Development of a multiplex system for determining 11 genetic markers of predisposition to obesity

A system has been developed to identify 11 genetic markers associated with the risk of obesity: rs10852521, rs11075990, rs1121980, rs1421085, rs1477196, rs17817449, rs3751812, rs7206790, rs8047395, rs9940128 (FTO gene) and rs1137101 (LEPR gene) by minisequencing (SNaPshot analysis). The conditions for carrying out the amplification and minisequencing reactions, as well as the compositions of the reaction mixtures, were optimized so that the analysis was carried out for all 11 markers simultaneously. The resulting system was tested and showed a high degree of reproducibility and sensitivity required for the detection of these polymorphisms.

Association of leptin receptor gene polymorphisms and meta-inflammation markers with metabolically unhealthy obesity in children

The aim: to study the contribution of single-nucleotide polymorphisms (SNP) of the leptin receptor (LEPR) gene and meta-inflammation markers to the formation of metabolically unhealthy obesity (MUO) in children. Materials and methods. A total of 109 obese children aged 6–18 years were examined. Based on the recommendations of the National Heart, Lung, and Blood Institute (NHLBI), 2 observation groups were formed. The main group (n = 56) was represented by patients with MUO. The control group (n = 53) comprised children with metabolically healthy obesity (MHO). Serum levels of interleukin-1β (IL-1β) were measured using a chemiluminescent immunoassay (CLIA) method, interleukin-6, leptin, adiponectin – by enzyme-linked immunosorbent assay (ELISA) and the serum level of C-reactive protein were quantified by latex turbidimetric method (Synevo, Ukraine). The method of next-generation sequencing (NGS) (CeXGat, Germany) was used to identify LEPR SNP. Statistical methods were used: analysis of variance, Spearman’s correlation analysis and multiple discriminant analysis. Results. In obese children aged 6 to 18 years, there was an increase in pro-inflammatory adipokines IL-6 and leptin and a decrease in anti-inflammatory adiponectin. Statistically significant changes in these indicators were more expressed in the main group: IL-6 – 7.4 ± 0.5 pg/ml (ρ = 0.65; P ≤ 0.001); adiponectin – 3.9 ± 0.8 μg/ml (ρ = -0.27; P = 0.007) among all the children examined, leptin in girls – 47.8 ± 4.4 ng/ml (ρ = -0.28; P = 0.003) compared with the results of patients in the control group: IL-6 – 4.3 ± 0.3 pg/ml, adiponectin – 7.7 ± 2.4 μg/ml, leptin in girls – 32.5 ± 4.3 ng/ml, P ≤ 0.05. The most important in the development of MUO were the following SNP of the LEPR gene: rs3790435 (CiMUO = 0.939), rs2186248 (CiMUO = 0.862), P < 0.05. A strong correlation was found between MUO and serum IL-6 level (ρ = 0.7), LEPR SNP rs3790435 (ρ = 0.7), basal hyperinsulinemia (ρ = 0.72); Р ≤ 0.001. The risk of IL-6-dependent meta-inflammation in the presence of SNP rs3790435 of the LEPR gene: OR = 17.11; 95 % CI 2.8–20.4. Conclusions. Meta-inflammation in MUO is IL-6-dependent. Among the 10 SNPs of the LEPR gene that we identified, SNP rs3790435 of the LEPR gene has a strong association with the formation of MUO. SNP rs2186248 LEPR was described by us for the first time when it was found in 94.1 % of obese children, but it was characterized by the presence of a weak association with MUO.

The role of LEPR, PPARG and PPARGC1A genes polymorphisms in the development of metabolic disorders in patients with vibration diseases

Introduction. Patients with vibration disease (VD) often have obesity and metabolic syndrome (MS), which increase the risk of developing type 2 diabetes, hypertension, and other conditions predisposing to a decrease in the quality and longevity of life. Genetic predisposition, overnutrition, environmental factors, including industrial ones, and others are factors influencing the formation of MS. The aim of the study is to identify associations between polymorphisms of the LEPR, PPARG and PPARGC1A genes, metabolic syndrome and obesity in VD patients caused by exposure to local vibration and the combined effects of local and general vibration. Material and methods. We examined 248 VD male patients exposed to local vibration and the combined impact of the local and general vibration. We have identified a subgroup of MS and obesity cases. The distribution of genotypes of the LEPR, PPARG and PPARGC1A genes in groups was studied. Results. In the group of VD and MS patients exposed to the combined effect of local and general vibration, the Gln/Gln genotype of the Arg223Gln polymorphic locus of the LEPR gene was less common, and the Arg / Gln genotype in MS cases was detected more often than in patients without it. Among patients with VD caused by exposure to local vibration with a waist circumference of more than 102 cm, the Gly / Ser genotype of the Gly482Ser polymorphism of the PPARGC1A gene was more common than among those with lower values of this indicator. Conclusion. In patients with VD caused by combined exposure to local and general vibration, the Gln/Gln genotype carrier of the Arg223Gln polymorphism of the LEPR gene may play a protective role in the formation of MS. Among individuals with VD caused by exposure to local vibration, carriers of the heterozygous genotype of the Gly482Ser polymorphism of the PPARGC1A gene may have a predisposition to the development of obesity. The obtained results are preliminary and require further research.

Association of Leu72Met Polymorphism of GHRL Gene and Gln223Arg of LEPR Gene With Hunger, Satiety, Biochemical, and Anthropometric Variables in Adults From Western Mexico

Objectives To determine the association of Leu72Met single nucleotide polymorphism (SNP) of GHRL gene and Gln223Arg SNP of LEPR gene with hunger, satiety, biochemical, and anthropometric variables in adults from Western Mexico. Methods Quasi-experimental study design with 132 participants of which 109 were women. Inclusion criteria were age 18–25 years old, BMI 18.5–24.9 kg/m2, 10 hours of fasting, and have the habit of eating breakfast. Exclusion criteria were subjects with a diagnosed disease, vegetarians or vegans, use of drugs that alter appetite or for weight loss, food allergies, elite athletes. Subjects were summoned twice. In the first one, medical history and anthropometric measurements were realized. A week later vital signs were taken, and blood sampling was obtained in fasting and at 120′ post breakfast. Anthropometrics and biochemical measurements were done with the InBody 370 and Vitros 350 analyzer, respectively. SNP´s were analyzed using the TaqMan® allelic discrimination assay in a real-time PCR thermocycler. Five visual analog scales to assess hunger, fullness, satiety, desire to eat, and prospective consumption were applied in fasting, after breakfast, and at 30′, 60′, 90′, and 120′ post-ingestion. Breakfast had an energy content of 526.5 kcal (36% lipids, 43% carbohydrates, and 21% protein). All variables were analyzed among genotypes considering the dominant model. Data were analyzed in SPSS version 20.0. Normality distribution was assessed with the Shapiro-Wilk test. Student T-test was applied for related (intra) or independent (inter) groups, respectively. Results BMI of participants was 22.0 ± 2.0 kg/m2 with a mean age of 20.6 ± 2.0 years. At 60′ post-ingestion hunger was lower and at 120′ glucose levels were lower in Leu72Leu carriers than in Leu72Met/Met72Met carriers. In fasting, total cholesterol, LDL-c, triglycerides, and desire to eat were higher in subjects with Gln223Gln genotype than in Gln223Arg/Arg223Arg genotype carriers. Conclusions The GHRL SNP was associated with higher hunger and glucose in the postprandial state; contrary, the LEPR SNP was associated with lower lipids levels and less desire to eat in fasting. Genetic variants could be involved with hunger, satiety, and metabolism biomarkers. Funding Sources "PROINPEP” and “PRO-SNI” from University of Guadalajara.

Severe Early-Onset Obesity in Three Adult Patients Harboring Inactivating Mutations of the Leptin Receptor Gene

Background: Leptin is secreted by white adipocytes in response to fat storage and binds to leptin receptors (LEPR) expressed all over the body particularly in hypothalamic neurons. This hormone regulates several physiologic functions including energy expenditure and appetite. Clinical Case: We describe the clinical and hormonal findings in 3 adult brothers with body mass index (BMI) of 36.7, 50.7, and 46.1 kg/m2, respectively. There is no history of consanguinity in the family and none of the patients exhibited dysmorphic features. A rapid weight gain was noticed during their first few months of life, associated with permanent hyperphagia. At 2 years old, their BMI was already above 25 kg/m2 (&gt; +3SD), and at 10 years old, it was over 40 kg/m2 (&gt; +3SD). The linear growth was within the expected target heights for their parents. They did not seem to present cognitive impairment. The pubertal development began at 16 to 18 years old, and since then they maintained levels and FSH and LH above the upper limit of normal (15.6±3.7mUI/mL and 12.3±2.2mUI/mL, reference range 1.5–12.4 and 1.7–8.6, respectively), but with normal sexual steroids (estradiol 36.4±16.1pg/mL and total testosterone 445±401ng/dL, reference range 11–44 and 249–836, respectively). The thyroid function was normal and none of the patients suffered from dyslipidemia or diabetes, despite high serum insulin levels 26.4±15.8 mU/L (normal 5–10). Genetic sequencing identified a homozygous mutation of the leptin receptor gene in the 3 brothers: c.2357T&gt; C, p. (Leu786Pro). Their parents were heterozygous for the mutation as well as the patients’ daughters. Homozygous carriers of the mutation presented a significantly higher BMI than their heterozygous family members, 44.5±7.1 Kg/m2 vs 32.2±1.7 Kg/m2 (p=0.023), a significantly increased leptin levels, 80±36.4 ng/ml vs 26.3±9.3 ng/ml (p=0.028), and significantly higher weight, 134.6±16.9 vs 89.2±15,2 kg (p=0.021), respectively. Women had higher BMI than men (42.0 ±12.2 Kg/m2 vs 37.9±7.5 Kg/m2, p=0.496) and also higher percentage of fat (46.4±3.1% vs 34.9±6.8%, p=0.108). Serum levels of leptin in homozygous patients were not significantly higher than those measured in 10 patients with adult-onset morbid obesity, 80± 36.4 ng/ml vs 53.8±24.1 ng/ml, respectively (p=0.149). Therefore, serum leptin is not a useful discriminative maker of LEPR gene mutations. Conclusion: Patients with severe early-onset obesity should have a genetic diagnosis workup. Firstly, because clinical trials with MC4R-agonists have raised expectations regarding the treatment of patients with mutations of the LEPR gene. Secondly, and in contradiction to other reports in the literature, our patients were fertile. Therefore, identification of the mutation allows genetic counseling of these patients and their families, including the possibility of Pre-Natal Diagnosis or Pre-Implantation Genetic Diagnosis.

EFFECT OF LEPTIN CONCENTRATIONS AND LEPTIN RECEPTOR GENE POLYMORPHISMS ON THE OUTCOME OF RENAL TRANSPLANTATION

IntroductionLeptin is a pro-inflammatory adipocytokine implicated in cardiovascular disease, insulin resistance, obesity and chronic kidney disease.Material and methodsIn a cohort of 236 renal transplant recipients, we aimed to determine whether circulating leptin concentrations and/or three polymorphisms in the leptin receptor (LEPR) gene, namely rs1137100, rs1137101 and rs1805094, were related to clinical outcomes in renal transplantation. Plasma leptin concentrations were measured by ELISA. Genetic variants were determined by conventional real-time PCR assays and statistical associations with clinical outcomes were obtained by logistic regression modelling.ResultsPatients with elevated leptin levels were at higher risk of acute rejection [OR=1.03 (1.01–1.05), p=0.03] and displayed worse renal clearance (p=0.001) than patients with lower levels. Leptin levels were not significantly affected by any of the three LEPR SNPs. The rs1137101 G variant showed an inverse association with the risk of delayed graft function (DGF) [OR=0.42 (0.22-0.81), p=0.009], whilst the homozygous rs1805094 CC genotype was associated with increased risk of acute rejection [OR=11.38 (2.15-60.16), p=0.004]. A statistically significant association was also observed between the rs1137100 GG genotype and better renal function [mean difference vs. AA/AG =20.20 (4.91-35.49) ml/min, p=0.010].ConclusionsOur results show that both leptin plasma concentrations and polymorphisms in the LEPR gene may affect clinical outcomes in renal transplant recipients, suggesting that the determination of these parameters could be useful in predicting post-transplant adverse events.

The LEPR Gene Is Associated with Reproductive Seasonality Traits in Rasa Aragonesa Sheep

The aim of this study was to characterize and identify causative polymorphisms in the leptin receptor (LEPR) gene responsible for the seasonal variation of reproductive traits in sheep. Three reproductive seasonality traits were studied: the total days of anoestrous (TDA), the progesterone cycling months (P4CM) and the oestrous cycling months (OCM). In total, 18 SNPs were detected in 33 ewes with extreme values for TDA and OCM. Six SNPs were non-synonymous substitutions and two of them were predicted in silico as deleterious: rs596133197 and rs403578195. These polymorphisms were then validated in 239 ewes. The SNP rs403578195, located in exon 8 and leading to a change of alanine to glycine (Ala284Gly) in the extracellular domain of the protein, was associated with the OCM trait, being the G allele associated with a decrease of 12 percent of the OCM trait. Haplotype analyses also suggested the involvement of other non-synonymous SNP located in exon 20 (rs405459906). This SNP also produces an amino acid change (Lys1069Glu) in the intracellular domain of the protein and segregates independently of rs403578195. These results confirm for the first time the role of the LEPR gene in sheep reproductive seasonality.

Investigasi Hubungan Keragaman Gen Leptin Receptor (LEPR) dengan Karakteristik Karkas dan Kualitas Daging Domba

Improvement of meat quality plays important role for sheep meat producer and costumer market.Leptin Receptor (LEPR) gene is speculated has important role in carcass and meat quality. This studyaims to investigate the relationship between the LEPR gene variation with characteristics of carcassand meat quality in sheep. A total 50 rams consisted 20 of Javanese fat tailed (JFT), 10 of garutcomposite (GCS), 10 of compass agrinak (CAS), and 10 of barbados cross (BCS) were used in this study.Polymorphism of LEPR gene were performed by Polymerase Chain Reaction-Restriction FragmentLength Polymorphism (PCR-RFLP) with Acil as restriction enzyme. The results showed that of theamplification product was 432 bp. The result of polymorphism of LEPR gene were polymorphic withthird genotype including AA, AC, dan CC. The SNP of LEPR gene with genotype AA was associated(P<0.05) with carcass characteristics including empty body weight, cold carcass and with genotype ACon meat quality including tenderness. It could be concluded that the SNP g.40854778 A>C of LEPR genemay contribute to the characterictic carcass and meat quality in sheep