genetic variations in the LEP-R gene in the absence of any qualitative or quantitative leptin abnormality [14, 15]. In the LEP-R gene, several mutations are reported that alter the normal functional capacity of the LEP-R protein, resulting in an apparent “leptin receptor deficiency,” hence preventing either the binding of leptin to the receptor or the receptor from responding to the bond leptin. The consequences include excessive hunger, weight gain, and hypogonadotropic hypogonadism [16]. Severe obesity due to excessive eating (hyperphagia) begins in early childhood in LEP-R deficiency disorders and the hypogonadotropic hypogonadism, is caused by alleviated secretion of hormones involved in direct sexual development. Affected individuals experience the absence of or delay in puberty and later may suffer from infertility disorder. In a recent investigation, a statistically significant association was found between polycystic ovarian syndrome (PCOS), obesity and leptin, where the overweight and obese women with PCOS had higher serum levels of leptin compared with controls, though they have no association between PCOS and other adipokines, such as resistin and adiponectin [17]. This should prompt the researcher to deeply investigate this issue also in the influence of leptin gene polymorphism. Several polymorphisms have been reported in the LEP-R gene, which has been linked to a number of different disorders. One of the non-synonymous polymorphisms rs1137101 (Gln223Arg), is linked to insulin resistance, metabolic syndrome, dyslipidaemias, diabetes mellitus, obesity, besides several cancers (breast, colorectal, thyroid, prostate), preterm delivery, osteoarthritis, atherosclerosis, and ischemic heart disease [18‐25]. We hypothesized that LEP-R gene polymorphisms might be associated with PCOS since the latter is associated with obesity, insulin resistance, hypogonadotropic hypogonadism, and infertility, all being common features of LEP-R deficiency [26]. Some associations have been reported, but the results have frequently been contradictory.

Among the Saudi women, PCOS is of frequent occurrence, though the exact frequency is yet not known (Babay Z; personal communication). In this study, the distribution of genotype and allele frequencies of the non-synonymous SNP rs1137101 (Gln233Arg) in the normal healthy Saudi females and Saudi PCOS patients, was investigated. We determined the effect of the different genotypes on the demographic and metabolic profiles. In this paper, we also present our results of association studies between rs1137101 polymorphism and PCOS and the effect of the polymorphism on demographic and metabolic profile in healthy females and those suffering from PCOS.

The study group included 122 healthy females of Saudi origin and another group of 130 women who were diagnosed as suffering from PCOS. The demographic, metabolic and hormonal parameters in the two groups were as presented in Daghestani et al. (28). The two groups matched for their ages and BMI but showed a significant difference in the W/H ratio, where the PCOS patients had a significantly higher ratio compared to the control group. Among the lipid parameters, all parameters were elevated in the PCOS compared to the control group except HDL, which was significantly lower. Insulin, glucose, and HOMA-IR were also significantly elevated in the PCOS, but leptin, ghrelin, and HOMA-S were not different significantly when compared with the control group.

The genotype and allele frequencies of rs1137101 in the control group were compared to the results in the PCOS patients. Table 1 presents the frequencies and shows that there were no significant differences between the two groups.

To determine differences in the genotype and allele frequencies in patients who were normal weight and obese, the control and patients were separated into two groups based on their BMI. Table 2 presents the genotype (AA, AG, GG) and allele (A and G) frequencies of rs1137101 in the normal weight and obese PCOS patients compared to normal weight and obese controls.

The PCOS and control group were separated into three groups based on the genotypes: AA, AG and GG and the demographic and metabolic data were separately calculated for each genotype. The results in the different genotypes were compared, and the significance of the difference in the results was recorded. In the control group, BMI, W/H ratio, leptin, Insulin, and HOMA-IR were significantly higher in the GG genotype compared to AA. In the PCOS group statistically significant differences were observed for W/H ratio and leptin in the AA vs. GG (p = < 0.05), in the PCOS, while in the AA vs. AG genotypes, the triglyceride levels were higher (p = 0.003). However, this genotype also associated with leptin and triglyceride level in the controls (p = 0.01). The results are presented in Table 3.

Subject’s distribution and statistical power were assessed by further tests, by evaluating the effect size on our sampling procedures. Table 4 describes the outputting results. Most of the comparisons (78.8%) showed a small non-overlapping percentage, calculated on Cohen’s d-effect size and demonstrate that for certain parameters (e.g., triglycerides SNP rs1137101 AA vs. AG) Cohen’s d and its co-related Gates’ delta and Hedges g, showed the same population overlapping between PCOS subjects and controls (see Table 4, 29.59 and 30.26%, respectively), assessing the sampling process performed in the study. Yet, this correspondence has not been observed for leptin (SNP rs1137101 AA vs. GG, see Table 4), what might also explain our negative data for leptin polymorphism association with PCOS, inducing out further research to improve cases and controls recruitment.

The LEP-R gene has been extensively investigated in several populations, and the association has been investigated with a number of diseases including obesity, preterm delivery, recurrent spontaneous abortion and different cancers [16‐25]. Several polymorphisms have been investigated, and rs1137101 has been commonly associated with several disorders. Our results showed that LEP-R gene SNP rs1137101 do not show any association with obesity either in the healthy normal Saudi population or the females suffering from PCOS. The only important association was reported for the waist/hip ratio (W/H ratio), while the association with lipidogram and leptin was also observed for non-PCOS individuals. Reports showing possible leptin-W/H ratio associations were suggested either for healthy people or several disorders and aging [31‐34].

However, despite some recent conclusions on obese patients [35, 36] and cancer [35, 37], no report was yet published about the possible association of the SNP rs1137101 at the leptin gene. Recent reports showed that obesity should play a major role in the pathogenesis of PCOS through the insulin, leptin and endocannabinoid receptor, and by an association between insulin receptor polymorphism and PCOS was observed [38]. Furthermore, an association of the leptin gene SNPs (Gln223Arg and Pro1019Pro) with PCOS was recently reported in a Korean population [39]. Despite this interesting evidence, our data seem to confirm previous reports showing no association between leptin gene single nucleotide polymorphism and PCOS, though PCOS is frequently associated with insulin resistance and obesity and these conditions are linked to leptin resistance and expression in the leptin receptor. Past analyses demonstrated that patients diagnosed with PCOS did not show any mutation of the leptin coding exons, while the amino acid variants in exon 2, 4 and 12, through a single-stranded conformational polymorphism (SSCP) analysis, followed by sequencing, did not show any association between leptin and PCOS [40], suggesting that much more insightful evidence should be gained by further studies. The results of this study highlight an important point, i.e. the polymorphic change resulting from the substitution of an A by G, changing the codon, CAG to CGG, and replacing Gln, a neutral amino acid by Arg, a basic amino acid, brings about an effect thereby affecting the value of different anthropometric and biochemical parameters, both in normal control and PCOS patients. The exact mechanism by which these changes are brought about needs functional and in-silico studies, whereby the mechanism of leptin-LER-R interaction can be evaluated.

Finally, since our study did not show any statistical association between rs1137101 and PCOS, further investigation, possibly by also evaluating obese patients should be needed to elucidate this issue better.