GENE ASSOCIATION OF α-B-CRYSTALLIN WITH R577X POLYMORPHISM FOR ACTN3 AND NOCICEPTION IN SUBJECTS WITH TMD-RELATED MYALGIA ________________________________________________________________________ A Thesis Submitted to The Temple University Graduate Board ________________________________________________________________________ In Partial Fulfillment Of the Requirements for the Degree MASTER OF SCIENCE in ORAL BIOLOGY ________________________________________________________________________ By Zhanna Konovalenko, DDS July 2016 Thesis Approval(s): Michael J. Horton, Ph.D Thesis Advisor, Temple U. Kornberg School of Dentistry, Dept. of Orthodontics James J. Sciote, D.D.S., M.S., Ph.D Committee Member, Temple U. Kornberg School of Dentistry, Dept. of Orthodontics Jeffrey H. Godel, D.D.S. Committee Member, Temple U. Kornberg School of Dentistry,Dept. of Orthodontics ABSTRACT Masseter muscle is one of the major muscles of mastication, and is comprised of actin and myosin myofibrils organized into sarcomeric contractile units. Structurally, sarcomeres are repeating portions of myofibrils between neighboring Z-lines (a.k.a. Z- disc, Z-band). The Z-line or Z-disc is composed of non-contractile proteins that provide mechanical stability to the sarcomere. One of the proteins of Z-disc is alpha-B-crystallin, a protein product of the gene CRYAB. Together with several other proteins of the Z-disc, CRYAB gene has been found to be up-regulated in Actn3 knock-out mice. In addition, CRYAB is suspected to be a pain mediator gene, having similar structure and function to CRYAA (alpha,A-crystallin) identified as one of the candidate genes from the Pain Research Panel, previously investigated in the Orofacial Pain: Prospective Evaluation and Risk Assessment (OPPERA) Study. Finally, in a microarray of global gene expression CRYAB was increased in subjects with facial asymmetry. We have examined CRYAB expression in masseter muscle of 64 orthognathic surgery patients to determine associations with skeletal malocclusions. Salivary DNA was genotyped for a single nucleotide polymorphism (SNP) for ACTN3 (rs1815739) and masseter muscle RNA isolated from an orthognathic surgery patient population. These genotyping and expression data have been used to identify differences in CRYAB expression in sub-groups of our patient population with Class II and III, normal, open and deep bite malocclusions who are null for ACTN3. In addition, we evaluated expression levels of CRYAB in patients with TMD-related myalgia. ii We found that relative quantities of CRYAB expression differed very significantly between sexes (p=0.005). ANOVA comparison between all subjects with and without TMD-myalgia indicated that males with TMD-myalgia had significantly greater (p<0.02) expression than other groups. An unpaired t-test showed that with TMD-related myalgia, CRYAB expression was significantly higher (p=0.03) in males than in females. ANOVA comparison between sexes with Class II and Class III malocclusions showed greater expression of CRYAB (p=0.005) in males with Class II. Expression was likewise greater in males with Class III malocclusion than in females with Class III (p<0.01). Among different age groups, subjects 25 years of age or younger had significantly (p value=0.025) increased expression of CRYAB gene. There were no significant differences for genotypes or facial asymmetry. iii ACKNOWLEDGEMENTS I would like to express my gratitude to everyone involved in this project for their help and support. I extend my appreciation to Dr. Horton, my thesis advisor as well as to Dr. Godel and Dr. Sciote for their mentorship. I am thankful to the Department of Orthodontics at Temple Kornberg School of Dentistry for kindly accommodating me and making the facilities available that were necessary for the success of this study. iv TABLE OF CONTENTS Page ABSTRACT ………………………………………………………………………. ii ACKNOWLEDGEMENTS ……………………………………………………….. iv LIST OF FIGURES ……………………………………………………………….. ix LIST OF TABLES ……………………………………………………………….... x CHAPTER 1. INTRODUCTION ………………………………………………………… 1 2. REVIEW OF THE LITERATURE ……………………………………….. 5 2.1 The models of inheritance of malocclusions ………………………. 5 2.2 Malocclusion is a Musculoskeletal Trait …………………………… 9 2.3 ACTN3 Polymorphisms and Associated Genotype ………………… 11 2.4 The Z-disc of Skeletal Muscle ……………………………………… 12 2.5 α-B-crystallin - a Multifunctional Protein of the Z-disc…………… 15 2.6 Classification and Pathophysiology of Temporomandibular Disorders 17 2.7 The Orofacial Pain: Prospective Evaluation and Risk Assessment Study 22 3. AIMS OF THE INVESTIGATION ………………………………………… 26 4. MATERIALS AND METHODS …………………………………………… 28 4.1 Study sample ………………………………………………………… 28 4.2 Genotyping …………………………………………………………... 30 4.3 Jaw and Function Analysis ………………………………………….. 31 4.4 RNA Isolation and RT-PCR ……………………………………….. 32 v 4.5 Statistical Analysis ………………………………………………..... 38 5. RESULTS …………………………………………………………………. 39 5.1 Sex-specific difference ……………………………………………… 39 5.2 Malocclusion ………………………………………………………… 40 5.2.A. Sagittal Malocclusion ……………………………………………… 40 5.2.B. Vertical Malocclusion ……………………………………………… 42 5.3 Genotyping ………………………………………………………..…. 45 5.4 Temporomandibular Joint Disorders ………………………………… 46 5.5 Facial Asymmetry …………………………………………………… 48 5.6 Age …………………………………………………………………... 48 6. DISCUSSION………………………………………………………………. 50 7. CONCLUSIONS……………………………………………………………. 55 BIBLIOGRAPHY……………………………………………………………… . 57 APPENDICES ………………………………………………………………… 69 APPENDIX A …………………………………………………………………. 69 Jaw Pain and Function Questionnaire APPENDIX B ………………………………………………………………… 70 Summary of Subject Sample APPENDIX C …………………………………………………………………… 73 Comparison of CRYAB Expression Between Class II and Class III Subjects APPENDIX D …………………………………………………………………… 74 Comparison of CRYAB Expression Between Male Subjects with Class II and Class III Malocclusion APPENDIX E……………………………………………………………………. 75 Comparison of CRYAB Expression Between Male Subjects with Normal, Deep and Open Bite vi APPENDIX F……………………………………………………………………………………………… 76 Comparison of CRYAB Expression Between Female Subjects with Normal, Deep and Open Bite APPENDIX G…………………………………………………………………….. 77 Comparison of CRYAB Expression Between Male and Female Subjects with Deep Bite APPENDIX H……………………………………………………………………. 78 Comparison of CRYAB Expression Between Male and Female Subjects with Open Bite APPENDIX I……………………………………………………………………… 79 Comparison of CRYAB Expression Between Male and Female Subjects with Normal Bite APPENDIX J……………………………………………………………………… 80 Comparison of CRYAB Expression in Subjects with Three Different Genotypes for R577X Polymorphism for ACTN3 APPENDIX K……………………………………………………………………. 81 Comparison of CRYAB Expression in Male and Female Subjects with Three Different Genotypes for R577X Polymorphism for ACTN3 APPENDIX L……………………………………………………………………. 82 Comparison of CRYAB Expression in Male Subjects with Three Different Genotypes for R577X Polymorphism for ACTN3 APPENDIX M…………………………………………………………………… 83 Comparison of CRYAB Expression in Female Subjects with Three Different Genotypes for R577X Polymorphism for ACTN3 APPENDIX N…………………………………………………………………… 84 Comparison of CRYAB Expression in Subjects with and without TMD-myalgia APPENDIX O…………………………………………………………………… 85 Comparison of CRYAB Expression in Male Subjects with and without TMD-myalgia APPENDIX P…………………………………………………………………… 86 Comparison of CRYAB Expression in Female Subjects with and without TMD-Myalgia APPENDIX Q…………………………………………………………………… 87 Comparison of CRYAB Expression in Subjects with and without Facial Asymmetry vii APPENDIX R…………………………………………………………………… 88 Comparison of CRYAB Expression in Male versus Female Subjects with and without Facial Asymmetry APPENDIX S…………………………………………………………………… 89 Comparison of CRYAB Expression in Male Subjects with and without Facial Asymmetry APPENDIX T…………………………………………………………………… 90 Comparison of CRYAB Expression in Female Subjects with and without Facial Asymmetry APPENDIX U…………………………………………………………………… 91 Comparison of CRYAB Expression in Male Subjects with and without TMD-myalgia And Facial Asymmetry APPENDIX V…………………………………………………………………… 92 Comparison of CRYAB Expression in Female Subjects with and without TMD-myalgia and Facial Asymmetry APPENDIX W ……………………………………………………………………… 93 Comparison of CRYAB expression in subjects younger or older than 18 years of age APPENDIX X ………………………………………………………………………. 94 Comparison of CRYAB expression between subjects by age: 18 years old versus 19-40 years old > 40 years old viii LIST OF FIGURES Figure Page 1. Anatomy of Human Skeletal Muscle ……………………………………… 12 2. Striated Muscle Sarcomere ……….………………………………………… 13 3. The Sarcomeric Z-disc- Multiple Functions and Links to Maladaptation … 14 4. Schematic representation of proteins involved in the cytoarchitecture of cardiomyocytes …….…………………………………………………… 16 5. Masseter Muscle Biopsy Site……..……………………………………….. 29 6. Amplification Plot of HPRT1 Standards …………………………………… 33 7. Amplification Plot of CRYAB Standards ………………………………….. 34 8. Standard curves for HPRT1 and CRYAB ……………………………………… 35 9. Comparison of relative quantitates of RNA expression for three genes: ADORA1, CRYAN, PPP3CC ……………………………………… 36 10. Sex-specific differences in expression of CRYAB …………………………… 39 11. The ANOVA analysis comparing CRYAB gene expression between ………. 40 sexes with Class II and Class III malocclusion. 12. Expression of CRYAB between subjects with different vertical malocclusions.. 42 13. Results of the ANOVA analysis of CRYAB expression in males and females with normal, deep and open