1. Molecular basis of obesity
University of Medicine and Pharmacy Tîrgu Mureş – Marisiensis 2014
Molecular basis of obesity
Tripon Florin
Crauciuc George Andrei , Mărginean Oana Maria
Coord.: Ass. Prof. Dr. Bănescu Claudia -Genetics departament
Prof. Dr. Mărginean Oana -Pediatrics department

5. Background
What is happening now?
(1-3)

8. In the Hypothalamus is found in a very large percentage the Fat mass and obesity (FTO) associated protein (4) also known as alpha-ketoglutarate-dependent dioxygenase. An enzyme that in humans is encoded by the FTO gene located on chromosome 16 (5). Dates from the literature demonstrate that FTO gene is associated with levels of Ghrelin and Leptin hormone (6) also with Body Mass Index (7).

9. Objective
The aim of this study was to evaluate, in a case-controlled study, if the polymorphisms of RS T/A and RS T/G FTO genes is associated with obesity.

10. Matherial and Methods
We obtained the approval of the Ethic Committee of The University of Medicine and Pharmacy Tîrgu Mureș.
Our study included a group control with 89 children whit age, gender and social status similar to those in the patients group, consisting of 90 young obesity patiens.
We obtained from the analysis results(1st Pediatrics Clinic of Clinical County Hospital Tîrgu Mures ) the values of followed markers: Leptin and BMIz (standard deviation)
Genetic DNA was extracted from peripheral leukocytes according to the protocol described by manufacturer (ZymoResearch DNA kit).

11. DNA was submitted to restriction fragment length polymorphism-chain polymerase reaction (RFLP-PCR Eppendorf Mastercycler) using the following primers ( Fermentas/Thermo Scientific)
Rs T/A FTO gene Rs T/G Fto gene
The results were seen by electrophoresis in 2% agarose gel and a source of UV light.
forward primer sequence: AACTGGCTCTTGAATGAAATAGGATTCAGA AGGACCTCCTATTTGGGACA
reverse primer sequence: AGAGTAACAGAGACTATCCAAGTGCAGTAC AGCTTCCATGGCTAGCATTA
PCR protocols
initial denaturation 95°C for 4min °C for 5 min
denaturation °C for 30sec °C for 1min
annealing °C for 35sec X °C for 1min
extension °C for 1min °C for 1min
final extension °C for 10min °C for 7min
Amplified products were digested with 
2U of Sca I Fast digest enzyme U of Alw NI Fast digest enzyme For 10 min at 37°C ( Thermo Scientific)

12. Results
Our study included a number of 98 women and 81 men, with ages between 2-17 year and an median age of 10 year.

13. Results Rs 9939609 T/A FTO gene Control Group
Sex distribution in the control group : 60% girls and the remaining 40% boys
The mean BMIz :
The mean Leptin level : ng/dl
In the Control group the Rs T/A gene was found in the following variants:
-homozygous normal (TT genotype) at 33 people,
-heterozygous (TA genotype) 42 and
-homozygous mutant (AA genotype) on 14.
T 182 pb
A 154 pb

14. Results Rs 9939609 T/A FTO gene Patients Group
Sex distribution : 53.33(3)% boys and 46.33(3)% girls
In the Patients group the Rs T/A gene was found in the following variants:
-homozygous normal (TT genotype) at 28 people
-heterozygous (TA genotype) 29 and
-homozygous mutant (AA genotype) on 33.
Genotype
Number of person
Sex distribution (percente)
Mean BMIz
Mean value of Leptin ng/dl TT 28
20.84% ♂
42.85% ♀
2.06
13.52 TA 29
39.58%
23.80%
2,19
15.40 AA 33
33.35%
2,23
18.24

15. Results Rs 17817449 T/G FTO gene Control Group
In the Control group the Rs T/G gene was found in the following variants:
-homozygous normal (TT genotype) at 0 people,
-heterozygous (GT genotype) 54 and
-homozygous mutant (GG genotype) on 35.
223 pb G
123 pb T
100 pb T

16. Results Rs 17817449 T/G FTO gene Patients Group
In the Patients group the Rs T/G gene was found in the following variants:
-homozygous normal (TT genotype) at 3 people,
-heterozygous (GT genotype) 60 and
-homozygous mutant (GG genotype) on 27.
Genotype
Number of person
Sex distribution (percente)
Mean BMIz
Mean value of Leptin ng/dl TT 3
4.16% ♂
2.38% ♀
1.71
6.57 GT 60
68.75%
64.28%
2.12
16.20 GG 27
27.09%
33.34%
2.35
16.67

17. PATIENST GROUP VS. CONTROL GROUP
Distribution of Rs T/A genotype and allele distribution in Patients group and Control group.
PATIENTS GROUP
CONTROL
GROUP
PATIENST GROUP VS. CONTROL GROUP
p value, OR, CI
Genotype TT TA AA 28 29 33 42 14
---
p=0,5998 OR 0,8138
95% CI (0,4077-1,624)
p=0,0184 OR 2.778
95% CI (1,, )
Allele frequency
T allele
Aallele 85 95
106 70
p=0,0148, OR 1,692
95% CI (1,111-2,577)

18. PATIENTS GROUP VS. CONTROL GROUP
Distribution of Rs T/G genotype and allele distribution in Patients group and Control group.
PATIENTS GROUP
CONTROL
GROUP
PATIENTS GROUP VS. CONTROL GROUP
p value, OR, CI
Genotype GG GT TT 27 60 3 35 54
---
p=0,0887, OR
95% CI (0,005-2,173)
p=0,2480 OR 0,1586
95% CI (0,008-3,142)
Allele frequency
G allele
T allele
114 66
124
p=0,2193, OR
95% CI (0,4842-1,169)

19. Conclusion
BMIz and leptin levels are directly proportional with the number of RS / Rs FTO allele, an maximum risk have the patients homozygous mutant for both genes.
Exist a correlation between obesity and the RS T/A FTO gene polymorphism with statistical and scientific significance.
The RS T/G FTO gene polymorphism in this study is not related to obesity, compared to other international studies ( 8-9)
The fact that we have not found associations between RS T/G FTO gene and susceptibility to obesity explains the complex etiology of this disease , with the involvement of multiple several factors: alimentation, social factors, economic conditions, environmental factors etc.

20. Conclusion
RR p=0.018 CI MEN RR P= CI

21. Protocols by

22. References Images from www.google.com
Association of genetic variation in FTOwith risk of obesity and type 2 diabetes with data from 96,551 East and South Asians, H. Li, T. O. Kilpeläinen et all., Diabetologia April 2012, Volume 55, Issue 4, pp
FTO gene associates to metabolic syndrome in women with polycystic ovary syndrome ,Redha Attaouaa, ,Samira Ait El Mkadem et all., Biochemical and Biophysical Research Communications,Volume 373, Issue 2, 22 August 2008, Pages 230–234
Genetic Variants of FTO Influence Adiposity, Insulin Sensitivity, Leptin Levels, and Resting Metabolic Rate in the Quebec Family Study, Ron Do,Swneke D. Bailey, et all., DIABETES, VOL. 57, APRIL 2008
The common rs variant of the fat mass and obesity-associated gene is associated with obesity risk in children and adolescents of Beijing,China,Bo Xi1†, Yue Shen et all., BMC Medical Genetics 2010, 11:107
Associations between BMI and the FTO Gene Are Age Dependent: Results from the GINI and LISA Birth Cohort Studies up to Age 6 Years ,Peter Rzehaka,b* André Scherag et all., Obes Facts 2010;3:173–180
Serological and somatometrical parameters to a Târgu Mureş obese children group,Monica Tarcea et all., Revista Română de Medicină de Laborator Vol. 2, Nr. 1, Martie 2006
Hypothalamic-Specific Manipulation of Fto, the Ortholog of the Human Obesity Gene FTO,Affects Food Intake in Rats. Tung Y-CL, Ayuso E, Shan X, Bosch F, O’Rahilly S, et al. (2010) PLoS ONE 5(1): e doi: /journal.pone
Variation in the FTO gene locus is associated with cerebrocortical insulin resistance in humans,O. Tschritter et all., Diabetologia (2007) 50:2602–2603
Childhood obesity: are genetic differences involved? Claude Bouchard, Am J Clin Nutr-2009
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