Masseter muscle gene expression in relation to various craniofacial deformities: A genotype-phenotype study Hadwah AbdelMatloub Moawad B.D.S Dental College/ King Saud University (Saudi Arabia) M.Sc in Orthodontics Eastman Dental Institute/ University of London (UK) A thesis submitted for the degree of Doctor of Philosophy Division of Craniofacial Developmental Sciences, Orthodontic Unit UCL Eastman Dental Institute, UK 2009 Dedication To my beloved parents My sisters: Ruba Anadel Ranad My brother: Mohamed You are the greatest treasure of my life and without your love, support and prayers I would have never produced this work ABSTRACT Craniofacial form is defined by a number of factors. A major contributor is the jaw musculature especially of the masseter muscle, as differences in transcription and translation of various genes have been documented from this tissue. Up to this point however, no reliable biological predictors of form have been identified. The aims of this study were therefore, to describe the transcriptome of the masseter muscle using microarray technology and to establish and correlate the expression levels of potential candidate and known “informative” genes in masseter muscle with selected clinical, radiographic and dental features of subjects with a variety of craniofacial morphologies. A total of 29 patients (18 deformity and 11 control) were selected from the orthodontic/orthognathic clinics at the Eastman Dental and Whipps Cross Hospitals, London, and Riyadh Military Hospital, Saudi Arabia. Microarray results indicated five “novel” genes not previously reported in relation to the masseter muscles of subjects with variable craniofacial morphologies. Two genes (KIAA1671 and DGCR6) were down-regulated in long face patients, one (SERGEF) was down-regulated in Class III patients and one (LOC730245) was up-regulated in Class II long faces and in all Class III subjects, compared to controls. Another gene (NDRG2) was down-regulated in Class II compared to Class III individuals. Subsequent quantitative Reverse Transcriptase PCR results strongly confirmed that the “novel” gene SERGEF was down-regulated in relation to the clinical, dental and radiographic features of subjects with Class III appearance. SERGEF gene had a positive relationship to the number of dental occlusal contacts and ANB angle. The “informative” gene MHC7 was strongly related to both vertical and horizontal facial deformities. These data suggest that the expression profiles of a number of genes can be analysed and used to make assessments as to their role in the primary aetiology and successful or unsuccessful treatment of patients with specific craniofacial morphologies. TABLE OF CONTENT ACKNOWLEDGEMENT VII DECLARATION IX LIST OF FIGURES X LIST OF TABLES XI LIST OF ABBREVIATIONS XII CHAPTER 1. BACKGROUND 1 1.1. INTRODUCTION 2 1.2. THE MASSETER MUSCLE 2 1.2.1. GENERAL MASSETER MUSCLE STRUCTURE AND FUNCTION 3 1.2.1.1. Intrauterine growth 3 1.2.1.2. Contraction of muscle fibres 4 1.2.1.3. Postnatal growth and development 5 1.2.1.4. Masseter muscle fibre types 6 1.2.1.5. Masseter muscle extracellular matrix 7 1.3. CRANIOFACIAL MORPHOLOGY 8 1.3.1. THE CRANIAL BASE 8 1.3.1.1. Intrauterine growth 8 1.3.1.2. Postnatal growth and development 9 1.3.2. THE MAXILLA 9 1.3.2.1. Intrauterine growth 9 1.3.2.2. Postnatal growth and development 9 1.3.3. THE MANDIBLE 10 1.3.3.1. Intrauterine growth 10 1.3.3.2. Postnatal growth and development 10 1.4. CRANIOFACIAL DEFORMITIES 12 1.4.1. DESCRIPTION OF JAW DEFORMITIES 14 1.4.2. BASIC CRANIOFACIAL PATTERNS 14 1.4.2.1. Vertical facial patterns 14 1.4.2.1.1. The long face 15 1.4.2.1.2. The short face 15 1.4.2.2. Horizontal facial patterns 15 1.4.2.2.1. Class II 15 1.4.2.2.2. Class III 16 1.4.3. PREVALENCE OF CRANIOFACIAL PATTERNS 17 1.4.4. AETIOLOGY OF CRANIOFACIAL PATTERNS 19 1.4.4.1. Genetic studies of non-syndromic craniofacial patterns 19 1.4.4.2. Environmental factors affecting craniofacial patterns 20 1.5. MASSETER MUSCLE STRUCTURE AND FUNCTION IN RELATION TO CRANIOFACIAL PATTERNS 20 1.5.1. MASSETER MUSCLE ACTIVITY 20 1.5.2. MASSETER MUSCLE SIZE AND SHAPE 21 1.5.3. MASSETER MUSCLE EFFICIENCY 21 1.5.4. MASSETER MUSCLE FIBRE TYPE COMPOSITION 22 1.6. EXPERIMENTAL STUDIES OF MUSCULO-SKELETAL FUNCTION 23 1.6.1. THE EFFECT OF DIET 23 1.6.2. THE EFFECT OF EXERCISE 23 1.6.3. THE EFFECT OF ORTHODONTIC APPLIANCES 23 1.6.4. THE EFFECT OF ORTHOGNATHIC SURGICAL TREATMENT 24 1.7. GENE EXPRESSION STUDIES OF THE MASSETER MUSCLE 25 1.8. THE SCOPE OF GENE EXPRESSION TECHNOLOGIES 27 i 1.8.1. GENE EXPRESSION MICROARRAYS IN OROFACIAL CLEFTS 27 1.8.2. GENE EXPRESSION MICROARRAYS IN CRANIOSYNOSTOTIC CONDITIONS 28 1.8.3. GENE EXPRESSION MICROARRAYS IN HEMIFACIAL MICROSOMIA 28 1.9. STATEMENT OF THE PROBLEM 29 1.10. AIMS OF THE PROJECT 30 1.11. LAYOUT OF THE THESIS 31 CHAPTER 2. GENERAL MATERIALS AND METHODS 32 2.1. INTRODUCTION 33 2.2. CLINICAL DESIGN 33 2.2.1. INCLUSION AND EXCLUSION CRITERIA 33 2.2.2. INITIAL SAMPLE SIZE ESTIMATION 34 2.2.3. THE EDH AUDIT 35 2.3. ETHICAL APPROVAL 36 2.4. RESEARCH SUBJECTS 36 2.4.1. THE RECRUITMENT PROCEDURE 36 2.4.2. RETROSPECTIVE RECALCULATION OF SAMPLE SIZE 36 2.4.3. SUBJECTS INCLUDED 37 2.4.4. LATERAL CEPHALOMETRIC RADIOGRAPHS 39 2.4.4.1. Cephalometric landmarks 39 2.4.4.2. Cephalometric variables 40 2.4.4.3. Cephalometric norms 42 2.4.4.4. Errors of the method 43 2.4.4.4.1. Magnification factor 43 2.4.4.4.2. Correction of the ANB angle 43 2.4.4.4.3. Assessment of reliability and reproducibility 44 2.5. TISSUE SAMPLES 45 2.5.1. BIOPSY PROCEDURE 46 2.5.2. SAMPLE COLLECTION 46 2.6. RNA EXTRACTION 46 2.7. MICROARRAY 47 2.7.1. DEFINITION 47 2.7.2. GENE EXPRESSION MICROARRAY ISSUES 47 2.7.3. PRINCIPLES OF GENE EXPRESSION MICROARRAY PLATFORMS 50 2.7.4. PROPERTIES OF AFFYMETRIX® GeneChips® 51 2.7.4.1. The substrate 51 2.7.4.2. The probe 51 2.7.4.2.1. Source of the probe 51 2.7.4.2.2. Length of the probe 51 2.7.4.2.3. Number of probes presenting each gene 52 2.7.4.3. The method of immobilising the probe onto the substrate 52 2.7.4.4. The hybridisation system 53 2.7.4.5. Affymetrix® GeneChip® generations 53 2.7.5. MICROARRAY MATERIALS AND METHODS 53 2.7.5.1. RNA samples used 54 2.7.5.2. Sample preparation 54 2.7.5.3. cDNA/cRNA synthesis and amplification 55 2.7.5.4. Synthesis of biotin-labelled cRNA 55 2.7.5.5. cRNA fragmentation 55 2.7.5.6. Hybridisation 55 2.7.5.7. Washing and staining 56 2.7.5.8. Scanning (data extraction) 56 2.7.6. GeneChip® QUALITY CONTROL 58 2.7.7. PRE-PROCESSING DATA 58 2.7.7.1. Background adjustment and normalisation 58 2.7.7.2. Summarisation of probe intensity value 58 2.7.8. MICROARRAY DATA ANALYSIS 59 ii 2.7.8.1. Grouping of patients for microarray data analysis 59 2.7.8.2. Generating differentially expressed gene lists 60 2.7.8.3. Filtering the data 60 2.8. QUANTITATIVE RT-PCR 61 2.8.1. INTRODUCTION 61 2.8.2. THE CONCEPT OF REVERSE TRANSCRIPTION (RT) AND THE POLYMERASE CHAIN REACTION (PCR) 61 2.8.3. QUANTITATIVE vs. END POINT TO MEASURE GENE EXPRESSION 62 2.8.4. TYPES OF qRT-PCR 63 2.8.5. qRT-PCR PROTOCOL 63 2.8.5.1. RNAsamples used 64 2.8.5.2. cDNA synthesis 64 2.8.5.3. Endogenous reference gene 64 2.8.5.4. Fluorescent dye chemistry 65 2.8.5.5. Normalisation and calculation of gene expression values 67 2.8.6. qRT-PCR DATA ANALYSIS 68 2.9. SUMMARY OF THE DESIGN OF THE STUDY 69 CHAPTER 3. OPTIMISATION OF TOTAL RNA EXTRACTION PROTOCOL FROM FRESH HUMAN MASSETER MUSCLE BIOPSIES 70 3.1. INTRODUCTION 71 3.2. STEPS TO ELIMINATE DNA CONTAMINATION 71 3.2.1. DISRUPTION AND HOMOGENISATION 71 3.2.2. THE PURIFICATION TECHNIQUE 73 3.2.3. DNase DIGESTION 73 3.3. MATERIALS AND METHODS 74 3.3.1. INHIBITION OF RNase ACTIVITY 74 3.3.2. SOURCES OF THE SAMPLES USED 75 3.3.3. THE AMOUNT OF STARTING MATERIAL 75 3.3.4. DISRUPTION AND HOMOGENISATION 75 3.3.4.1. Chemical disruption 75 3.3.4.2. Mechanical homogenisation 75 3.3.5. RNA PURIFICATION 77 3.3.5.1. Binding 77 3.3.5.2. Elimination of DNA contamination 77 3.3.5.3. Washing 77 3.3.5.4. Elusion 77 3.3.6. RNA QUALITY CONTROL 78 3.3.6.1. RNA quantity 78 3.3.6.2. RNA purity 78 3.3.6.3. RNA quality 78 3.4. RESULTS 81 3.4.1. THE EFFECT OF DIFFERENT BEADS AND MACHINE SETTINGS ON THE DISRUPTION AND HOMOGENISATION OF MASSETER MUSCLE BIOPSIES 81 3.4.2. THE EFFECT OF DNase DIGESTIONS ON RNA PURIFICATION 83 3.5. QUALITY CONTROL OF TOTAL RNA SAMPLES 84 3.6. SUMMARY AND CONCLUSIONS 88 CHAPTER 4. DISCOVERY OF MASSETER MUSCLE CANDIDATE GENES IN RELATION TO NON-SYNDROMIC CRANIOFACIAL DEFORMITIES: A MICROARRAY ANALYSIS 89 4.1. INTRODUCTION 90 4.2. AFFYMETRIX® GENECHIP® QUALITY CONTROL 90 4.3.