Open Access

Leptin receptor gene polymorphisms and morbid obesity in Mexican patients

  • Martin Edgardo Rojano-Rodriguez1,
  • Jose Luis Beristain-Hernandez1,
  • Beatriz Zavaleta-Villa2,
  • Pablo Maravilla3,
  • Mirza Romero-Valdovinos2 and
  • Angelica Olivo-Diaz2Email author
Hereditas2016153:2

DOI: 10.1186/s41065-016-0006-0

Received: 7 September 2015

Accepted: 24 January 2016

Published: 22 February 2016

Abstract

Background

Human obesity is due to a complex interaction among environmental, behavioral, developmental and genetic factors, including the interaction of leptin (LEP) and leptin receptor (LEPR). Several LEPR mutations and polymorphisms have been described in patients with early onset severe obesity and hyperphagic eating behavior; however, some contradictory findings have also been reported. In the present study we explored the association of six LEPR gene polymorphisms in patients with morbid obesity.

Findings

Twenty eight patients with morbid obesity and 56 non-obese Mexican Mestizo individuals were included. Typing of rs1137100, rs1137101, rs1805134, Ser492Thr, rs1805094 and rs1805096 LEPR polymorphisms was performed by PCR and allele specific hybridization. The LEPR Ser492Thr polymorphism was monomorphic with the presence of only the Ser492Thr-G allele. Allele C and genotype T/C for rs1805134 polymorphism were associated with susceptibility to morbid obesity (p = 0.02 and p = 0.03, respectively). No association was observed with any haplotype. Linkage disequilibrium (LD) showed that five polymorphisms (rs1137100, rs1137101, rs1805134, rs1805094 and rs1805096) were in absolute (D’ = 1) but none in perfect (r2 = 1) LD.

Conclusions

Our results suggest that rs1805134 polymorphism could be involved in the development of morbid obesity, whilst none of the alleles of the LEPR gene, rs1137100, rs1137101, rs1805094 and rs1805096 were associated as risk factors. However, more studies are necessary to confirm or reject this hypothesis.

Keywords

Genetic susceptibility LEPR Morbid obesity Single nucleotide polymorphisms

Findings

Human obesity is due to a complex interaction among environmental, behavioral, developmental and genetic factors, the latter contributing to 40–70 % of the obese phenotype [13]. Leptin (LEP) is a hormone specifically produced by adipocytes, and its serum concentration is proportional to body fat mass which, in turn, has its amount regulated by the hypothalamic effects of LEP. Intravenous administration of LEP reduces appetite; while its deficiency increases food intake [4]. Its action occurs through the leptin receptor (LEPR), which is encoded by the LEPR gene. LEPR is a single-transmembrane-domain receptor of the cytokine-receptor family with widespread tissue distribution and several alternatively spliced isoforms [5].

Several LEPR mutations have been described in patients with early-onset of severe obesity and hyperphagic eating behavior [6, 7]. In contrast, a protective influence of two polymorphisms (rs1137100 and rs1137101) to higher blood pressure levels in men has been identified, increasing the protection when the carriers have the arginine allele in the two single nucleotide polymorphisms (SNPs) [8]. Thereby, several SNPs have been studied, and their replication in detail across different populations, for their potential association with obesity and its complication, has been stated. However, some contradictory findings have also been reported, adding the fact that there are scarce studies in non-Caucasian populations; therefore, the lack of data on this subject emphasizes the need for studies among and across different ethnic groups [9, 10]. In this work we explored the association of several LEPR gene polymorphisms with morbid obesity compared with non-obese Mexican Mestizo adults.

Blood samples were obtained from 28 patients with morbid obesity (mean age 39.6 ± 6.6 years, mean body mass index [BMI] 42.7 ± 6.5 Kg/m2) and 56 non-fat (mean age 32.7 ± 14.4, mean BMI 21.5 ± 1.5 Kg/m2) healthy unrelated volunteers. The sex of patients and controls was 89.7 and 93.3 % females, respectively. All participants were unrelated, with no consanguinity at all, and none couple was included. In addition to obesity, two patients presented hypothyroidism, one patient presented type 2 diabetes mellitus and arterial hypertension and one patient presented fat liver. This work complies with the current health laws of Mexico, and was approved by the Ethics and Research Committees of the Hospital General “Dr. Manuel Gea Gonzalez” with the reference number 04-92-2009. All participants were informed about the objectives of the study and were included only after providing written informed consent.

DNA was obtained from 10 ml of EDTA-peripheral blood using proteinase K and phenol/chloroform extraction [11]. The LEPR polymorphisms rs1137100 (Lys109Arg), rs1137101 (Gln223Arg), rs1805134 (Ser343Ser), Ser492Thr and rs1805096 (Pro1019Pro) were detected by PCR-restriction fragment length polymorphism (RFLP) technique, using primers described by Gotoda et al. [12] and Matsuoka et al. [13]. For allele determination of rs8179183 (Lys656Asn) SNP, a dot–blot format and the chemiluminescence method was used, employing the specific probes Lys656Asn-G: 5′-CTATGAAAAAGGAGAAAAATG-3' and Lys656Asn-C: 5'-CTATGAAAAACGAGAAAAATG-3′ ddUTP-Digoxigenin labelled [14, 15].

Allele frequencies (AF) and genotype frequencies (GF) were calculated by direct counting and were compared between patients and controls of each group. Chi-square analysis with Yate’s correction, considering p ≤ 0.05 as the minimum level of significance, was performed; exact Fisher test was used when appropriate. Relative risk was calculated as an odds ratio (OR). Ninety-five percent confidence intervals (95 % CI) were obtained by using Cornfield’s approximation. Haplotypes and linkage disequilibrium (LD) blocks were determined by confidence interval method using Haploview 4.2 software [16].

Table 1 summarizes the significant associations found between alleles, genotypes and haplotypes observed in patients with morbid obesity and in controls. Polymorphism Ser492Thr was monomorphic with the only presence of Ser492Thr-G allele. Allele C and genotype T/C for rs1805134 SNP were associated with susceptibility to morbid obesity (p = 0.02 and p = 0.03, respectively). Regarding the haplotype frequency, no association was observed; however, when shorter combinations were analyzed, haplotype rs1137101G-rs1805134C becomes associated with susceptibility (p = 0.036; OR [95 % CI] 3.4 [1.02–11.14]). Finally, according to LD plots generated in Haploview 4.2 (Fig. 1), the LEPR gene showed that five SNPs (rs1137100, rs1137101, rs1805134, rs1805094 and rs1805096) were in absolute but none in perfect LD.
Table 1

Alleles, genotypes and haplotypes frequencies of LEPR SNPs in a Co-dominant model

 

Patients (n = 28)

Controls (n = 56)

p valuea

OR (95 % CI)b

Alleles

 rs1137100-C (Lys109Arg)

0.45

0.43

0.99

1.08 (0.51–2.30)

 rs1137100-T

0.55

0.57

0.99

0.92 (0.43–1.96)

 rs1137101-G (Gln223Arg)

0.43

0.39

0.79

1.18 (0.58–2.39)

 rs1137101-A

0.57

0.61

0.79

0.85 (0.41–1.72)

rs1805134 - C (Ser343Ser) c

0.17

0.06

0.02

3.41 (1.11–10.49)

 rs1805134-T

0.83

0.94

0.02

0.29 (0.09–0.90)

 Ser492Thr-G

1

1

 Ser492Thr-C

0

0

 rs1805094-G (Lys656Asn)

0.22

0.12

0.13

2.02 (0.89–4.59)

 rs1805094-C

0.78

0.88

0.13

0.49 (0.22–1.13)

 rs1805096-G (Pro1019Pro)

0.40

0.40

0.93

0.97 (0.48–1.95)

 rs1805096-A

0.60

0.60

0.93

1.03 (0.51–2.07)

Genotypes

 rs1137100 C/C

0.05

0.10

0.87

0.66 (0.07–6.01)

 rs1137100 C/T

0.79

0.65

0.42

1.85 (0.19–0.59)

 rs1137100 T/T

0.16

0.25

0.65

0.64 (0.16–2.57)

 rs1137101 A/A

0.32

0.30

0.88

1.09 (0.38–3.16)

 rs1137101 A/G

0.50

0.61

0.54

0.65 (0.24–1.76)

 rs1137101 G/G

0.18

0.09

0.45

2.28 (0.55–9.43)

rs1805134 T/T d

0.65

0.89

0.03

0.24 (0.07–0.83)

rs1805134 T/C d

0.35

0.11

0.03

4.01 (1.20–13.41)

 Ser492Thr G/G

1.00

1.00

 rs1805094 C/C

0.59

0.77

0.13

0.43 (0.17–1.13)

 rs1805094 C/G

0.38

0.22

0.17

2.19 (0.83–5.77)

 rs1805094 G/G

0.03

0.01

0.82

2.09 (0.13–34.61)

 rs1805096 A/A

0.33

0.33

0.84

1.05 (0.37–2.92)

 rs1805096 A/G

0.54

0.54

0.82

1.01 (0.38–2.66)

 rs1805096 G/G

0.13

0.13

0.80

0.99 (0.23–4.20)

Haplotypese

 CGTGCA

0.266

0.271

0.94

0.97 (0.47–2.01)

 TATGCG

0.173

0.223

0.47

0.74 (0.32–1.69)

 TATGCA

0.108

0.106

0.98

1.01 (0.36–2.88)

 CATGCG

0.078

0.117

0.44

0.64 (0.20–2.00)

 TATGGA

0.084

0.088

0.92

0.94 (0.30–3.01)

 TGCGCA

0.073

0.058

0.70

1.28 (0.36–4.62)

 CATGCA

0.037

0.041

0.91

0.91 (0.17–4.92)

 TGTGCG

0.037

0.029

0.79

1.27 (0.22–7.53)

 TATGGG

0.047

0.024

0.39

2.12 (0.37–12.03)

 TACGGG

0.032

0.005

0.13

7.97 (0.34–185.4)

 TGTGCA

0.005

0.016

0.56

0.34 (0.01–15.99)

* p value with Yates correction; bOdds ratio (95 % confidence interval); cStatistical power = 0.632; dStatistical power = 0.679; e LEPR haplotypes: rs1137100-rs1137101-rs1805134-Ser492Thr-rs1805094-rs1805096. Characters in bold and italics indicate statistically significant values

https://static-content.springer.com/image/art%3A10.1186%2Fs41065-016-0006-0/MediaObjects/41065_2016_6_Fig1_HTML.gif
Fig. 1

Linkage disequilibrium (LD) in LEPR polymorphisms between obese and control groups. The pairwise LD plot was created by Haploview 4.2. Within each diamond the pairwise standardized coefficient of LD (D’) or the correlation coefficient (r2) is presented in percentage. Standard color coding was used for the Haploview LD plots with the confidence bounds color scheme, for the D’ plot, numbers are representative LD and the logarithm of the odds are in color: white = strong evidence of recombination; light grey = uninformative; dark grey = strong evidence of LD; for r2 LD plots white (r2 = 0), shades of grey (0 < r2 < 1), black (r2 = 1). A marker pair is said to show moderate or usable LD if |D’| is between 0.33 and 0.5, and strong LD if |D’| is 0.5 or above (i.e. at least half the maximum value)

There are data pointing to the fact that the Hispanic population exhibits predisposition towards obesity ([17, 18], http://www.who.int/topics/obesity/en/). Therefore, it is important to study the genetic mechanisms that predispose individuals to this pathology. In the present study, of the six SNPs analyzed, only allele C and genotype T/C for rs1805134 (Ser343Ser) SNP was associated with morbid obesity. Interestingly, this polymorphism has been scanty studied in obesity; when association between serum lipids and LEP/LEPR gene polymorphisms in obese Japanese children was studied, the rs1805134 SNPs showed a significant relationship with serum lipid profile, since lower triglyceride levels were obtained in rs1805134 C/C homozygotes [19]. In another study performed in Spanish adults with obesity, no significant case–control differences were found in allele/genotype frequencies for rs1137100, rs1137101, rs1805134 and rs8179183 SNPs [20]. Since rs1805134 SNP addresses a synonymous amino acid change and, according to our results, it was in absolute LD with both flanking SNPs (rs1137100 and rs1805096) in the LEPR gene, it suggests that SNPs have not been affected by recombination events, which is in concordance with the literature. The present observations allow us to speculate about the presence of others polymorphisms, probably located in introns, close to rs1805134 SNP in absolute and perfect LD, which might regulate the expression of exon 9 of LEPR gene, as it has been described for other Eukaryotes genes [21].

Regarding rs1137100, rs1137101 and rs8179183 SNPs, our data are similar with those reported in others Mexican populations; Guizar-Mendoza et al. [22] assessed rs1137101 and rs1805096 LEPR polymorphisms in Mexican adolescents from Guanajuato state, finding no differences in the genotype frequencies of these SNPs between obese and non-obese participants. Furthermore, another study in Mexican children and adolescents from Colima state, analyzed rs1137100, rs1137101 and rs8179183 SNPs, and no statistically significant association with obesity was found in any of the alleles [18].

The main limitation of the present study was the small sample size; nevertheless we were able to demonstrate statistically significant differences with an adequate power between case and control groups. Rosmond [10] has made harsh criticisms on the association with risk factors of case–control genetic studies, highlighting that the current literature linking central obesity to genetic variants has many reports of associations that either cannot be reproduced or corroborated. The present study, as opposed to Rosmond’s criticism, does replicate the findings reported by other researchers in the same ethnic group, supporting the consistency of allele and genotype frequency for some LEPR polymorphisms in a specific group of participants; therefore, we propose that the rs1805134 SNP is interesting and might be involved in morbid obesity; however, more studies are necessary to confirm or reject this hypothesis.

Abbreviations

LEP: 

leptin

LEPR: 

leptin receptor

SNP: 

single nucleotide polymorphism

BMI: 

body mass index

DNA: 

deoxyribonucleic acid

EDTA: 

ethylenediaminetetraacetic acid

PCR: 

polymerase chain reaction

RFLP: 

restriction fragment length polymorphism

AF: 

allele frequency

GF: 

genotype frequency

LD: 

linkage disequilibrium

OR: 

odds ratio

CI: 

confidence interval

Declarations

Acknowledgments

The authors would like to thank Rocio Jimenez-Lucio and David Sierra-Barrera for technical support.

Funding

No financial support was received during the present study.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Departamento de Cirugia Endoscopica, Hospital General “Dr. Manuel Gea Gonzalez”
(2)
Departamento de Biologia Molecular e Histocompatibilidad, Hospital General “Dr. Manuel Gea Gonzalez”
(3)
Departamento de Ecologia de Agentes Patogenos, Hospital General “Dr. Manuel Gea Gonzalez”

References

  1. Guh DP, Zhang W, Bansback N, Amarsi Z, Birmingham CL, Anis AH. The incidence of co-morbidities related to obesity and overweight: a systematic review and meta-analysis. BMC Public Health. 2009;9:88.View ArticlePubMedPubMed CentralGoogle Scholar
  2. Malis C, Rasmussen EL, Poulsen P, Petersen I, Christensen K, Beck-Nielsen H, et al. Total and regional fat distribution is strongly influenced by genetic factors in young and elderly twins. Obes Res. 2005;13:2139–45.View ArticlePubMedGoogle Scholar
  3. Marti A, Martínez-González MA, Martínez JA. Interaction between genes and lifestyle factors on obesity. Proc Nutr Soc. 2008;67:1–8.View ArticlePubMedGoogle Scholar
  4. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature. 1994;372:425–32.View ArticlePubMedGoogle Scholar
  5. Stefan N, Vozarova B, Del Parigi A, Ossowski V, Thompson DB, Hanson RL, et al. The Gln223Arg polymorphism of the leptin receptor in Pima Indians: influence on energy expenditure, physical activity and lipid metabolism. Int J Obes Relat Metab Disord. 2002;26:1629–32.View ArticlePubMedGoogle Scholar
  6. Clément K, Vaisse C, Lahlou N, Cabrol S, Pelloux V, Cassuto D, et al. A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature. 1998;392:398–401.View ArticlePubMedGoogle Scholar
  7. Farooqi IS, Wangensteen T, Collins S, Kimber W, Matarese G, Keogh JM, et al. Clinical and molecular genetic spectrum of congenital deficiency of the leptin receptor. N Engl J Med. 2007;356:237–47.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Rosmond R, Chagnon YC, Holm G, Chagnon M, Pérusse L, Lindell K, et al. Hypertension in obesity and the leptin receptor gene locus. J Clin Endocrinol Metab. 2000;85:3126–31.PubMedGoogle Scholar
  9. Fan SH, Say YH. Leptin and leptin receptor gene polymorphisms and their association with plasma leptin levels and obesity in a multi-ethnic Malaysian suburban population. J Physiol Anthropol. 2014;33:15.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Rosmond R. Association studies of genetic polymorphisms in central obesity: a critical review. Int J Obes Relat Metab Disord. 2003;27:1141–51.View ArticlePubMedGoogle Scholar
  11. Sambrook J, Fitsch EF, Maniatis T. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor Laboratory Press; New York 2001.
  12. Gotoda T, Manning BS, Goldstone AP, Imrie H, Evans AL, Strosberg AD, et al. Leptin receptor gene variation and obesity: lack of association in a white British male population. Hum Mol Genet. 1997;6:869–76.View ArticlePubMedGoogle Scholar
  13. Matsuoka N, Ogawa Y, Hosoda K, Matsuda J, Masuzaki H, et al. Human leptin receptor gene in obese Japanese subjects: evidence against either obesity-causing mutations or association of sequence variants with obesity. Diabetologia. 1997;40:1204–10.View ArticlePubMedGoogle Scholar
  14. Bignon JD, Fernández-Viña M, Arnaiz-Villena A. Technical handbook of twelfth international histocompatibility workshop. 1st ed. France: HLA et Medicine Paris; 1996.Google Scholar
  15. Olivo-Diaz A, Romero-Valdovinos M, Gudiño-Ramirez A, Reyes-Gordillo J, Jimenez-Gonzalez DE, Ramirez-Miranda ME, et al. Findings related to IL-8 and IL-10 gene polymorphisms in a Mexican patient population with irritable bowel syndrome infected with Blastocystis. Parasitol Res. 2012;111:487–91.View ArticlePubMedGoogle Scholar
  16. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005;21:263–5.View ArticlePubMedGoogle Scholar
  17. Wauters M, Mertens I, Rankinen T, Chagnon M, Bouchard C, Van Gaal L. Leptin receptor gene polymorphisms are associated with insulin in obese women with impaired glucose tolerance. J Clin Endocrinol Metab. 2001;86:3227–32.
  18. Angel-Chávez LI, Tene-Pérez CE, Castro E. Leptin receptor gene K656N polymorphism is associated with low body fat levels and elevated high-density cholesterol levels in Mexican children and adolescents. Endocr Res. 2012;37:124–34.View ArticlePubMedGoogle Scholar
  19. Okada T, Ohzeki T, Nakagawa Y, Sugihara S, Arisaka O, Study Group of Pediatric Obesity and Its related Metabolism. Impact of leptin and leptin-receptor gene polymorphisms on serum lipids in Japanese obese children. Acta Paediatr. 2010;99:1213–7.View ArticlePubMedGoogle Scholar
  20. Marti A, Santos JL, Gratacos M, Moreno-Aliaga MJ, Maiz A, Martinez JA, et al. Association between leptin receptor (LEPR) and brain-derived neurotrophic factor (BDNF) gene variants and obesity: a case–control study. Nutr Neurosci. 2009;12:183–8.View ArticlePubMedGoogle Scholar
  21. Parenteau J, Durand M, Morin G, Gagnon J, Lucier JF, Wellinger RJ, et al. Introns within ribosomal protein genes regulate the production and function of yeast ribosomes. Cell. 2011;147:320–31.View ArticlePubMedGoogle Scholar
  22. Guízar-Mendoza JM, Amador-Licona N, Flores-Martínez SE, López-Cardona MG, Ahuatzin-Trémary R, Sánchez-Corona J. Association analysis of the Gln223Arg polymorphism in the human leptin receptor gene, and traits related to obesity in Mexican adolescents. J Hum Hypertens. 2005;19:341–6.View ArticlePubMedGoogle Scholar

Copyright

© Rojano-Rodriguez et al. 2016

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