Association study of ATG16L1 gene polymorphism with osteopenia and osteoporosis Author: Radu Anghel Popp, MD, PhD Co-author: Marius Florin Farcas, MD,

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

1. Johnell O, Kanis J. Epidemiology of osteoporotic fractures. Osteoporos Int. 2005;16:S3–7.2. Chao EY, Inoue N, Koo TK, Kim YH. Biomechanical considerations of fracture treatment and bone

quality maintenance in elderly patients and patients with osteoporosis. Clin Orthop Relat Res. 2004;425:12–25.

3. Muller ME, Webber CE, Bouxsein ML. Predicting the failure load of the distal radius. Osteoporos Int. 2003;14:345–352.

4. Weichetova M, Stepan JJ, Haas T, Michalska D. The risk of Colles’ fracture is associated with the collagen I a1 Sp1 polymorphism and ultrasound transmission velocity in the calcaneus only in heavier postmenopausal women. Calcif Tissue Int. 2005;76:98–106.

5. Tse KY, Macias BR, Meyer RS, Hargens AR. Heritability of bone density: regional and gender differences in monozygotic twins. J Orthop Res. 2009;27:150–154.

6. Duncan EL, Cardon LR, Sinsheimer JS, Wass JA, Brown MA. Site and gender specificity of inheritance of bone mineral density. J Bone Miner Res. 2003;18:1531–1538.

7. Cuddihy MT, Gabriel SE, Crowson CS, O’Fallon WM, Melton LJ 3rd. Forearm fractures as predictors of subsequent osteoporotic fractures. Osteoporos Int. 1999;9:469–475.

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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