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Original Article

Verónica Loera-Castañeda (0000-0003-4158-1078)a; Raquel Sida-Medinab; G. Antonio Loera-Castañedab; Ignacio Villanueva-Fierrob; Ismael Lares-Asseffa; F. Daniel Hernández-Velázquezc; Heriberto Rodríguez-Hernándezd; Horacio Almanza-Reyese; Leslie Padrón-Romeroe; Susana Vázquez-Simentalf; Irene Leal-Berúmeng; Martha Rodríguez-Moránd; Fernando Guerrero-Romerod; J. Alonso Gándara-Mirelesb; Leslie C. Cerpah; Luis A. Quiñonesi.
aInstituto Politécnico Nacional (IPN), Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional (CIIDIR) Unidad Durango, Latin American Network for the Implementation and Validation of Pharmacogenomic Clinical Guidelines (RELIVAF-CYTED), Madrid, Spain; bInstituto Politécnico Nacional (IPN), Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional (CIIDIR) Unidad Durango; cNucleus for Research and Clinical Diagnosis, NIDIAC-Institute; dUnidad de Investigación Biomédica, Instituto Mexicano del Seguro Social (UIB-IMSS) Durango; eFacultad de Medicina y Psicología, Universidad Autónoma de Baja California; fDiabetes Clinic Service, Clínica de Medicina Familar, ISSSTE Durango; gFacultad de Medicina y Ciencias Biomédicas, Universidad Autónoma de Chihuahua; hLatin American Network for the Implementation and Validation of Pharmacogenomic Clinical Guidelines (RELIVAF-CYTED), Laboratory of Chemical Carcinogenesis and Pharmacogenetics (CQF), Department of Basic and Clinical Oncology (DOBC), Facultad de Medicina, University of Chile; iLatin American Network for the Implementation and Validation of Pharmacogenomic Clinical Guidelines (RELIVAF-CYTED), Laboratory of Chemical Carcinogenesis and Pharmacogenetics (CQF), Department of Basic and Clinical Oncology (DOBC), Facultad de Medicina, Universidad de Chile.
Corresponding Author: , . Tel: ; e-mail: [email protected]

Citation: Loera Castañeda V, Sida Medina R, Loera Castañeda GA, Villanueva Fierro I, Lares Asseff I, Hernández Velázquez, FD, et al. Influence of the polymorphisms leptin rs7799039, leptin receptor rs1137101, biochemical, and somatometric parameters as risk factors for developing type 2 diabetes in Mexican patients.
Lat Am J Clin Sci Med Technol. 2022 Nov; 4:162-172.
Received: July 21st, 2022.
Accepted: October 26th, 2022.
Published: November 3rd, 2022.
Views: 57
Downloads: 3
ABSTRACT

The role of leptin in humans is an essential factor in the pathophysiology of obesity and type 2 diabetes mellitus (T2D); and it is also related to their comorbidities. The objective of this study was to determine the influence together of rs7799039 (-2548G>A) LEP and rs1137101 (223Gln>Arg) LEPR polymorphisms, as well as biochemical and somatometric parameters, as risk factors for T2D. Material and Methods. A cross-sectional case-control study with a control group and a case group. Both groups were matched by age, sex, and body mass index. The genes were amplified by conventional PCR and were genotyped by restriction fragment length polymorphism (RFLP) analysis. Polymorphisms were validated using real-time PCRand TaqMan® probesin triplicate. Sample size was determined using an equation for cases and controls. Genomic DNA was isolated from leukocytes of peripheral blood samples using the DTAB/CTAB method and gene amplification wasperformed by conventional PCR. The presence of the polymorphisms was validated using real-time PCR and Applied Biosystems® TaqMan® probes C_3001671_10 for leptin and C_8722581_10 for leptin receptorpolymorphisms. Results. The polymorphism LEP rs7799039 in genotypes G/A, and A/A, was associated with T2D [OR=2.23 C =1.13 to 4.37 and OR=2.62 CI=1.27 to 5.40, respectively]. Additionally, polymorphism rs1137101 in genotypes A/G and G/G was associated with T2D (OR=0.35, IC=0.17 to 0.72 and OR=4.7, CI=2.29 to 9.62, respectively). Analyzed together, the combined genotypes GA-2548/GG223 (OR=7.61 CI=2.02 to 28.67) and AA-2548/GG223 (OR=3.53 CI=1.14 to 10.92) were found to be strongly associated with T2D. Remarkably, the combined genotype GG-2548/AA223 and AA-2548/AA223 tended to be a protective factor against T2D (OR=0.28, CI=0.08 to 0.47 and OR=0.26, CI=0.08 to 0.81). Conclusions.LEP and LEPR polymorphisms are associated with T2D in this population. Notably, the combined genotypes GG-2548/AA223 and AA-2548/AA223 are factors associated with a reduced risk of T2D, while the GA-2548/GG223 and AA-2548/GG223 genotypes combined conform a risk factor for T2D. Our findings warrant future cohort studies.

Keywords: type 2 diabetes, polymorphisms, leptin, leptin receptor, risk factor
RESUMEN

Introducción. El papel de la leptina en humanos es un factor importante en la fisiopatología de la obesidad y la diabetes mellitus tipo 2 (DT2), así como sus comorbilidades relacionadas. El objetivo de este estudio fue determinar la influencia conjunta de los polimorfismos rs7799039 (-2548G>A) LEP y rs1137101 (223Gln>Arg) LEPR, así como parámetros bioquímicos y somatométricos, como factores de riesgo para DT2. Material y métodos. Estudio transversal de casos y controles, con un grupo control y un grupo de casos. Ambos grupos fueron pareados por edad, sexo e índice de masa corporal. Los genes se amplificaron mediante PCR convencional y se genotipificaron mediante análisis de polimorfismo de longitud de fragmentos de restricción (RFLP). Los polimorfismos se validaron mediante PCR en tiempo real y sondas TaqMan® por triplicado. El tamaño de la muestra se determinó mediante una ecuación para casos y controles. El ADN genómico se aisló de leucocitos de muestras de sangre periférica mediante el método DTAB/CTAB y la amplificación de genes se realizó mediante PCR convencional. La presencia de los polimorfismos se validó mediante PCR en tiempo real y sondas TaqMan® de Applied Biosystems® C_3001671_10 para leptina y C_8722581_10 para polimorfismos del receptor de leptina. Resultados. El polimorfismo LEP rs7799039 en los genotipos G/A y A/A se asoció con T2D [OR=2.23, C =1.13 a 4.37 y OR=2.62, IC=1.27 a 5.40, respectivamente]. Además, el polimorfismo rs1137101 en los genotipos A/G y G/G se asoció con T2D (OR=0.35, IC=0.17 a 0.72 y OR=4.7, IC=2.29 a 9.62, respectivamente). Analizados juntos, se encontró que los genotipos combinados GA-2548/GG223 (OR=7.61, CI=2.02 a 28.67) y AA-2548/GG223 (OR=3.53, CI=1.14 a 10.92) estaban fuertemente asociados con la diabetes tipo 2. Sorprendentemente, el genotipo combinado GG-2548/AA223 y AA-2548/AA223 tendió a ser un factor de protección contra T2D (OR = 0.28 IC = 0.08 a 0.47 y OR = 0.26, IC = 0.08 a 0.81). Conclusiones. Los polimorfismos LEP y LEPR están asociados a DT2 en esta población. En particular, los genotipos combinados GG-2548/AA223 y AA-2548/AA223 son factores asociados con un riesgo reducido de DT2, mientras que los genotipos GA-2548/GG223 y AA-2548/GG223 combinados conforman un factor de riesgo para DT2. Nuestros hallazgos justifican futuros estudios de cohortes.

Palabras clave: diabetes tipo 2, polimorfismos, leptina, receptor de leptina, factor de riesgo

INTRODUCTION

Type 2 diabetes (T2D) is a public health problem worldwide due to its high prevalence and morbidity.1,2 Among the environmental risk factors associated with the development of T2D are those related to high carbohydrate consumption, sedentary lifestyle, and obesity.2-5

Leptin and its receptor have been associated with an increased risk of developing hypertension, preeclampsia, polycystic ovary syndrome, mammary cancer, and nonalcoholic fatty liver disease.6-12 Polymorphisms in the LEP and LEPR genes, such as rs7799039 (-2548G>A) and rs1137101 (223Gln>Arg), respectively, are associated with the development of obesity in different populations.9,13-16

LEP gene encodes the role of leptin in humans, and it is an important factor in the obesity and T2D pathophysiology, as well as in their related comorbidities.6,7,14,16-21 The half-life of endogenous leptin in serum is approximately 25 minutes, while that of exogenous leptin is 90 minutes.

Kidneys remove leptin from the body20, which increases exponentially with increasing fat mass. Mutations in the LEP gene cause leptin/receptor interaction abnormalities resulting in the disruption of energy balance, excessive fatty tissue accumulation, and impairment of glucose metabolism.22-24 Among the most frequent and noteworthy polymorphisms of LEP and LEPR genes in the Mexican population are rs7799039 in the LEP gene, which is associated with overweight and obesity.14,25-27

Objective

The objective was to determine the influence together of rs7799039 (-2548G>A) LEP and rs1137101 (223Gln>Arg) LEPR polymorphisms, as well as biochemical and somatometric parameters, as risk factors for T2D. Therefore, it is relevant to carry out association studies of the rs7799039 and rs1137101 polymorphisms in the LEP and LEPR genes separately and simultaneously, and to perform intentional searches for combined genotypes to estimate the risks for T2D.

MATERIAL AND METHODS

Study Design

A cross-sectional case-control study of 206 participants was carried out: a control group (n=103) and a case group with T2D (n=103). Both groups were matched by age, sex, and body mass index (BMI). The genes were amplified by conventional PCR and were genotyped by restriction fragment length polymorphism (RFLP) analysis.

To ensure reliability, the polymorphisms were validated using real-time PCR and TaqMan® probes in triplicate. The population sample was obtained by public invitation for candidates to attend the Primary Medical Care Offices at ISSSTE Durango and Unidad de Investigación Biomédica from Instituto Mexicano del Seguro Social.

The Research Ethics Committee from Hospital General de Durango and “Santiago Ramón y Cajal” Hospital ISSSTE Durango, Mexico, approved the study. All subjects signed an informed consent form to participate in the study.

Data Collection

Based on the T2D prevalence of T2D in Mexico (16%, ENSANUT 202028), we calculated the sample size29 using an equation for cases and controls, considering a power of 80%, a confidence level of 95%, and an odds ratio (OR) estimated of 3.

A total of 56 cases and 56 controls were obtained with a calculation of 10% more to avoid the expected loss, resulting in 66 participants in each group. The study was conducted with volunteers from the mestizo population.

Subjects with T2D30 (International Diabetes Federation, 2014) (n=103) were allocated to the case group and compared to healthy (n=103) subjects in the control group. BMI, waist circumference (C), sex, and age were used as matching criteria. Familial history of diabetes (FHD) was noted.

The inclusion criteria were:

  • men and women over 18 years of age,
  • non-pregnant women,
  • no history of blood transfusion in the last two months,
  • patients with a history of polycystic ovarian syndrome,
  • cardiovascular or kidney disease,
  • and stroke were excluded from the study.

Clinical Assessment

Detailed physical examinations and clinical histories were conducted. Weight and height were assessed with an automatic electric stadiometer (TANITA WB-3000); waist circumference was assessed using a certified measuring tape. Blood pressure was determined using a semiautomatic manometer (Omron). The somatometric variables were weight, height, BMI, waist circumference, hip circumference, systolic blood pressure, diastolic blood pressure, glucose, total cholesterol, triglycerides, and HDL cholesterol; the sociodemographic variables, age, sex, and family history of diabetes.

Other Laboratory Assessments

Blood samples were drawn from the antecubital vein at Instituto Mexicano del Seguro Social and Hospital General de Durango, Mexico. Molecular analysis was performed at the Pharmacogenomics and Molecular Biomedicine Laboratory of CIIDIR-IPN Durango. Blood samples were obtained under fasting conditions (8-10 hours), and the biochemical parameters analyzed included fasting glucose and lipid profiles.

Molecular Studies

Genomic DNA was isolated from leukocytes of peripheral blood samples using the DTAB/CTAB method.31 After quantification control of DNA samples (Nanodrop 2000), gene amplification was performed by conventional PCR using the following primers for the LEP gene, forward 5'-TTTCCTGTAATTTTCCCGTGAG-3' and reverse 5'-AAAGCAAAGACAGGCATAAAAA-3', which generated a 242-bp fragment, under the following reaction conditions, initial denaturing at 95 °C x 5 minutes followed by 30 amplification cycles of 94 °C, 58.5 °C, and 72 °C for 1 minute and a final extension at 72 °C for 5 minutes.

For the LEPR gene, the following primers were used: forward 5'-AAACTCAACGACACTCTCCT-3' and reverse 5'-TGAACTGACATTAGAGGTGAC-3', yielding an 80-bp fragment using the following reaction conditions: 94 °C x 2 minutes of initial denaturing, followed by 32 cycles of 94 °C for 1 min, 50 °C for 45 s, and 72 °C for 30 s, with a final extension of 7 min.

After PCR analysis, all products were stored at 4 °C. The enzyme Cof I from Promega® (Madison, WI, USA) was used to detect the rs7799039 polymorphism in the LEP gene, as it generates a restriction pattern of 61 bp and 181 bp when the cleavage site is present.

The enzyme Msp I from Sigma® (Saint Louis, MO. USA) was used to detect the rs1137101 polymorphism of the LEPR gene, as it generates a restriction pattern of 22-bp and 58-bp fragments. RFLPs were then visualized through 2% agarose electrophoresis stained with ethidium bromide.

The presence of the polymorphisms was validated using real-time PCR and Applied Biosystems® TaqMan® probes C_3001671_10 for leptin and C_8722581_10 for leptin receptor polymorphisms. Every sample was analyzed in triplicate to ensure reliability (Laboratory Protocol dx.doi.org/10.17504/protocols.io.bfuujnww).32

We randomly chose 20% of the samples for quality assurance purposes to repeat the analysis. When analyses were not coincident twice, we excluded the samples.

Statistical Analysis

We performed descriptive statistics for sociodemographic variables, measures of central tendency, and dispersion (means, medians, standard deviation, and percentiles).

We carried out comparison tests between groups using Student's t test (means and standard deviation) or Mann-Whitney U testt (median and percentiles). Allelic and genotypic frequencies were calculated.

The associations between polymorphisms rs7799039 of the LEP gene and rs1137101 of the LEPR gene with T2D were estimated by calculating of the OR.33

The log-linear logit analysis was carried out using the Statistica AX Release 7.0 software package.33,34

Multivariate logistic regression analysis evaluated the association between polymorphisms and T2D through inheritance models. Statistical significance was established at p < 0.05 with a 95% confidence interval (CI). PASW Statistics 18.0.0 software35 was used for statistical analysis.

RESULTS

The results of the data analysis showed no significant differences by sex, age, and BMI, given that these were matched criteria; no differences were found for waist circumference, total cholesterol, HDL-cholesterol, and LDL-cholesterol between the case participants and the control group.

Subjects in the case group showed higher diastolic blood pressure (DBP), systolic blood pressure (SBP), serum glucose, triglyceride, and VLDL-cholesterol levels than those in the control group, besides a family history of the disease (Table 1).

Table 1. Population characteristics
Cases n=103Control n=103P
Family history of diabetes n (%)78 (75.7)60 (58.2)<0.01

Age, years52.0 ± 11.049.9 ± 9.40.07

Body mass index, kg/m229.9 ± 5.728.4 ± 4.30.04

Waist circumference, cm96.7 ± 26.693.7 ± 17.60.07

Systolic blood pressure, mmHg116.3 ± 6.6109.9 ± 9.8<0.001

Diastolic blood pressure, mmHg75.4 ± 7.471.6 ± 7.9<0.001

Glucose, mg/dL159.6 ± 52.986.0 ± 9.7<0.001

Cholesterol, mg/dL210.5 ± 44.5203.1 ± 31.20.16

Triglycerides, mg/dL239.5(146-292)*175.2 (111-229.5)*<0.001**

HDL - cholesterol, mg/dL45.3 ± 13.142.6 ± 9.80.09

LDL - cholesterol, mg/dL117.3 ± 45.6125.5 ± 37.10.17

VLDL - cholesterol, mg/dL47.9 ± 31.435.0 ± 18.3<0.001

P < 0.05 is considered as significant. Means ± standard deviation compared using Student’s t-test; *median and interquartile range (25 and 75); ** data compared using Mann-Whitney’s U test|

The allele and genotype frequencies of the polymorphisms rs7799039 in the LEP gene and rs1137101 in the LEPR gene are shown in Table 2. The heterozygous (G/A) state was the most frequent genotype in the case group, whereas the wild-type homozygous (G/G) state was the most frequent in the control group. Both polymorphisms are in Hardy-Weinberg equilibrium. Furthermore, regarding the rs1137101 polymorphism in the LEPR gene, the homozygous mutant (G/G) state was the most common genotype in the case group. In contrast, the wild homozygous state (A/A) was the most common genotype in the control group (Table 2).

Table 2. Genotypic and allelic frequency in the study groups
Genotypic frequency n (%)Allelic frequency n (%)Hardy-Weinberg equilibrium
rs7799039 (-2548G>A)
G/AG/GA/AGA

Case44 (42.71)23 (22.33)36 (34.95)90 (43.68)116 (56.31)X2 = 31.2

Control36 (34.95)42 (40.77)25 (24.27)120 (58.25)92 (44.66)p > 0.05
rs1137101 (223Gln>Arg (A>G))
A/GA/AG/GAG

Case31 (30.09)24 (23.30)48 (46.60)79 (38.34)127 (61.65)X2 = 23.1

Control36 (34.95)47 (45.63)20 (19.41)130 (63.10)76 (36.89)p > 0.05

As shown in Table 3, inheritance models can be analyzed to evaluate the effect of the variation in a gene on an event or when the genotype of risk of disease in a specific population is unknown. In the codominant model, the analysis for the rs7799039 polymorphism showed that the heterozygous genotype (HT) had a statistically significant association with the risk of T2D (OR = 2.23, CI = 1.13 to 4.37, p = 0.01).In addition, the mutated genotype (HM) presented a statistically significant association with the risk of developing T2D (OR = 2.62, CI = 1.27 to 5.40, p = 0.008), and each genotype provided a different and nonadditive risk of T2D.

Table 3. Association of rs7799039 (2548G>A) of the LEP gene and rs1137101(223Gln>Arg) of the LEPR gene, in relation to the risk of developing T2D
Cases
(n=103)
Control
(n=103)
ORICP
rs7799039
Codominant Model
GG23421

GA44332.2319
1.1776
1.1390 - 4.3734
0.5621 - 2.4476
0.01
0.76

AA36252.6296
1.1782
1.2795 - 5.4040
0.6004 - 2.3119
0.008
0.63
Dominant Model
GG23420.41760.2274 - 0.76680.004

GA + AA8061
Additive Model
GA + AA59670.72050.4105 - 1.26460.25

GA4436

G901200.55600.3763 - 0.82170.003

A11686

A116861.79881.2171 - 2.65760.002

G90120
Recessive Model
AA36251.67640.9147 - 3.07230.09

GG + GA6778

rs1137101
Codominant Model
AA24471
1.6863
0.8481 - 3.35320.136

AG31360.35880.1766 - 0.72910.004

GG48204.7000
2.7869
2.2949 - 9.6258
1.2985 - 6.0476
0.001
0.007
Dominant Model
AA24470.36200.1988 - 0.65900.009

GA + GG7956
Dominant Model
AA + AG55830.27710.1481 - 0.51480.001

GG4820

A791300.36370.2441 - 0.54180.001

G12776

G127762.74981.8456 - 4.09700.001

A79130
Recessive Model
GG48203.62181.9423 - 6.75360.001

AA + AG5583

P< 0.05 is considered as significant; OR: odds ratio; CI: 95 % confidence interval

It is interesting to note that the major allele (G) by itself showed a statistically significant protective association with T2D (OR = 0.55, CI = 0.37 to 0.82, p = 0.003), while the minor allele (A) shows a statistically significant association with the risk of T2D (OR = 1.79, CI = 1.21 to 2.65, p = 0.002).

The additive effect of the major allele (G) can be observed since, in the dominant model, the WT (GG) genotype had a statistically significant association with T2D protection (OR = 0.41, CI = 0.22 to 0.76, p = 0.004).

For the rs1137101 polymorphism, the codominant model showed that the HT (GA) genotype had a statistically significant association with T2D protection (OR = 0.35, IC = 0.17 to 0.72, p = 0.004), and the HM genotype (GG) presented a statistically significant risk association with T2D (OR = 4.70, CI = 2.29 to 9.62, p = 0.001).

The major allele (A) by itself showed a statistically significant protective association with T2D (OR = 0.36, IC = 0.24 to 0.54, p = 0.001), while the minor allele (G) showed a statistically significant risk association (OR = 2.74, CI = 1.84 to 4.09, p = 0.001) with the development of T2D.

Therefore, we observed an additive effect of the alleles; a single copy of the dominant allele "A" will be enough to grant protection to T2D, while for the recessive allele "G", two copies will be necessary to grant a risk effect to T2D.

The dominant model shows us that the WT (AA) genotype had a statistically significant association with T2D protection (OR = 0.36, IC = 0.19 to 0.65, p = 0.009), while the recessive model showed that there was an association with the risk of statistically significant T2D in the HM (GG) genotype (OR = 3.62, IC = 1.94 to 6.75, p = 0.001).

As shown in Table 4, the GA-2548/GG223 and AA-2548/GG223 genotypes (heterozygous/mutated and mutated/mutated alleles, respectively) were significantly associated with the presence of T2D (OR = 7.61, CI = 2.02 to 28.67, p = 0.002 and OR = 3.53, CI = 1.14 to 10.92, p = 0.028, respectively).

Table 4. Combined risk association analysis to T2D
Genotypes LEP/LEPRCasesControl# cases# controlsORCIP
GG-2548/AA22341819240.28070.0813 to 0.96940.0445

GG-2548/AG22381315291.18970.4044 to 3.50000.7523

GG-2548/GG223111112312.58330.8871 to 7.52310.0818

GA-2548/AA223131731190.46870.1867 to 1.17680.1067

GA-2548/AG223131631200.52420.2083 to 1.31910.1701

GA-2548/GG22318326337.61542.0223 to 28.67750.0027

AA-2548/AA22371229130.26150.0837 to 0.81670.0210

AA-2548/AG22310726180.98900.3171 to 3.08500.9848

AA-2548/GG22319617193.53921.1465 to 10.92580.0280

P< 0.05 is considered as significant; OR: odds ratio; CI: 95 % confidence interval

Furthermore, the OR calculation of the logit model showed that the presence of the GG-2548/AA223 and AA-2548/AA223 genotypes showed a trend as a protective factor (OR = 0.28, CI = 0.08 to 0.47, p = 0.044 and OR = 0.26, CI = 0.08 to 0.81, p = 0.021) by T2D. The remaining genotypes had nonsignificant relationships.

We observed a neuronal network multilayer, where we later found 96.7% network training, with a sensitivity and specificity of 0.996 for correct diagnosis with this test.

The normalized importance of variables present in our populations with T2D showed glucose as the main factor in the T2D diagnosis. That enables the association of the variables VLDL, SBP, BMI, HDL-cholesterol, age, waist circumference, triglycerides, and total cholesterol, to lead to the last genetic factor.

When detecting the genetic polymorphisms of leptin and leptin receptor, it is essential to determine the correlations between genotype and the risk or importance of the development of T2D.

DISCUSSION

T2D is a multifactorial disease in a large portion of the Mexican population. Obesity is a chronic metabolic disease affecting adults and children and the leading risk factor for many non-communicable diseases, in particular type 2 diabetes.36,37

Approximately 24.27% (25) of people in this study had T2D and normal weight, and their BMI was below 25. Within this group, the lowest patient BMI was 21.16, the highest reaching 24.9, with an average of 23.36.

Regarding genotypes, 4% (4) of the subjects in this group had a complete wild-type genotype (i.e., both polymorphisms showed the wild-type state).

The percentages of patients with 1, 2, 3, or 4 mutated alleles were 20% (21), 32% (33), 28% (29), and 16% (16), respectively. Specifically, 25 (24%) individuals in this group had homozygous mutations for polymorphism rs7799039 in the LEP gene and 21 (20%) with mutations homozygous for the rs1137101 polymorphism in the LEPR gene.

Most association studies carried out in various populations worldwide have included the analysis of a single polymorphism. Still, none have simultaneously analyzed both polymorphisms in the Mexican population and determined the influence of combined genotypes in the association with T2D.

A risk factor for T2D has been established for the heterozygous/mutated and mutated/mutated LEP/LEPR genotypes using the logit model and inheritance models. In this study, we found that 96% of subjects with T2D and normal weight (26 cases) had at lea The major or wild-type allele and showed a protective effect on the genotypes GG-2548/AA223 and AA-2548/AA223.

The statistically significant differences between the study groups regarding clinical analyses (such as DBP, SBP, glucose, triglycerides, VLDL-cholesterol, and HFD [high-fat diet]) are noteworthy and evidenced. The loss of metabolic control is also present in our study population, and the a strong association of a positive family history of T2D with disease development.

In this study, T2D risk increased up to 2.23 times (p = 0.02) in patients with only one copy of the mutated allele for the rs7799039 polymorphism. In contrast, the presence of two mutated copies (homozygous mutant, A/A) increased the risk to 2.62 times (p = 0.01). That is likely to a decrease in total leptin production since this polymorphism is in the gene promoter, affecting its transcription rate.9,11,16,20

Polymorphism rs7799039 has also been associated with obesity and extreme obesity in various Brazilian, French, and Japanese populations. However, contrasting results have been found in another region in Brazil, where no association with obesity was found9,14,38,39, suggesting that the allele distribution vs. disease has regional variations. These findings emphasize the need to identify other genetic changes that affect the leptin/receptor interaction or environmental conditions, such as high-stress levels, to trigger pathology.9,38,40 This study showed the association between obesity and T2D in a group of Mexican populations.

A study of polymorphism rs1137101 in healthy Caucasians in Los Ángeles, CA23 described an independent association of this polymorphism with an increase in the risk of developing T2D; such an association was absent in our population.

In contrast, in the present study, the presence of the homozygous mutant genotype (G/G) (polymorphism rs1137101) was associated with a 4.7-fold increase in the risk of developing T2D, while the heterozygous condition increased the risk only 2.78-fold.

Clearly, these findings suggest that in the studied population, the presence of a single mutated copy of this polymorphism increases the risk for T2D. Nevertheless, the variability in the findings of previous studies suggests the presence of a covariant risk factor that could be either genetic or environmental.

The results of the analysis of the combined genotypes suggest a significant interaction between polymorphisms rs7799039 and rs1137101 since the presence of the combined genotypes GA-2548/GG223 and AA-2548/GG223 (rs7799039 and rs1137101, respectively) increases the likelihood that an individual will develop T2D (Figure 1). This analysis even showed that the GG-2548/AA223 and AA-2548/AA223 genotypes (wild type/wild type and homozygous mutant/wild type) tended to decrease T2D risk, as shown in Table 4.

The main strength of this study lies in the combined genotype association analysis of polymorphisms rs7799039 and rs1137101 with T2D. To date, there is scarce information available on the concurrent association of polymorphisms rs7799039 and rs1137101 with the risk for T2D phenotypes.

Fletcher B et al. (2002)40 mention the importance of focusing on current interventions to prevent or delay T2D. Such interventions should be aimed at modifying environmental risk factors and reducing obesity.

Laakso M. (2019)41 mentionsin a meta-analysis how environmental factors trigger genetic factors involved in the development of T2D. In turn, this disease generates other entities that worsen the state of those afflicted by this disease.

In our work, we used multilayer neural network analysis to observe the order of importance of the variables in our study group. It is interesting how factors such as VLDL-cholesterol (40.7%) and SBP (30.6%) are among the study group's variables with the most significant importance.

Beddhu et al. (2018)42 refer to the importance of SBP management in patients with T2D. Moreover, Pulgaron et al. (2014)43, mention the influence of obesity, high BMI, and metabolic syndrome as risk factors for developing T2D. In our study, we observed through the analysis of neural networks how BMI has an important influence on the development of T2D (28.1).

Boumaiza et al. (2012)44 state the important influence of the LEP and LEPR genes in the development of obesity and metabolic syndrome in a Tunes population.

In our study (Figure 1), we found that the WT genotype of the LEP gene presents a strong association with the population in both the cases and controls studied (relative inertia = 0.07), whose average BMI is 29.15. These findings likely indicate that our population could have, as a consequence of this genetic variation, a tendency to increase BMI, which could, together with other factors, increase the risk of developing T2D.

In our study, we found that the HM genotype of the LEPR gene has the most significant association with T2D of all the genotypes of the two polymorphisms (Figure 1) (relative inertia = 0.08).In addition, this polymorphism has a risk associated with the development of T2D seen through inheritance models (OR = 4.70, p = 0.001).

This study stresses the association of the polymorphisms rs7799039 in the leptin gene and rs1137101 in the leptin receptor gene with T2D in a Mexican population.

The association of combined genotypes GA-2548/GG223 and AA-2548/GG223 with T2D was a major finding of this study. The combined genotypes GG-2548/AA223 and AA-2548/AA223 showed a tendency to be protective factors.

a) Association between variables and T2D including cases and controls. b) Association between genotype and T2D including only cases
CONCLUSIONS

LEP and LEPR polymorphisms are associated with T2D in this population. Notably, the combined genotypes GG-2548/AA223 and AA-2548/AA223 are factors associated with a reduced risk of T2D, while the GA-2548/GG223 and AA-2548/GG223 genotypes combined conform a risk factor for T2D. Our findings warrant future cohort studies.

ACKNOWLEDGMENTS

We thank Ilse Mijares Velazquez for the professional revision of this manuscript and the and proofreading of the English version.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

REFERENCES

1.World Health Organization. Global report on diabetes. 2016;6-88.Available from URL: https://www.who.int/publications/i/item/9789241565257
2.American Diabetes Association. Classification and diagnosis of diabetes: Standards of medical care in diabetes-2018. Diabetes Care. 2018,41(Suppl 1):S13-S27.
3.Kolb H, Martin S. Environmental/lifestyle factors in the pathogenesis and prevention of type 2 diabetes. BMC Med. 2017;15(1):131. DOI: 10.1186/s12916-017-0901-x
4.Dendup T, Feng X, Clingan S, Astell-Burt T. Environmental risk factor for developing type 2 diabetes mellitus: A systematic review. Int J Environ Res Public Health. 2018;15(1):78.
5.Raynor HA, Champagne CM. Position of the Academy of Nutrition and Dietetics: Interventions for the treatment of overweight and obesity in adults. J Acad Nutr Diet. 2016;116(1):129-147.
6.de Faria AP, Ritter AMV, Sabbatini AR, Modolo R, Moreno H. Effects of leptin and leptin receptor SNPs on clinical- and metabolic-related traits in apparent treatment-resistant hypertension. Blood Pressure. 2017;26(2):74-80
7.Wiedemann A, Vocke F, Fitzgerald JS, Jeschke U, Markert U, Lohse P, Toth B. Leptin gene (TTTC)(n) microsatellite polymorphism, as well as leptin receptor R223Q and PPARgamma2 P12A substitutions are not associated with hypertensive disorders in pregnancy. Am J Reprod Immunol. 2010;63(4):310-7
8.Liang J, Lan J, Li M, Wang F. Associations of leptin receptor and peroxisome proliferator-activated receptor gamma polymorphisms with polycystic ovary syndrome: A meta-analysis. Ann Nutr Metab. 2019;75(1):1-8.
9.Liu CR, Li Q, Hou C, Li H, Shuai P, Zhao M, et al. Changes in body mass index, leptin, and leptin receptor polymorphisms and breast cancer risk. DNA Cell Biol. 2018;37(3):182-188
10.Méndez-Hernández A, Gallegos-Arreola MP, Moreno-Macías H, Espinoza-Fematt J, Pérez-Morales R. LEP re7799039, LEPR rs1137101, and ADIPOQ rs2241766 and 150299 polymorphisms are associated with obesity and chemotherapy response in Mexican women with breast cancer. Clin Breast Cancer. 2017;17(6):453-62.
11.Pan X, Zheng M, Zou T, Liu W, Gu X, Zhang X, et al. The LEPR K109R and Q223R might contribute to the risk of NAFLD: A meta-analysis. Curr Mol Med. 2018;18(2):91-99
12.An BQ, Lu LL, Yuan C, Xin YN, Xuan SY. Leptin receptor gene polymorphisms and the risk of non-alcoholic fatty liver disease and coronary atherosclerosis in the Chinese Han population. Hepat Mon. 2016;16(4):e35055
13.Zhang L, Qin Y, Liang D, Li L, Liang Y, Chen L, et al. Association of polymorphisms in LEPR with type 2 diabetes and related metabolic traits in a Chinese population. Lipids Health Dis. 2018;17(1):2.
14.Li YY, Wang H, Yang XX, Wu JJ, Geng HY, Kim HJ, et al. LEPR gene Gln223Arg polymorphism and type 2 diabetes mellitus: a meta-analysis of 3,367 subjets. Oncotarget. 2017;8(37):61927-61934.
15.Arlier S. Endothelial cell leptin receptors, leptin, and interleukin-8 in the pathogenesis of preeclampsia: An in-vitro study. Turk J Obstet Gynecol. 2017;14(4):220-27.
16.Wang TN, Huang MC, Chang WT, Ko AM, Tsai EM, Liu CS, et al. G-2548A polymorphism of the leptin gene is correlated with extreme obesity in Taiwanese aborigines. Obesity (Silver Spring). 2006;14(2):183-7.
17.Dagdan B, Chuluun-Erdene A, Sengeragchaa O, Malchinkhuu M, Janlav M. Leptin gene G2548A polymorphism among Mongolians with metabolic syndrome. Med Sci (Basel). 2019;7(1):3.
18.Lakka TA, Rankinen T, Weisnagel SJ, Chagnon YC, Lakka HM, Ukkola O, et al. Leptin and leptin receptor gene polymorphisms and changes in glucose homeostasis in response to regular exercise in nondiabetic individuals: The HERITAGE family study. Diabetes. 2004;53(6),1603-8. DOI: 10.2337/diabetes.53.6.1603
19.D’souza AM, Neumann UH, Glavas MM, Kieffer TJ. The glucoregulatory actions of leptin. Mol Metab. 2017;6(9):1052-1065.
20.Acosta Hernández ME, Ramos Morales FR, Escobar Henríquez JBH, López Muñóz JJD, Escobar Castillo P, García Rodríguez RV, et al. Mecanismos bioquímicos de la leptina implicados en el desarrollo de la obesidad. Rev Med Uiv Veracruzana. 2015;2(15):103-113.
21.Simeoni U, Osmond C, Garay R, Buffat C, Boubred F, Chagnaud C, et al. Leptin and insulin in young adulthood are associated with weight in infancy. J Endocrinol. 2020;244(2):249-59.
22.Tabassum R, Mahendran Y, Dwivedi OP, Chauhan G, Ghosh S, Marwaha RK. et al. Common variants of IL6, LEPR, and PBEF1 are associated with obesity in Indian children. Diabetes. 2012;61(3):626-31.
23.Martínez-Martínez MD, Mendieta-Zerón H, Celis L, Layton-Tovar CF, Torres-García R, Gutiérrez-Pliego LE, et al. Correlation of the homeostasis model assessment index and adiponectin, leptin and insulin levels top body mass index-associated gene polymorphisms in adolescents. Sultan Qaboos Univ Med J. 2018;18(3): e291-e298.
24.Kroll C, Mastroeni SSBS, Veugelers PJ, Mastroeni M.F. Associations of ADIPOQ and LEP gene variants with energy intake: A systematic review. Nutrients. 2019;11(4): 750.
25.Samodien E, Pheiffer C, Erasmus M, Mabasa L, Louw J, Johnson R. Diet-induced DNA methylation within the hypothalamic arcuate nucleus and dysregulated leptin and insulin signaling in the pathophysiology of obesity. Food Sci Nutr. 2019;7(10):3131-45.
26.Romero-Martínez M, Shamah Levy T, Cuevas Nasu L, Méndez Gómez-Humarán I, Gaona-Pineda, Gómez-Acosta LM, et al. Diseño metodológico de la Encuesta Nacional de Salud y Nutrición de Medio Camino 2016. Salud Pública Mex. 2017;59(3):299-305. Disponible en URL: https://www.scielo.org.mx/pdf/spm/v59n3/0036-3634-spm-59-03-00299.pdf
27.Cui H, López M, Rahmouni K. The cellular and molecular bases of leptin and ghrelin resistance in obesity. Nat Rev Endocrinol. 2017;13(6):338-351.
28.ENSANUT. Encuestra Nacional de Salud y Nutrición 2020 sobre Covid-19. Resultados Nacionales. Instituto Nacional de Salud Pública. 2021. Disponible en URL: https://ensanut.insp.mx/encuestas/ensanutcontinua2020/doctos/informes/ensanutCovid19ResultadosNacionales.pdf
29.Mejía Aranguré JM, Fajardo Gutiérrez A, Gómez Delgado A, Cuevas Urióstegui ML, Hernández Hernández DM, Garduño Espinoza J, et al. El tamaño de muestra: un enfoque práctico en la investigación clínica pediátrica. Biol Med Hosp Infant Mex. 1995;52(6): 381-91.
30.International Diabetes Federation Guideline Development Group. Global guidelines for type 2 diabetes. Diabetes Res Clin Pr. 2014;104(1):1-52.
31.Gustincich S, Manfioletti G, Del Sal G, Schneider C, Carninci P. A fast method for high-quality genomic DNA extraction from whole human blood. Biotechniques. 1991;11(3):298-300.
32.Martínez M. Martinez Protocol. Laboratory Protocol. Available from URL:https://www.protocols.io/view/martinez-protocol-bfuujnww
33.Agresti A. An introduction to categorical data analysis. 2nd ed. Wiley Series in Probability and Statistics. New York: Wiley-Interscience; 2002.394 p. Available from URL: https://mregresion.files.wordpress.com/2012/08/agresti-introduction-to-categorical-data.pdf
34.Haberman SJ. Maximum likelihood estimates in exponential response models. Ann Statist. 1977;5(5),815-41.
35.PASW Statistics 18.0.0. Available from URL: https://www.malavida.com/es/soft/pasw/#gref
36.Leitner DR, Frühbeck G, Yumuk V, Schindler K, Micic D, Woodward E, et al. Obesity and type 2 diabetes: Two diseases with a need for combined treatment strategies - EASO can lead the way. Obesity Facts. 2017;10(5):483-492.
37.Wasim M, Awan FR, Najam SS, Khan AR, Khan HN. Role of leptin deficiency, inefficiency, and leptin receptor in obesity. Biochem Genet. 2016;54(5): 565-72
38.Zhang L, Yuan LH, Xiao Y, Lu MY, Zhang LJ, Wang Y. Association of leptin gene –2548 G/A polymorphism with obesity: A meta-analysis. Ann Nutr Metab. 2014;64(2):127-36
39.Wu J, Zhuo Q, Chen X, Tian Y, Piao J, Yang X. Association of leptin receptor gene polymorphism with metabolic syndrome in older Han adults from major cities in China. Wei Sheng Yan Jiu. 2016;45(3):376-82.
40.Fletcher B, Gulanick M, Lamendola C. Risk factors for type 2 diabetes mellitus. J Cardiovasc Nurs. 2002;16(2):17-23
41.Laakso M. Biomarkers for type 2 diabetes. Mol Metab. 2019;27S(Suppl): S139-S146.
42.Beddhu S, Chertow GM, Greene T, Whelton PK, Ambrosius WT, Cheung AK, Cutler J, et al. Effects of intensive systolic blood pressure lowering on cardiovascular events and mortality in patients with type 2 diabetes mellitus on standard glycemic control and in those without diabetes mellitus: Reconciling results from ACCORD BP and SPRINT. J Am Heart Assoc. 2018;7(18): e009326.
43.Pulgaron ER, Delamater AM. Obesity and type 2 diabetes in children: Epidemiology and treatment. Curr Diab Rep. 2014;14(8):508.
44.Boumaiza I, Omezzine A, Rejeb J, Rebhi L, Ouedrani A, Ben Rejeb N, et al. Relationship between leptin G2548A and leptin receptor Q223R gene polymorphisms and obesity and metabolic syndrome risk in Tunisian volunteers. Genetic Test Mol Biomark. 2012;16(7), 726–733. doi.org/10.1089/gtmb.2011.0324


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Latin American Journal of Clinical Sciences and Medical Technology,
Año 1, No. 1, octubre, 2019 es una publicación contínua editada por Vesalio S.C.; https://www.lajclinsci.com/    Editor responsable: Gilberto Castañeda Hernández.    Reserva de Derechos al Uso Exclusivo: 04-2019-062013242000-203; ISSN: 2683-2291; ambos otorgados por el Instituto Nacional del Derecho de Autor.    Responsable de la última actualización de este número, Web Master Hunahpú Velázquez Martínez,
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All Rights Reserved® 2019

Latin American Journal of Clinical Sciences and Medical Technology,
Año 1, No. 1, octubre, 2019 es una publicación contínua editada por Vesalio S.C.; https://www.lajclinsci.com/    Editor responsable: Gilberto Castañeda Hernández.    Reserva de Derechos al Uso Exclusivo: 04-2019-062013242000-203; ISSN: 2683-2291; ambos otorgados por el Instituto Nacional del Derecho de Autor.    Responsable de la última actualización de este número, Web Master Hunahpú Velázquez Martínez,
Calle San Luis Potosí #182-1, Col. Roma, Alcaldía Cuauhtémoc, C.P. 06700, Ciudad de México; teléfono: 55 64 40 41    Fecha de última modificación, 30 de marzo de 2020.