Utilising Clinical Exome Sequencing in Patients with Rare Genetic Disease and Regions of Homozygosity Detected by SNP Microarray
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Utilising clinical exome sequencing in patients with rare genetic disease and regions of homozygosity detected by SNP microarray A thesis submitted to The University of Manchester for the degree of Doctor of Clinical Science In the faculty of Biology, Medicine and Health 2020 Lewis P Darnell School of Biological Sciences, Division of Cell Matrix Biology and Regenerative Medicine List of Contents Description Page Number Word count 7 List of figures 8 List of tables 9 List of abbreviations 10 Abstract 11 Declaration 12 Copyright statement 12 Acknowledgements 14 The author 15 1 Introduction 16 1.1 Introduction to Rare Genetic Disease 17 1.1.1 Rare Genetic Disorders 17 1.1.2 Diagnosing Rare Genetic Disorders 20 1.1.3 Consanguinity and Genetic Disease 22 1.2 Genetic Testing Methods 27 1.2.1 Microarray 27 1.2.2 DNA Sequencing 30 1.2.3 Whole Exome Sequencing 34 1.2.4 Clinical Exome Sequencing 38 1.2.5 Whole Genome Sequencing 41 1.2.6 Genetic Testing Summary 42 1.3 Variant Analysis 45 1.3.1 Variant Prioritisation 45 2 1.3.2 Variant Analysis 48 1.4 Genetic Testing for Rare Disease in the East Midlands 49 1.5 The Importance of this Research and Controversial Issues 53 1.6 Research Hypothesis 58 1.6.1 Research Question 58 1.6.2 Overarching Hypothesis 58 1.6.3 Specific Hypothesis 59 1.7 Detailed Project Aims 60 1.8 Evaluation of the Methodology Decision 62 1.9 Relevance to Research Area 64 1.10 Summary 65 2 Materials and Methods 67 2.1 Participants and Phenotypes 67 2.1.1 Participant Referral 67 2.1.2 Ethics and Consent 68 2.1.3 Participant Phenotypes, Previous Genetic Tests and Family Relationships 69 2.2 DNA Extraction and Storage 70 2.3 Microarray and Loss of Heterozygosity 70 2.4 Next-Generation Sequencing 71 2.4.1 Library Preparation 71 2.4.2 DNA Sequencing 72 2.5 Variant analysis 72 2.5.1 Bioinformatics 72 2.5.2 Autosomal Recessive Variant Filtering 73 2.5.3 Autosomal Dominant Variant Filtering 76 2.5.4 Assigning Pathogenicity 78 3 2.6 Variant Confirmation 81 2.6.1 Primer Design 81 2.6.2 Sanger Sequencing 83 2.7 Reporting 84 2.8 Cost and Practicality Analysis 87 3 Results 89 3.1 Participant Results 89 3.1.1 Family 1 89 3.1.2 Family 2 94 3.1.3 Family 3 95 3.1.4 Family 4 96 3.1.5 Family 5 97 3.1.6 Family 6 102 3.1.7 Family 7 102 3.1.8 Family 8 107 3.1.9 Family 9 112 3.1.10 Family 10 113 3.1.11 Family 11 113 3.2 Next-Generation Sequencing 119 3.2.1 Mid-Output vs High-Output 119 3.2.2 NA12878 Analysis 123 3.2.3 Variant Detection 125 3.2.4 Diagnostic Yield 127 3.3 Regions of Homozygosity 127 3.4 Cost and Practicality Analysis 130 4 4 Discussion and Conclusion 132 4.1 Participant Results 132 4.1.1 Patient Benefits 137 4.1.2 Diagnostic Yield 139 4.1.3 Incidental Findings 139 4.2 Performance of the SOPHiA GENETICS Clinical Exome Solution 142 4.2.1 Mid-output vs High-output 142 4.2.2 Variant Detection 143 4.2.3 Variant Filtering 145 4.3 Practicality and Cost in an NHS Laboratory 148 4.3.1 Practicality of the Method 148 4.3.2 Cost Effectiveness 150 4.4 Limitations of the Method 152 4.5 Research Implications and Future Work 154 4.6 Conclusion 156 5 References 159 6 Appendix 178 6.1 Patient Consent Form 178 6.2 Full Cost Analysis 178 6.2.1 Staff Costs 178 6.2.2 General Reagent Costs 178 6.2.2.1 Pipette Tip Costs Cost 179 6.2.2.2 Sample Reception Labels Cost 179 6.2.2.3 Stock DNA Label/Dilution label Cost 179 5 6.2.3 General Laboratory Costs 179 6.2.3.1 Fume Hood Maintenance Cost 180 6.2.3.2 Nanodrop maintenance Cost 180 6.2.3.3 Pipette Maintenance Cost 180 6.2.3.4 Thermocylcer Maintenance Cost 180 6.2.3.5 Centrifuge Maintenance Cost 180 6.2.3.6 Balance Maintenance Cost 181 6.2.3.7 Q-pulse Software Cost 181 6.2.3.8 StarLIMS Software Cost 181 6.2.3.9 Alamut Software Cost 181 6.2.3.10 Gloves Cost 181 6.2.3.11 Sharps Bins Cost 181 6.2.4 Sample Reception 182 6.2.4.1 Sample Collection and LIMS Entry Cost 182 6.2.4.2 Second Checking Cost 182 6.2.4.3 Duty Scientist Cost 182 6.2.4.4 Administrative Review Cost 183 6.2.5 DNA Extraction 183 6.2.5.1 Chemagic Maintenance Cost 183 6.2.5.2 DNA Extraction Reagent Cost 183 6.2.5.3 Extraction Staff Cost 184 6.2.6 SOPHiA GENETICS CES Kit and NGS 184 6.2.6.1 SOPHiA GENETICS Reagent Cost 184 6.2.6.2 Illumina NextSeq Reagents Cost 185 6.2.6.3 SOPHiA GENETICS and Illumina NextSeq Plastics Cost 186 6.2.6.4 Illumina NextSeq Leasing and Maintenance Cost 188 6.2.6.5 Staff Time Cost 188 6 6.2.7 Sanger Sequencing 189 6.2.7.1 Sanger Sequencing Reagent Cost 189 6.2.7.2 Sanger Sequencing Plastics Cost 191 6.2.7.3 ABI Reagents Cost 192 6.2.7.4 ABI Plastics Cost 193 6.2.7.5 ABI and Robot Maintenance Cost 193 6.2.7.6 Sanger Sequencing Staff Time Cost 194 6.2.8 Reporting 195 6.3 Rare Variants Detected Using Autosomal Recessive Filter 196 6.3.1 Family 1 196 6.2.2 Family 2 196 6.2.3 Family 3 196 6.2.3 Family 4 197 6.2.3 Family 5 197 6.2.3 Family 6 197 6.2.3 Family 7 198 6.2.3 Family 8 198 6.2.3 Family 9 198 6.2.3 Family 10 199 6.2.3 Family 11 200 6.4 A Units and C1 Credits 201 6.5 C1 Innovation Proposal 202 Word count: 38,678 7 List of Figures Description Page Number 1. Example inheritance patterns 19 2. Consanguinity across the world 25 3. Autosomal recessive inheritance in a consanguineous family 25 4. Variant filter for rare homozygous variants 75 5. Variant filter for rare heterozygous variants 77 6. Laboratory workflow 86 7. SRD5A3 c.57G>A p.(Trp19*) Sanger sequencing trace 92 8. SRD5A3 c.57G>A p.(Trp19*) gene diagram 93 9. CUL7 c.3898-715_3943del variant 100 10. CUL7 c.3898-715_3943del gene diagram 101 11. Family 7 TRAPPC9 c.1708C>T p.(Arg570*) variant 105 12. TRAPPC9 c.1708C>T p.(Arg570*) gene diagram 106 13. Family 8 DHTKD1 c.2185G>A p.(Gly729Arg) variant and pedigree 110 14. DHTKD1 c.2185G>A p.(Gly729Arg) gene diagram 111 15. UNC80 c.409C>T p.(Arg137*) Sanger sequencing trace 116 16. Flowchart of family outcomes 136 8 List of Tables Description Page Number 1. Degrees of relatedness 26 2. Commercial clinical exome library generation kits 40 3. Genetic testing methods 44 4. EMRGL test requests 52 5. Rare genetic disease testing location 52 6. ACMG variant classification criteria for pathogenicity 80 7. Sanger sequencing primer sequences 82 8. Summary of the recruited families 117 9. Summary of genes with pathogenic variants 118 10. Mid-Output vs High-Output sequencing kit metrics 121 11. NA12878 precision and recall 124 12. Number of variants detected 126 13. Variants in the ROH 129 9 List of abbreviations Abbreviation Explanation ACMG American College of Medical Genetics bp Base pair(s) BAC Bacterial artificial chromosome CDG Congenital disorder of glycosylation CES Clinical Exome Solution CGH Comparative genomic hybridisation ChAS Chromosome Analysis Software EMRGL East Midlands Regional Genetics Laboratory ESP Exome Sequencing Project Gb Giga base pair(s) HGMD Human Gene Mutation Database HGVS Human Genome Variation Society HRA Health Research Authority IF Incidental finding IGV Integrative Genomics Viewer INDEL Insertion/deletion variant Kb Kilo base pair(s) MAF Minimum allele frequency Mb Mega base pair(s) MDT Multi-disciplinary team MLPA Multiplex ligation-dependent probe amplification MRI Magnetic Resonance Imaging NGS Next-generation sequencing NHS National Health Service NMD Nonsense mediated decay NUH Nottingham University Hospitals OMIM Online Mendelian Inheritance in Man PCR Polymerase chain reaction PGD Pre-implantation genetic diagnosis PND Prenatal diagnosis REC Research Ethics Committee ROH Region of homozygosity SNP Single nucleotide polymorphism SNV Single nucleotide variant UK United Kingdom UKGTN United Kingdom Genetic Testing Network UPD Uniparental disomy VUS Variant of uncertain significance WES Whole exome sequencing 10 Abstract Patients from consanguineous backgrounds are at increased risk of having very rare, recessive genetic disorders making them susceptible to a diagnostic odyssey where it can take many years to achieve a genetic diagnosis. There is a need to update genetic testing methods in the National Health Service (NHS) to support patients with rare genetic diseases while taking into account the time and budget constraints imposed on the NHS. Research question: Can SNP microarray and clinical exome sequencing in a clinical diagnostic laboratory expedite a genetic diagnosis for patients with suspected rare genetic disorders who have consanguineous parents and form the basis of a cost-effective testing service? Overarching hypothesis: By highlighting cases most at risk of having homozygous pathogenic variants, prioritising gene agnostic testing and tailoring the analysis to the situation the chance of finding the causative variant would improve.