1. Gene Expression Differences in Young Male and Female Ruptured ACL Tissue Jeffrey S Johnson, MD Sports Medicine Fellow May 28, 2013 Jeffrey Johnson MD, Susan Moen MD, Robin Jacquet MS, Dennis Weiner MD, Kerwyn Jones MD, Melanie Morscher BS, William Landis PhD

2. DISCLOSURES This study was supported by a grant from:

3. ACL INJURY  >200,ooo ACL injuries in US/year  ~120,000 reconstructions/year  Total costs per injury:  $17,000-25,000  Popular topic in the literature  As of ‘99 >3500 articles  Since ‘99: many more

4. ACL INJURY  ACL anatomy  Collagen fascicles 0.25-3.0 mm in diameter  Multiple types of collagen (I, III, V, XII)  Enveloped in synovium  Blood supply:  Primarily middle geniculate artery  Also med/lat geniculate vessels through fat pad

5. ACL INJURY  ACL anatomy cont’d  Innervation: Posterior articular nerve (Tibial n.)  Size:  ~33mm in length  ~11 mm in diameter

6. ACL INJURY  ACL anatomy cont’d  Divided into 2 main bundles (Girgis et al)  Anteromedial bundle-tight in flexion  Posterolateral bundle-tight in extension  Intermediate bundle?

7. ACL INJURY  ACL anatomy cont’d  Ligament strength-2500 Newtons  Carries only 20% load during normal daily activity  70% of injuries-non contact injuries  30% of injuries-contact injuries

8. ACL INJURY  Greater incidence of ACL injuries in females than males  2-8 x greater incidence 1,2  Reasons for gender difference:  Anatomy, strength, joint laxity, neuromuscular activation, and possible hormonal influence?

9. ACL INJURIES  Other contributing factors?

10. LITERATURE- ACL Gene Expression  Estrogen and progesterone receptors in male and female ACL tissue 3  Alterations in receptor expression in pregnancy 3  Incubation of ACL tissue with estrogen down regulates type I collagen synthesis 3

11.  Type I collagen α 1 Sp 1 transcription factor binding site variant is associated with decreased ACL injury 4  Type V collagen genotype variant associated with increased risk ACL injury 5 LITERATURE- ACL Gene Expression

12. PROBLEM No one has compared gene expression between male and female ACL tissue

13. HYPOTHESIS

14. METHODS  14 participants: 7 female, 7 male  Injury documented by exam & MRI  Underwent ACL Reconstruction  Average age at surgery : 16.8 ± 2.5  Type of injury: Primarily non-contact

15. METHODS  ACL sample obtained intraoperatively  RNA extracted/purified  Microarray analysis of samples

16. METHODS Microarray Analysis Powerful genetic research tool Compares gene expression across genome (>20,000 genes) Qualitative tool

17. METHODS Analogy for Microarray:

18. METHODS After Microarray: Bioinformatic analysis of gene expression Functional Gene Classification Review of available literature on gene products Verification of Results with RT-qPCR

19. STATISTICS  Microarray  Significance Analysis of Microarray—FDR and Adjusted p-values  (p < 0.05 )  RT-qPCR  Student’s T-test  (p < 0.05 )

20. Genes for Analysis X- and Y- chromosome linked Genes Significant Gene Differences All Genes Microarray > 20,000 Insignificant Differences EXCLUDED Significant Differences X- Y-chromosome linked differences EXCLUDED Non X-Y- chromosome linked differences INGENUITY RT-qPCR RESULTS (Number of genes analyzed) >20,000 n=32 n=14 n=3

21. UP-REGULATED GENES (9) SymbolGene NameFCAdj p ACANAggrecan 2.20.05 CELF4CUGBP, Elav-like family member 3.20.01 COL8A2Collagen type VIII, alpha 2 2.30.01 CRABP2Cellular retinoic acid binding protein 2 2.10.02 FMODFibromodulin 2.10.02 GLYATL2Glycine-N-acyltransferase-like 2 2.60.03 HSD387Hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2.20.04 ITGA11Integrin, alpha 11 2.00.04 STXBP6Syntaxin binding protein 6 (amisyn) 2.20.03

22. DOWN-REGULATED GENES (5) SymbolGene NameFCAdj p CLEC2B C-type lectin domain family 2, member B -3.60.03 TDRD10Tudor domain containing 10 -2.00.05 TFPITissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) -2.10.06 TSTD1Thiosulfanate sulfur transferase-like domain containing 1 -3.00.001 WISP2Wnt1 inducible signaling pathway protein 2 -2.30.05

23. GENES OF CHOICE (PCR ANALYSIS) SymbolGene NameFCAdj p ACANAggrecan2.20.05 FMODFibromodulin2.10.02 WISP2Wnt1 inducible signaling pathway protein 2 -2.30.05

24. RT-qPCR * * * * p < 0.05 1 2 3

25. RESULTS

26. DISCUSSION ↑FMOD FMOD: proteoglycan; inhibits collagen fibrils Sequesters TGF-β, inhibiting cell signaling Up regulation may lead to ↓ ligament strength ↑ACAN ACAN: proteoglycan; ligament structural integrity, ECM regulation Up regulation may lead to ↓ ligament strength ↓ WISP 2 WISP 2 : Wnt 1 pathway; found in connective tissues Stimulates mitosis, apoptosis, ECM production Down regulation may lead to ↓ ligament strength

27. LIMITATIONS  Ruptured ACL tissue (may differ from normal)  Menstrual cycle info in females was unreliable  Gene expression studied, not protein secretion/function

28. CLINICAL RELEVANCE Novel way to study the genetics of a musculoskeletal disease Novel study showing genetic differences between torn male and female ACL’s One of the first studies to show this Provides direction for further research into ACL gene expression

29. CONCLUSION  Significant differences in expression of ECM genes found between male and female ruptured ACL tissue  Genetic differences may help account for increased incidence of ACL injuries in females compared to males

30. REFERENCES 1Griffin LY, Agel J, Albohm MJ, et al. J Am Acad Orthop Surg. 2000;8:141-50 2Hewett TE, Ford KR, Myer GD. Am J Sports Med. 2006;34:490-8 3Sciore P, Frank CB, Hart DA. J Orthop Res. 1998;16:604-10 4Khoschnau S, Melhus H, Jacobson A, et al. Am J Sports Med. 2008;36:2432-6 5Posthumus M, September AV, O’Cuinneagain D, et al. Am J Sports Med. 2009;37:2234-40

31. Thank You!