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Endpoint Genotyping with the LightCycler® 480 Real-Time PCR System

 

Featured project: Association of cannabinoid receptor variants with adiposity in obese men

Armand Peeters1, Sigri Beckers1, Ilse Mertens2, Wim Van Hul1, and Luc Van Gaal2
1Department of Medical Genetics, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Antwerp University
2Department of Diabetology, Metabolism and Clinical Nutrition, Antwerp University Hospital, Antwerp, Belgium

 

Read in this article:

Introduction
Materials and Methods
Results and Discussion
Conclusions
References


Introduction

The epidemic rise in obesity has generated large amounts of research into the factors underlying this complex disease as well as to mechanisms linking its metabolic and cardiovascular complications [1]. Recent data suggest that the endocannabinoid system is a key circuit contributing to central appetitive regulation and that its aberrant activation may contribute to obesity [2]. These findings have been corroborated by studies in which the cannabinoid receptor (CB1) has emerged as a possible target for the treatment of obesity [3].

We aimed at evaluating, by means of a genetic association research study, whether genetic variation at the cannabinoid receptor locus (CNR1) could have an effect on adiposity and fat distribution. The synonymous G/A single nucleotide polymorphism (SNP) at position 1422 of the CNR1 gene was selected because it resides in the gene's coding region (threonine at position 453).

 

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Materials and Methods

For research purposes, 1064 research samples were collected from obese subjects, and 251 samples were collected from non-obese individuals for controls. G1422A (rs1049353) genotypes were determined by a commercial hydrolysis-probe based genotyping assay on the LightCycler® 480 System (Figure 1). Blank samples and samples with known genotype were included as negative and positive controls. We achieved 100% concordance in the analysis of duplicate samples. Statistical analyses were performed using SPSS software, version 12.0 (SPSS, Chicago, IL, USA). Linear regression was used to adjust anthropometric parameters for age and BMI.

 

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Results and Discussion

The prevalence of the G1422A variant was not significantly different between cases and controls (OR=1.056; p=0.626). In obese men (but not women), homozygosity for the variant A allele was significantly associated with highest waist-to-hip ratio (WHR) and increased waist circumference (p=0.009 and p=0.008, respectively; values adjusted for age and body-mass-index). A trend for an association with increased fat mass was also observed (p=0.033).

Figure 1: Typical results for a hydrolysis-probe assay for CNR1 gene variant G1422A (rs1049353), using the LightCycler® 480 Instrument. A fragment of the human CNR1 gene was amplified using the LightCycler® 480 Probes Master and subjected to endpoint analysis. Scatter plots consistently revealed wild-type (GG), mutant (AA) and heterozygote (GA) variants.

Phenotype Genotype Mean ± SEM * n   P (adjusted for age and body-mass-index)
Waist (cm) G/G+G/A
A/A
123.3 ± 0.6
127.3 ± 2.2
422
33
0.008
Waist-to-Hip ratio G/G+G/A
A/A
1.09 ± 0.006
1.15 ± 0.022
420
32
0.009
Fat mass (kg) G/G+G/A
A/A
52.2 ± 0.8
57.5 ± 3.2
432
36
0.033
Fat mass % G/G+G/A
A/A
42.1 ± 0.3
44.7 ± 1.0
432
36
0.218

Table 1: Associations under a recessive model between G1422A genotypes and anthropometric phenotypes in obese men. (*standard error of the mean)

 

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Conclusions

Comparative anthropometric analysis of the samples of adult obese individuals stratified by gender revealed meaningful differences between the samples of obese men with distinct genotypes of the CNR1 G1422A variant. Under a recessive model, we found three significant associations in the same direction namely 1422A/A homozygotes are more abdominally obese, which could indicate that these are true associations. Our data indicate that the absence of a CNR1 gene with the G-allele at position 1422 might increases the risk for obesity in males. The fact that the associations were only seen in samples of obese men can potentially be explained by general gender specific discrepancies in eating-habitsand fat ingestion in particular. In conclusion, our data indicate that the G1422A polymorphism in the cannabinoid receptor-1 gene might be associated with increased abdominal adiposity in obese men.

 

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References

  • [1] Van Gaal, L., Mertens, I. and De Block, C. (2006). Nature. 444, 875-80.
  • [2] Cota, D., Marsicano, G., Tschop, M., Grubler, Y., Flachskamm, C., Schubert, M., Auer, D., Yassouridis, A., Thone-Reineke, C., Ortmann, S., Tomassoni, F., Cervino, C., Nisoli, E., Linthorst, A. C., Pasquali, R., Lutz, B., Stalla, G. K. and Pagotto, U. (2003). J Clin Invest. 112, 423-31.
  • [3] Engeli, S., Bohnke, J., Feldpausch, M., Gorzelniak, K., Janke, J., Batkai, S., Pacher, P., Harvey-White, J., Luft, F. C., Sharma, A. M. and Jordan, J. (2005). Diabetes. 54, 2838-43.

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