Association study of ATG16L1 gene polymorphism with osteopenia and osteoporosis Author: Radu Anghel Popp, MD, PhD Co-author: Marius Florin Farcas , MD, PhD Student Iuliu Hatieganu University of Medicine and Pharmacy Medical Genetics Department
Dec 31, 2015
Association study of ATG16L1 gene polymorphism with osteopenia and
osteoporosis
Author: Radu Anghel Popp, MD, PhD
Co-author: Marius Florin Farcas , MD, PhD Student
Iuliu Hatieganu University of Medicine and Pharmacy Medical Genetics Department
Osteoporosis - systemic skeletal disease
Low bone mass Microarchitectural deterioration of bone tissue Increase of bone fragility Susceptibility to fractures (Johnell et al., 2005)
A complex disease - interplay of environmental factors and genetics (Zheng et al., 2011) (several possible pathways)
Introduction
Autophagy
Autophagy is a highly conserved housekeeping function of eukaryotic cells.
Failure of autophagy – associated with the demise of long-lived postmitotic cells in many organs, including brain, heart, muscle, and kidney (Cuervo et al., 2008).
ATG16L1
Role in the pathogenesis of Crohn’s disease (Murdoch, Wang,Thachil), tuberculosis (Kleinnijenhuis), psoriazis (Douroudis).
ATG16L1 T300A shown to play a critical role in Crohn’s disease .
Aim of the study
To investigate the possible association between the rs2241880 (T300A) polymorphism in the ATG16L1 autophagy related gene and the risk of reduced bone mass.
Group I consisting of 55 osteoporotic patients (-2.5 or below ) ~66.5y
Group II consisting 55 osteopenic patients (between -1.0 and -2.5 ) ~63.7y
Group III consisting 55 healthy controls (-1 or greater) ~ 62.1y
Clinical and imagistical diagnosis was performed at the Second Medical Clinic, Cluj Napoca.
Written and informed consent was obtained from each participant
Molecular Genetics techniques – PCR-RFLP
DNA isolation
Amplification (PCR reaction)
Enzymatic digestion LweI (FermentasTM)
Electrophoresis of digested fragments
Statistical analysis GraphPadTM - Fisher’s Exact test
In-house PCR protocol (developed in the Medical Genetics department by the author)
Electrophoresis of ATG16L1 polymorphism in 3%Metaphor
10 – water11 – DNA ladder (50bp)2, 6 – variant homozygous (GG)1,3, 4, 8 – heterozygous (AG)5, 7, 9 – wild-type homozygous (AA)
182 bp 129 bp
53 bp
Results
Painting by Francisco José de Goya
Analysis model p-value (95%) OR CI
AD osteoporosis – controls
0.82 1.2 0.51- 2.86
AD osteopenia – controls
0.5 1.5 0.62 - 3.64
AD osteoporosis - osteopenia
0.82 0.8 0.33 - 2
AR osteoporosis – controls
0.26 1.8 0.75 - 4.54
AR osteopenia – controls
0.12 2.2 0.9 - 5.31
AR osteoporosis - osteopenia
0.83 0.8 0.37 - 1.89
HWE (Hardy Weinberg
equilibrium)0.45
Not consistent with HWE if p<0.05
Discussions
Osteocytes (high lifetime spam)
Homing of osteoclasts/osteoblasts to the site of remodeling Influencing osteoblast and osteoclast generation Mineral homeostasis, mechanical loading Control of matrix mineralization
Other possible mechanisms cannot be excluded: oxidative stress, nuclear pore leakiness, excess glucorticoids or deficient sex hormones.
From a total of 963 biologic pathways/gene sets analyzed the regulation-of-autophagy (ROA) pathway achieved the most significant result for association with UD BMD (Zhang).
Several other gene pathways have also been proposed as responsible for the degeneration of bone architecture and mass (Zheng).
ATG16L1 T300A might not influence basal authopagy levels (Kubala, 2008).
Future plans: to perform studies on the IRGM related autophagy gene, VDR and COL1A1 genes.
Conclusion
First association study between the ATG16L1 T300A variant and osteoporosis or osteopenia.
No statistically significant differences were observed in the genotype distribution, hence the studied polymorphism is not a risk factor for reduced bone mass in our Romanian population groups.
Selected references
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8. Gardsell P, Johnell O, Nilsson BE, Gullberg B. Predicting various fragility fractures in women by forearm bone densitometry: a follow- up study. Calcif Tissue Int. 1993;52:348–353.
9. Mallmin H, Ljunghall S, Persson I, Naessen T, Krusemo UB, Bergstrom R. Fracture of the distal forearm as a forecaster of subsequent hip fracture: a population-based cohort study with 24 years of follow-up. Calcif Tissue Int. 1993;52:269–272.
10. Styrkarsdottir U, Halldorsson BV, Gretarsdottir S, et al. Multiple genetic loci for bone mineral density and fractures. N Engl J Med. 2008;358:2355–2365.
Acknowledgements
This study was financially supported by a grant offered by the “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj Napoca.
Special thanks to MD, PhD Daniela Fodor from the Second Medical Clinic, Cluj Napoca
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