Genetic Mechanisms in
Colorectal Polyposis
Emma L. Short2018
A thesis submitted in candidature for the degree of Doctor of Philosophy
Division of Cancer and Genetics
School of Medicine
Cardiff University

Declarations
This work has not been submitted in substance for any other degree or award at thisor any other university or place of learning, nor is being submitted concurrently incandidature for any degree or other award.
Signed……………………………………(candidate)

Date…………………………

This thesis is being submitted in partial fulfilment of the requirements for the degreeof PhD.
Signed……………………………………(candidate)

Date…………………………

This thesis is the result of my own independent work/ investigation, except whereotherwise stated, and the thesis has not been edited by a third party beyond what ispermitted by Cardiff University’s policy on the Use of Third Party Editors by
Research Degree Students. Other sources are acknowledged by explicit
references. The views expressed are my own.
Signed……………………………………(candidate)

Date…………………………

I hereby give consent for my thesis, if accepted, to be available online in the
University’s Open Access repository and for inter-library loan, and for the title andsummary to be made available to outside organisations.
Signed……………………………………(candidate)

Date…………………………

i

Acknowledgements
First, I want to thank the patients, relatives and healthy volunteers who have taken
part in this study. Without you, this work would not have been possible. I reallyappreciate the time you have taken to talk to me and the blood/ tissue samples you
have donated. I sincerely hope that the results from this study will have a positive
impact on you, your families and future generations.
I’d like to thank my supervisors Professor Julian Sampson, Dr. Karen Reed and Dr.
Laura Thomas for giving me the opportunity to do this project. Thank you for yoursupport and for teaching me the skills and techniques that have allowed me tobecome a scientist as well as a clinician.

Thank you to The Pathological Society of Great Britain and Ireland for giving me thegrants that have allowed me to purchase necessary equipment and consumables.

Thank you to the Inherited Tumour Syndromes Research Group: to Iris and Hala forrecruiting patients and obtaining blood samples; to Hannah for her suggestions
about my thesis write-up and to Elena: you are a fantastic scientist and I reallyappreciate your help and advice.
Thanks go to Professor Geraint Williams – I’ve never come across someone so
fiercely intelligent and yet also so kind, helpful and approachable. You’re myinspiration for becoming an academic pathologist. Thank you for all yourencouragement, not just during this project but over the 10 years that I have been in
Cardiff.

Thanks also go to Drs. Daniel Dancer and Claudia Consoli for their help with qPCR;
to Professor Ian Tomlinson, Dr. Claire Palles and Laura Chegwidden for making me
so welcome in their laboratory group; to Dr. Ian Frayling for his advice about themismatch repair genes; to Dr. Pierre Rizkallah for his help with crystal modelling ofproteins; to the All Wales Medical Genetics Service, especially Justyna Tull for
arranging DNA extractions and Nelson Cottrim for doing the somatic APC
screening; to Dr. Andy Tee for his help with Western blotting; to Dr. Matthew Mortiifor his assistance with statistics and to all the staff in the Cardiff and Vale Cellular
Pathology Department for making so many slides for me.
I’d like to thank our international collaborators/ advisors Professor Eric Fearon,
Professor Raquel Seruca and AmbryGen. Thank you for being so helpful, and forsharing information and resources which have allowed the care of the patient to be
the number one priority.

Huge thanks go to Professor Trevor Dale, Dr. Anika Offergeld, Dr. Ken Ewan, Eider
Valle-Encinas and all of the Dale laboratory group for allowing me to join them forthe final phase of my project. Their help with the AXIN2 functional studies has been
invaluable, and I am extremely grateful for their time and support.
Most importantly, I’d like to give my heartfelt thanks to all my wonderful family for a
lifetime of love, support, encouragement and inspiration. I wouldn’t be where I am
today without you, and I am truly grateful for everything. Finally, I want to say a
special thank you to Steve, Bella and Bea, the most incredible husband and children
anyone could wish for. Thank you for being my everything and for filling every day
with happiness and joy. You mean the world to me.

iii

iv

Summary
Individuals with >/= 10 colorectal adenomas have traditionally been referred forgenetic testing to identify APC and MUTYH mutations which cause Familial
Adenomatous Polyposis (FAP) and MUTYH-Associated Polyposis (MAP)
respectively. Mutations are found in most patients with >100 adenomas but in only
a minority of those with 10-100 adenomas. The research described in this thesis
focuses on polyposis patients with ‘no mutation identified’ (NMI).
The aim of this project was to identify novel genetic mechanisms causing polyposis
in a cohort of 60 unrelated NMI patients. Genetic variants were sought outside of
the open reading frames of APC and MUTYH, at a low frequency in APC, in otherknown and candidate ‘polyposis genes’ and in whole exomes. Novel variants werecharacterised genetically and functionally to provide evidence for or against theirclinical significance.
Rare variants were found in the 5’UTR of APC, the mismatch repair (MMR) genes,
POLE, POLD1 and AXIN2. The 5’UTR APC variant, c.-190A>G, was associatedwith reduced transcript levels and segregated with FAP in a multiplex family,
allowing translation to diagnostic testing. Three additional patients had reduced
APC transcript levels, but the cause was not determined by deep sequencing of
their genomic APC loci. The MMR gene variants were deemed unlikely to bepathogenic as associated tumours were microsatellite stable with normal MMR
protein immunohistochemistry. Studies into the pol gene variants are ongoing. The
AXIN2 mutation, c.1642G>T, p.Glu548*, was identified in a family with polyposis
and ectodermal dysplasia. Most of their adenomas appeared to lack APC mutations
and in vitro studies suggested that the mutation may impair inhibition of Wntsignalling.
Gene panel testing using next-generation sequencing technologies may improve
molecular genetic diagnosis of previously NMI patients but additional
characterisation of novel variants is likely to be required for clinical translation, withthe ultimate aim of preventing colorectal cancer.

1

2

Table of Contents
Declarations ............................................................................................................... i
Acknowledgements .................................................................................................. ii
Summary.................................................................................................................... 1
Table of Contents ..................................................................................................... 3
List of Figures.......................................................................................................... 11
List of Tables ........................................................................................................... 13
Abbreviations .......................................................................................................... 15
Chapter 1

General Introduction ................................................................... 19

1.1

Background ............................................................................................. 19

1.2

Colorectal Carcinoma ............................................................................ 19

1.2.1

Chromosomal Instability (CIN), APC and the Wnt-Pathway............ 20

1.2.1.1 Adenomatous Polyposis Coli (APC) .................................................... 21
1.2.1.2 The Wnt pathway.................................................................................... 21
1.2.1.3 KRAS, SMAD4 and P53 ........................................................................ 23
1.3

Microsatellite Instability (MSI) ............................................................... 25

1.4

CpG Island Methylator Phenotype (CIMP) ......................................... 26

1.5

The Colorectal Polyposis Syndromes ................................................. 26

1.5.1

Colorectal Adenomas ............................................................................ 29

1.5.1.1 Familial Adenomatous Polyposis (FAP) ............................................. 29
1.5.1.2 MUTYH-Associated Polyposis (MAP) ................................................. 31
1.5.1.3 Polymerase Proofreading Associated Polyposis (PPAP) ................ 33
1.5.1.4 NTHL1-Associated Polyposis ............................................................... 34
1.5.1.5 MSH3-Associated Polyposis ................................................................ 35
1.5.2

Hyperplastic Polyps (HPPs).................................................................. 35

1.5.2.1 Serrated Polyposis Syndrome .............................................................. 36
1.5.3

Hamartomatous Polyps ......................................................................... 38

1.5.3.1 Peutz-Jegher’s Syndrome (PJS).......................................................... 38
1.5.3.2 Familial Juvenile Polyposis Syndrome................................................ 39
1.5.3.3 Cowden’s Syndrome .............................................................................. 39
1.5.3.4 Hereditary Mixed Polyposis Syndrome (HMPS) ................................ 40
1.6

Clinical Management of Patients with Colorectal Polyposis ............ 41

1.6.1

Why Might Some Genetic Variants Be Missed? ................................ 413

1.6.1.2 Promoter Variants and Allelic Imbalance (AI) .................................... 42
1.6.1.3 Intronic Variants...................................................................................... 44
1.6.1.4 Untranslated Region (UTR) Variants .................................................. 44
1.6.1.5 Mosaicism................................................................................................ 45
1.6.1.6 The Involvement of Additional Genes ................................................. 46
1.7

Summary ................................................................................................. 47

1.8

Aims of Thesis ........................................................................................ 47

Chapter 2Materials and Methods ............................................................... 49

2.1

Materials, Equipment and their Suppliers .......................................... 49

2.2

Patient Information ................................................................................. 52

2.2.1

Inclusion Criteria ..................................................................................... 52

2.2.2

Exclusion Criteria ................................................................................... 53

2.2.3

Individuals Participating in this Study.................................................. 53

2.2.3.1 No Mutation Identified (NMI) Polyposis Patients ............................... 53
2.2.3.2 Control Samples ..................................................................................... 54
2.2.3.4 Demographic Details and Clinical Phenotypes ................................. 54
2.3

General Techniques .............................................................................. 65

2.3.1

Agarose Gel Electrophoresis................................................................ 65

2.3.2

Germline DNA Extraction ...................................................................... 65

2.3.3

RNA Extraction ....................................................................................... 66

2.3.3.1 Assessment of RNA Quality ................................................................. 66
2.3.3.2 RNA Conversion to cDNA ..................................................................... 67
2.3.4

DNA Extraction from FFPE Tissue ...................................................... 67

2.3.5

Quantification of Nucleic Acids ............................................................ 68

2.3.6

Primer Design ......................................................................................... 68

2.3.7

Bisulfite Conversion of DNA ................................................................. 68

2.3.8

Standard PCR and Sanger Sequencing ............................................. 69

2.3.8.1 Standard Reagents for PCR ................................................................. 70
2.3.8.2 Reagents for PCR for FFPE Tissue DNA .......................................... 70
2.3.8.3 Reagents for Fast-COLD-PCR............................................................. 70
2.3.8.4 Reagents for Full-COLD-PCR .............................................................. 70
2.3.8.5 Standard PCR Reaction Conditions: ................................................... 71
2.3.8.6 Reaction Conditions for Fast-COLD PCR: ......................................... 71
2.3.8.7 Reaction Conditions for Full-COLD-PCR: .......................................... 71
2.3.9

Gel Electrophoresis................................................................................ 72

2.3.10

ExoSap PCR Purification ...................................................................... 72

4

2.3.11

Big Dye Reaction .................................................................................... 72

2.3.12Isopropanol Clean-Up Protocol ............................................................ 73

2.3.13

Sequencing and Data Interpretation.................................................... 73

2.3.14

Methylation-Specific PCR (MSP) ......................................................... 73

2.3.14.1 Reagents for APC Promoter 1A MSP ................................................. 73
2.3.14.2 Reaction Conditions for APC Promoter 1A MSP .............................. 73
2.3.14.3 Reagents for APC Promoter 1B MSP ................................................. 74
2.3.14.4 Reaction Conditions for APC Promoter 1B MSP .............................. 74
2.3.15

Quantitative PCR (qPCR) ..................................................................... 74

2.3.15.1 qPCR Reagents ...................................................................................... 75
2.3.15.2 qPCR Reaction Conditions ................................................................... 75
2.3.16

APC and MUTYH Expression in Healthy Controls............................ 75

2.3.16.1 qPCR in a Healthy Control Cohort ....................................................... 76
2.3.16.2 Data Analysis .......................................................................................... 76
2.4

Next Generation Sequencing (NGS) ................................................... 77

2.4.1

Haloplex Assay ....................................................................................... 77

2.4.2

Target Gene Capture and Sequencing ............................................... 77

2.4.3

Bioinformatic Analysis Following UDS ................................................ 78

2.4.4

Whole Exome Sequencing (WES) ....................................................... 78

2.4.5

Bioinformatic Analysis Following Whole Exome Sequencing ......... 79

2.4.6
Single Molecule Molecular Inversion Probe Gene Capture,
Sequencing and Bioinformatic Analysis.............................................................. 80
2.5

Molecular Biology Techniques ............................................................. 84

2.5.1

Plasmid Retrieval from Filter paper ..................................................... 84

2.5.2

Preparation of Lysogeny Broth (LB) .................................................... 84

2.5.3

Preparation of Lysogeny Broth (LB) Agar .......................................... 84

2.5.4

Bacterial Transformation of XL1-Blue Competent Cells .................. 85

2.5.5

Plasmid Extraction from Transformed Bacteria ................................. 86

2.5.5

Sequencing of Plasmid DNA ................................................................ 86

2.5.6
Site Directed Mutagenesis (SDM) and Transformation of XL-1 Blue
Competent Cells ..................................................................................................... 86
2.5.7

Defrosting HEK293 TCF-Luc Cells ...................................................... 88

2.5.8

Splitting Cells .......................................................................................... 88

2.5.9

Plating the Cells for Transfection ......................................................... 89

2.5.10Transfection ............................................................................................. 89

2.5.11 Stimulation with Recombinant Human Wnt-3a and Human RSpondin-1 ................................................................................................................ 91
2.5.12

WST and Luciferase Assays ................................................................ 915

2.5.13

Cell Lysis and Protein Extraction ......................................................... 91

2.5.14

Protein Quantification using a Bicinchoninic Acid (BCA) Assay ..... 92

2.5.15Western Blotting ..................................................................................... 93

2.5.16

Stripping Nitrocellulose Membranes and -Actin Detection ............ 94

Chapter 3

APC and MUTYH in Colorectal Polyposis ............................. 95

3.1

Introduction.............................................................................................. 95

3.2

Methods ................................................................................................... 97

3.2.1

Quantitative PCR (qPCR): APC and MUTYH Transcription ........... 97

3.2.2

Karyotype Analysis................................................................................. 99

3.2.3

Promoter Methylation Studies .............................................................. 99

3.2.4

RNA Studies: Allelic Imbalance (AI) and Splicing Abnormalities .. 99

3.2.4.1 gDNA analysis ...................................................................................... 100
3.2.4.2 RNA Analysis ........................................................................................ 100
3.2.4.3 APC and MUTYH Splicing Abnormalities ......................................... 100
3.2.4.3.1 APC cDNA Sequencing ...................................................................... 100
3.2.4.3.2 MUTYH cDNA Sequencing................................................................. 101
3.2.5

APC and MUTYH Capture and Ultradeep Sequencing (UDS) ..... 101

3.2.5.1 Variant Analysis and Selection .......................................................... 102
3.2.5.2 Validation of Identified Variants ......................................................... 103
3.3

Results ................................................................................................... 103

3.3.1

Quantitative PCR (qPCR): APC and MUTYH Expression ............. 103

3.3.2

Karyotype Analysis............................................................................... 107

3.3.3

Promoter Methylation Studies ............................................................ 107

3.3.4

Allelic Imbalance Studies .................................................................... 109

3.3.4.1 APC Allelic Imbalance ......................................................................... 109
3.3.4.2 MUTYH Allelic Imbalance ................................................................... 110
3.3.5

APC and MUTYH Splicing Abnormalities ......................................... 110

3.3.5

APC and MUTYH Capture and Ultradeep Sequencing (UDS) ..... 110

3.3.5.1 Coverage of Ultradeep Sequencing .................................................. 110
3.3.5.2 Control Results ..................................................................................... 111
3.3.5.3 Patient Results...................................................................................... 112
3.4

Discussion ............................................................................................. 116

3.4.1

Gene Expression Studies and Allelic Imbalance ............................ 116

3.4.2

Abnormal Splicing ................................................................................ 120

3.4.3

Ultradeep Sequencing ......................................................................... 120

3.4.3.1 Validation of Low Frequency Variants .............................................. 120

6

3.4.3.2 Validated Variants ................................................................................ 121
3.4.3.2.1 Validated Variants in APC ................................................................... 121
3.4.3.2.2 Validated Variants in MUTYH ............................................................. 123
3.4.3.3 Summary of Gene Capture and Ultradeep Sequencing ................ 123
3.4.4

Chapter Conclusions............................................................................ 124

Chapter 4 Exome Sequencing in Polyposis Patients using Targeted
and Whole Exome Approaches ...................................................................... 127
4.1

Introduction ............................................................................................ 127

4.2

Targeted Exome Screening and Sequencing .................................. 127

4.2.1.1 NTHL1 .................................................................................................... 128
4.2.1.2 BMPR1A and SMAD4.......................................................................... 128
4.2.1.3 CDH1 ...................................................................................................... 128
4.2.1.4 CHEK2 ................................................................................................... 129
4.2.1.5 EPCAM and the MMR Genes: MLH1, MSH2, MSH6, PMS2 ....... 129
4.2.1.6 POLE and POLD1 ................................................................................ 130
4.2.1.7 PTEN ...................................................................................................... 130
4.2.1.8 STK11 .................................................................................................... 130
4.2.1.9 TP53 ....................................................................................................... 130
4.2.1.10 AXIN2 ..................................................................................................... 131
4.2.2

Aims and Objectives: Targeted Exome Sequencing ..................... 131

4.2.3

Methods ................................................................................................. 131

4.2.3.1 Screening NTHL1 ................................................................................. 132
4.2.3.2 Haloplex Assay and Sequencing ....................................................... 132
4.2.3.2.1 Targeted Exome Capture, Sequencing and Data Analysis ........... 132
4.2.3.3 Validation of Identified Variants.......................................................... 133
4.2.3.4 Confirmation of the Variants in cDNA ............................................... 133
4.2.4

Results ................................................................................................... 133

4.2.4.1 Screening for the common mutations in NTHL1 ............................. 133
4.2.4.2 Haloplex Assay and Sequencing ....................................................... 133
4.2.4.2.1 Metrics of UDS ...................................................................................... 134
4.2.4.2.2 Variants Selected for Validation ......................................................... 135
4.2.4.2.3 Confirmation of the Variants in cDNA ............................................... 140
4.2.5

Further Assessment of Validated Variants ....................................... 140

4.2.5.1 CHEK2 ................................................................................................... 142
4.2.5.2 POLE and POLD1 ................................................................................ 143
4.2.5.3 CDH1 ...................................................................................................... 143

7

4.2.5.4 MSH2, MSH6, MSH6, MLH1, EPCAM ............................................. 143
4.2.5.5 AXIN2 ..................................................................................................... 143
4.2.5.6 TP53 ....................................................................................................... 144
4.3

Whole Exome Sequencing ................................................................. 145

4.3.1

Methods ................................................................................................. 148

4.3.1.1 Sequencing ........................................................................................... 148
4.3.1.2 Sequence Analysis .............................................................................. 149
4.3.1.3 Variant Analysis and Selection .......................................................... 149
4.3.1.4 Validation of Identified Variants ......................................................... 151
4.3.2

Results ................................................................................................... 151

4.3.2.1 Coverage ............................................................................................... 151
4.3.2.2 Variants Selected for Validation ......................................................... 153
4.4

Discussion ............................................................................................. 167

4.4.1

Targeted Exome Sequencing ............................................................. 167

4.4.2

Whole Exome Sequencing ................................................................. 169

4.5

Summary ............................................................................................... 173

Chapter 5 Functional Characterisation of Variants Identified in the
Mismatch Repair Genes, POLE, POLD1 and AXIN2 ................................. 175
5.1

Introduction............................................................................................ 175

5.2

The Mismatch Repair (MMR) Genes ................................................ 175

5.2.1

Introduction............................................................................................ 175

5.2.2

Database Interrogation ........................................................................ 176

5.2.3

MMR Immunohistochemistry (IHC) ................................................... 177

5.2.4

Microsatellite Instability (MSI) Testing .............................................. 177

5.2.5

Methods ................................................................................................. 177

5.2.5.1 Database Interrogation ........................................................................ 178
5.2.5.2 MMR IHC ............................................................................................... 178
5.2.5.3 MSI Testing ........................................................................................... 179
5.2.6

Results ................................................................................................... 179

5.2.6.1 Database Interrogation ........................................................................ 179
5.2.6.2 MMR IHC ............................................................................................... 180
5.2.6.3

MSI Testing .......................................................................................... 181

5.2.7

Conclusions of MMR Gene Investigations ....................................... 181

5.3

POLE and POLD1 ................................................................................ 183

5.3.1

Introduction............................................................................................ 183

5.3.2

Methods ................................................................................................. 186

8

5.3.2.1 In Silico Modelling of Pol Variants ..................................................... 186
5.3.2.2 Somatic ‘Hot Spot’ Mutation Screening in APC and KRAS ........... 186
5.3.2.3 MMR IHC and Microsatellite Stability of Tumours in Carriers of Pol
Gene Variants ....................................................................................................... 187
5.3.2.4 Segregation Analysis ........................................................................... 187
5.3.2.5 Investigation into the Mutation Signature of Tumours .................... 188
5.3.3

Results ................................................................................................... 189

5.3.3.1 Modelling of Pol Variants .................................................................... 189
5.3.3.2 Somatic ‘Hot Spot’ Mutation Screening in APC and KRAS ........... 191
5.3.3.3 MMR IHC and Microsatellite Stability of Tumours in Carriers of Pol
Gene Variants ....................................................................................................... 191
5.3.3.4 Segregation Analysis ........................................................................... 191
5.3.3.5 Investigation into the Mutation Signature of Tumours .................... 191
5.3.4

Conclusions of POLE/ POLD1 Gene Interrogation ......................... 192

5.4

AXIN2 and AXIN2-Associated Polyposis (AxAP) ............................ 195

5.4.1

Introduction ............................................................................................ 195

5.4.2

Halo47 and Halo68: Family History and Clinical information ....... 197

5.4.3

AXIN2: c.1642G>T:p.Glu548* ............................................................ 202

5.4.4

Studies to Investigate the Mutation ................................................... 202

5.4.4.1 Family Segregation Studies ................................................................ 202
5.4.4.2 Protein Analysis to Confirm that the AXIN2 Mutation Produces a
Truncated Protein ................................................................................................. 202
5.4.4.3 β-catenin IHC on FFPE Tumours to Assess Somatic Activation of
the Wnt Pathway .................................................................................................. 203
5.4.4.4 Somatic APC Screening ...................................................................... 203
5.4.4.5 AXIN2 Loss of Heterozygosity (LOH) Analysis ................................ 204
5.4.4.6 Functional Characterisation of AXIN2 c.1642G>T:p.Glu548* ....... 205
5.4.5

Methods ................................................................................................. 205

5.4.5.1 Family Segregation Studies ................................................................ 205
5.4.5.2 Protein Analysis: Confirmation of a Truncated Protein .................. 208
5.4.5.3 β-Catenin Immunohistochemistry (IHC)............................................ 208
5.4.5.4 Somatic APC Mutations in Colorectal Neoplasms .......................... 209
5.4.5.5 AXIN2 Loss of heterozygosity Analysis (LOH) ................................ 209
5.4.5.6 Functional Characterisation of the AXIN2 Variant .......................... 209
5.4.5.6.1 AXIN2-Containing Plasmids ............................................................... 209
5.4.5.6.2 Site Directed Mutagenesis (SDM) ..................................................... 210
5.4.5.6.3 The Luciferase Reporter Assay in HEK293 TCF-Luc Cells ........... 210
5.4.6

Results ................................................................................................... 210

95.4.6.1 Family Segregation Studies................................................................ 210
5.4.6.2 Protein Analysis .................................................................................... 211
5.4.6.3 β-Catenin Immunohistochemistry (IHC) ........................................... 212
5.4.6.4 Somatic APC mutations in colorectal neoplasms ........................... 219
5.4.6.4.1 Correlation between β-Catenin Immunohistochemistry (IHC) and
Somatic APC Mutations ...................................................................................... 222
5.4.6.5 AXIN2 Loss of Heterozygosity Analysis (LOH) ............................... 222
5.4.6.6 Functional Characterisation of the AXIN2 Variant .......................... 223
5.4.7

Conclusions of Studies into AXIN2 c.1642G>T:p.Glu548* ............ 225

5.5

Chapter Conclusions ........................................................................... 231

Chapter 6

Thesis Discussion ..................................................................... 233

6.1

APC and MUTYH Studies ................................................................... 234

6.1.1

Strengths and Weaknesses of the Study of APC and MUTYH .... 235

6.1.2

Clinically Translatable Outcomes ...................................................... 236

6.2

Targeted and Whole Exome Sequencing ........................................ 237

6.2.1
Strengths and Weaknesses of Targeted and Whole Exome
Sequencing in NMI Polyposis Patients ............................................................. 238
6.2.2

Recommendations Resulting from Exome Sequencing ................ 239

6.3
Functional Characterisation of Variants Identified in the Mismatch
Repair Genes, POLE, POLD1 and AXIN2 ....................................................... 239
6.3.1

The MMR Genes .................................................................................. 239

6.3.2

POLE and POLD1 ................................................................................ 240

6.3.3

AXIN2 c.1642G>T:p.Glu548* ............................................................. 240

6.4

Thesis Conclusions .............................................................................. 242

References ............................................................................................................ 245
Appendices are available in the attached CD.

10

List of Figures
1.1

Genes involved in the malignant progression of colorectal tumours

21

1.2

The Wnt-pathway

22

1.3

Genes and signalling pathways in inherited polyposis syndromes

28

1.4

Actions of MUTYH in the repair of oxidative DNA damage

31

3.1

Studies employed in the interrogation of APC and MUTYH

96

3.2

MSP for APC promoter 1A

108

3.3

MSP for APC promoter 1B

108

3.4

Family tree of Halo46

117

4.1

WES variant analysis and selection

150

5.1

Representative images of MMR IHC

181

5.2

Family tree of Family A

199

5.3

Patient phenotypes

200

5.4

Family tree of Family A illustrating recruited individuals

206

5.5

Western blot to demonstrate truncated AXIN2

212

5.6

-catenin IHC: FAP control patient

214

5.7

-catenin IHC: patient results in hyperplastic lesions

215

5.8

-catenin IHC: patient results for tubular adenomas

216

5.9

-catenin IHC: Individual 1.2 results in VA

217

5.10

-catenin IHC: Halo47 results in CRC

218

5.11

-catenin IHC: Individual 1.1 results in CRC

219

11

5.12

Graph to show the effects of different AXIN2 mutations on Wnt-

223

pathway inhibition

12

List of Tables
2.1

Demographic details/ clinical phenotypes of NMI patients and55

their relatives
2.2

Further control samples included in this study

63

2.3

Taqman assays used in qPCR experiments

76

2.4

Healthy control samples used in qPCR experiments

77

2.5

BSA protein standards

92

3.1

FAP and MAP control samples used in transcription studies

98

3.2

Results for APC and MUTYH gene transcription studies in

104

control samples
3.3

Phenotypes of patients with reduced transcription of APC or

106

MUTYH
3.4

Control samples used for validating Haloplex gene capture and

112

UDS
3.5

Summary of validated APC and MUTYH variants

114

3.6

Summary of the key results from Chapter 3

115

4.1

Coverage of the 15 genes on the Haloplex assay

134

4.2

Variants selected for validation following targeted exome capture136

and ultradeep sequencing
4.3

AmbryGen classification of validated variants

141

4.4

Patients and relatives undergoing whole exome sequencing

145

4.5

Patient-specific depth of coverage for whole exome sequencing

151

4.6

Whole exome sequencing validation

154

13

5.1

Variants in the MMR genes identified through targeted exome

176

sequencing
5.2

Results of MMR gene database interrogation

180

5.3

Variants in the pol genes identified through targeted exome

184

sequencing
5.4

Three-dimensional location of variants in POLE/ POLD1 and190

their predicted functional consequences
5.5

Demographic and phenotypic information about recruited family

206

members
5.6

Somatic APC sequencing results

220

5.7

Correlation between -catenin IHC and somatic APC mutations

222

14

Abbreviations°C

Degrees Celsius

µ

Micro

µg

Microgramµl

Microlitreµm

Micrometre

8-oxodG8-hydroxyguanine

AD

Autosomal Dominant

AFAP

Attenuated Familial Adenomatous Polyposis

AI

Allelic Imbalance

APC

Adenomatous Polyposis ColiAR

Autosomal Recessive

AWMGS

All Wales Medical Genetics Service

BCA

Bicinchoninic Acid

BER

Base Excision Repair

BMP

Bone Morphogenetic Protein

Bp

Base Pair

BR

Broad Range

BSA

Bovine Serum Albumin

CADD

Combined Annotation Dependent Completion

cDNA

Complementary Deoxyribonucleic Acid

CHRPE

Congenital Hypertrophy of the Retinal Pigment Epithelium

CIMP

CpG Island Methylator Phenotype

CIN

Chromosomal Instability

COOT

Crystallographic Object-Oriented Toolkit

CRC

Colorectal CancerdAMP

Deoxyadenosine MonophosphatedbSNP

Single Nucleotide Polymorphism Database

DDD

Digital Differential Display

DGGE

Denaturing Gradient Gel Electrophoresis

DHPLC

Denaturing High Performance Liquid Chromatography

DNA

Deoxyribonucleic AciddNTP

Deoxyribose Nucleoside TriphosphatedsDouble Stranded

15

Dsh

Dishevelled

EB1

End Binding Protein 1

EDTA

Ethylenediaminetetraacetic Acid

FAP

Familial Adenomatous Polyposis

FBS

Fetal Bovine Serum

FFPE

Formalin Fixed Paraffin Embedded

gDNA

Genomic DNA

GDP

Guanosine Diphosphate

GSK3βGlycogen Synthase Kinase 3β

GTP

Guanosine Triphosphate

HA

Heteroduplex Analysis

HF

High Fidelity

HGD

High Grade Dysplasia

HGMD

Human Gene Mutation Database

HMPS

Hereditary Mixed Polyposis Syndrome

HP/ HPP

Hyperplastic PolypHPSHyperplastic Polyposis Syndrome

HS

High Sensitivity

IGV

Integrative Genomics Viewer

IMG

Institute of Medical Genetics

IVSP

In Vitro Synthesised Protein Assay

JPS

Juvenile Polyposis Syndrome

k

Kilo

Kb

Kilobase

kDa

Kilodalton

LB

Lysogeny BrothLDS

Lithium Dodecyl Sulphate

LEF

Lymphoid Enhancer Factor

LGD

Low Grade Dysplasia

LOH

Loss of Heterozygosity

LS

Lynch Syndrome

M

Milli

MAP

MUTYH-Associated Polyposis

MCR

Mutation Cluster Region

MES

2-(N-morpholino) ethanesulphonic acidmiRNA

Micro Ribonucleic Acid

16

ml

Millilitre

MLPA

Multiplex Ligation-Dependent Probe Analysis

MMG

Megamix Gold

MMR

Mismatch Repair

M-PVA

Polyvinyl Alcohol Particle Magnetic Beads

mRNA

Messenger Ribonucleic Acid

MSI

Microsatellite Instability

MSP

Methylation Specific PCR

n

Nano

NER

Nucleotide Excision Repair

NFQ

Non-Fluorescent Quencher

Ng

Nanogram

NGS

Next Generation Sequencing

NHS

National Health Service

NMD

Nonsense Mediated Decay

NMI

No Mutation Identified

NTC

No Template Control

ORF

Open Reading Frame

PBS

Phosphate Buffered Saline

PCR

Polymerase Chain reaction

PDB

Protein Databank

PI3K

Phosphatidylinositol-4,5-bisphosphate 3-kinase

PJS

Peutz-Jegher’s Syndrome

Poly

Polymorphism

PPAP

Polymerase Proofreading Associated Polyposis

PTT

Protein Truncation Test

Px

Patient

QC

Quality Control

qPCR

Quantitative Polymerase Chain Reaction

Rcf

Relative Centrifugal ForceREC

Research Ethics Committee

RNA

Ribonucleic Acid

RNase

Ribonuclease

RNAsinRibonuclease Inhibitor

ROS

Reactive Oxygen Species

Rpm

Revolutions Per Minute

17

RT

Reverse Transcription

S

Seconds

SDM

Site Directed Mutagenesis

SDS

Sodium Dodecyl SulphatesmMIPsSingle Molecule Molecular Inversion Probes

SNP

Single Nucleotide Polymorphism

SOC

Super Optimal Broth with Catabolite Repression

SOP

Standard Operating Procedure

SPS

Serrated Polyposis Syndrome

ss

Single Stranded

SSA

Sessile Serrated Adenoma

SSCP

Single Strand Conformation Polymorphism

SSP

Sessile Serrated PolypTA

Tubular Adenoma

Ta

Annealing Temperature

TAE

Tris Base, Acetic Acid and Ethylenediaminetetraacetic AcidTBS

Tris-Buffered SalineTC

Tissue Culture

TCF

T Cell Factor

TE Buffer

Tris EDTA Buffer

TGFβTransforming Growth Factor βTmMelting Temperature

TSA

Traditional Serrated Adenoma

TVA

Tubulovillous Adenoma

U

Units

UCSC

University of California Santa Cruz

UDS

Ultra-Deep Sequencing

UNG

Uracil-N-Glycosylase

UTR

Untranslated Region

UV

Ultraviolet

V

Volts

VLB

Variant Likely Benign

VLP

Variant Likely Pathogenic

VUS

Variant of Unknown Significance

VA

Villous Adenoma

WGP

Wales Gene Park18

Chapter 1
1.1

General Introduction

BackgroundIn 2016, cancer was the leading cause of mortality in England and Wales,
accounting for 28.5% of all deaths (Office for National Statistics, 2017). It isestimated that the lifetime risk of developing any malignant neoplasm, excluding
non-melanoma skin cancer, is 40% for males and 37% for females (Sasieni et al2011). Cancer refers to the process in which cells grow uncontrollably. The
transformation of a normal cell into a malignant one is a multistep process, in which
several genetic alterations occur, allowing the cell to escape from normal control
mechanisms (Hanahan and Weinberg, 2000). The genetic changes involve theactivation of oncogenes and the silencing of tumour suppressors (Hanahan andWeinberg, 2011). These changes allow the cell to attain a growth advantage, forexample through self-sufficiency in growth signals, insensitivity to growth inhibitory
signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis,
tissue invasion/ metastasis, reprogramming of cellular energy metabolism andimmune evasion (Hanahan and Weinberg, 2011; Hanahan and Weinberg, 2000).

In order to acquire these advantageous traits, there is a succession of alterations in
the genomes of neoplastic cells (Hanahan and Weinberg, 2011). Mutations may
occur spontaneously or may be triggered by external factors such as viruses,
radiation and chemicals. In addition to mutation events, gene expression can bealtered through epigenetic mechanisms such as DNA methylation, histone
modification and micro-RNA (miRNA) expression (Berdasco and Esteller 2010;
reviewed in Jones and Baylin 2007). Ultimately, most cancers develop as a resultof a complex interaction between an individual’s genetic make-up and theirenvironment.

1.2

Colorectal Carcinoma

Colorectal carcinoma (CRC) is the fourth most common cancer in the United
Kingdom (UK) and the second most common cause of cancer death (Cancer
Research UK 2015). Between 2013- 2015, approximately 41 700 people werediagnosed with CRC, which is more than 110 people every day (Cancer Research
UK 2015). An individual’s lifetime risk of developing CRC is 5%, but this figure

19increases dramatically with age (reviewed in Fodde 2002). The incidence of CRC isgenerally high in developed countries, with a 20-fold difference in incidence rates
between high- and low-risk geographical areas (reviewed in Fodde 2002). The
difference is thought to largely result from environmental factors, in particulardifferences in diet (Fodde 2002).

The majority of CRCs occur sporadically, but in 15-35% of patients, hereditary
factors are important (reviewed in Mishra and Hall 2012; Burt 2007). In
approximately 5% of cases, the disease is caused by a highly penetrant dominantly
inherited syndrome (reviewed in Mishra and Hall 2012). The most common is
Lynch Syndrome, due to inherited defects in the mismatch repair (MMR) system.
This accounts for 2-5% of cases of CRC (reviewed in Mishra and Hall 2012).
Familial Adenomatous Polyposis (FAP), due to germline mutations in the APC gene,is responsible for <1% of the disease burden and non-syndromic familial
presentations comprise 20% of cases (reviewed in Mishra and Hall 2012).

CRCs result from the progressive accumulation of genetic and epigenetic alterations
which cause normal colonic epithelium to transform into adenocarcinoma (Grady
and Carethers 2008). Recent work based on gene expression profiling (Guinney etal 2015) suggests that CRC can be classified into 4 molecular subtypes: MSI
Immune, Canonical, Metabolic and Mesenchymal. However, the traditional
approach has been to categorise tumours into 3 groups: those with chromosomal
instability (CIN), those with microsatellite instability (MSI) and those with ahypermethylated phenotype (CpG Island Methylator Phenotype or ‘CIMP’). There isconsiderable overlap between the latter two groups.

1.2.1 Chromosomal Instability (CIN), APC and the WntPathway
The vast majority of CRCs develop from pre-existing adenomas. Such tumours arecharacterised by chromosomal instability, which is seen in 80-85% of colorectal
tumours (reviewed in Grady and Carethers 2008). There are certain key genetic
mutational events which occur, allowing the progression from normal epithelium, todysplasia, and finally invasive malignancy. The loss of adenomatous polyposis coli
(APC) gene function seems to be the initiating event, followed by mutations in
KRAS, SMAD4 and p53 (Fodde 2002). In keeping with Knudson’s two hit
hypothesis of tumourigenesis, two mutational events are required to knock out the20

functioning of a tumour suppressor gene (Knudson 1971), whilst activation of an
oncogene requires only one mutation.

Figure 1.1: Image from Walther et al (2009) illustrating the progressive accumulation ofgenetic changes involved in the malignant progression of colorectal tumours

1.2.1.1

Adenomatous Polyposis Coli (APC)

The APC gene is found on chromosome 5 (5q22.2). It consists of 8535 coding basepairs, encoding a 2843 amino acid multidomain protein. Exon 15 is responsible formore than 75% of the coding sequence of the gene and is the most common site forgermline and somatic mutations (reviewed in Fearnhead et al 2002; reviewed in
Fearnhead et al 2001). 95% of CRC-associated APC mutations are nonsense orframeshift mutations, creating a truncated protein with abnormal function (Bodmer
1999).
The APC protein is a 312kDa tumour suppressor, which is involved in many cellular
processes including intercellular adhesion, signal transduction, proliferation,
apoptosis and migration. One of its major roles is in regulating cytoplasmic levels of
β-catenin, thus negatively regulating Wnt signalling (Mishra and Hall 2012; reviewed
in Fearnhead et al 2002; Fodde 2002; Fearnhead et al 2001).

1.2.1.2

The Wnt pathway

The Wnt proteins are a family of signalling proteins which are involved indevelopmental events during embryogenesis and in adult tissue homeostasis(Logan and Nusse 2004). They have multiple effects within a cell, including

21

triggering cell division, cell fate specification and differentiation (Logan and Nusse
2004).

Wnt proteins bind to Frizzled/ low density lipoprotein receptor-related proteins,which are found on cell surface membranes (Logan and Nusse 2004). Thistransduces a signal to intracellular proteins, including Dishevelled (Dsh), GlycogenSynthase Kinase-3β (GSK-3), AXIN, APC and β-catenin (Logan and Nusse 2004).
In the absence of Wnt signalling, β-catenin levels are usually low: a complex
composed of GSK-3, APC and AXIN targets it for ubiquitin-mediated degradation(Logan and Nusse 2004; reviewed in Fearnhead et al 2002; reviewed in Fearnhead
et al 2001). When cells receive Wnt signals, the degradation pathway is inhibited.
This allows β-catenin to accumulate in the cytoplasm and nucleus. In the nucleus, itcomplexes with one of the T cell factor (TCF) or lymphoid enhancer factor (LEF)
transcription factors, to initiate transcription of a range of genes, including c-myc
and cyclin D1 (Logan and Nusse 2004; reviewed in Fearnhead et al 2002; reviewed
in Fodde 2002; reviewed in Fearnhead et al 2001). Myc and cyclin D1 are both
relevant to tumourigenesis as they have roles in proliferation, apoptosis and cellcycle progression (reviewed in Fodde 2002).

Figure 1.2: Image from Logan and Nusse (2004) illustrating the Wnt pathway in both theabsence and presence of Wnt stimulating molecules

In the normal intestinal epithelium, nuclear β-catenin expression is higher in the
proliferative component, and APC levels are higher in post-replicative cells(reviewed in Fodde 2002). These findings support β-catenin signalling having a role22

in maintaining stem cell properties and controlling differentiation in the bowel(reviewed in Fodde 2002). As cells move along the crypt-villous axis, increasinglevels of APC counteract β-catenin signalling and allow differentiation to occur(reviewed in Fodde 2002). APC mutations hence allow increased numbers of stem
cells and reduced cellular differentiation (Fodde 2002).

In addition to their roles in the initial stages of the adenoma-carcinoma sequence,
APC mutations remain important throughout malignant progression. Nuclear βcatenin staining strongly correlates with tumour size and dysplasia, and high levelsof nuclear β-catenin have been found at the invasive fronts of adenocarcinomas(reviewed in Fodde 2002). The APC protein is also involved in chromosomal
stability at mitosis: it has an EB1-binding domain in its C-terminal end, whichassociates with the growing ends of cytoplasmic and spindle microtubules, as wellas centrosomes. APC mutant cells are hence characterised by chromosomal
instability which is observed in the majority of CRCs (reviewed in Grady andCarethers 2008; reviewed in Fodde 2002).

APC mutations can therefore be seen to have a key role in both initiating andpromoting CRC: activation of the Wnt pathway affects cell proliferation, apoptosis,
and possibly differentiation of intestinal stem cells (Fodde 2002) and at later stagesof carcinogenesis, CIN resulting from APC mutations can accelerate tumour
progression (Fodde 2002).

1.2.1.3

KRAS, SMAD4 and P53

As illustrated in Figure 1.1, further genes involved in colorectal tumourigenesis
include KRAS, SMAD4, and p53. The importance of ras gene mutations incolorectal carcinogenesis was first reported in 1987 (Bos et al 1987; Forrester et al1987). The K-ras oncogene has been found to be mutated in 10-15% of adenomas<1cm, and in 30-60% of adenomas >1cm and carcinomas (reviewed in Brink et al
2003; reviewed in Fearon and Vogelstein 1990). The gene encodes a 21kDA
protein located in the inner plasma membrane, with intrinsic GTPase activity. It isinvolved in the transduction of mitogenic signals (reviewed in Brink et al 2003). It is
activated by a diverse spectrum of extracellular stimuli, such as growth factors,
cytokines and hormones (reviewed in Brink et al 2003; Shields et al 2000). Once
activated, it stimulates a multitude of downstream signalling cascades, including the23Raf serine/ threonine kinases, phosphoinositide 3-kinases (PI3Ks) and a family of
GDP-GTP exchange factors (reviewed in Shields et al 2000).
Mutant KRAS has impaired GTPase activity, meaning that it is constitutively active(reviewed in Brink et al 2003). This can cause uncontrolled cell growth andproliferation. A KRAS mutation in a colonic epithelial cell which already has APC
mutations results in a clonal expansion and increased risk of progression to cancer(reviewed in Vogelstein and Kinzler 2004).

The SMAD4 gene is on chromosome 18q. It was first identified as a tumour
suppressor gene in pancreatic cancer in 1996 (Hahn et al 1996). SMAD4 mediates
the TGFβ signalling pathway to suppress epithelial growth (reviewed in Miyaki andKuroki 2003). The SMAD4 protein acts as a trimer and forms complexes withadditional SMAD proteins: receptor-phosphorylated SMAD2 and SMAD3(Woodford-Richens et al 2001). These complexes then translocate from thecytoplasm to the nucleus and associate with DNA binding factors to facilitate thetranscription of target genes, including cyclin-dependent kinase inhibitors such asp15(ink4B) and the inhibitory SMAD7 (Woodford-Richens et al 2001). Loss of
SMAD4 function may result in the loss of transcription of genes necessary for cellcycle control (Woodford-Richens et al 2001). Cells may therefore become TGF-βresistant and escape from TGF-β-mediated growth control and apoptosis(Woodford-Richens et al 2001). SMAD4 is mutated in a significant proportion ofcolorectal tumours, with the frequency of mutational events increasing with theprogression of carcinogenesis: it is mutated in 0% of adenomas, 10% of‘intramucosal carcinomas’, 7% of carcinomas without metastases and 35% ofcarcinomas with distant metastases (reviewed in Miyaki and Kuroki 2003).
In 1988, it was reported that 73% of CRCs, 47% of ‘advanced adenomas’ and 1113% of ‘early stage adenomas’ had a deletion of part of chromosome 17 (Vogelstein
et al 1988). This region was subsequently shown to include the p53 gene (17p13.1)(Baker et al 1989). The p53 protein is a transcription factor which has a vital role inmaintaining genomic stability (reviewed in Sarasqueta et al 2013). Following DNA
damage, p53 activation causes arrest of the cell cycle to allow DNA repair (reviewed
in Sarasqueta et al 2013). If the damage is too extensive, p53 can drive a cell
towards senescence or apoptosis (reviewed in Sarasqueta et al 2013; reviewed in
Vogelstein and Kinzler 2004). The functional loss of p53 is a key event in themalignant progression of a colorectal adenoma to CRC (reviewed in Iacopetta 2003;
Vogelstein et al 1988).

24

1.3

Microsatellite Instability (MSI)

The human mismatch repair system (MMR) involves 7 key genes: MSH2, MSH6,
MSH3, MLH1, PMS1, PMS2 and MLH3. Their protein products are able torecognise and repair nucleotide mismatches which have escaped the normal editing
function of DNA polymerase (reviewed in Grady and Carethers 2008). If suchmismatches are not repaired, nucleotide transitions or transversions result, allowing
potentially oncogenic mutations to occur more frequently, leading to a
‘hypermutable phenotype’ (reviewed in Grady and Carethers 2008).

Lynch Syndrome (LS) is an autosomal dominant (AD) disease, which accounts forapproximately 5% of cases of CRC (reviewed in Mishra and Hall 2012). Patients
also have an inherited predisposition to a range of other malignancies, in particularendometrial carcinoma (Lynch et al 2015). LS occurs due to inherited mutations of
MSH2, MSH6, MLH1 or PMS2 (reviewed in Grady and Carethers 2008). In 15-20%of sporadic colon cancers, inactivation of the mismatch repair (MMR) systemoccurs, either though methylation of MLH1 or point mutations in MLH1/ MSH2/ othermembers of the MMR family (reviewed in Grady and Carethers 2008). This leads tomicrosatellite instability (MSI).

It is thought that certain key tumour suppressor genes drive the pathogenesis of
MSI tumours, and these are different to those which are mutated in CIN tumours(reviewed in Grady and Carethers 2008). In around 85% of colorectal tumours with
MSI, a repeat of 10 adenines undergoes a frameshift mutation in the TGFBR2 gene.
This allows tumour cells to escape the growth suppressing effects of TGF-β1(reviewed in Grady and Carethers 2008). Another gene commonly mutated in MSI
CRC is BAX, which plays a role in apoptosis. It is mutated in 50% of MSI CRC(reviewed in Grady and Carethers 2008). Interestingly, frameshift mutations within
coding mononucleotide repeats are also seen in APC (reviewed in Lynch et al2015).

MSI CRCs tend to have a certain clinical and pathological phenotype. They
generally occur in the right side of the colon, and microscopically they have amucinous appearance with large numbers of tumour-infiltrating lymphocytes(reviewed in Grady and Carethers 2008).

25

1.4

CpG Island Methylator Phenotype (CIMP)

Epigenetic regulation of gene expression can be achieved through methylation of
CpG islands found in gene promoters, causing silencing of the downstream gene.
Such silencing of tumour suppressors and/ or DNA repair genes is a common
feature of human neoplasia (reviewed in Hughes et al 2012). Widespread CpG
island promoter methylation is referred to as the CpG island methylator phenotype(CIMP) and was first described in 1999 (Toyota et al 1999). The cause of CIMP
remains to be elucidated but may potentially result from aberrant de novo
methylation or through the loss of protection against de novo methylation (reviewed
in Toyota et al 1999). Environmental factors, such as anthropometry and physicalactivity, smoking and alcohol may also play a role (reviewed in Hughes et al 2012).
CRC which exhibit CIMP are thought to arise via the ‘serrated pathway ofneoplasia’. The precursor lesions are hyperplastic polyps, rather than adenomas,
and an early event is a mutation of the BRAF oncogene (reviewed in Guarinos et al2012). BRAF is a component of the MAPK signalling pathway. The pathway
involves activation of cell membrane signalling molecules with subsequent
stimulation of cytoplasmic protein kinases (Seger and Krebs 1995). The transmitted
signals eventually activate cellular processes such as proliferation, differentiationand development (Seger and Krebs 1995). Activating mutations of BRAF increase
its kinase activity, which drives the proliferation of malignant cells (reviewed in
Bollag et al 2012).

Most CIMP CRCs have epigenetic silencing of MLH1, leading to microsatellite
instability (reviewed in Hughes et al 2012), and may have silencing of tumour
suppressor genes such as p16 (Toyota et al 1999).

Typically, CIMP tumours are associated with older age, female sex and occurrencein the right side of the bowel (reviewed in Hughes et al 2012), as is seen with MSI
CRC.

1.5

The Colorectal Polyposis Syndromes

Colorectal polyps are masses of tissue which are found projecting from the mucosaof the large bowel. They are classified according to their microscopic appearance,
and include adenomas, hyperplastic polyps and hamartomatous polyps. Most
polyps occur sporadically, but some are seen as part of a genetic ‘polyposis

26

syndrome’ (Figure 1.3). Colorectal polyps are benign but are clinically significant asthey may confer a risk of malignancy.

27

Figure 1.3: Diagram illustrating relationships between genes and signalling pathways involved in inherited colorectal polyposis syndromes. FAP, Familial
Adenomatous Polyposis; HMPS, Hereditary Mixed Polyposis Syndrome; JPS, Juvenile Polyposis Syndrome; LS, Lynch Syndrome; MAP, MUTYH-Associated
Polyposis; NTHL1, NTHL1-Associated Polyposis; PJS, Peutz-Jegher’s Syndrome; PPAP, Polymerase Proofreading-Associated Polyposis. Image modified
from Short et al (2015)28

1.5.1 Colorectal Adenomas
Colorectal adenomas are common. They are found in between 1.72% and 63% of
autopsies (Pendergrass et al 2008; Paspatis et al 2001; Correa et al 1977; Arminski
and McLean 1964; Chapman 1963). In asymptomatic patients undergoingcolonoscopies, adenoma prevalence is between 6.3% and 41% (Chung et al 2010;
Rundle et al 2008; Lin et al 2006; Strul et al 2006; Soon et al 2005; reviewed in
Giacosa 2004; Yamaji et al 2004; DiSario et al 1991). They occur more frequently
in males than in females, and their prevalence increases with age (Chung et al
2010; Pendergrass et al 2008; Lin et al 2006; Strul et al 2006; Soon et al 2005;
Yamaji et al 2004; Paspatis et al 2001; DiSario et al 1991; Correa et al 1977;
Chapman 1963).

The significance of colorectal adenomas is that they are pre-malignant lesions. The
majority of colorectal CRCs are thought to develop from pre-existing adenomas.
The probability that an adenoma will become malignant depends upon its size,
morphology and degree of dysplasia. Large villous lesions harbouring high grade
dysplasia confer the highest risk (Terry et al 2002; O’Brien et al 1990; Shinya and
Wolff 1979; Muto et al 1975).

Most colorectal adenomas occur sporadically. However, there are syndromes ofcolorectal polyposis, in which patients develop multiple polyps as a result of anunderlying genetic mutation. These include Familial Adenomatous Polyposis (FAP),
MUTYH-Associated Polyposis (MAP), Polymerase Proofreading-Associated
Polyposis (PPAP), NTHL1-Associated Polyposis/ CRC and MSH3-Associated
Polyposis/ CRC.

1.5.1.1

Familial Adenomatous Polyposis (FAP)

FAP is a dominantly inherited Mendelian trait, in which patients develop hundreds tothousands of colorectal adenomas during adolescence or the third decade of life
(reviewed in Fearnhead et al 2002; Bodmer 1999). All such patients will invariably
develop CRC if they are left untreated (Bodmer 1999; reviewed in Fearnhead et al2002).
The first case of histologically verified adenomatous polyposis was published in1881 by Sklifasowski (reviewed in Bülow et al 2006). The following year Harrison-29

Cripps described ‘disseminated polypus of the rectum’ in two teenage siblings, bothof whom had 20-30 colorectal polyps (reviewed in Bülow et al 2006; Harrison-Cripps
1882). In the late 1800s there were numerous case reports describing patients withmultiple colorectal adenomas, and an association with colorectal malignancy was
noted (reviewed in Bülow et al 2006). In 1925, Lockhart-Mummery stated that the‘condition of multiple adenomata was invariably antecedent to carcinoma’ and that‘the condition of multiple adenomata is often hereditary in a marked degree’(Lockhart-Mummery 1925).

It is now known that FAP, and an attenuated form of the disease, AFAP, are due togermline or somatic mosaic mutations in APC. Over 1500 different mutations in
APC have been identified to date (Kadiyska et al 2013). The majority of mutations
are inherited. It used to be thought that approximately one quarter of cases
occurred de novo (reviewed in Fearnhead et al 2001), but this is an overestimate,as this figure included apparent de novo patients who actually had MUTYHAssociated Polyposis (MAP). A third of all germline mutations occur at codons 1061
and 1309, with the remainder spread relatively uniformly between codons 200 and
1600 (reviewed in Fearnhead et al 2001). The nature of the germline mutation
determines the nature of the second somatic hit to APC (reviewed in Fearnhead etal 2002; reviewed in Fearnhead et al 2001). Germline mutations occurring between
codons 1194 and 1392 tend to be followed by allelic loss of APC as a second hit(loss of heterozygosity, LOH), whereas germline mutations lying outside of thisregion tend to be associated with truncating mutations in the mutation cluster region(MCR) between codons 1286 and 1513 (reviewed in Fearnhead et al 2002). The
reason for this may be related to the resultant level or functional characteristics of
APC protein produced: it is proposed that to allow efficient tumourigenesis, thefunction of APC must be impaired sufficiently to allow a certain level of nuclear βcatenin accumulation, but that β-catenin levels must not be too great, or this canresult in apoptosis (Albuquerque et al 2002).

The incidence of FAP is approximately 1 per 8000, and it accounts for around 0.5%
- 1% of CRC (Mishra and Hall 2012; reviewed in Fearnhead et al 2002; reviewed in
Bodmer 1999). In addition to colorectal adenomas, FAP patients may develop
extra-intestinal manifestations of their disease, for example congenital hypertrophyof the retinal pigment epithelium (CHRPE), duodenal and peri-ampullary tumours,
desmoid tumours, papillary carcinoma of the thyroid, medulloblastoma,30

hepatoblastoma, osteomas and epidermoid cysts (Mishra and Hall 2012; reviewed
in Fearnhead et al 2002).

1.5.1.2

MUTYH-Associated Polyposis (MAP)

Prior to 2002, inherited defects of base excision had not been associated with any
human genetic disorder (Al-Tassan et al 2002). That year, mutations in the MUTYH
gene were shown to cause an inherited predisposition to colorectal tumours (AlTassan et al 2002).

The MUTYH gene is located on the short arm of chromosome 1 (1p32.1-p34.3)(Poulsen and Bisgaard 2008). It consists of 16 exons and encodes a protein of 535
amino acids, the MUTYH glycosylase (Poulsen and Bisgaard 2008). MUTYH
glycosylase is part of the base excision repair (BER) system. It is involved inrepairing DNA mismatches occurring as a result of oxidative DNA damage (Mazzei
et al 2013). Each human cell metabolises approximately 1012 molecules of oxygen
per day (reviewed in Nohmi et al 2005). About 1% of oxygen metabolism results inthe production of reactive oxygen species (ROS) which include superoxide,hydrogen peroxide, hydroxyl radicals and singlet oxygen (reviewed in Nohmi et al2005). ROS can damage DNA, producing 8-hydroxyguanine (8-oxodG). Thisfrequently pairs with dAMP. Under normal circumstances, this mispairing would be
repaired by MUTYH, to create C: 8-oxodG base pairs. Another enzyme, OGG1 willthen remove the 8-oxodG. Hence the combined effects of MUTYH and OGG1 will
prevent GC > TA transversions (Mazzei et al 2013).

Figure 1.4: Image from David et al (2007) illustrating the actions of MUTYH in the repair ofoxidative DNA damage

31Patients with biallelic germline MUTYH mutations are predisposed to mutations ingenes including APC and KRAS. The clinical manifestation of this is MAP. MAP is
an autosomal recessive (AR) disease, in which patients develop multiple colorectal
adenomas. The mean age of diagnosis is 45-50 years, and patients typically have
between 10 and 100 polyps (reviewed in Mazzei et al 2013; reviewed in Poulsen
and Bisgarrd 2008; Croitoru et al 2007; Nielsen et al 2007; Gismondi et al 2004;
Wang et al 2004; Sampson et al 2003; Sieber et al 2003). Some patients do notdevelop polyps but present with cancer (Farrington et al 2005; Wang et al 2004;
Enholm et al 2003). Although the majority of polyps are adenomas, hyperplastic
polyps (HPPs) and sessile serrated adenomas (SSAs) are also seen (reviewed in
Mazzei et al 2013; Boparai et al 2008; Lipton et al 2003a).

Patients with MAP have an increased risk of developing CRC. Malignancy has
been reported with varying frequencies: one paper reported a prevalence of
19.47% at 50 years, and 42.89% at 60 years (Lubbe et al 2009); another found that
48% of patients with MAP developed CRC with a mean age of diagnosis of 49.7
years (Sampson et al 2003). It has been suggested that biallelic inactivation of
MUTYH imparts an overall 93-fold excess risk and that all homozygotes/ compound
heterozygotes will develop CRC by age 60 (Farrington et al 2005). Win et al (2014)
estimated that males carrying biallelic MUTYH mutations had a 75.4% risk ofdeveloping CRC by age 70, and females had a 71.7% risk (Win et al 2014).

MAP may also have extra-colonic manifestations, although these are generally notpart of the characteristic phenotype (Poulsen and Bisgaard 2008). The lesions
which have been reported include duodenal adenomas and carcinoma, fundic gland
polyps, stomach cancer, CHRPE, osteomas and breast cancer (reviewed in
Venesio et al 2012; reviewed in Poulsen and Bisgaard 2008). The incidence of
extra-intestinal malignancies is almost double that of the general population, with asignificant increase in ovarian, bladder and skin cancers (reviewed in Venesio et al2012).

By 2013, >300 MUTYH variants among MAP patients and/ or controls had been
described (Ruggieri et al 2013). The mutations observed in MUTYH vary according
to the ethnic group studied, suggesting population specific ancestral variants(Dolwani et al 2007; Sieber et al 2003). In Caucasian populations, P.Tyr165Cys
and p.Gly382Asp are the common mutations (Jones et al 2002; Sampson et al32

2003; Enholm et al 2003; Sieber et al 2003). These account for 73% of all
mutations reported and have been described in Swiss (Russell et al 2006), Italian(Gismondi et al 2004; Venesio et al 2004), French (Küry et al 2007), Swedish(Kanter-Smoler et al 2006; Zhou et al 2005), Canadian (Croitoru et al 2007),
Australian (Kairupan et al 2005), Portuguese (Isidro et al 2004), Czech (Šulová et al2007), British, American and Dutch populations (reviewed in Cheadle and Sampson
2007). There is evidence for strong founder effects for these mutations: it issuggested that they derive from ancestors who lived between 5-8000 years and 69000 years BC respectively (Aretz et al 2014).

Numerous other pathogenic variants in MUTYH have been reported, includingc.1395delGGA in Italians (Gismondi et al 2004), p.Glu466* and p.TyrY90* in Asians(Sampson et al 2003; Jones et al 2002) and p.Arg231Cys in the Japanese (Miyaki
et al 2005).

1.5.1.3

Polymerase Proofreading Associated Polyposis(PPAP)
PPAP is a relatively recently defined clinical entity. POLE and POLD1 code for
DNA polymerases with exonuclease (proofreading) activity. Mutations in thesegenes are thought to cause a defect in correcting mispaired bases inserted during
DNA replication (Palles et al 2013). In 2012, Palles et al undertook whole-genome
sequencing of probands who had at least 10 colorectal adenomas by age 60, who
had previously had known Mendelian cancer syndromes excluded in a clinicaldiagnostic setting. They also sequenced several affected relatives (Palles et al2013). The group found that a genetic variant, POLE p.Leu242Val, was associatedwith multiple colorectal adenomas and carcinoma (Palles et al 2013). The trait
showed dominant inheritance, with high penetrance (Palles et al 2013). Anothervariant, POLD1 p.Ser478Asn, predisposed to colorectal tumours, endometrial
cancer, and possibly brain tumours (Palles et al 2013).

In 2014, Valle et al sought to determine the prevalence of these mutations in 858
patients with unexplained familial/ early-onset CRC or polyposis. They didn’t
identify either mutation in any of their CRC cases. However, the POLE p.Leu424Val
mutation was found in a polyposis family, in which case it had occurred as a denovo mutation in the proband. This accounted for 0.52% of the polyposis cases.

33

The group also reported a novel variant, POLD1 c.1421T>C (p.Leu474Pro) in a
MMR-proficient family. This mutation occurs in the proofreading domain of a DNA
polymerase and was predicted to be pathogenic (Valle et al 2014).

Valle’s group sought to further characterise the phenotypic spectrum of patients
carrying germline POLE/ POLD1 mutations (Bellido et al 2016). They sequenced
the entire exonuclease domains of POLE/ POLD1 in 544 CRC cases from 529
families, including those from Valle’s original paper (Valle et al 2014). Although noadditional POLE mutations were identified, 4 of 6 novel/ rare nonsynonymous
POLD1 variants detected were believed to be pathogenic: p.Asp316His,
p.Asp316Gly, p.Arg409Trp and p.Leu474Pro. The group reviewed the phenotypic
data from all 69 carriers of POLE/ POLD1 mutations that had been reported to date.
They observed that the associated phenotype was characterised by attenuated/oligo- adenomatous polyposis, with >80% of POLE and >60% of POLD1 mutation
carriers being diagnosed with >/= 2 adenomas, with an average of 19 lesions. CRC
was diagnosed in 60-64% of carriers, and brain tumours in 5.8%. Gastroduodenal(mostly duodenal) adenomas were identified in 57.1% of carriers who underwent
gastroduodenoscopies. For patients harbouring POLD1 mutations, the phenotypic
spectrum was extended to include endometrial tumours (57.1% of carriers) andbreast tumours (14.3% of carriers).

1.5.1.4

NTHL1-Associated Polyposis

A further gene involved in the pathogenesis of colorectal neoplasia is NTHL1. In2015 Weren et al carried out whole exome sequencing on 51 patients with multiple
colorectal adenomas +/- CRC, who had tested negative for APC and MUTYH
mutations (Weren et al 2015). The group found that 7 individuals were homozygous
for a NTHL1 nonsense mutation, c.268C>T, which triggers nonsense-mediateddecay (NMD). The patients harbouring the mutations all had multiple colorectal
adenomas, ranging from 8-50, and 4 also had multiple CRCs. All 3 affected womendeveloped complex endometrial hyperplasia or endometrial cancer.

NTHL1 is a base excision repair gene, and homozygous mutations cause anincrease in C:G>T:A changes in genes such as APC, p53, KRAS and PI3K (Werenet al 2015).

34

1.5.1.5

MSH3-Associated Polyposis

The most recently identified polyposis syndrome is MSH3-Associated Polyposis(Adam et al 2016). Adam’s group performed whole exome sequencing on germline
DNA extracted from 102 unrelated individuals with unexplained adenomatous
polyposis. They found two different individuals with different compound
heterozygous mutations in the mismatch repair gene, MSH3. Both index persons
had an affected sibling carrying the same mutations. The mutations wereassociated with tumours which displayed Elevated Microsatellite Alterations At
Selected Tetranucleotide Repeats (EMAST), a type of microsatellite instability.

The phenotypic spectrum in MSH3 mutation carriers was reported to include
colorectal and duodenal adenomas, CRC, gastric cancer and an early onset
astrocytoma (Adam et al 2016).

1.5.2 Hyperplastic Polyps (HPPs)
HPPs are a frequent finding, seen in between 1% and 73% of autopsies (Paspatis
et al 2001; Williams et al 1982; reviewed in Correa et al 1977). In asymptomatic
individuals undergoing colonoscopy, HPPs are observed in 21% - 34% of cases(Forsberg et al 2012; DiSario et al 1991). They are more common in males thanfemales, and their prevalence increases with age (Williams et al 1982; Williams et al
1980; Correa et al 1977).
Until approximately 1990, hyperplastic (or ‘metaplastic’) polyps were regarded as ahomogeneous group of tumours with no malignant potential (Rosty et al 2013a).
Since that time, it has been increasingly recognised that hyperplastic lesions are nota single entity – they differ in their morphology and their clinical significance, inparticular their risk for progressing to carcinoma. In 2010, the World HealthOrganisation published a classification system which subdivides hyperplastic
lesions into 3 groups based upon their microscopic appearance: Hyperplastic
Polyps (HPPs), Sessile Serrated Adenomas/ Polyps with or without cytologicaldysplasia (SSA) and Traditional Serrated Adenomas (TSA) (reviewed in Rosty et al2013a; reviewed in Leggett and Whitehall 2010).

It is now known that hyperplastic lesions may be the precursors to CRC developing
along the serrated pathway of carcinogenesis, which accounts for approximately

3510% of CRCs (Yamane et al 2014). In contrast to the CRC which follow thetraditional ‘adenoma carcinoma’ pathway, tumours arising from the serrated
pathway tend not to display CIN, but instead exhibit MAPK pathway activation,
through BRAF mutations, and they commonly develop CIMP (Yamane et al 2014;
Rosty et al 2013a; Leggett and Whitehall 2010).

1.5.2.1

Serrated Polyposis Syndrome

Although most HPPs are sporadic lesions, there is a condition in which patientsdevelop multiple and/ or large lesions. One of the first descriptions was in 1980 byWilliams et al (Williams et al 1980). They observed 7 patients, with a mean age of
37.4 years, who each had at least 50 lesions throughout their large bowel. At thattime, the authors concluded that ‘it is impossible to deduce whether or not‘metaplastic polyposis’ is a distinct entity. There is no good evidence that it isfamilial in this small series, but the appearance of numerous metaplastic polyps ofan unusually large size and configuration, predominantly in young males, mightsuggest a specific ‘disease’’.

As increasing evidence came to light that there seemed to be a syndrome in which
patients developed numerous hyperplastic lesions throughout their large bowel, it
was named ‘hyperplastic polyposis syndrome’ (HPS). In 2000, a definition of HPS
was proposed by Jass and Burt in the World Health Organisation classification oftumours (Jass and Burt 2000). This definition was modified in 2010, and thedisease was officially renamed Serrated Polyposis Syndrome (SPS). It appears in
the 2010 World Health Organisation classification of tumours of the digestivesystem (Snover DC et al 2010). In order to meet the diagnostic criteria for SPS,
patients must fulfil at least one of the following criteria:

1. At least 5 serrated (hyperplastic) polyps proximal to the sigmoid colon, 2 ofwhich are >10mm diameter
2. Any number of serrated polyps proximal to the sigmoid colon in an individual
who has a first degree relative with serrated polyposis
3. More than 20 serrated polyps of any size distributed throughout the colon(reviewed in Rosty et al 2013a; reviewed in Guarinos et al 2012; reviewed inLeggett and Whitehall 2010; Snover DC et al 2010)36

It was thought that SPS affected 1 in 3000 asymptomatic individuals between theages of 55 and 64 years (reviewed in Rosty et al 2013b). However, most studies
report a broad age distribution of the disease (17 to 85 years) with a mean age ofdiagnosis of 47.7-56 years, so it is possible that the prevalence of SPS in thegeneral asymptomatic population is higher than 1/3000 (Rosty et al 2013b). Recent
data suggest that the prevalence may be as high as 1/151 patients who have acolonoscopy following a positive faecal occult blood test (reviewed in Rosty et al2013a).

SPS shows no sex predilection, and the mean age of diagnosis is 55 years(Guarinos et al 2012; Kalady et al 2011). As well as hyperplastic lesions, up to 85%
of patients also have conventional adenomas present in the bowel (Rosty et al2013a; Rosty et al 2012). Patients with SPS have an increased risk of developing
CRC, which generally occurs between 50 and 60 years of age (reviewed in
Guarinos et al 2012). Malignancy is associated with a larger number of polyps, thepresence of dysplasia (reviewed in Guarinos et al 2012; Yeoman et al 2007) and thepresence of conventional adenomas in addition to HPPs (Rosty et al 2013b). The
incidence of CRC in SPS patients varies from 14% to 58% (reviewed in Rosty et al2013b; Yeoman et al 2007; Hyman et al 2004; Lage et al 2004) and the incidenceis greater in females than in males, with a ratio of 2.4:1 (Rosty et al 213b). Whencarcinoma develops in an SPS patient, it is likely to have a proximal location: 64%
of CRC are identified proximal to the descending colon (Rosty et al 2013b).
Interestingly, a large proportion of CRCs seen in patients with SPS do not develop
through the ‘serrated pathway of carcinogenesis’ driven by BRAF mutation (Rosty etal 2013b). The tumours show various molecular changes, including those more
likely to be associated with the traditional adenoma-carcinoma pathway, forexample β-catenin activation and/ or overexpression of p53 (Rosty et al 2013b).

SPS is thought to be a genetic disease, but the mode of inheritance is unclear(reviewed in Guarinos et al 2012). There are papers which report germline
mutations in the Wnt inhibitor RNF43, an E3 ubiquitin ligase, in individuals andfamilies with features of SPS (Yan et al 2017; Taupin et al 2015; Gala et al 2014).
However, Buchanan et al note that mutations in RNF43 may account for only asmall proportion of SPS, and that additional genetic risk factors are yet to be
identified (Buchanan et al 2017).

37

1.5.3 Hamartomatous Polyps
Hamartomas are overgrowths of the tissue which is native to the site of the lesion.
Polyposis syndromes which are characterised by hamartomas include PeutzJegher’s Syndrome (PJS), Juvenile Polyposis Syndrome (JPS) and Cowden’s
Disease.

1.5.3.1

Peutz-Jeghers Syndrome (PJS)

In 1921, a Dutch physician, Dr. Peutz, first described the combination ofgastrointestinal polyps and mucocutaneous pigmentation (Peutz 1921). In 1949, Dr.
Jeghers published an article describing 10 patients who had a combination ofpigmentation of the oral mucosa/ lips/ digits, and intestinal polyps (Jeghers et al1949). The observations made by Peutz and Jeghers led to the definition of an AD
syndrome characterised by gastrointestinal polyposis and mucocutaneous
pigmentation, now known as Peutz-Jeghers Syndrome (PJS) (Westerman et al1999).

PJS is inherited in an AD manner and has variable penetrance. It is a rare
condition, with a prevalence of approximately 1 in 200 000 (reviewed in Omundsen
and Lam 2012). The disease is characterised by hamartomatous polyps throughout
the gastrointestinal tract and mucocutaneous pigmentation. Patients present at a
median age of 11 years, and this is often as a result of a complication of their GI
polyps, for example intussusception, small bowel obstruction, rectal bleeding or
volvulus (reviewed in Omundsen and Lam 2012).

Approximately 50% of cases of PJS are caused by germline mutations in the
nuclear serine threonine kinase gene LKB1/ STK11 (Jenne et al 1998; reviewed in
Omundsen and Lam 2012). This gene regulates cell polarisation, growth andmetabolism. Most mutations are small insertions or deletions, resulting in atruncated protein with no kinase activity (reviewed in Omundsen and Lam 2012).

Patients with PJS are at increased risk of developing cancer, both at gastrointestinal
and extra-intestinal sites. The most common tumours are CRCs, but there is also
an increased risk of other gastrointestinal (GI) carcinomas (oesophageal, gastric,
small bowel and pancreas), breast cancer, cervical cancer and sex cord tumours(Hizawa et al 1993; reviewed in Omundsen and Lam 2012).

38

1.5.3.2

Familial Juvenile Polyposis Syndrome

In 1914, Hertz described four family members who had rectal polyps in childhood,the youngest being only 8-years-old (Hertz 1914). That report is regarded as thefirst instance of juvenile polyposis in the medical literature (Calva and Howe 2009).
Juvenile polyps are common hamartomatous lesions which occur in the large bowel.
They are usually solitary and sporadic. If multiple juvenile polyps are present, thepatient may have Juvenile Polyposis Syndrome (JPS). JPS is an AD disease withvariable penetrance and which has an incidence of 1 per 100 000 births (reviewed
in Omundsen and Lam 2012). Patients can present in infancy with GI bleeding,intussusception, rectal prolapse or a protein losing enteropathy. Around 15% will
have an associated congenital birth defect, such as gut malrotation, cardiac andcranial abnormalities, cleft palate, polydactyly or genitourinary defects. If
presentation is as an adult, the patient is likely to suffer from GI bleeding (reviewed
in Omunsden and Lam 2012).

For a diagnosis of JPS, patients must fulfil one of the following criteria:
1. More than 5 juvenile polyps of the colon or rectum
2. Juvenile polyps in other parts of the GI tract
3. Any number of juvenile polyps and a positive family history(reviewed in Omundsen and Lam 2012)Germline mutations in SMAD4 and BMPR1A are seen in JPS, and it is suggested
that ENG mutations may also have a role (Sweet et al 2005), although this is notcertain (Howe et al 2007). All of these genes are involved in transforming growth
factor β (TGFβ) signalling. The TGFβ family of cytokines are growth inhibitors, andloss of sensitivity to these factors promotes tumourigenesis (reviewed in Fleming et
al 2013). As such, patients with JPS are at increased risk of developing CRC(Rozen and Baratz 1982; Järvinen and Franssila 1984; Giardiello et al 1991). In
additional to colorectal malignancies, patients are also at risk of developing gastricand duodenal cancer (reviewed in Omundsen and Lam 2012).

1.5.3.3

Cowden’s Syndrome

In 1963 Lloyd and Dennis reported a 20-year old female with multiple pathologies,
including multiple thyroid adenomas, extensive fibrocystic change of both breasts

39and ‘space occupying lesions in the liver and bone’ (Lloyd and Dennis, 1963). At
that time, it was noted that ‘whether this case represents a new familial disease …
has not been established’ (Lloyd and Dennis, 1963), although they named the
syndrome ‘Cowden’s Disease’. It is now established that Cowden’s Syndrome is an
AD disease, affecting approximately 1 in 200 000 births. It is commonly diagnosedin the second decade of life, but age of onset may vary from 4 to 75 years (reviewed
in Lam-Himlin et al 2014). Patients develop multiple hamartomas in multiple organsystems. Lesions can be found in the skin, GI tract, breast, thyroid gland andcentral nervous system (reviewed in Omunsden and Lam 2012; Hanssen and Fryns
1995).

80% of patients have a germline mutation in the PTEN gene (reviewed in
Omunsden and Lam 2012). PTEN is a tumour suppressor: its product is a
phosphatase which negatively regulates the phosphatidylinositol 3-kinase-AKT andmammalian target of rapamycin (mTOR) signalling pathways, which are involved incell growth and proliferation, cell cycle progression and apoptosis (reviewed in LamHimlin et al 2014). PTEN mutations are involved in the pathogenesis of several
carcinomas, including breast, endometrial, thyroid, large bowel and kidney(reviewed in Lam-Himlin et al 2014). As such, patients with Cowden’s Syndromeare at increased risk of these malignancies (Hanssen and Fryns 1995).

1.5.3.4

Hereditary Mixed Polyposis Syndrome (HMPS)

Hereditary Mixed Polyposis Syndrome (HMPS) is a relatively recently defined entity.
In 1971, Kaschula described an 11-year-old girl who had profuse diarrhoea mixed
with blood and mucus. She was found to have polyps throughout her large bowel,
and the polyps had both adenomatous and juvenile morphologies (Kaschula 1971).
Over a decade later, in 1987, the term ‘mixed familial polyposis syndromes’ wasused as the title of a report by Sarles et al (Sarles et al 1987). This article described
3 patients, including a father and son, who all had multiple polyps of different
histopathological types.

In HMPS, patients develop multiple polyps with mixed morphologies. This is an AD
disease, and patients may have adenomas, hyperplastic polyps and hamartomatous
polyps. There is a high risk of developing CRC (Jaeger et al 2012). The disease iscaused by a duplication spanning part of the SCG5 gene and a region upstream of40

the GREM1 locus. This duplication causes increased expression of GREM1, whichacts as a bone morphogenetic protein (BMP) antagonist. The subsequent reductionin BMP signalling is thought to play a role in tumourigenesis (Jaeger et al 2012).

1.6

Clinical Management of Patients with Colorectal

Polyposis
This thesis is focused on patients with multiple colorectal adenomas. In view of thepossible genetic diagnoses which may underlie such a phenotype it has beencommon clinical practice for patients with >10 colorectal adenomas to be referred toa regional genetics centre for genetic counselling and for consideration of diagnostic
analysis of the APC and/ or MUTYH genes. The diagnostic testing carried out will
depend upon the individual’s phenotype and their family history.

Up to 90% of patients with a phenotype of typical FAP have a pathogenic APC
germline mutation identified through sequencing of coding exons and deletion/
duplication analysis via multiplex ligation-dependent probe amplification (MLPA)(Spier et al 2012). Of those with a phenotype of AFAP, APC or biallelic MUTYH
germline mutations are detected in only 20-50% of cases (Spier et al 2012).

1.6.1 Why Might Some Genetic Variants Be Missed?
Since 1991, when APC was recognised as the causative gene of FAP (Groden et al
1991; Joslyn et al 1991; Kinzler et al 1991; Nishisho et al 1991), several screening
and diagnostic strategies have been developed to identify pathogenic APC
mutations (Scott et al 2001) in patients with multiple colorectal adenomas. These
have included denaturing gradient gel electrophoresis analysis (DGGE) (Scott et al
2001; Olschwang et al 1993), ribonuclease protection analysis (Miyoshi et al
1992a), single strand conformation polymorphism analysis (SSCP) (Cottrell et al
1992; Groden et al 1993), heteroduplex analysis (HA) (Cottrell et al 1992) and theprotein truncation test (PTT)/ in vitro synthesised protein assay (IVSP) (Powell et al1993). The characterisation of a genetic mutation identified through screening
requires DNA sequencing. Sanger sequencing has been the gold standard ofsequencing for several decades, and until very recently was the main approach
used for the molecular diagnosis of colorectal polyposis. In the last few years, next-41

generation sequencing (NGS) technologies have progressively replaced Sanger
sequencing.

Genetic diagnostic protocols used in patients with polyposis typically include
sequencing of APC and/ or MUTYH and dosage analysis of the genes using atechnique such as MLPA. These approaches could miss pathogenic variants
located in promoter regions, deep within introns (Spier et al 2012) or in untranslated
regions (UTRs), which may have effects on gene expression mediated througheffects on transcription, mRNA splicing or mRNA stability. Similarly, diagnostic
protocols may miss low frequency variants in patients with somatic mosaicism.
Protocols used in the National Health Service (NHS) in Wales would also not
identify epigenetic phenomenon such as promoter methylation, they would notdetect mutations in genes which are established but rare causes of polyposis (e.g.
POLE/ POLD1) and they would not identify novel polyposis genes.

1.6.1.2

Promoter Variants and Allelic Imbalance (AI)The APC gene has two promoter regions, 1A and 1B (reviewed in Rohlin et al2011). The major transcript is initiated by the major promoter, 1A (reviewed in
Charames et al 2008). It is possible that genetic variants occurring in thesepromoters could lead to reduced gene expression, therefore predisposing to tumour
formation. Such variants might not be identified through standard genetic
diagnostics as the promoter is not typically included in diagnostic sequencing
protocols.

There are several reports of APC promoter mutations in the literature. However,these typically describe deletions, which would be detected through diagnostic
MLPA (Yamaguchi et al 2016; Pavicic et al 2014; Rohlin et al 2011; Charames et al2008). At the time of writing, there is a paucity of literature describing APC
promoter point mutations or methylation as a cause of colorectal polyposis.

Allelic imbalance (AI) refers to a situation in which the two alleles of a given geneare expressed at different levels in a given cell (Wagner et al 2010). It can occur
due to epigenetic inactivation of one of the alleles, or because of genetic variation in
regulatory regions (Wagner et al 2010). AI can involve complete inactivation of one
allele, for example in parent-of-origin imprinting, when a specific allele at a given

42

locus is silenced through epigenetic mechanisms depending on whether it wasinherited from the mother or father (Wagner et al 2010). Allele expression can also
be partially reduced, which can occur when different alleles have differing affinities
for transcription factors (Wagner et al 2010), or through cis- acting genetic variants,
for example in promoters (Wagner et al 2010).

As well as being important in normal phenotypic variation, AI can also contribute totumourigenesis. There is some evidence that BRCA1 and BRCA2 AI plays a role in
the pathogenesis of ovarian and breast cancer (Chen et al 2008; Shen et al 2011).
Whilst only a small number of studies have considered APC AI in the context ofcolorectal neoplasia, those which have been performed have found it may make animportant contribution. As early as 1993, Powell et al (Powell et al 1993) used anallele-specific expression assay to show that 3/11 APC NMI patients with clinical
FAP had significantly reduced expression of one APC allele. In 1999 Laken et al(Laken et al 1999) used monoallelic mutation analysis (MAMA) to reveal that
7/9 APC NMI patients had reduced/ no expression from one of their APC alleles.
More recently Yan et al (2002b) identified a patient with colorectal tumours who was
known to have reduced levels of the APC protein. The group quantified the relative
levels of mRNA transcripts from each APC allele using Digital-SNP. They foundthat gDNA yielded the expected 50% allelic ratio, but that cDNA fromlymphoblastoid cells showed a skewed distribution, with a ratio of approximately
66% (Yan et al 2002b). Linkage analysis showed that the allele whose mRNA wasexpressed in lower amounts was the one linked to disease (Yan et al 2002b).
Further work confirmed that the skewed allelic ratio was also present in 4 affectedfamily members, but that the ratio was normal in 24 unrelated unaffectedindividuals. The group continued to investigate expression levels of APC: in four
patients with clinical FAP who had no abnormalities with the in vitro synthesised
protein assay (IVSP) or allele sequencing, one was found to have an abnormal 71%
allelic ratio in cDNA. When tumours from the patients with AI were studied, 30/38
had LOH of APC, and in 29 of these cases, it was the normal allele which had beenlost. Yan et al concluded that an allele which causes a decrease in transcript levels
can result in a predisposition to severe disease, but that there needs to be a second
hit to the normal allele for a disease to manifest (Yan et al 2002b). Interestingly thecause of the decreased expression was not determined: the sequences of thecoding regions, promoter and 3’UTR were normal, so the group assumed that thepathogenic variant must lie within an intron or upstream of the gene (Yan et al
2002b). These early findings regarding APC AI have been supported by43Castellsagué et al (2010). Of 23 APC/ MUTYH NMI polyposis families who were
heterozygous for rs2229992, 2 were shown to harbour APC AI. The AI in one family
was suggested to result from promoter variants (Castellsagué et al 2010).

1.6.1.3

Intronic Variants

Intronic mutations may affect RNA splicing and introns are not screened as part ofmost routine genetic diagnostic protocols. In 2000 Su et al sought to identify novel
intragenic rearrangements of APC in patients with a clinical phenotype of FAP or
AFAP. They found four germline APC mutations, one of which was a deletion 271627bp downstream of exon 14, which was replaced with a novel sequence ofabout 180bp. The deletion was completely within intron 14, but it affected thesplicing of exon 14 (Su et al 2000). In 2010 Tuohy et al used Southern Blot analysisof the APC gene to identify a 1.4kb deletion within intron 14 in a family with AFAP.
Subsequent PCR amplification from exon 13 to exon 15 of cDNA showed that the
intronic deletion resulted in abnormal splicing, and that exon 14 was deleted. This
caused a frameshift and protein truncation at codon 673 of the normal reading
frame resulting in a truncated product that lacked all of the β-catenin, microtubuleand EB-1 binding domains (Tuohy et al 2010).

In 2012 transcript analysis in a sample of 125 mutation negative patients withcolorectal adenomatous polyposis found that 8% had a reproducible aberrant
transcript pattern, suggesting an intronic mutation at a genomic level (Spier et al2012). 80% of these were found to have transcript insertions between two exons
originating from exonised sequences deep within the corresponding intron (Spier et
al 2012). All pseudoexons were predicted to result in out-of-frame transcripts withpremature stop codons (Spier et al 2012). In those patients who had insertions, theunderlying genomic mutations were identified: they comprised 3 different
heterozygous point mutations (c.532-941G>A, c.1408+731C>T, c.1408+735A>T)
which activated cryptic splice sites. A pre-existing complementary cryptic splice site
was predicted at the other end of the insertion (Spier et al 2012).

1.6.1.4

Untranslated Region (UTR) Variants

A pre-RNA molecule undergoes several steps of processing before it becomes afunctional mRNA molecule (Mignone et al 2002). Mature mRNA consists of a 5’

44

untranslated region (5’UTR), a coding region, and a 3’ UTR (Mignone et al 2002).
UTRs, particularly 3’UTRs, have multiple roles in the post-transcriptional regulationof gene expression, including effects on mRNA transport out of the nucleus,translation efficiency, subcellular localisation and mRNA stability (reviewed in
Mignone et al 2002). In addition to normal physiological intracellular effects, there isincreasing evidence that UTR variants can be involved in disease, for example anexpanded number of trinucleotide repeats in the 3’UTR of the DMPK gene isthought to play a role in the pathogenesis of in myotonic dystrophy (Conne et al2000), and a somatic 5’UTR variant has been reported to reduce translation
efficiency of BRCA1 in a highly aggressive sporadic breast cancer (Signori et al2001).
In the context of colorectal neoplasia, Wilding et al have shown that in microsatellite
unstable cancer, deregulation of mRNA stability due to mutations in regulatory
3’UTR sequences can lead to a marked difference in gene expression profiles whencompared to microsatellite stable tumours (Wilding et al 2010).

1.6.1.5

Mosaicism

A mosaic is an individual who has at least two genetically different cell lines despite
developing from a single zygote. Mosaic mutations may be missed with standard
mutation diagnostic techniques, for example if they occur at a low frequency within
the individual or if they do not occur in the part of the body which is being analysed.

It is reported that somatic mosaicism can occur in 10-20% of sporadic cases of FAP(reviewed in Rohlin et al 2009; Hes et al 2008; Aretz et al 2007). The timing at
which an APC mutation occurs will have an important bearing on the patient’s
phenotype: if it arises in a single colonic epithelial stem cell, the only consequence
will be adenomatous polyps in the segment of the colon that becomes populatedwith descendants of the stem cell (Tuohy and Burt 2008). At the other extreme, if
the mutation occurs early in embryogenesis, it may be found in all three germ cell
layers. This would result in multiple clinical manifestations seen throughout multiple
organ systems, potentially including mutations in reproductive cells, which could
then be passed on to future generations (Tuohy and Burt 2008).

Depending on the frequency of the mutation, it is likely that a significant proportion
will be missed using current diagnostic sequencing protocols and DNA extracted

45

from whole blood. Rohlin et al (2009) carried out a study to evaluate the different
mutation screening/ diagnostic techniques in terms of their sensitivity in detecting
mosaicism. They looked at Sanger sequencing, single-strand conformation
polymorphism (SSCP)/ heteroduplex analysis (HA), the protein truncation test
(PTT), denaturing high performance liquid chromatography (DHPLC) and massively
parallel sequencing. A total of 9 mutations were addressed – 8 in APC and 1 in
NF2. The group constructed 7 artificial mosaics in APC through serial dilutions ofDNA, with a non-mosaic heterozygous mutation being defined as 100%. The tworemaining samples were from naturally occurring mosaics. All of the dilutions of allof the mutations were analysed with SSCP, DHPLC and Sanger sequencing. Three
were included in the PTT assay (these were mutations in exon 18, which is readily
screened by PTT). Only four artificial mosaics, at various concentrations, and bothnatural mosaics underwent massively parallel sequencing, due to cost limitations.
The group found that SSCP and DHPLC were able to detect mutant alleles atfrequencies between 5% and 25%, whereas Sanger sequencing required
frequencies between 15% and 50% for detection. The mutations included in the
PTT assay were detected at frequencies between 10 and 100%. The Genome
Sequencer FLX was used for massively parallel sequencing, and this achieved
coverage between 648 and 8313 reads. Mutation frequencies as low as 1% could
be detected, but this required a high coverage (Rohlin et al 2009).

The results from this study showed that Sanger sequencing, which has beencommonly used in a diagnostic setting, was the least sensitive method at detecting
mosaics. Dependent upon the type of mutation being analysed, this technique may
require mutation frequencies as high as 50% in order for them to be detected(Rohlin et al 2009).

Genetic testing in the diagnostic setting is usually carried out on DNA extracted fromwhole blood. For some patients, a mosaic mutation may only be present in colonictissue so will not be detectable through testing blood-derived DNA (Jansen et al2017).

1.6.1.6

The Involvement of Additional Genes

Standard diagnostic protocols carried out in the context of adenomatous colorectal
polyposis typically examine the APC and MUTYH genes, although an increasing

46

number of laboratories are screening DNA for the recurrent mutations in POLE and
POLD1. It is only in recent years that the pathogenic effects of mutant POLE,
POLD1, NTHL1 and MSH3 have been discovered. It is therefore feasible that there
may be further genes involved in the development of heritable colorectal polyposis,
which are yet to be identified.
Furthermore, mutations in genes such as APC, MUTYH, POLE, POLD1, NTHL1
and MSH3 are all highly penetrant. It may be the case that some patients with
unexplained colorectal polyposis have a phenotype which results from the complex
interplay of several low/ moderate penetrance genetic variants.

1.7

Summary

Colorectal carcinoma (CRC) is the fourth most common cancer in the United
Kingdom (UK) and the second most common cause of cancer death (Cancer
Research UK 2015). Most CRCs occur sporadically, but in approximately a third ofpatients, hereditary factors are important (reviewed in Mishra and Hall 2012; Burt
2007). Some patients with an inherited predisposition to CRC will be diagnosed
with a ‘genetic polyposis syndrome’ such as FAP, MAP, PPAP, NTHL1-Associated
Polyposis, MSH3-Associated Polyposis or a hamartomatous polyposis syndrome. It
is important to identify these patients, and to define the mutations causing theirpolyposis, so that the individuals and their relatives can be managed appropriately.

1.8

Aims of Thesis

The genetic bases for several ‘polyposis syndromes’ are known and have been
described above. However, there are numerous patients with a ‘polyposis
phenotype’ who have had no genetic mutation identified (NMI) through standard
genetic diagnostic protocols.

The aim of this thesis is to:

1. Identify novel genetic variants/ molecular mechanisms responsible forcolorectal polyposis using a cohort of polyposis patients who have had no
mutation identified during clinical genetic diagnostic testing. This will
involve:47

a. searching for APC and MUTYH variants which are outside of theopen reading frame (ORF)
b. searching for mosaic APC mutations
c. searching for novel variants in the other known or candidate‘polyposis genes’
d. searching for new polyposis genes
e. characterising novel variants using genetic and functional
approaches, to provide additional evidence for or against their clinicalsignificance48

Chapter 2Materials andMethods
2.1

Materials, Equipment and their Suppliers

2-Propanol

Sigma Aldrich

2X iproof HF Mastermix

BioRad25cm2 flask

ThermoFisher

96-well Assay plate

Fisher Scientific96-well PCR plates

4titude96-well PCR seals

4titudeABI Prism Genetic Analyser

Applied Biosystems

Acetic Acid

Sigma Aldrich

Agarose

ThermoFisher

Agarose plates

Millipore

Ampligase

Illumina

Ampligase DNA Ligase Buffer

Illumina

Anti--Actin Antibody (A5411)

Sigma

Anti-Mouse Antibody (NXA931)

GE Healthcare

Anti-Myc-Tag Antibody (9B11)

Cell Signalling Technologies

-mercaptoethanol

Stratagene

BCA

ThermoScientific

Big Dye Terminator Reaction Mix v1.1

ThermoFisher

BioDoc-IT Imaging System

AnalytikJena

BSA

Promega

Cell Scraper

ThermoFisher

Centrifuge PIC017

ThermoScientific

Chemagic STAR DNA Extraction Kit

ThermoFisher

Complete protease inhibitor

Roche

Control Methylated DNA

QiagenControl Unmethylated DNA

QiagenDMEM (1X)-GlutaMax

Life Technologies

dNTPs

ThermoFisher

DTT

ThermoFisher

49

EDTA

Sigma Aldrich

Epitect Bisulfite Kit

QiagenEpitect Mastermix

QiagenEppendorf Tubes

ThermoFisher

Exonuclease I/III

New England Biolab

Falcon Tubes

ThermoFisher

FBS

ThermoFisher

FS Buffer

ThermoFisher

G Storm Labtech Model GS0002M

LabtechGel RedCambridge Bioscience

Gel Tank

ThermoScientific AB0708 100V/
Clever Scientific

Gene Read FFPE Kit

QiagenGene Ruler 1kb Plus Ladder

ThermoFisher

Glo-Lysis Buffer and Substrate

Promega

GlycerolSigma Aldrich

Haloplex Assay

Agilent

Hemo Klen Taq DNA Polymerase

New England Biolab

HiDi Formamide

ThermoFisher

HiSeq 2500

Illumina

Iblot

Invitrogen

IGEPAL CA630

Sigma Aldrich

Incubator

Millipore

Light Microscope

Both Olympus BX43 and Leica models
have been used

Lipofectamine

Life Technologies

LB Agar Lennox

Invitrogen

LB Broth Base Lennox

Invitrogen

Milk Powder

MarvelMiniPrep Kit

QiagenMiSeq

Illumina

MLPA Kit P043-B1

MRC Holland

MLPA Kit P378-A2

MRC Holland

MMG

Microzone

NaCl Solution

Sigma Aldrich

Nextera Rapid Capture Exome v1.2

Illumina

50

Novex Sharp Pre-Stained Protein Standard

Invitrogen

NuPAGE 4-12% Bis Tris Mini Gel

Invitrogen

NuPAGE MES SDS Running Buffer

Invitrogen

NuPAGE LDS Sample Buffer

Invitrogen

Orange G

Sigma Aldrich

Orange G Solution (20ml)

4 ml 0.5 M EDTA, 8 g Sucrose, pinch of
Orange G, Water

Optimem

Life Technologies

PBS

Santa Cruz Biotech

PhosphoSTOP Phosphatase Inhibitor

Roche

Pipettes and Tips

RaininPlatinum PFX PCR Kit

Invitrogen

Power Supply

BioRad 200/2.0

QBD2 Heat Block

Grant

Quantstudio 12K Real Time PCR System

Life Technologies

Qubit dsDNA BR Assay Kit

ThermoFisher

Qubit dsDNA HS Assay Kit

ThermoFisher

Qubit 2.0 Fluorometer

ThermoFisher

Qubit RNA Assay Kit

ThermoFisher

QuikChange II SDM Kit

Agilent

Random Primers

ThermoFisher

Restore Plus Western Blot Stripping Buffer

ThermoFisher

RNASinThermoFisher

R-Spondin

PeproTech

Shaking Incubator

KühnerShrimp Alkaline Phosphatase

Affymetrix

SOC Medium

Invitrogen

SucroseSigma Aldrich

Superscript II

ThermoFisher

Supersignal West Pico Chemiluminescent
Substrate
TAE (1L)

ThermoFisher
48.4 g Tris, 3.72 g EDTA, 17 ml Acetic
Acid, Water

Taqman Gene Expression Mastermix

ThermoFisher

TBS

Fisher BioReagents

TC Hood

HeraSafe51

TC Incubator (37˚C, 5% CO2, 21% O2)

BinderTE Buffer

Invitrogen

Tempus Blood RNA Tube

Life Technologies

Tempus Spin RNA Isolation Reagent Kit

Applied Biosystems

Tris

Sigma Aldrich

Trypsin (0.05%)

Life Technologies

Trypan Blue (0.4%)

Life Technologies

Vortex Genie 2

Scientific Industries

Waterbath

Clifton

Wnt3a

R&D Systems

WST-1

Sigma Aldrich

XL-1 Blue Cells

Agilent

Zymoclean Gel DNA Recovery Kit

Zymo Research

2.2

Patient Information

This study is approved by the Research Ethics Committee for Wales (REC 3, study
12/WA/0071). To date, 306 patients of the 350 target have been recruited. A subsetof these patients was used for the work described in this thesis.

2.2.1 Inclusion Criteria
Eligible participants included:
1) adult patients who are affected by polyposis (>/= 10 polyps)/ other phenotypes
consistent with APC mutations who have had genetic testing to determine thegenetic cause of their disease that proved negative by routine tests in NHS genetic
diagnostic laboratories or2) adult patients who are affected by polyposis who have previously had genetic
testing to determine the genetic cause of polyposis of the bowel that found an
uncharacterised variant of unknown pathogenicity by routine tests by NHS genetic
diagnostic laboratories or3) adult patients who have been identified as carrying a known polyposis-causing
gene and who were having a screening or a surgical procedure at which polyp orother tumour or cancer tissue may be removed or524) unaffected adult (aged 18 years or over) family members of these patients whose
DNA could serve as a control to assess the association of genetic variants with
polyposis

Participants were recruited from a pool of patients who had previously been referredfor diagnostic genetic testing and/ or genetic counselling at the Institute of Medical
Genetics, Cardiff, or at other regional clinical genetics centres. They were identified
by consultant clinical geneticists or genetic counsellors.

2.2.2 Exclusion Criteria
The study exclusion criteria were as follows:
1) unaffected family members under the age of 18 years of age

2.2.3 Individuals Participating in this StudyOf the 306 patients recruited to the Genetic Mechanisms of Polyposis study, 60
affected individuals were included in the work described within this thesis. Whenthe project started in 2013, these were the recruited patients for whom phenotypic
information was available. I have included 45 patients with a predominantly
adenomatous phenotype (>/= 10 adenomas); 8 patients with a mixed adenoma/
HPP phenotype; and 7 patients for whom histopathological information was scanty.

2.2.3.1

No Mutation Identified (NMI) Polyposis PatientsThe 60 NMI polyposis patients included in this thesis have not had a pathogenic
genetic mutation identified by standard genetic diagnostic protocols in the NHS(although the cohort does include two patients who are carriers of monoallelic
MUTYH mutations). In the genetic diagnostic service, patients have undergone
APC sequence analysis of all the coding exons and up to 20 bases of flanking
intronic sequences. Dosage analysis for all coding exons and the promoter had
been completed using the MRC-Holland MLPA kit P043-B1. All the patients havealso undergone MUTYH analysis. This involved a real time allelic discrimination
assay for the two common European MUTYH mutations (c.536A>G (p.Tyr179Cys)
and c.1187G>A (p.Gly396Asp)) and Sanger sequencing of exons 3 and 14 for the53

two common Asian MUTYH mutations (c.312C>G, p.Tyr104* and c.1432G>T,
p.Glu480*). If this failed to identify any mutations, patients had sequence analysisof all coding exons and up to 20 bases of flanking intronic sequence. Dosage
analysis for all coding exons was undertaken with the MRC-Holland MLPA kit P378A2.

Blood samples for DNA were obtained for all 60 NMI patients. Blood samples for
RNA were obtained for 45 of the NMI patients.

2.2.3.2

Control Samples

The control samples in this study included:

13 unaffected relatives of NMI patients

10 control FAP patients, with known APC mutations



5 control MAP patients, with known MUTYH mutations

37 healthy individuals recruited through a local study: Causes of Bowel
Polyps: Recruitment of Healthy Controls (approved by Cardiff University
School of Medicine Ethics Committee). The inclusion criteria were being
over the age of 18 with no personal/ family history of colorectal polyposis/
CRC

DNA was available for all unaffected relatives, FAP controls and MAP controls.
RNA was prepared from blood samples for 7 unaffected relatives, 3 FAP patients, 4
MAP patients and 37 healthy individuals.

2.2.3.4

Demographic Details and Clinical Phenotypes

The demographic details and clinical phenotypes of the participants are in Tables
2.1 (patients and their unaffected relatives) and 2.2 (further controls). The age ofthe participants refers to the age at which they were recruited to the study. Thiswas deemed the most unambiguous way to describe age, as it was often unclear atwhat age the patients had initially developed polyposis, and the DNA samples usedthroughout this project had frequently been obtained at multiple historical time
points. The phenotypic information was obtained from the patient’s genetic files/

54hospital records, if they had been recruited from Wales, or was supplied by referral
centre if they had been recruited from another region.

UniqueIdentifier

Age atRecruitment

Clinical Phenotype

RNA

Relative(s)and Sex
Halo02

Halo0430-year-old
female(mother):>100 adenomas in 30s

Yes

unaffected,
negativecolonoscopy

Halo05

Halo06

Halo0746-year-old
femaleAged 39 had multiple
adenomas (at least 19): one
contained pT1 CRC

77-year-old

11 polyps: 10 adenomas, 1

male

HPP

70-year-old
male

No

17 adenomas

Yes

Yes
S_Halo08(son) 42-yearold male, over
100 polyps.
He has notbeen includedin the NMI

Halo0870-year-old
femalepatient total as
629 adenomas

Yes

he wasrecruited at alate stage ofthe project, so
DNA was notavailable formost of the
study
protocols

55

Halo1373-year-old
male>10 adenomas

Yes

Subtotal colectomy and

Halo09ileorectal anastomosis abroad
Halo1433-year-old

for adenomatous polyposis.

male

Has had a further 40 polyps(sister):Yes

negative

removed from rectal stumpcolonoscopy?morphology
Halo1584-year-old
male

12 adenomas

unaffected,

No

25 mixed polyps: adenomas
Halo1766-year-old

and hyperplastic lesions (exact

male

numbers of each is not clear).

Yes

2 CRC aged 63 (pT2, pT3)

Halo18

Halo1972-year-old
male

Referred from screening
programme. 27 polyps:

Noadenomas with 4 HPPs

62-year-old>10 adenomas and CRC (nofemalefurther information available)

Yes

Referred from screening
Halo2071-year-old

programme. 12 adenomas,

maleplus additional hyperplastic

Yeslesions (?number and type)
Referred from screening
Halo2273-year-old

programme. Aged 72 had 40+

femaleadenomas and 2 CRC

No(?stage)
Halo23

Halo24

Halo2573-year-old
female68-year-old
male
66-year-old
male>20 adenomas

Yes>10 adenomas (?19)

Yes

12 adenomas

Yes

56

Halo26

Halo2785-year-old
female67-year-old
female21 adenomas

Yes

17 adenomas

Yes

1990: sigmoid colectomy for aprobable diverticularperforation. Multiple 'benign

Halo2861-year-old
male

metaplastic polyps' were noted?number. 2012: subtotal

No

colectomy for >30 polyps. The
majority of the polyps were TA;
2 were serrated adenomas
with LG dysplasia

Halo29Halo30

Halo31

Halo3280-year-old

17 polyps: at least 9 confirmed

male

adenomas and 7 HPPs

73-year-old

20 polyps: at least 13

maleadenomas

70-year-old
male
66-year-old
femaleYes

Yes

12 TA with LGD, 3 HPP

Yes

pT2 CRC + 12 adenomas

Yes

Heterozygous for a nonpathogenic APC variant:c.6363_6365dupTGC.
pT3N1M1 CRC, 4 adenomas
and the patient notes describe
Halo3336-year-old
female"multiple polyps in the sigmoid,smaller numbers of polyps inNo

the proximal left colon, a
couple of tiny polyps in thetransverse colon and anumber of small polyps in theright colon" but there is no
more detail57

Halo34

Halo35

Halo3669-year-old

11 adenomas plus 6 polyps ofmale

unknown morphology

68-year-old
male
67-year-old
male

Yes

15 adenomasYes

At least 10 adenomas

Yes

18 adenomas, 3 HPPs
Halo4070-year-old

Carrier of monoallelic MUTYH

femalemutation (MUTYH c.920G>A,

Yes

pR307G)
10 adenomas, 10 HPPs
Halo4151-year-old

Carrier of a monoallelicmale

MUTYH mutation (MUTYH

Yes

c.1187G>A G396D)
Referred from bowel
screening. Colonoscopy
showed 18 polyps and CRC.

Halo4367-year-old
male

It is not clear how many polyps
were examinedNo

microscopically (at least 5) but
these all showed adenoma.
The CRC was moderately
differentiated ?stage

Halo44

Halo45

Halo4672-year-old

8 TAs, 2 HPPs, one 'mixed

femaleHPP/ TA'77-year-old
male

27 adenomas, 7 HPP

44-year-old

Clinical FAP – has had afemalecolectomy and proctectomy

Yes

Yes

Yes

Colonoscopy found in excess

Halo4751-year-old
femaleHalo68of 50 sessile polyps
throughout the colon. 2 ofthese were biopsied: TVA.
The patient had a subtotalYes(sister): 54year-old
female. 14 TA

58

colectomy - ?4 polyps werewith LGD andsampled: 2 TA, 2 serrated

6 HPPsadenomas. Subsequent
completion colectomy
identified a CRC (pT4N2Mx)

Halo4871-year-old
male

Referred from screening. 30
polyps: 4 have been biopsied:Yes

all adenoma
Rectal cancer age 32 (?stage),

Halo4957-year-old

ameloblastoma age 38. 22

femalepolyps: mixture of adenomasNoand hyperplastic lesions
Halo5064-year-old
male

10 adenomas

No
Halo77(mother),
Halo78(father):
unaffected
Both aredescribed as
unaffected by

Halo5143-year-old
femaleSt. Mark’s
208 adenomas

Yes

Hospital,
where acolonoscopy
waspresumably
performed, but
this isunconfirmed.
RNA available
for bothHalo5259-year-old
maleOver 1000 adenomas

Yes

59

Halo75(mother),
Halo76(father):
Halo5351-year-old

Approximately 400 adenomas,

male

CRC age 27 (Duke’s C)

Yes

unaffected.
Both have had
negativecolonoscopies.
RNA available
for bothHalo5470-year-old
male

Approximately 260 polyps,
rectal cancer age 46 (Duke’s

NoA)
M_Halo55(mother),
F_Halo55

Halo5554-year-old

18 polyps: 9 adenomas, 9

maleserrated lesions(father):Yes

unaffectedaccording toclinical history.
RNA available
for bothHalo73(mother),
Halo74(father):
unaffected.
Mother had

Halo5638-year-old
female57 adenomas

Yes

one polyp in2002 but 2
clear
colonoscopies
subsequently.
Father had 2
polyps atcolonoscopy

60

Halo57

Halo58

Halo61

Halo62

Halo6340-year-old

Approximately 3500

male

adenomas

64-year-old
male

Approximately 100 polyps:
mixture of adenomas and

11 polyps: 2 SAs, 7 TA, 1

female

TVA, and 1 HPP

femaleYes

HPPs

60-year-old

76-year-old

No

Yes

Ileorectal anastomosis, but theindication for prior surgery is

Yesunclear. 10 rectal adenomas

68-year-old

At least 24 polyps. 5 biopsies

male

show 4 adenomas and 1 HPP

Yes

Thousands of colorectal

Halo6454-year-old
femalepolyps. Those which havebeen biopsied showedYesadenomas. CRC aged 23(?stage)

Halo65

Halo66

Halo6733-year-old
male
70-year-old
male
73-year-old
male

117 adenomas

Yes

23 adenomas, 4 HPPs

Yes

At least 15 adenomas

Yes

At age 72 had CRC, pT4, andHalo6973-year-old
femaleat least 24 polyps. It is notclear how many wereNo

removed, but they included
adenomas, HPPs and one SA

Halo7071-year-old

15 polyps. 11 shown to bemaleadenomas on microscopy

Yes

61

Halo71

65-year-old
female14 adenomasYes

At least 10 adenomas, one of

Halo7261-year-old
malewhich contained moderately
differentiated CRC (?stage).

Yes

Additionally, had Duke’s A
sigmoid carcinoma age 53

Halo79

Halo8080-year-old

Colectomy for polyposis. No

maleother information

76-year-old
female38 adenomas

No

Yes

17 polyps: 8 adenomas (4 TA
Halo8165-year-old

with LGD, 3 TVA with LGD, 1

male

TVA arising in a SSL) and 9

No

HPPs

Table 2.1: Table describing the demographic details and clinical phenotype of the NMI
polyposis patients and their relatives included in this study

62

Unique

SampleIdentifier

Details

Halo01

Control FAP

F014_M_
004_1
F014_M_
005_1

Control FAP

Control FAP

Genotype

APC c.36313632delAT

APC c.2805 C>A

APC c.43934394delGARNA

Further Information

No

No

No

FAP mosaic: 5%
F014_M_
006_1

Control FAP

mosaic APC
c.4393-

No4394delGA
APC
Halo10Control FAP

c.3183_3187delA

No

CAAA

Halo12Control FAP

Halo16

Control FAP

FAPPol51

Control FAP

FAPPol14
1

Control FAP

Control FAP,
Halo42

monoallelic
MUTYH
mutation

APC c.39273931delAAAGA
APC c.3408delA
APC promoter 1B
deletion
Deletion of APC
exons 11 and 12

No

No

Yes

Yes

APC c.1187dupA,
MUTYH

Yes

c.536A>G

CompoundHalo37

Control MAP

heterozygous for

Yes

MUTYH

63

c.536A>G andc.649C>T
Compoundheterozygous for
Halo39Control MAP

MUTYH

No

c.303G>T andc.312C>A
MUTYH
Halo59Control MAP

c.1187G>A

Yes

homozygote
Compoundheterozygous for
MAPPol71

Control MAP

MUTYH

Yes

c.303G>T andc.312C>A
Compoundheterozygous for
MAPPol90

Control MAP

MUTYH

Yes

c.1187G>A andc.536A>G
Father of a patient

89-year-old
Halo03

male
Healthywith jaw osteomasNobut not included incontrol

this thesis
Mother of a patient

79-year-old
Halo11

femaleHealthy
controlrecruited to the study

with jaw osteomasNo

recruited to the study
but not included inthis thesis6462-year-old
Halo95femaleHealthy
control

Mother of a patientYeswith HPPs recruited tothe study but notincluded in this thesis

Table 2.2: Table describing the sample details and the genotype of control samples included inthis study.

2.3

General Techniques

2.3.1 Agarose Gel Electrophoresis
Agarose gels were made with 1x TAE buffer to a concentration of 0.8-1.5%. The
appropriate mass of agarose was added to 100 ml of 1x TAE and heated in a
microwave for 2 minutes to allow it to melt. The liquid was cooled by running it
under a cold tap for 2-5 minutes. 5 µl of Gel Red was added, and the liquid poured
into a gel mould to set. The gel was completely submerged in 1x TAE buffer in an
AB0708 100 V gel tank or a Cleaver Scientific gel tank. Unless otherwise stated,
5 µl of Orange G loading dye was added to 5 µl of sample, and the entire volume
was loaded onto the gel. A 1Kb plus DNA ladder (ThermoFisher) was loaded
alongside the samples. Electrophoresis was performed at 100 V for 30 minutes
using the BioRad 200/2.0 power supply. Following electrophoretic separation of thesamples, visualisation was achieved using the BioDoc-It Imaging System Benchtop
UV Transilluminator.

2.3.2 Germline DNA Extraction
Genomic DNA was extracted from venous blood using the Chemagic Star
Automated Extraction Method by the All Wales Medical Genetics Service(AWMGS). At least 4 ml of peripheral blood was collected in an EDTA tube. The
blood was then transferred into a 50 ml tube and lysis buffer added for up-front DNA
extraction. The Chemagic Star instrument uses polyvinyl alcohol particles (M-PVA
Magnetic Beads), which have a hydrophilic surface, to bind to nucleic acids. Oncebound, the nucleic acids were transferred with a magnetisable rod to different wash
buffers and finally to an elution buffer65

(http://www.hamiltonrobotics.com/fileadmin/user_upload/Standard_Solutions/B1110-03_Chemagic_STAR_web.pdf. Accessed 21/5/14). DNA was stored at 20⁰C.

2.3.3 RNA Extraction
RNA was extracted from whole blood using a Tempus Spin RNA Isolation Reagent
Kit (Applied Biosystems). 3 ml peripheral blood was collected in a Tempus blood
RNA tube (Life Technologies) and shaken vigorously for 30 seconds. The blood
sample was poured into a clean 50 ml conical tube. 1x Phosphate Buffered Saline(PBS) was added to bring the volume up to 12 ml. The tube was vortexed for 30
seconds, then centrifuged at 4⁰C for 30 minutes at 3000 x g. The supernatant waspoured into a disinfectant solution. The tube was inverted on absorbent paper for 1
to 2 minutes, and any remaining liquid was blotted from the rim of the tube withabsorbent paper. 400 μl of RNA purification re-suspension solution was added tothe tube, and the tube was vortexed briefly to re-suspend the RNA pellet. RNA wasthen purified according to the manufacturer’s protocol. Briefly, this involved prewetting a filtration membrane with RNA purification wash solution 1, pipetting the resuspended RNA onto the filter, centrifuging the sample for 30 seconds at 16 000 x
g, discarding the liquid waste, pipetting 500 µl wash solution 1 onto the filter andcentrifuging. The liquid waste was discarded, then the process was repeated twicewith wash solution 2. The filter was centrifuged to dry it, then 90 µl elution solution
was added. The tube underwent centrifugation, and the eluted RNA wassubsequently pipetted back onto the filter, which was centrifuged again. All
centrifugation steps were performed at 16 000 x g for 30 seconds, apart from thefinal centrifugation, which was carried out for 2 minutes. Eluted RNA wastransferred to a fresh tube and stored at -80⁰C.

2.3.3.1

Assessment of RNA Quality

RNA integrity was assessed by electrophoresing 3 µl RNA, along with 8 µl Orange
G loading dye, though a 0.8% agarose gel for 40 minutes at 120 V. Images of thegel were taken using the BioDoc-It Imaging System Benchtop UV Transilluminator.
Any degraded samples were omitted from further analysis. If the RNA sample

66

contained a significant amount of genomic DNA, it was omitted from further
analysis.

2.3.3.2

RNA Conversion to cDNA

Following RNA extraction, conversion to cDNA was carried out by making a 12 µl
reaction mix containing 2 µl random primers (100 ng/µl), 1 µl dNTPs (10 mM) and 9µl RNA (2.5 µg/µl). This was incubated for 5 minutes at 65⁰C on the G Storm to
disrupt any template secondary structures. It was then placed on ice to prevent the
re-formation of secondary structures. To the reaction mixture, 1 µl of reverse
transcriptase was added (Superscript II 200 U/µl), and the mixture incubated for 2
minutes at 25⁰C on the G Storm. Subsequently, 4 µl 5x first strand buffer, 2 µl
0.1 M DTT and 1 µl RNAsin was added, and the reaction mix was incubated for 10
minutes at 25⁰C, 50 minutes at 42⁰C and 15 minutes at 70⁰C on the G Storm.
cDNA was stored at -20⁰C.

2.3.4 DNA Extraction from FFPE Tissue
To extract DNA from FFPE tissue, the GeneRead FFPE Kit (Qiagen) was used. 10
μm tissue sections were cut by the Cellular Pathology Department, University
Hospital of Wales. Excess paraffin was trimmed using a sterile scalpel, and thetissue was placed in a 1.5 ml Eppendorf tube. 160 μl deparaffinisation solution wasadded, and the tube was vortexed vigorously for 10 seconds then centrifuged briefly
to bring the sample to the bottom of the tube. DNA extraction was carried out
following the manufacturer’s protocol. Briefly, this involved incubating the solution
at 56⁰C for 3 minutes, then allowing it to cool to room temperature. 55 μl RNasefree water, 25 μl Buffer FTB, and 20 μl proteinase K were added to the solution,
which was vortexed and briefly centrifuged. The solution was incubated at 56⁰C for
1 hour, then at 90⁰C for 1 hour. The solution was briefly centrifuged to remove
drops from inside the lid. The lower, clear phase was transferred to a new
microcentrifuge tube. 115 μl RNase-free water was added and the solution mixed.
35 μl UNG was added, and the solution was vortexed and incubated at 50⁰C for 1
hour in a thermomixer. Again, the mix was briefly centrifuged to remove drops frominside the lid. 2 μl RNase A (100 mg/ml) was added, and the solution was mixed
and incubated for 2 minutes at room temperature. 250 μl Buffer AL was added tothe sample, which was mixed thoroughly by vortexing. 250 μl ethanol (96–100%)67

was added and the mixture vortexed and centrifuged. Subsequently 700 μl of lysate
was transferred to the QIAamp MinElute column, in a 2 ml collection tube. The lid
was closed and the tube centrifuged. The flow-through was discarded and thecollection tube re-used. The process of transferring the lysate/ centrifugation/
discarding the flow-through was repeated until the lysate was used up. 500 μl
Buffer AW1 was added to each spin column and the mixture centrifuged. The flowthrough was discarded and the collection tube re-used. The process was repeatedusing 500 μl Buffer AW2, then 250 μl ethanol. The spin column was then placed
into a new 2 ml collection tube and centrifuged to remove any residual liquid. The
QIAamp MinElute column was transferred into a clean 1.5 ml microcentrifuge tube.
40 μl Buffer ATE was pipetted on to the centre of the membrane. The tube wasincubated at room temperature for 5 minutes, then centrifuged. All centrifugations
were performed for 1 minute at full speed. DNA was stored at -20⁰C.

2.3.5 Quantification of Nucleic Acids
Two platforms were used to quantify nucleic acids – the Nanodrop and the Qubit 2.0
Fluorometer. In both cases, 2 µl of sample was used for quantification, following the
manufacturer’s protocol.

2.3.6 Primer Design
Unless otherwise stated, all primers were designed using Primer3 v0.4.0 (Rozen
and Skaletsky 2000) and were supplied by Eurogentec or Eurofins. The specificity
of primer pairs was determined using UCSC In Silico PCR software(https://genome.ucsc.edu/cgi-bin/hgPcr).

2.3.7 Bisulfite Conversion of DNA
Sodium bisulfite conversion of DNA is a technique which allows the detection of
unmethylated versus methylated cytosines. It involves the deamination of
unmodified cytosines to uracil, leaving the methylated cytosines intact.
DNA was bisulfite-converted using the Epitect Bisulfite Kit (Qiagen) following the
manufacturer’s protocol. Briefly, this involved making a mixture of 2 µg DNA, 85 µl
Bisulfite Mix, 35 µl DNA protect buffer and an appropriate volume of RNase-free

68water to make the total volume 140 µl. The mixture was vortexed. The bisulfite
conversion was carried using the G Storm thermal cycler for 5 minutes at 95⁰C, 25
minutes at 60⁰C, 5 minutes at 95⁰C, 85 minutes at 60⁰C, 5 minutes at 95⁰C and 175
minutes at 60⁰C. The PCR tubes were briefly centrifuged, then the complete
reaction was transferred to a clean tube. 560 µl Buffer BL containing 10 µg/ml
carrier RNA was added to each sample. This was then vortexed and briefly
centrifuged. The entire reaction mix was transferred into an Epitect spin column,
which was centrifuged at maximum speed for 1 minute. The flow-through was
discarded and the spin column placed back in the collection tube. The process was
repeated with 500 µl Buffer BW. 500 µl Buffer BD was added and the mixture
incubated for 15 minutes at room temperature. The spin columns were centrifugedat maximum speed for 1 minute. The flow-through was discarded, and the spincolumns were placed back in the collection tubes. The process was repeated two
further times, using 500 µl Buffer BW. The spin columns were then placed in clean
collection tubes and centrifuged at maximum speed for 1 minute to remove any
residual liquid. The spin columns were placed with the lids open into clean
microcentrifuge tubes and incubated for 5 minutes at 56⁰C in a heating block. Spincolumns were then placed into clean microcentrifuge tubes. 20 µl Buffer EB was
pipetted onto the centre of each membrane, and the purified DNA was eluted bycentrifugation for 1 minute at 15 000 x g. To complete the process, 20 µl of Buffer
EB was pipetted to the centre of each membrane and centrifuged for 1 minute at
maximum speed. The resultant bisulfite-converted DNA was stored at -20⁰C.

2.3.8 Standard PCR and Sanger Sequencing
The standard steps for PCR and Sanger Sequencing are:
1. PCR
2. Gel Electrophoresis
3. ExoSap PCR Purification
4. Big Dye Reaction
5. Isopropanol Clean Up
6. Sequencing and Data Analysis

These shall be described in sections 2.3.8.1-2.3.13.

69

2.3.8.1

Standard Reagents for PCR

Water

11 µl

Forward primer (10 µM)

0.25 µl

Reverse primer (10 µM)

0.25 µl

Megamix Gold (MMG)

12.5 µl

DNA/ cDNA (5 ng/µl)

1 µl

2.3.8.2

Reagents for PCR for FFPE Tissue DNA

Water

Variable

Forward primer (10 µM)

0.25 µl

Reverse primer (10 µM)

0.25 µl

Megamix Gold (MMG)

15 µl

DNA

10 ng

2.3.8.3

Reagents for Fast-COLD-PCR

Water

11 µl

Forward primer (10 µM)

0.25 µl

Reverse primer (10 µM)

0.25 µl

Megamix Gold (MMG)

12.5 µlDNA (5 ng/µl)

1 µl

2.3.8.4

Reagents for Full-COLD-PCR

Water

11 µl

Forward primer (10 µM)

0.25 µl

Reverse primer (10 µM)

0.25 µl

Megamix Gold (MMG)

12.5 µlDNA (5 ng/µl)

1 µl

70

2.3.8.5

Standard PCR Reaction Conditions:

95⁰C for 5 minutes
95⁰C for 1 minute
Annealing temperature: 58⁰C for 1 minuteElongation temperature 72⁰C for 1 minute
Number of cycles: 35
72⁰C for 5 minutes

2.3.8.6

Reaction Conditions for Fast-COLD PCR:

95⁰C for 5 minutes
70⁰C - 95⁰C for 1 minute
58⁰C for 1 minute
72⁰C for 1 minute
Number of cycles: 35
72⁰C for 5 minutes

2.3.8.7

Reaction Conditions for Full-COLD-PCR:

Cycle 195⁰C for 5 minutes
95⁰C for 1 minute
58⁰C for 1 minute
72⁰C for 1 minute

Number of cycles: 10
72⁰C for 5 minutes

Cycle 2:

95⁰C for 1 minute
70⁰C for 5 minutes
70-95⁰C for 1 minute
58⁰C for 1 minute
72⁰C for 1 minute

Number of cycles: 25
72⁰C 5 for minutes

71For both the fast and the full protocols, the initial COLD-PCR protocol allowed thedetermination of the lowest denaturation temperature over a wide range oftemperatures (70-95°). Once this was ascertained, the protocol was repeated using
a narrower temperature gradient to further specify the lowest denaturation
temperature.

2.3.9 Gel Electrophoresis
To confirm that PCR reactions had worked and that the PCR products were thecorrect size, products were electrophoresed through a 1.5% agarose gel, with acurrent of 120 V for 30 minutes. Images of the gel were taken using the BioDoc-It
Imaging System benchtop UV transilluminator (see 2.3.1).

2.3.10

ExoSap PCR Purification

The ExoSap PCR purification reaction involves an exonuclease enzyme degrading
excess primers and any ssDNA present, and an alkaline phosphatase enzyme
degrading any dNTPs. The ExoSap stock was made from Exonuclease and Shrimp
Alkaline Phosphatase at a ratio of 1:2. 1 μl of ExoSap was added to PCR products.
These were incubated on the G Storm for one hour at 37⁰C , then for 15 minutes at
85⁰C .

2.3.11Big Dye Reaction

The Big Dye Terminator v3.1 Sequencing Kit was used for the sequencing reaction.
1 μl of PCR product was added to a reaction mixture containing:

Big Dye v.3.1

0.75 μl

Water

5.25 μl

Primer (10 μM)

1 μl

BD Buffer

2 μlThis then underwent thermal cycling on the G Storm: 25 cycles of 95⁰C for 10
seconds, 50⁰C for 5 seconds, 60⁰C for 3 minutes and 30 seconds.

72

2.3.12Isopropanol Clean-Up Protocol

Isopropanol was used to precipitate DNA to allow purification. 40 μl of 75%
isopropanol was added to each sample, which were incubated for 30 minutes atroom temperature before being centrifuged for 45 minutes at 4000 revolutions per
minute (rpm). The samples were inverted, placed into the centrifuge upside down
and spun for 30 seconds at 500 rpm. Once this was complete, they were left in thedark to dry for 10 minutes before 10 μl of Hi-Di was added to re-suspend the DNA.

2.3.13

Sequencing and Data Interpretation

The samples were sequenced on the ABI Prism 3100 Genetic Analyser.
Sequencing data was read using Sequencher 5.2.4 software.

2.3.14

Methylation-Specific PCR (MSP)

Methylation-specific PCR is a technique used to detect DNA methylation. Primers
are designed to bind to bisulfite-converted DNA dependent upon whether theoriginal DNA molecule was methylated/ unmethylated. The presence or absence ofa PCR product will therefore indicate the methylation status of the original DNA. In
this thesis, MSP was used to determine the methylation status of the APC 1A and1B promoters.

2.3.14.1

Reagents for APC Promoter 1A MSP

Epitect Mastermix

25 µl

Forward primer

500 ngReverse primer

500 ngWater

Variable

DNA

50 ng

Total:

50 µl

2.3.14.2

Reaction Conditions for APC Promoter 1A MSP

95⁰C

10 minutes73

35 cycles of:
95⁰C

30 seconds

53.9⁰C

30 seconds

72⁰C

30 seconds

72⁰C

10 minutes

2.3.14.3

Reagents for APC Promoter 1B MSP

Epitect Mastermix

25 µl

Forward primer

250 ngReverse primer

250 ngWater

VariableDNA

100 ngTotal:

50 µl

2.3.14.4

Reaction Conditions for APC Promoter 1B MSP

95⁰C

10 minutes

35 cycles of:
95⁰C

30 seconds

50.1⁰C

30 seconds

72⁰C

30 seconds

72⁰C

10 minutes

2.3.15

Quantitative PCR (qPCR)

Quantitative PCR (qPCR) is a PCR technique in which the reaction is monitored inreal time. This allows the quantification of the input sample, as higher sample
concentrations will result in PCR products which can be detected at an earlier stage.
This study used Taqman technology (ThermoFisher). The principles underpinning it
are that sample DNA is denatured, at the appropriate annealing temperature
primers bind, along with a Taqman probe which includes a fluorescent dye and a
non-fluorescent quencher (NFQ). During DNA synthesis, using the primers and the74template DNA, the 5’nuclease activity of the Taq polymerase will cleave the probe,
separating the dye from its quencher. The dye will then emit a signal which isdetected. With each cycle of PCR amplification, increasing amounts of dye arereleased, therefore the fluorescent signal will increase.

Prior to performing qPCR reactions, the basic foundations for this work were
established, i.e.

1. Determination of optimal RNA input
2. Determination of reaction efficiencies, to ensure that that ΔΔCt
method could be used for data analysis

The details of this are described in Appendix 2.1.

2.3.15.1

qPCR Reagents

20X Taqman Gene Expression Assay

1 µl

2X Taqman Gene Expression Master Mix

10 µl

cDNA template

4 µl

Water

5 µl

2.3.15.2

qPCR Reaction Conditions

Hold 50⁰C for 2 minutes
Hold 95⁰C for 10 minutes
Cycle (40 cycles): 95⁰C for 15 seconds, 60⁰C for 1 minute

The thermal cycler used was the QuantStudio 12K Flex Real-Time PCR System(Life Technologies). qPCR reactions were carried out in triplicate for each sample.
A no-template control was included for each assay.

2.3.16

APC and MUTYH Expression in Healthy Controls

This study involved comparing the cDNA levels of APC and MUTYH in NMI
polyposis patients to a cohort of healthy controls.

75

2.3.16.1

qPCR in a Healthy Control Cohort

Venepuncture was performed on the healthy control cohort after each volunteer had
given informed consent. RNA was extracted (2.3.3, 2.3.3.1) and reverse
transcribed to cDNA (2.3.3.2). A total of 44 cDNA samples from healthy relatives
and healthy controls underwent qPCR. The reagents and reaction conditions aredescribed above (2.3.15.1, 2.3.15.2). The Taqman assays used are listed in Table
2.3:

Gene

Designation

Taqman Assay

GAPDH

Endogenous control

HS02758991_g1

ACTB

Endogenous control

HS99999903_m1

APC

Target

HS01568269_m1

MUTYH

TargetHS01014856_m1

Table 2.3: Taqman assays used for qPCR

2.3.16.2

Data Analysis

Results were interpreted using ThermoFisher Cloud software(https://apps.thermofisher.com/apps/dashboard/#/). The first stage of analysis wasa quality assessment. Any results which were flagged as outliers were examined.
This usually referred to results which had a CT value which deviated significantly
from the CT values in the associated replicate group, or whole replicate groups
which had a significant deviation within them.

The individual replicate results which deviated significantly from their replicate groupwere excluded as this was likely due to experimental error and would unfairly skew
results. Those replicate groups which had a significant deviation within them were
excluded as the results were not tight enough for robust analysis. If a sample had acomplete replicate group for one of the endogenous controls excluded, that sample
was removed from further analysis as we were seeking to normalise results to two
different endogenous controls. Therefore, some samples needed to undergo qPCR
a second time to obtain usable results.

The final 40 samples included in this study are listed in the Table 2.4:

76

Sample ID

Sample group

C2-C27, C30-C33, C35, C37, C39

Healthy Controls

M_Halo55

Healthy Controls

F_Halo55

Healthy Controls

Halo75

Healthy Controls

Halo76

Healthy Controls

Halo77

Healthy Controls

Halo78

Healthy Controls

Halo79Healthy Controls

Table 2.4: Healthy control samples used for qPCR studies

2.4

Next Generation Sequencing (NGS)

2.4.1 Haloplex Assay
The probes for the Haloplex assay were designed using Agilent’s Sure Design
software (https://earray.chem.agilent.com/suredesign/home.htm). The probes
generate read lengths of 250 base pairs.

2.4.2 Target Gene Capture and Sequencing
The concentration of the DNA samples to be examined were determined using the
Qubit 2.0 Fluorometer. The DNA was diluted to a concentration of 5 ng/μl.
Sequencing was carried out by Dr. James Colley, WGP, using the HiSeq 2500
(Illumina). The protocol involved digesting gDNA with 16 different restriction
enzymes to create a library of restriction fragments. Digested DNA was hybridised
to Haloplex probes for target enrichment and sample indexing. The DNA-Haloplex
probe hybrids were captured by streptavidin beads, prior to the ligation of thecircularised fragments. DNA was eluted with sodium hydroxide and underwent PCR
to amplify the target libraries. Target libraries were purified, quantified and pooled
before sequencing.

772.4.3 Bioinformatic Analysis Following UDS
Bioinformatic analysis was performed by Dr. Peter Giles, WGP.
Reads were analysed using the following procedure:

1. Raw reads (in fastq format) were transferred from the sequencer to the local
computer cluster
2. Reads were mapped against the hg19 genome using bwa-mem, producing a
BAM file
3. Quality control was reviewed using 10% of reads using a combination of in-house
scripts, FastQC and circos for visualisation
4. GATK was then used to recalibrate quality scores and to apply a localised
realignment around indels before filtering for artefactual duplicates using samtools
5) Variant calling was performed using GATK (Unified Genotyper) and varscan (forlow frequency variants) producing VCF files
6) Variants were annotated, including the following parameters:- Coverage- Allele balance- UCSC genes- 1000 genomes- dbsnp137- Gene description
7) Variants were visualised using Integrative Genomics Viewer (IGV)

2.4.4 Whole Exome Sequencing (WES)
Sequencing was performed by the WGP. Briefly, 50 ng of gDNA was used as the
input template. Sequencing libraries were prepared using the Illumina Nextera
Rapid Capture Enrichment kit. Subsequent steps included tagmentation of thegDNA, clean-up of the tagmented DNA, amplification of DNA, clean-up of amplified
DNA, hybridisation of probes, capture of the hybridised probes, second hybridisationof probes, second capture, clean-up of the captured library, amplification of enrichedlibrary, clean-up of the enriched library, validation of the complete library.
The manufacturer’s instructions were largely followed with extra quantitation steps
prior to the hybridisation of the probes to ensure that close to 50 ng of each sample
was pooled. The libraries were validated using the Agilent 2100 Bioanalyser and ahigh-sensitivity kit (Agilent Technologies) to ascertain the insert size, and the Qubit782.0 Fluorometer was used for quantitation. Following validation, the libraries werenormalised to 4 nM, pooled together and clustered on the cBot™2 following the
manufacturer’s recommendations. The pool was sequenced using a 75-basepaired-end (2x75bp PE) dual index read format on the Illumina HiSeq 2500 in highoutput mode according to the manufacturer’s instructions.

2.4.5 Bioinformatic Analysis Following Whole Exome
Sequencing
Bioinformatic analysis was performed by Dr. Kevin Ashelford (WGP).
Reads were analysed following the steps outlined below:

1. The raw output from the HiSeq 2500 were BCL files. These, once transferredfrom the sequencing rig, were converted to fastq.gz using the Illumina tool bcl2fastq(version 2.17.1.14; https://support.illumina.com/content/dam/illuminasupport/documents/documentation/software_documentation/bcl2fastq/bcl2fastq_letterbooklet_15038058brpmi.pdf)
2. Initial quality control was performed on 10% of the data (for speed) using a
combination of an in-house script and FastQC (version
0.11.2; http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The in-house
script performed a quick mapping of the data using BWA-MEM (version
0.7.4; http://bio-bwa.sourceforge.net) and then calculated percentage mapped andinsert size
3. Reads were trimmed with Trimmomatic (version
0.35; http://www.usadellab.org/cms/?page=trimmomatic) to remove any adapter andpoor-quality base calls from the ends of reads
4. Trimmed reads were then mapped with BWA-MEM (version 0.7.10). Mapping
was performed against standard human reference hg19 (Feb. 2009 (GRCh37/hg19)— http://genome-euro.ucsc.edu/cgibin/hgGateway?db=hg19&redirect=manual&source=genome.ucsc.edu)
5. Mapping outputs were then post-processed using several GATK (version
3.3.0; https://software.broadinstitute.org/gatk/) tools: (i) RealignerTargetCreatorand IndelRealigner to realign reads around indels, (ii) BaseRecalibrator
and PrintReads to recalibrate raw Quality Values. The Picard toolkit (version
1.118; https://broadinstitute.github.io/picard/) tool MarkDuplicates was then used toflag duplicate reads

79

6. After post-processing a further round of quality control was performed using
FastQC as before and an in-house script to calculate on-target mapping metrics
7. Raw variant calls were called on each sample separately using GATK
HaplotypeCaller in GVCF mode followed by GenotypeGVCFs to produce a final
combined vcf file. Raw variant calls were then filtered using VariantRecalibrator toprovide Variant Quality Score Recalibration (VQSR) which allows for dynamic
filtering of variants using machine learning techniques
8. Variants were then annotated against databases dbSNP version 138,
1000Genomes, COSMIC and SIFT using ANNOVAR (version
20150322; http://annovar.openbioinformatics.org). In-house scripts were used toformat the output and extract additional filtering criteria including coverage depths,
allele balance, and gene descriptions

2.4.6 Single Molecule Molecular Inversion Probe Gene
Capture, Sequencing and Bioinformatic Analysis
1. A reaction mixture composed of the following reagents was prepared:DNA (5 ng/µl)

10.0 µlHemo Klen Taq DNA Polymerase (10 U/µl)

0.32 µlAmpligase DNA ligase (100 U/µl)

0.01 µl

smMIP

3.49 µl

10X Ampligase DNA ligase buffer

2.5 µldNTPs (0.25 mM)

0.32 µlWater

8.36 µl

To make the reaction mix, the ligase, buffer and dNTPs were combined. To
this, the smMIPs and water were added, then finally the DNA
2. The mix was incubated at 95⁰C for 10 minutes, and then at 60⁰C for 18 hours
3. To ensure that the gene capture was successful, 5 µl of the reaction mixture
was electrophoresed through a 3% agarose gel for 30 minutes. Images of thegel were taken with the Bio Rad Gel Doc XR+ System
4. The reaction mixture was cooled and kept on ice. An exonuclease reaction
mixture was added to each sample. This was composed of:EXOI

0.5 µl

EXOIII

0.5 µl

Ampligase buffer

0.2 µl

80



Water

0.8 µl

5. The mixture was incubated at 37⁰C for 45 minutes and then at 95⁰C for 10
minutes
6. A test PCR was carried out on a subset of the reactions, to ensure that PCR
amplification would be successful. The following reagents were used:

2X iProof HF Mastermix

12.5 µlIllumina forward primer* (100 µM)

0.125 µl

Barcoded reverse primer PGM* (100 µM)

0.125 µlWater

7.25 µlTemplate MIP reaction

5 µl(*Test PCR primers: SLXA_PE_MIPBC_FOR:
AATGATACGGCGACCACCGAGATCTACACATACGAGATCCGTAATCGGGAAGCTGAAG;SLXA_PE_MIPBC2_REV_001: CAAGCAGAAGACGGCATACGAGATGTTAAGACACACGCACGATCCGACGGTAGTGT Bold: sample barcode)
7. The reaction mixture underwent the following reaction:
98⁰C for 30 seconds

Cycles of: tumour tissue: 24 cycles, normal tissue 21 cycles:98⁰C for 10 seconds



60⁰C for 30 seconds



72⁰C for 2 seconds

8. 5 µl of PCR products were electrophoresed through a 3% agarose gel for 30
minutes. The PCR bands were visualised using the Bio Rad Gel Doc XR+
System. Provided that PCR had been successful, all samples underwent thereaction. The reagents for each reaction were as follows:

2X iProof HF Mastermix

37.5 µlIllumina forward primer* (100 µM)

0.375 µl

Barcoded reverse primer PGM* (100 µM)

5.0 µl

Water

18.375 µlTemplate MIP reaction

15 µl

9. Reaction conditions are as described in Step 7
10. 5 µl of PCR products were electrophoresed through a 3% agarose gel for 30
minutes. The PCR bands were visualised using the Bio Rad Gel Doc XR+
System
11. Each sample was given a score based on the intensity of its PCR band: High,
Medium, Low

81

12. The samples were pooled so that approximately equivalent amounts of eachsample were included. There were separate pools for tumour DNA andgermline DNA
13. The pools were run on a 3.5% agarose gel for 30 minutes. Images of the gelwere taken with the Bio Rad Gel Doc XR+ System
14. The PCR products were extracted from the gel using a Zymoclean Gel DNA
Recovery Kit
15. PCR products were assessed using the TapeStation to confirm that products ofthe correct size were present. If the TapeStation was suggestive of >1 productin the reaction mix, steps 13-15 were repeated to try and purify the desired
product
16. The samples were quantified using the Qubit Fluorometer 2.0, following the
manufacturer’s protocol
17. All pools were combined into a megapool, and the concentration was quantified
using the Qubit Fluorometer 2.0
18. The megapool was diluted to 2nM
19. Sequencing was performed using the MiSeq (Illumina)

Following sequencing, bioinformatic analysis was carried out by Laura Chegwidden,
Oxford University/ Birmingham University. The protocol is outlined below:
Samples were demultiplexed using basespace. The FASTQ files were downloaded
for each sample. Each FASTQ file was processed using Perl scripts from Roland
Arnold to:
1. remove MIP backbones from read 1 (R1) and read 2 (R2)
2. remove reads that were <55bp long after processing
3. remove reads whose mate had been filtered out to leave paired reads only
Next, tools were used as suggested by the MIPGEN pipeline builder(https://github.com/shendurelab/MIPGEN/tree/master/tools):
1.

Use PEAR to join R1 and R2

2.

Use a python script to move the single molecule tag to the FASTQ header

82

Mapping and basic quality control was performed following the processes described
below:
1. BWA mem was used to align reads to hg19
2. Python script was employed to collapse reads into single molecule tag
defined read groups
3. Statistics were generated on the number of panel targets covered at 20X to
identify samples that had failed. A sample was considered as having“passed” basic sequence QC if >50% of targets were covered at 20x
Samtools was used to make BAM files.
For Indel realignment, pre-processing was carried out as per the lofreq website.
This involved Viterbi realignment, adding indel quality scores to files and adding
alnqual. Variants were called using Lofreq-star:

call-indelsm 60 (filtering on mapping quality >60)



no-default-filter (these were set at a later stage)

Following calling, variants were filtered according to the following criteria:

cov-min 10 (min coverage 10)

sb-thresh 60 (strand bias phred 60)



a 0.01 (MAF>0.01)

Formatting/ further filtering involved:


Filtering the data so that calls with >5 reads in either Forward OR Reverse
remained. Those with <5 reads were omitted

Assigning genotypes (VAF<0.75=HET; VAF>=0.75=HOM)



MAF>0.05

Assessing strand bias: if there were reference reads in Forward/Reverse,
but there were no variant reads in the opposite strand, the variant wasomitted

Annotation using VEPRemoving likely germline variants

Filter "ExAC.r0.3.nonTCGA_AF > 0.01"83Filter "UK10K_AF > 0.01"

Filter "EUR_MAF > 0.01"

Adjusting formats & layouts so that vcf (original lofreq call) columns werereported first, then VEP annotations

Filtering to remove intronic, synonymous and splice region variants

Variants were removed if they were part of a homopolymer repeat >4 inlength

Variants were removed if they were discovered as part of a search for panel
artefacts- poorly performing molecular inversion probe/ hard to sequence
region

2.5

The high confidence call sets had a depth > 20 and VAF support > 5%

Molecular Biology Techniques

2.5.1 Plasmid Retrieval from Filter paper
The demarcated spot containing plasmid was cut out from the filter paper using aclean razor blade. It was immersed in 30 µl TE and mixed with a pipette. It was left
for at least 10 minutes at room temperature prior to bacterial transformation. If it
was not used immediately, DNA was stored at -20C.

2.5.2 Preparation of Lysogeny Broth (LB)
To prepare LB, 20 g LB Broth Base was added to 1 L distilled water. It was
autoclaved at 126⁰C for 45 minutes. An appropriate antibiotic, at 1:1000 dilution,
was added when the solution had cooled down. The broth was stored at room
temperature.

2.5.3 Preparation of Lysogeny Broth (LB) Agar
To prepare LB agar, 10.5 g LB Agar was added to 300 ml distilled water. It was
autoclaved at 126⁰C for 45 minutes. It was allowed to cool slightly, prior to adding
an appropriate antibiotic, at 1:1000 dilution, when hand-warm. It was poured into84

plates next to a naked flame. Once the agar had set, it was turned upside down toprevent condensation from dripping onto the agar. The plates were stored at 4⁰C .

2.5.4 Bacterial Transformation of XL1-Blue Competent Cells
All steps of the protocol were carried out next to a naked flame, and the equipment
openings/ lids were quickly waved through the flame. Bacterial transformation
followed the protocol detailed below:

1. The waterbath was warmed to 42⁰C
2. SOC medium was warmed to room temperature
3. The agarose plates containing the desired antibiotic were warmed to37⁰C in an incubator
4. Competent E. Coli cells were thawed on ice
5. 25 µl bacteria was transferred into a pre-cooled 1.5 ml Eppendorf
tube
6. 0.4 µl -mercaptoethanol was added to each tube, which were kept
on ice7. 1 µl circularised DNA was added and the solution and mixed gently
8. The mix was incubated on ice for 30 minutes
9. The Eppendorf was placed into the water bath at 42⁰C for 45
seconds to allow transformation
10. The Eppendorf was then put back onto ice for 2 minutes, to reduce
damage to the bacteria
11. 250 µl SOC medium (without antibiotics) was added
12. The tubes were incubated at 37⁰C for one hour while shaking using ashaking incubator
13. Approximately 200 µl of the culture was spread onto LB plates withthe appropriate antibiotic. It was grown overnight at 37⁰C in an
incubator
14. After 12-16 hours, one clone was picked up using a pipette tip. The
tip was placed into a 25 ml Falcon tube with 5 ml LB + antibiotic
15. The Falcon tube was incubated at 37⁰C overnight in an incubator
prior to DNA extraction85

2.5.5 Plasmid Extraction from Transformed Bacteria
Plasmid extraction from bacteria was achieved using the MiniPrep Kit (Qiagen)following the manufacturer’s protocol. Briefly, the bacterial culture was pelleted bycentrifugation at >8000 rpm for 3 minutes at room temperature. The pellet was resuspended in 250 µl Buffer P1 and transferred to a microcentrifuge tube. 250 µl
Buffer P2 was added, and the solution mixed by inverting the tube 6 times. 350 µl
Buffer N3 was added, and the solution mixed by inverting the tube 6 times. The
solution was centrifuged at 13000 rpm for 10 minutes. The supernatant was added
to a QIAprep spin column and centrifuged at 13000 rpm for 1 minute. The flowthrough was discarded. 500 µl Buffer PB was applied to the membrane and thetube was spun at 13000 rpm for 1 minute. The flow-through was discarded. 750 µl
Buffer PE was added and the tube was spun at 13000 rpm for 1 minute. The flowthrough was discarded. The tube was spun again at 13000 rpm for 1 minute, andthe flow-through discarded. The QIAprep column was placed into a clean
microcentrifuge tube. To elute the DNA, 50 µl Buffer EB was added. The tube wasleft to stand for 1 minute and was then spun for 1 minute at 13000 rpm.

2.5.5 Sequencing of Plasmid DNA
Sequencing of plasmid DNA was carried out by Dundee Sequencing(https://www.dnaseq.co.uk/). 600 ng of DNA in 20 µl double distilled water was
submitted to the laboratory, which use plasmid-appropriate sequencing primers.

2.5.6 Site Directed Mutagenesis (SDM) and Transformation of
XL-1 Blue Competent Cells
Site directed mutagenesis was performed using the QuikChange II Kit (Agilent). Allprimers were designed using Agilent’s online primer-design tool
(www.agilent.com/genomics/qcpd). Briefly, the protocol involved carrying out amutant-strand synthesis reaction, using the following reagents:

10X Reaction Buffer

5 µl

dsDNA Template

5-50 ng

125 ng Forward primer

Variable

125 ng Reverse primer

Variable86

dNTP mix

1 µl

Water

Variable to a final volume of 50 µl

1 µl of PfuUltra HF DNA polymerase (2.5 U/µl) was added and the reaction mix
underwent thermal cycling using the conditions outlined as follows:

95⁰C

30 seconds

16 cycles of:
95⁰C

30 seconds

55⁰C

1 minute

68⁰C

7 minutes

After thermal cycling, the reaction mixes were placed on ice for 2 minutes.
1 µl of Dpn1 restriction enzyme was added.

The mutant dsDNA was subsequently used to transform XL-1 Blue Competent cells
using the following protocol:

1. XL1-Blue cells were thawed on ice. 50 µl of cells were transferred to a prechilled 14 ml BD Falcon polypropylene round-bottom tube
2. 1 µl of the SDM reaction mixture was added to the cells
3. The cells underwent a heat pulse for 45 seconds at 42⁰C in the waterbath
and were then placed on ice for 2 minutes
4. SOC media was preheated to 42⁰C in the waterbath, and 0.5 ml was added
to the cells. The cells were incubated at 37⁰C for 1 hour whilst shaken at225-250 rpm in the shaking incubator
5. 250 µl of each transformation reaction was then plated onto ampicillincontaining agar plates
6. The transformation plates were incubated at 37⁰C for at least 16 hours in an
incubator

Following the transformation reactions, colonies were selected and grown as
described in section 2.5.4, steps 14 and 15. DNA was extracted using the MiniPrep
Kit (2.5.5).

87

2.5.7 Defrosting HEK293 TCF-Luc Cells
HEK293 TCF-Luc cells were kept in 1 ml aliquots at -80⁰C .

They were defrosted following the protocol detailed below. Where possible, allsteps were performed using a sterile technique in a TC hood:

1.

DMEM (1X)-GlutaMax-10% FBS media was warmed to 37⁰C in thewaterbath

2.

The HEK293 TCF-Luc cells were thawed at 37⁰C in the waterbath for 1-3
minutes3.

9 ml of pre-warmed media and 1ml cells were pipetted into a 15 ml
Falcon tube

4.

Mixing was carried out by gentle inversion

5.

The cells underwent centrifugation for 5 minutes at 250 x g

6.

The supernatant was discarded

7.

The pellet was re-suspended with 10 ml media

8.

The cells underwent centrifugation for 5 minutes at 250 x g

9.

The supernatant was discarded

10.

The pellet was re-suspended in 5 ml media

11.

The suspension was transferred to a 25 cm2 flask

12.

It was kept at 37⁰C in an incubator

2.5.8 Splitting Cells
Cells were kept in an incubator at 37⁰C with 5% CO2. Once they reachedapproximately 70% confluence, they were split as described below. Where
possible, all steps were performed using a sterile technique in the TC hood.
Prior to the reactions, DMEM-Glutamax-10% FBS, PBS and Trypsin were prewarmed to 37⁰C in the waterbath.

1.

The media was removed from the flask

2.

10 ml PBS was added, and the flask was gently agitated to wash the
cells

3.

The PBS was removed

4.

3 ml 0.05% Trypsin was added

5.

The flask was incubated at 37⁰C for 1 minute in the incubator

88

6.

7 ml DMEM-Glutamax-10% FBS was added

7.

The solution was transferred to a 15 ml Falcon tube

8.

The Falcon tube was centrifuged for 5 minutes at 250 x g to create apellet9.

The cells were re-suspended in 10 ml DMEM-Gluatamax-10% FBS

10.

To maintain cells, they were diluted an appropriate amount (e.g. 1:4, 1:6,
1:10 depending on when they were required for the next reaction. Cells
were diluted in DMEM-Glutamax-10% FBS) and kept in a 25 cm2 flask

11.

The flask was incubated at 37⁰C in an incubator

2.5.9 Plating the Cells for Transfection
Once the cells reached approximately 70% confluence, they were plated fortransfection. Steps 1-6 from Section 2.5.8 were followed. Once the DMEMGlutamax-10% FBS had been added to the cells, they were counted.
20 µl cells were mixed with 20 µl Trypan Blue. 20 µl of the solution was pipetted
onto a haemocytometer, and the cells were counted using a light microscope(Leica).

For a full 96-well assay plate, 1 500 000 cells were pipetted from the solution andadded to a 15 ml Falcon tube. 10 ml DMEM-Glutamax-10% FBS was added, andthe solution was centrifuged for 5 minutes at 250 x g. Following centrifugation, thesupernatant was discarded. 10 ml DMEM-Glutamax-10% FBS was added to resuspend the cells. 100 µl of cell solution (15 000 cells) was added to each well of a
96-well Assay plate. The plate was stored at 37⁰C in an incubator.

2.5.10Transfection

Once the cells were 70-80% confluent they were transfected.

For the Luciferase assays, transfection was performed with the following plasmids:

WT-AXIN2, Fearon-AXIN2 (c.1989G>A), Short-AXIN2 (c.1642G>T), WT/ShortAXIN2. The WT-AXIN2 and Fearon-AXIN2 had kindly been supplied by Professor
Eric Fearon, University of Michigan, and were N-terminal 6xmyc-tagged AXIN2 in89

pCS2+MT. Short-AXIN2 was generated by SDM of the WT-AXIN2 as described in
2.5.6. For the WT/ Short mix, WT and Short plasmids were used at a ratio of 1:1.

To carry out the reactions, Lipofectamine was diluted in Optimem and incubated for
5 minutes at room temperature. For each well of 15 000 cells, the following
volumes were required:

Lipofectamine

0.3 µlOptimem

24.7µl

A total of 100 ng/well of plasmid was used for transfection.
For each AXIN2 plasmid, a series of 3-fold dilutions were made, with a maximum
amount of 90 ng plasmid (90 ng, 30 ng, 10 ng, 3.33 ng, 1.11 ng, 0.37 ng, 0.12 ng,
0.04 ng). A control with no AXIN2-plasmid was also employed. To make the total
plasmid mass 100 ng, pcDNA was added to AXIN2 plasmid.
The reaction volume for each well was made up to 25 µl with Optimem:

AXIN2-Plasmid

Variable: 90-0 ng

pcDNA

Variable: 10-100 ng

Total plasmid:

100ng

OptimemVariable to make volume up to 25 µl

The Lipofectamine solution was added to the plasmid solution and incubated atroom temperature for 20 minutes. The entire reaction mix (50 µl) was subsequently
added to a well of cells, and incubated overnight at 37⁰C.

For the Luciferase assays, each of the transfection reactions underwent 3 biologicalreplicates, separated in time, and each of the biological replicates underwent threetechnical replicates. Every experiment included control samples: cells weretransfected with empty plasmid, and subsequently did or did not undergo Wnt3a/ RSpondin stimulation (2.5.11).

90

2.5.11

Stimulation with Recombinant Human Wnt-3a andHuman R-Spondin-1
Following transfection, the cells were stimulated with Recombinant Human Wnt3aand Human R-Spondin-1. 100 µl media was initially removed from each well. 50 µl
DMEM-Glutamax-10% FBS was added, containing 20ng/ml R-Spondin and 200
ng/ml Wnt3a.

The cells were incubated at 37⁰C overnight.

2.5.12

WST and Luciferase Assays

10 µl WST-1 was added to each well, and the cells were incubated at 37⁰C for one
hour. WST-1 activity was measured using the Fluorostar Optima. Readings weretaken at 450 nm and 590 nm.

The assay plate was subsequently inverted, and all liquid tapped out. 35 µl GloLysis buffer was added to each well, and the assay plate was mixed using theStuart Gyro Rocker for 15 minutes at 45 rpm. 35 µl Glo-Substrate was then added
to each well. Luciferase activity was read using the Fluorostar Optima. Luciferase
activity was normalised to the WST-Score (Luciferase score/ (WST 450/WST 590)).

The normalised luciferase activity in the cells which had been transfected withempty vector and which had been stimulated with R-Spondin/ Wnt3a was given ascore of 1. The normalised luciferase activity in the cells transfected with AXIN2
plasmid was compared to this.

2.5.13Cell Lysis and Protein Extraction

The cell culture dish was placed on ice and the media removed. Cells were washedwith ice cold PBS, which was then removed. 150 µl of cell lysis buffer was added toeach well. 10 ml buffer was composed of:

NaCl (5 M)

0.3 mlTris (1 M)

0.5 mlIGEPAL CA630

0.05 ml91

Roche Complete protease inhibitor (1 tablet per 10 ml)
Roche PhosphoSTOP phosphatase inhibitor (1 tablet per 50 ml)
Glycerol (10%)

1 ml

Water

8.15 ml

Adherent cells were scraped off the dish using a cold cell scraper. The cellsuspension was transferred to a pre-cooled microcentrifuge tube and was agitated
at 4⁰C for 30 minutes. The microcentrifuge tubes were subsequently centrifuged at
4⁰C for 30 minutes at 13 000 rpm. The tubes were then placed on ice. The
supernatant was aspirated and placed into a fresh microcentrifuge tube. Proteincontaining lysate was stored at -80⁰C.

2.5.14

Protein Quantification using a Bicinchoninic Acid

(BCA) Assay
A BSA protein standard was prepared according to the following volumes:

Standard (µg/ml)µl of 10 mg/ml BSAµl H2O

500

25

475

400

20

480

300

15

485

200

10

490

100

5

495

50

2.5

497.5

0

0

500

Table 2.5: BSA protein standards

50 µl of each standard was added in duplicate to a 96-well plate.

1 µl of protein lysate was added to 199 µl water, to make a 1:200 dilution. 50 µl of
this was added in duplicate to the 96-well plate. The stock solution was then diluted
by a factor of 2 to give a 1:400 dilution. 50 µl of this was added in duplicate to the
96-well plate.

92

The BCA reagent was prepared by mixing components A and B at a ratio of 50:1.
75 µl of BCA reagent was added to each well of standard or sample. The plate wasincubated at room temperature for 30 minutes with gentle agitation.

The absorbance was read at 590 nm using the Fluorostar Optima, and a standard
curve was used to calculate the protein content in each sample.

2.5.15Western Blotting

677 µg of each protein in 15 µl water was mixed with 5 µl NuPAGE LDS Sample
Buffer. Samples were heated for 5 minutes at 95⁰C using the QBD2 heating block.
They were loaded onto a NuPAGE 4-12% Bis Tris Minigel. The protein marker
used was the Novex Sharp Pre-Stained Protein Standard. The samples were
electrophoresed for 1.5 hours at 200 V using NuPAGE MES SDS running buffer.

The protein was transferred from the gel to a nitrocellulose membrane using theiblot system (Invitrogen).

The membrane was blocked with 5% non-fat powdered milk in TBS for one hour atroom temperature, with constant agitation. The membranes were then incubatedwith anti-myc-tag primary monoclonal antibody (9B11 New England Biolabs) diluted
in TBS-Milk overnight at 4⁰C with constant agitation.

The membrane was washed twice in water, and was incubated with anti-mousesecondary antibody (NXA931 Sigma Aldrich) diluted in TBS-milk, for one hour atroom temperature with constant agitation.

The membrane was washed twice in water. It was incubated for 5 minutes at room
temperature with 8 ml Supersignal West Pico Chemiluminescent Substrate.

Antibody detection was achieved using the BioRad ChemiDoc MP Imaging System.

93

2.5.16

Stripping Nitrocellulose Membranes and -Actin

Detection
The nitrocellulose membrane was washed with TBS. It was immersed in Restore
Plus Western Blot Stripping Buffer and incubated for 15 minutes at room
temperature. The buffer was removed, and the membrane washed with TBS.

The membrane was blocked with 5% non-fat powdered milk in TBS for one hour atroom temperature, with constant agitation. To detect -actin, the primary antibody
used was A5441 (Sigma Aldrich), and the further steps followed are those describedabove (2.5.15).

94

Chapter 3

APC and MUTYH in

Colorectal Polyposis
3.1

Introduction

The aim of this chapter was to identify missed or novel genetic variants in APC or
MUTYH responsible for the polyposis phenotype in NMI patients. Such variants
may occur outside of the open reading frame (ORF) of the genes, in promoters,
untranslated regions (UTRs) or introns, or they may be conventional mutations
which occur at a low frequency within the patients (mosaicism). They could
underpin the pathogenesis of colorectal neoplasia through mechanisms such asabnormal gene transcription, reduced stability of mRNA and abnormal mRNA
splicing. I sought to identify variants and their mechanisms of action through a
combination of approaches (Figure 3.1):
Quantitative PCR (qPCR) to interrogate APC and MUTYH transcription

gDNA/ cDNA sequencing to screen for allelic imbalance (AI)

cDNA sequencing of the coding sequences of APC and MUTYH toscreen for splicing abnormalities

Ultradeep sequencing (UDS) of the whole of the APC and MUTYH
genomic loci to identify variants outside of the ORF and low frequency
variants, i.e. patients with generalised mosaicism

95

NMI patients(n=60)

RNA: reverse
transcribed to cDNA
(n=45)qPCR toassess APC
and MUTYH
transcription

gDNA/cDNA
comparison toscreen for APC/
MUTYH AI

APC and
MUTYH cDNA
sequencing toscreen for
splicing
abnormalities

DNA(n=60)

Haloplex captureof the entire APC
and MUTYH
genomic loci.
UDS using the
HiSeq 2500

Bioinformatic
analysisIf reduced expression of
APC: karyotype analysis
and promoter
methylation studies

Data interrogated.
Variants validatedby Sanger
sequencing

Figure 3.1 Flow chart of the studies employed in the interrogation of APC and MUTYH

96

3.2

Methods

3.2.1 Quantitative PCR (qPCR): APC and MUTYH
Transcription
To determine whether any NMI polyposis patient had evidence of reduced
transcription of APC or MUTYH compared to a cohort of healthy controls, RNAlevels within a control cohort needed to be determined. To date, no study has
characterised the normal range of APC and MUTYH RNA levels in RNA derived
from venous blood samples from a healthy control cohort.

High quality RNA was available for 40 healthy control samples (2.3.16) and 45 NMI
patients (2.2.3.4). 3 affected FAP control patients and 4 affected MAP control
patients were also included (Table 3.1). FAPPol51 was used as a positive control
for reduced APC transcription due to the confirmed presence by the NHS diagnostic
service of an APC promoter deletion known to impair transcription. The remaining
FAP and MAP patients have mutations which are not predicted to affecttranscription, so were used as negative controls. Although some of these mutations
may result in nonsense mediated decay (NMD) or could potentially affect transcript
stability, the Taqman probes generate such short amplicons that it is highly likely
they will be able to bind to fragmented RNA/ cDNA so that levels will appear to bewithin the normal range. Unfortunately, MUTYH promoter mutations resulting in
reduced gene transcription are thus far unreported in the literature, so no positive
controls for reduced MUTYH transcription were available.

97

SampleSampleGroup

Expected Result for APC
Mutationand MUTYH Gene
Transcription

Compound heterozygous
Halo37MAP

for MUTYH c.536A>G &Normalc.649C>T

Halo59MAP

MUTYH c.1187G>A
homozygote

NormalCompound heterozygous
MAPPol71

MAP

for MUTYH c.303G>T and

Normalc.312C>A
Compound heterozygous
MAPPol90

MAP

for MUTYH c.1187G>A

Normaland c.536A>GFAPPol51

FAP

FAPPol141 FAP

Halo42FAP

APC promoter 1B deletion

APC exons 11 and 12
deleted
APC c.1187 dup A,
MUTYH c.536A>G

Reduced APC
transcription

NormalNormalTable 3.1: FAP and MAP controls used in gene transcription studies

RNA was converted to cDNA following standard protocols (2.3.3.2). cDNA
underwent qPCR using standard reagents and reaction conditions (2.3.15) andTaqman assays for ACTB, GAPDH, APC and MUTYH (2.3.16.1, Table 2.3).
Results were analysed using ThermoFisher Cloud software (2.3.16.1.1). Any
patient with an Rq </= 0.6 when compared to the healthy control cohort was
identified for further characterisation.

983.2.2 Karyotype Analysis
Chromosomal rearrangements can affect gene expression through mechanisms
such as disrupting genes or regulatory elements, producing fusion genes, placing
genes in anomalous chromatin environments, placing genes near to telomeres andaberrant nuclear positioning (Harewood and Fraser 2014). Karyotype analysis was
performed by the AWMGS following their Standard Operating Procedure (SOP).

3.2.3 Promoter Methylation Studies
MSP was carried out to determine the methylation status of the APC 1A and 1B
promoters (2.3.14). MSP for the 1A promoter used primers described by Arnold etal (2004) (Appendix 3.1). MSP for the 1B promoter used primers desribed byEsteller et al (2000) (Appendix 3.2).

3.2.4 RNA Studies: Allelic Imbalance (AI) and Splicing
Abnormalities
45 NMI patients and 4 unaffected relatives (Halo75, Halo76, Halo77 and Halo78)
underwent studies on DNA and RNA to screen for APC +/- MUTYH RNA AI. All of
the work done with MUTYH was carried out by myself. I carried out the APC AI
work on 22 samples. The remaining work was completed by Alice Davies and Alice
Bolton, intercalating medical students (Davies A 2015, Bolton A 2016), and Julie
Maynard, research assistant, Cardiff University.

To screen for AI, common non-pathogenic exonic SNPs were identified in APC and
MUTYH, using UCSC Genome Bioinformatics (http://genome.ucsc.edu/cgibin/hgTracks?db=hg19&position=chr1%3A4579491445806142&hgsid=414232457_bzuux4uOZeA1wBLT5vBhLtb6YSg9) and NCBI
dbSNP software (http://www.ncbi.nlm.nih.gov/projects/SNP/) (accessed
26/11/2014). The APC SNPs were rs2229992, rs351771, rs41115, rs42427,
rs866006 and rs465899. The MUTYH SNPs were rs3219489 and rs3219497 (fulldetails in Appendix 3.3).
Comparison was made of the sequence at each of these SNPs in the patient’s
gDNA and their cDNA. If a patient was heterozygous for a common SNP in the99germline but was found to be hemizygous for either allele in cDNA, this was
suggestive of allelic imbalance.

3.2.4.1

gDNA analysis

gDNA analysis was initially carried out by examining the UDS results (3.2.5). Any
heterozygous SNPs identified were validated with Sanger Sequencing. One of the
SNPs, rs465899, was not covered adequately through UDS, therefore Sanger
sequencing was performed on all patients for this locus. Sanger sequencing andsequence analysis were undertaken using standard reagents and reaction
conditions (2.3.8.1, 2.3.8.5, 2.3.9-2.3.13). Primers were those used by the AWMGS(Appendix 3.4). Two samples required optimisation (Appendix 3.5).

3.2.4.2

RNA Analysis

RNA analysis involved reverse transcription to cDNA (2.3.3.2), PCR with gene
specific primers (Appendix 3.6), and sequence analysis. Standard protocols andreaction conditions were used (2.3.8.1, 2.3.8.5, 2.3.9-2.3.13). Only those SNPs
which were confirmed to be heterozygous in the germline were examined in theRNA, as homozygous SNPs do not yield any discriminating information. PCR
conditions were optimised as shown in Appendices 3.7 (APC) and 3.8 (MUTYH).

3.2.4.3

APC and MUTYH Splicing Abnormalities

45 NMI patients underwent sequencing of APC +/- MUTYH cDNA to screen for
splicing abnormalities. All MUTYH sequencing was carried out by myself. I carriedout the APC cDNA sequencing for 17 samples. The remaining work was completedby Alice Davies and Alice Bolton, intercalating medical students (Davies A 2015,
Bolton A 2016), and Julie Maynard, research assistant, Cardiff University.

3.2.4.3.1

APC cDNA Sequencing

RNA was reverse transcribed (2.3.3.2) and cDNA underwent PCR with gene
specific primers (Appendix 3.9). Standard reagents and reaction conditions were100

employed for PCR and sequencing (2.3.8.1, 2.3.8.5, 2.3.9-2.3.13). The details forthe samples which required optimisation are in Appendix 3.10.

3.2.4.3.2

MUTYH cDNA Sequencing

MAP is an autosomal recessive (AR) disease. Even if abnormal splicing is found in
one allele, there would need to be a pathogenic variant present in the other allele for
manifestation of polyposis. In view of this, a small study was initially performed on 9
NMI polyposis patients, in order to optimise reaction conditions. These were thefirst study participants that RNA was available for. Subsequently, MUTYH cDNA
sequencing was completed on all those patients who were known to have/potentially have a pathogenic variant present affecting MUTYH:Known monoallelic carriers of a MUTYH mutationoHalo40o

Halo41

Potentially reduced expression of MUTYH (3.3.1)
oHalo36oHalo63oHalo71

RNA was reverse transcribed (2.3.3.2) and cDNA underwent PCR with gene
specific primers (Appendix 3.11). Standard reagents and reaction conditions were
employed for PCR and sequencing (2.3.8.1, 2.3.8.5, 2.3.9-2.3.13). The details forthe samples which required optimisation are in Appendix 3.12.

3.2.5 APC and MUTYH Capture and Ultradeep Sequencing(UDS)
UDS was performed for the whole genomic loci of APC and MUTYH(chr5:112042936-112186350, chr1:45794202-45807013) to identify mutations
outside of the ORF and to identify APC mutations which occur at a low frequency
within an individual. It was carried out on DNA extracted from whole blood (2.3.2).
Target sequence capture was achieved using the Haloplex assay (2.4.1).

101Sequencing was performed by the Wales Gene Park (WGP) using the HiSeq 2500
(2.4.2).
82 individuals underwent UDS:

60 NMI polyposis patients (Table 2.1)



11 healthy controls (Tables 2.1 and 2.2): Halo02, Halo03, Halo09, Halo11,
Halo73, Halo74, Halo75, Halo76, Halo77, Halo78, and Halo95



11 positive control samples (Table 2.2): 8 FAP patients (Halo01,
F014_M_004_1, F014_M_005_1, F014_M_006_1, Halo10, Halo12, Halo16,
Halo42 (also carries a monoallelic MUTYH mutation) and 3 MAP patients(Halo37, Halo39, Halo59)

Bioinformatic analysis was performed by Dr. Peter Giles, WGP (2.4.3).

3.2.5.1

Variant Analysis and Selection

Variants of interest were selected using the following criteria:

Rare variants, with an allele frequency </= 1% according to dbSNP data or
The 1000 Genomes Project dataVariants which were present in all affected family members



Synonymous variants were excluded



Variants which had been identified previously by the AWMGS diagnostic
service and which were known to be non-pathogenic were excluded



Variants found in unaffected relatives/ healthy controls were excluded, taking
into account the mode of inheritance (i.e. if a patient was found to behomozygous for a MUTYH variant, it was only excluded if it was also
homozygous in an unaffected relative/ healthy control).



Exonic missense variants present in <2% of reads which were identified in
multiple samples were excluded as they were likely to represent sequencing
artefacts

All shortlisted variants were then analysed using CADD software(http://cadd.gs.washington.edu/home) and were assessed using the Integrative
Genomics Viewer (IGV) (https://www.broadinstitute.org/igv/). Variants which had a
CADD score >/= 15 were validated with Sanger sequencing.

1023.2.5.2

Validation of Identified Variants

Variants were validated with Sanger Sequencing, using standard reagents andreaction conditions (2.3.8.1, 2.3.8.5, 2.3.9-2.3.13). Primers are listed in Appendix
3.13. Variants present at a low frequency (<20%) were validated with COLD-PCR,
using the full- or fast- standard protocol depending on the nature of the variant(2.3.8.3, 2.3.8.4, 2.3.8.6, 2.3.8.7).

3.3

Results

3.3.1 Quantitative PCR (qPCR): APC and MUTYH Expression
A total of 92 samples underwent successful qPCR to determine levels of APC and
MUTYH transcription: 40 healthy controls, 45 NMI polyposis patients, 3 FAP
controls and 4 MAP controls (2.3.16, 2.2.3.5, Table 3.1). One of the FAP controls,
FAPPol51, had an APC promoter deletion known to impair APC transcription. The
sample was used as a positive control to ensure that the assay was capable ofdetecting reduced APC transcription. The remaining FAP and MAP controls all had
genetic variants which did not affect gene transcription: they were included as
negative controls to confirm that the assay identified that APC and MUTYH RNA
levels were within the normal range.

The results for the control samples were largely as anticipated, although one of the
FAP controls who also carries a monoallelic MUTYH mutation, Halo42, appeared tohave reduced MUTYH transcription, although this was not statistically significant(Table 3.2).

103

104

The individual results for all the NMI patients are in Appendix 3.14 .
Four patients were found to have APC Rq values below 0.6: Halo46, APC Rq=
0.563 (p=0.09); Halo52, APC Rq=0.368 (p=0.003); Halo53, APC Rq=0.545(p=0.07); Halo64, APC Rq=0.417 (p=0.01). These results were confirmed byrepeating the experiment from the initial reverse transcription reaction (Table 3.3).

Three patients had MUTYH Rq values below 0.6. Although these results areinteresting, they do not have the same clinical implications as do the APC genetranscription results. MAP is an AR disease, therefore reduced transcription of one
allele alone would not explain a clinical phenotype. In addition to this, a negative
control was also observed to have an apparently reduced MUTYH Rq value(although this was not statistically significant), a positive control was not available,
and only the Rq value for Halo36 reached statistical significance, with p=0.03. The
results were not repeated, but these patients were selected for further RNA studies(cDNA sequencing, 3.2.4.3.2) to determine whether they had any further potentiallydisease-causing genetic aberrations.

The phenotype of those patients with an Rq value </= 0.6 is summarised in Table
3.3:

105

Patient

APC/ MUTYH

Identification

Rq Value

Repeat Result

Clinical Phenotype

44-year-old female who
clinically has FAP and
Halo46APC Rq = 0.563*

who has undergone acolectomy andproctectomy

Halo52APC Rq = 0.368

APC Rq = 0.400

59-year-old male with
>1000 adenomas
51-year-old male with

Halo53APC Rq = 0.545

APC Rq = 0.527

approximately 400
adenomas and CRC atage 2754-year-old female with

Halo64APC Rq = 0.417

APC Rq = 0.291

thousands of colorectal
adenomas and a caecal
carcinoma at age 23

Halo36

MUTYH

67-year-old male with >10

Rq = 0.41

adenomas
68-year-old male with at

Halo63

MUTYH Rq =least 24 polyps 5 biopsies

0.509

show 4 adenomas and 1
HPP

Halo71

MUTYH Rq =65-year-old female with

0.508

14 adenomas

Table 3.3: Phenotypes of patients with evidence of reduced transcription of APC/ MUTYH*Repeat testing was not carried out for Halo46, as the causative mutation was identified(3.3.5.3).

106

3.3.2 Karyotype Analysis
Karyotype analysis was performed on Halo52 and Halo53, both of whom appeared
to have evidence of reduced APC expression. Both had normal chromosome
complements. Unfortunately, patient Halo64 has been lost to follow up, so it was
not possible to obtain a fresh blood sample. Halo46 did not undergo karyotyping asa pathogenic mutation was identified (see section 3.3.5.3).

3.3.3 Promoter Methylation Studies
Methylation analysis of the APC 1A and 1B promoters was performed on 3 patientswith reduced APC transcription (Halo52, Halo53 and Halo64) and the unaffectedmother of Halo53. It was not done for Halo46 as the pathogenic mutation was
identified (section 3.3.5.3). Methylation studies were also performed for NMI
patients with any evidence of APC AI (section 3.3.4.1): Halo08 and her affected
son, Halo51 and her unaffected parents.

There was no evidence of promoter methylation for either promoter:107M primers U primers M primers U primers
LadderLadder1.

Halo53

9.

2.

Halo53 Mother

10. Control M DNA

3.

Halo52

11. Control U DNA

4.

Halo645.

Halo51

6.

Halo51 Mother7.

Halo51 Father

Halo08 Son

8. Halo08

Figure 3.2. MSP for APC promoter 1A. All samples, apart from the control methylated DNA(M DNA: red arrow), produce a PCR product following MSP using primers specific for
unmethylated DNA (U primers). This is demonstrated by the high intensity of the bands.
The only PCR product obtained when MSP was undertaken using primers specific formethylated-DNA (M primers) was that from the control methylated DNA sample (M DNA:yellow arrow), as shown by the high intensity band.
M primers

M primers

U primers

U primers
Ladder

1.
2.
3.
4.
5.
6.
7.
8.

LadderHalo53
9. Halo08 Son
Halo53 Mother 10. Control M DNA
Halo52
11. Control U DNA
Halo64
Halo51
Halo51 MotherHalo51 Father
Halo08Figure 3.3 MSP for APC promoter 1B. All samples, apart from the control methylated DNA(M DNA: yellow arrow), produce a PCR product following MSP using primers specific for
unmethylated DNA (U primers) (there was a product for Halo52, although the PCR band was
fainter than those obtained for the other samples). The only PCR product obtained when
MSP was undertaken using primers specific for methylated DNA (M primers) was that fromthe control methylated DNA sample (M DNA: red arrow). The bands which are smaller than
the PCR products represent primer-dimers.

1083.3.4 Allelic Imbalance Studies
Allelic Imbalance (AI) studies were completed on 45 NMI patients and 4 unaffected
relatives for whom high quality RNA samples were available (Halo75, Halo76,
Halo77, Halo78).

3.3.4.1

APC Allelic Imbalance

Of the 45 patient samples, 22 had informative results, i.e. the presence of at least
one heterozygous SNP in the germline. A total of 4 patients displayed AI: 18% of
the informative cohort, or 9% overall. The patients were Halo08 and Halo51 (bothhad AI at the final SNP and visual inspection of the sequencing traces weresuggestive of one allele having a weaker signal at some of the preceding SNPs),
Halo52 (AI at first SNP only. The subsequent SNPs were uninformative) andHalo53 (AI throughout). All of the individual patient results are in Appendix 3.15.
The sequencing results for the SNPs exhibiting AI are in Appendix 3.16.

Patients Halo52 and Halo53 had results which would be consistent with absent
expression from one allele, which may result from a promoter lesion (Charames etal 2008). This was concordant with their gene expression results (3.3.1). It is notedthat patient Halo53 did display tiny peaks of the alternate allele at SNPs rs41115
and rs866006 (Appendix 3.16). Whilst this may represent a sequencing artefact, it
is possible that this allele was being expressed at such low levels that it was notreliably detected with Sanger sequencing.

Patients Halo08 and Halo51 displayed complete loss of signal for one allele at the
final SNP only. This could possibly result from allele specific transcription
termination or mRNA degradation, the presence of an allele specific alternate
transcription start site (Wagner et al 2010) or it could represent a sequencing
artefact. There also appeared to be an abnormal allelic expression ratio at some ofthe preceding SNPs. These findings were confirmed by repeating the experiments(Appendix 3.17). The significance of these findings was not confirmed.

The parents of Halo51 and Halo53 did not exhibit AI.

109

3.3.4.2

MUTYH Allelic Imbalance

45 NMI polyposis patients underwent MUTYH AI studies. 16 had informative
results. There was no evidence of AI in any sample. The full results are in
Appendix 3.18.

3.3.5 APC and MUTYH Splicing Abnormalities
APC cDNA sequencing was completed on 45 patients. There was no evidence ofany splicing abnormalities.
MUTYH cDNA sequencing was completed on 14 patients, including those with amonoallelic MUTYH mutation and those with potentially reduced MUTYH
expression. There was no evidence of any splicing abnormalities.

3.3.5 APC and MUTYH Capture and Ultradeep Sequencing(UDS)
The Haloplex assay was used for targeted capture of the whole of the APC and
MUTYH genomic loci, followed by UDS using the HiSeq 2500 (Illumina) (Threesamples had undergone sequencing using the MiSeq (Illumina) before the HiSeq
2500 was being utilised: F014_M_004_1, F014_M_005_1, F014_M_006_1).

3.3.5.1

Coverage of Ultradeep Sequencing

The average depth of coverage of APC and MUTYH using the HiSeq 2500 was
1665.5x and 2575.2x respectively. The mean on-target coverage for APC was
97.2% and for MUTYH it was 97.3%. For APC, regions with consistently low
coverage (<50 reads) occurred in exons 3 (65 bp), 5 (65 bp, 14bp), 6 (27 bp) and 15(9 bp, 11 bp, 15 bp, 14 bp, 65 bp, 23 bp). There were 2 small areas withconsistently no coverage: 16 bp of exon 5 and 52 bp of exon 15. For MUTYH there
were 9 bp of exon 12 with low coverage. The detailed coverage for each individual
patient is recorded in Appendices 3.19 (APC) and 3.20 (MUTYH).

110

3.3.5.2

Control Results

The Haloplex assay followed by UDS identified all 15 known variants in APC or
MUTYH. This included 8 FAP controls, one of whom also carried a MUTYH variant(Halo42), 3 MAP controls, a previously confirmed APC variant in Halo33 andpreviously confirmed MUTYH variants in Halo40 and Halo41. These variants andthe coverage are described in Table 3.4:

111

SampleMean Coverage of

Gene

Variant

F014_M_004_1

APC

c.2805C>A

2410

F014_M_005_1

APC

c.4393-4394delGA

3118

F014_M_006_1

APC

Halo01

APC

c.3631-3632delAT

723

Halo10APC

c.3183-3187delACAAA

328

Halo12APC

c.3927-3931delAAAGA

182

Halo16

APC

c.3408delA

236

Halo33APC

c.6363-6365dupTGC

235

Halo42APC

c.1133dupA

408

Halo37

MUTYH

Halo39MUTYH

Halo40MUTYH

c.920G>A heterozygote

2443

Halo41

MUTYH

c.1187G>A heterozygote

7636

Halo42

MUTYH

c.536A>G heterozygote

604

Halo59MUTYH

c.1187G>A homozygote

3387

Identification

5% mosaic APC c.43934394delGA

Compound heterozygote:c.536A>G & c.649C>T
Compound heterozygote:c.303G>T and c.312C>AGene with Variant

2421

3994

638

Table 3.4: Control samples used for validating Haloplex capture and UDS

3.3.5.3

Patient Results

60 NMI polyposis patients underwent UDS of APC and MUTYH. 15 variants
selected for validation/ further investigation are listed in Appendix 3.21. Variants112

present in >20% of reads were validated with standard Sanger sequencing following
standard PCR protocols (Appendix 3.22). Low frequency variants, present in <20%of reads, were amplified with COLD-PCR prior to Sanger sequencing (Appendix
3.23). Validation was not carried out if the variant had been previously identified by
the AWMGS.

Table 3.5 summarises the variants which were validated.

113

SampleID

Gene

Halo23

APC

Halo30APC

Halo35APC

Halo46APC

Halo51

APC

Halo56APC

Halo62APC

Halo66

APC

Halo71APC

Position

5:112043492

5:112102960

5:112104652

5:112043225
5:112095775
5:112043282
5:112162474
5:112163697
5:112102960

Variant

C>A

Additional

CADD

Information

Score

N/A

None

21.1

Misse

Validated by

nse

NHS

Location Effect

5’UTR/
Exon 1

C>T

Exonic34

T>A

Intronic

N/A

None

18.02

G>A

5'UTR

N/A

None

22.4

T>A

Intronic

N/A

None

16.36

C>CG

5’UTR

N/A

None

16.52

C>T

Intron

N/A

None

18.98

A>C

ExonicMisse

Validated by

nse

NHS

C>T

ExonicMisse

Validated by

nse

NHS

25.4

34

Validated by

Halo40MUTYH

1: 45797851

C>T

ExonicMisse
nse

NHS. Patient
only has one

20.4

pathogenic
mutation

Table 3.5 Validated APC and MUTYH variants

114

All the variants which appeared to be approximately heterozygous were validated.
None of the low frequency variants were definitely real, although one was uncertain,a missense exonic variant in sample Halo45, present in 4% of reads. A truncating
APC exonic variant in Halo63 was not validated with Sanger sequencing. However,there was low coverage at this locus with NGS, with the variant being present in only
2/11 reads, so it is likely to be an artefact. The validated variants will be appraised in
the Chapter Discussion (3.4.3.2).

Table 3.6 summarises the key positive findings from Chapter 3:

UniqueqPCR: Karyotype APC

Identifier APC

Analysis

Rq

Promoter

APC AIAPC cDNA
Sequencing

APC UDS

Methylation
Chr5:

Halo46

0.563

N/A

N/A

Uninformative Normal

112043225
G>A

AI at first

Halo52

0.368

NormalNormalNormalNil

Halo530.545

NormalNormalAI throughout

NormalNil

Halo640.417

Not doneNormalUninformative Normal

Nil

SNP

Table 3.6 Key positive results from all APC and MUTYH studies

115

3.4

Discussion

This study involved investigating patients with multiple colorectal adenomas (>10)
who had no mutation identified (NMI) in APC or MUTYH during routine clinicaldiagnosis. The aim of this chapter was to identify missed or novel genetic variantsin APC or MUTYH responsible for tumourigenesis. I sought to identify such variants
through a combination of:

Quantitative PCR (qPCR) to interrogate APC and MUTYH transcription

gDNA/ cDNA sequencing to screen for allelic imbalance (AI)

cDNA sequencing of the coding sequences of APC and MUTYH toscreen for splicing abnormalities

Ultradeep sequencing (UDS) of the whole of the APC and MUTYH genes
to identify variants outside of the ORF and APC low frequency variants

DNA samples were available for all 60 patients, and RNA samples were available
for 45 NMI polyposis patients.

3.4.1 Gene Expression Studies and Allelic Imbalance
RNA studies yielded interesting results for 7 patients: 4 appeared to have reduced
APC transcription and 3 appeared to have reduced MUTYH transcription. The 4
patients with reduced APC transcription all had phenotypes in keeping with a clinicaldiagnosis of FAP. The gene transcription results were concordant with the AI
results in Halo52 and Halo53, while AI studies were uninformative in Halo46 andHalo64.
The cause of the reduced APC expression was determined in Halo46. UDS
identified a 5’UTR variant (G>A at 5:112043225), at position c.-190G>A. Thisvariant is predicted to be pathogenic, with a CADD score of 22.4. The patient has astrong family history of polyposis/ CRC, with the inheritance pattern suggestive ofan autosomal dominant trait:

116

Key
Clinical FAP

Figure 3.4 Family tree of Halo46. Halo46 is marked with a black circle

At the time this variant was identified in the index case, a paper was publishedwhich describes the exact same variant (Li et al 2016). The variant occurred in a
family with 5 affected patients, over 3 generations, with polyposis and/ or CRC. The
group performed electromobility shift assays (EMSA) to show that the mutation led
to reduced protein binding, and that the protein likely to be affected was thetranscription factor YY1. Further work demonstrated that carriers of the mutation
also showed AI of APC. The work described by Li’s paper supports thepathogenicity of the variant identified in Halo46. Furthermore, we were able torecruit the patient’s father and one cousin, both whom have clinical FAP. Both were
found to carry the same mutation. qPCR of APC cDNA levels was also performed:
the Rq value was 0.55 when compared to the healthy control cohort for the father,and 0.63 for the cousin. I tried to recruit further family members to further
investigate whether the variant was segregating with the disease phenotype, but
unfortunately was unable to do so during the time course of this project. However,this research finding is being translated into clinical practice, and the AWMGS areestablishing predictive genetic testing for this specific mutation in members of theextended family. As far as we are aware, Halo46 does not have a gastricphenotype. This is an interesting observation, as point mutations in the APC 1B
promoter are reported to result in gastric neoplasia (Li et al 2016). It is possible that
Halo46 actually does have such a phenotype, but that we do not know about - forexample if she has not attended upper gastrointestinal (UGI) endoscopic
surveillance, or if she has attended UGI endoscopy but the associated endoscopic
reports/ histopathological reports were not sent to the AWMGS for inclusion in the117genetics notes. Alternatively, she may lack gastric neoplasia if her gastric mucosa
is 'protected' in some way. The APC 1B transcript is usually more important than
the 1A transcript in the stomach (reviewed in Li et al 2016) but it is possible that inthis family, the 1A transcript plays a significant role. Gastric protection could also be
the result of environmental factors such as the biome. It is also feasible that thisspecific mutation has a low/ incomplete penetrance in the stomach or has variable
expressivity, mediated through the effects of modifier genes.

I sought to determine the cause of the reduced gene transcription in the remaining 3
samples. Karyotype analysis was normal for Halo52 and Halo53, and there was noevidence of promoter methylation for any of the 3 patients. If further evidence
regarding the lack of promoter methylation was required, additonal techniques toconfirm this would include pyrosequencing or qPCR. UDS results did not identify
any variants which could underpin abnormal transcription. The cause of the
reduced gene transcription was therefore not apparent. There are several possible
explanations for this phenomenon, including upstream/ enhancer variants whichwere not captured by the Haloplex assay, abnormalities in transcription factor(s),variants in further genes which are modulating gene expression and epigenetic
effects. All 3 patients underwent whole exome sequencing (Chapter 4) but further
investigation into the reduced gene transcription was beyond the scope of thisproject. We shall recommend that the patients undergo whole genome sequencing
with the aim of searching for variants in the regions of the genome which have notyet been interrogated.

Three patients appeared to have a reduction in MUTYH transcription (Halo36,
Halo63 and Halo71), although only the result for Halo36 was statistically significant.
All AI results were uninformative. However, even if the reduced transcription is real,
this alone would not explain the patients’ phenotypes, as MAP is an AR disease,and there would have to be a pathogenic change in the second allele: in all threecases MUTYH cDNA sequencing showed no splicing abnormalities, and UDS did
not reveal any pathogenic MUTYH variants. Furthermore, reduced MUTYH
transcription was also observed in a negative control sample, and it was notpossible to identify a positive control, so the results are weak and are unlikely to beclinically significant.

118

To investigate transcription levels of APC and MUTYH it was necessary to
determine the range of transcription of these genes in a healthy control cohort.
Gene transcription studies were carried out on 40 unaffected individuals to generate
this data. To improve the reliability of the results, it would be necessary to increase
the population size of healthy individuals. If large-scale data were obtained, its
validity would be more certain, and this could offer the possibility of genetranscription studies being performed in a diagnostic setting. At the moment, it iscautiously suggested that gene transcription studies could be considered as ascreening test in NMI patients, but that they need to be interpreted in the context ofadditional investigations such as AI studies and whole gene sequencing.

Gene transcription studies were carried out using one probe for each gene. For
APC, the probe spanned the junction between exons 12 and 13 (HS01568269_m1)
and for MUTYH the probe spanned exons 14 and 15 (HS01014856_m1). For any
clinical screening test, it is suggested that more than one probe would need to be
used. This was not possible during this project because of financial implications. A
cut-off value of Rq = 0.6 was chosen to try and identify patients with clinicallysignificant reduced gene transcription. It is important to be aware that this is anarbitrary value, and more accurate determination of a clinically significant thresholdwould need to be determined through large-scale studies.

The AI studies identified APC AI in 4 samples: 18% of the informative cohort. In 2
patients, Halo52 and Halo53, it appeared to be across the whole allele. In 2
patients, Halo08 and Halo51, it was definite at the final SNP only. When patients
appeared to have lost a complete allele, this correlated with their gene expression
results (Halo52 and Halo53). However, there was no correlation for those patients
who appeared to have lost the 3’ end of the allele. The APC Rq values for patients
Halo08 and Halo51 were 0.869 and 0.826 respectively, so even if there is areduction in transcription from one allele, the overall amount of cDNA present
appears to be within the normal range. It is interesting to note that both Halo08 andHalo51 both have relatively strong phenotypes: Halo08 has 629 adenomas andHalo51 has 208 adenomas. In view of this, it might be expected that there could be
reduced transcription from one allele, however the qPCR results did not support thisfinding, and the clinical implications of loss of the final SNP are uncertain.

1193.4.2 Abnormal Splicing
cDNA analysis did not identify any splicing abnormalities in APC or MUTYH.
However, the sample size was relatively small (45 patients for APC and 14 for
MUTYH) and the patients’ phenotypes were highly variable. It has previously beenreported that 8% of NMI polyposis patients have an aberrant APC transcript (Spieret al 2012). However, all of Spier’s index patients had at least 20 synchronous or 40
metanchronous adenomas. In the patient cohort for this study, it is not often clear
from the patient notes whether the polyps were synchronous or metanchronous.
The RNA samples obtained account for a maximum of 22 patients with at least 20
adenomas, so the phenotype of this study cohort is weaker.

cDNA sequencing for APC is a cheap technique to perform, so it is suggested that it
forms part of the investigative protocol for NMI patients with an appropriate
phenotype, for example those with at least 20 adenomas. MUTYH cDNA
sequencing proved difficult to optimise. MAP is an AR disease, so even if any
samples with abnormal MUTYH splicing had been identified, this alone would not be
sufficient to explain the phenotype. Whilst MUTYH cDNA sequencing may be auseful tool in patients who are already known to carry one pathogenic MUTYH
variant, it is unlikely to be cost and time efficient if it were performed on all NMI
polyposis patients.

3.4.3 Ultradeep Sequencing
A total of 15 variants were identified for further investigation/ validation, 14 in APC
and 1 in MUTYH. 4 had been previously validated by the NHS, including themonoallelic MUTYH mutation, which is known to be pathogenic.
Four of the APC variants were of very low frequency (present in <5% of reads), andone was at low frequency (present in 18% of reads). None of the 5 low frequency
reads were validated. All of the variants which appeared to be approximately
heterozygous were validated.

3.4.3.1

Validation of Low Frequency Variants

The Haloplex assay followed by UDS identified 5 very low/ low frequency variants.
Whilst such variants may represent sequencing artefacts, they could also result120

from the patient exhibiting generalised mosaicism. This study sought to validate
such variants using COLD-PCR, which is a technique that can be used to enrichsamples for low-abundance DNA mutations (Milbury et al 2011). The techniqueselectively amplifies low-abundance variants from mixtures of wild-type and mutantcontaining sequences by using a critical denaturation temperature. A lower
denaturation temperature allows the selective denaturation of amplicons whichcontain mutant/ wild type heteroduplexes or which contain Tm-reducing variants(Milbury et al 2011), so that they are preferentially amplified. It is reported that
COLD-PCR followed by Sanger sequencing can be used to identify mutant fractions
down to 0.1%, but it is noted that enrichment potential is highly dependent upon thetype of mutation being analysed, the initial mutation abundance, and the PCR
platform utilised (Milbury et al 2011).

None of the low frequency APC variants were definitely validated, although theresult was ambiguous for sample Halo45. It is likely that they are sequencing
artefacts from the UDS protocol, however it is also possible that the COLD-PCR
reactions employed were not sufficiently sensitive to detect them. Fortunately, mostof the variants examined were missense changes. Even if they had been real, such
low frequency missense APC variants in an individual are unlikely to have a clinicalimpact (Cleary et al 2008). There was a stop-gain variant present in Halo63 which
was not validated. However, it was only present in 2/11 reads and there was noevidence of it at all with Sanger sequencing, so it highly likely to be a sequencing
artefact. If more sensitive validation of low frequency variants was required, it would
be necessary to consider an alternative technique such as digital droplet PCR.

3.4.3.2

Validated Variants

A total of 10 potentially pathogenic APC or MUTYH variants were identified by UDS
and validated with Sanger sequencing (Table 3.5) .

3.4.3.2.1

Validated Variants in APC

All exonic variants occurring in APC had previously been identified by the NHS
diagnostic service. They were all putative missense variants which were of
uncertain pathogenicity. Pathogenic APC mutations are usually truncating121

mutations, and there is currently minimal evidence that missense variants areclinically significant (Cleary et al 2008).

Sample Halo23 has a variant in the untranslated region of exon 1. She has noevidence of AI, gene expression studies were normal with APC Rq = 0.804 andthere was no evidence of abnormal splicing. Although the CADD score for thisvariant was 21.1, it has recently been classified as a benign variant on ClinVar(http://www.ncbi.nlm.nih.gov/clinvar/variation/133505/ accessed 3/5/16).

Halo35 has an APC intronic variant. Its CADD score is relatively low, at 18.02.
There is no evidence of abnormal expression, with APC Rq = 0.894, and there was
no evidence of abnormal splicing or allelic imbalance, so the variant is regarded as
likely non-pathogenic.
Sample Halo46 has a 5’UTR variant and evidence suggestive of reduced APC
transcription. She has been discussed above and this variant is believed to bepathogenic.

Sample Halo51 has an intronic APC variant, rs4705624. This has a CADD score of
16.36, and there is one submission on ClinVar(http://www.ncbi.nlm.nih.gov/clinvar/variation/82836/ accessed 01/03/16) in which it
is given a classification of ‘other’ in the context of familial colorectal cancer. The
patient exhibited loss of the final APC SNP, but there was no evidence of abnormal
splicing. DNA was initially only available for the patient’s mother: the variant was
not present. However, once DNA was obtained for the patient’s unaffected father,
the same variant was present, so the variant has been regarded as non-pathogenic.
Sample Halo56 has an APC 5’UTR variant with a CADD score of 16.52. There isno evidence of reduced APC expression, with APC Rq = 0.793. AI studies are
uninformative. Once the patient’s parents had been recruited to the study thevariant was identified in the unaffected father, so it has been regarded as nonpathogenic.

Sample Halo62 has an intronic variant with a CADD score of 18.98. There is noevidence of reduced APC expression, with APC Rq = 0.766, and AI studies show noevidence of AI. There is no abnormal splicing. Taking into account these findings,
the variant has been assumed to be non-pathogenic.

1223.4.3.2.2

Validated Variants in MUTYH

The final variant was identified in MUTYH in sample Halo40. This is one of therecurrent MUTYH mutations known to cause MAP, p.Gly396Asp. The patient is acarrier of a monoallelic mutation, so this by itself cannot explain the polyposis
phenotype. The patient had no evidence of MUTYH AI, reduced gene expression orabnormal splicing.

3.4.3.3

Summary of Gene Capture and Ultradeep

Sequencing
The Haloplex assay followed by UDS was used to look for variants throughout thewhole of the APC and MUTYH genomic loci, and to identify any patients who might
exhibit mosaicism. There are several other available techniques for targeted gene
capture, including long-range PCR (LR-PCR). LR-PCR was initially trialled for use
in this project. However, it failed to amplify the known mutant alleles from two FAP
control patients. Despite carrying out a series of investigations to determine why
this had occurred, the explanation was not clear. It is possible that certain
mutations alter the structure of the DNA in such a way that the polymerase cannot
replicate the mutant strand. Instead of LR-PCR, the Haloplex Assay was employed.
The assay costs approximately £100 per patient sample, which is not prohibitive foruse in a diagnostic setting. 60 patients underwent UDS of APC and MUTYH. All
exonic variants which we filtered for had previously been identified by the NHS, so
this study did not identify any mutations which had been ‘missed’ in the diagnostic
setting. There were 6 variants outside of the ORF which were validated, one ofwhich is believed to be pathogenic (the APC 5’UTR variant in Halo46). The
remaining variants are regarded as unlikely to be clinically significant once the gene
expression results, cDNA sequencing and AI studies are taken into consideration.
This highlights the importance of using a range of investigative techniques, so that
results are never viewed in isolation.

None of the patients had evidence of APC mosaicism. The depth of coverage was
excellent with UDS, so if there were any true cases, it is unlikely to have been
missed. It is reported that somatic mosaicism can occur in 10-20% of sporadic

123

cases of FAP (reviewed in Rohlin et al 2009; Hes et al 2008; Aretz et al 2007). 28
of the patients included in this study are thought to be sporadic polyposis cases, but
the actual figure may differ from this as the family histories obtained are often
vague/ missing.
It is also important to note that analysis has been performed in DNA extracted fromwhole blood. It is clear from the literature that some patients have mosaic mutations
which are only present in colonic tissue (Jansen et al 2017), so we would not haveidentified these.

NGS inevitably generates large amounts of data, and a challenge is to identify what
is clinically significant. In this thesis, CADD scoring was used as a tool to predict
pathogenicity. CADD scoring gives a standardised genome-wide, variant scoring
metric that incorporates the weighted results of widely used in silico pathogenicity
prediction tools, such as SIFT and PolyPhen, and of genomic annotation sources
like ENCODE (reviewed in van der Velde et al 2015). The resulting CADD scores
are expressed as a measure of deleteriousness (selection pressure bias) for singlenucleotide variants (SNVs) and small indels (van der Velde et al 2015). Although
CADD scores are useful in assisting the prioritisation of identified variants (van der
Velde et al 2015), it is important to be aware that their sensitivity and specificity will
never be 100%. CADD scores should be used in combination with other available
data such as family segregation studies, functional analysis and literature reviews(Schiemann and Stowell 2016) and must not be regarded as the gold standard ofvariant appraisal.

3.4.4 Chapter Conclusions
60 NMI polyposis patients underwent interrogation of APC and MUTYH to try anddetermine the cause of their genetically unexplained phenotype. The study
employed qPCR, AI screening, cDNA sequencing, karyotype analysis, MSP and
UDS as research tools. Whilst the gene transcription and AI studies gave
interesting results suggestive of reduced APC transcription for 4 patients, Halo46,
Halo52, Halo53 and Halo64, the underlying cause for this was only determined for
Halo46: UDS identified a 5’UTR variant in APC.

Although pathogenic variants/ molecular mechanisms of disease were not identified
for the majority of the patients, the techniques used as part of this study are all124relatively cost-effective and could be considered for use in future diagnostic
protocols for NMI polyposis patients, although further validation in clinical cohorts
would be necessary.

In Chapter 4 of this thesis, all 60 patients who underwent UDS of APC and MUTYH
will be subject to UDS of a further 15 genes possibly implicated in the pathogenesisof colorectal neoplasia to further investigate the cause of their phenotype. A subset
of 24 patients will undergo whole exome sequencing (WES).

125

126

Chapter 4 Exome Sequencing in
Polyposis Patients using Targeted
and Whole Exome Approaches
4.1

Introduction

After a detailed examination of APC and MUTYH, the next phase of this study
focused on exome sequencing. The initial strategy was to use a targeted approach.
Targeted exome sequencing of 15 genes associated with colorectal/ intestinal
neoplasia was performed. Subsequently, whole exome sequencing (WES) was
undertaken for a subset of patients.

60 NMI patient samples and 11 relatives/ healthy controls underwent targeted
exome sequencing of 15 genes: AXIN2, BMPR1A, CDH1, CHEK2, EPCAM, MLH1,
MSH2, MSH6, PMS2, POLD1, POLE, PTEN, SMAD4, STK11 and TP53. The
relatives/ healthy controls were included to assess whether variants identified in
NMI patients were occurring de novo and to allow variants to be assigned as likely
non-pathogenic if they were found in a healthy control, once the mode of inheritance
was taken into consideration. Gene capture was achieved using the Haloplex
Assay and sequencing was performed using the HiSeq 2500 (Illumina). The
patients were also screened for recurrent pathogenic NTHL1 mutation, c.268A>T ,p.Gln90* (Weren et al 2015). NTHL1 had not been included in the Haloplex assay
as the assay been designed prior to the publication of Weren’s paper. Putatively
pathogenic variants were validated with Sanger Sequencing and confirmed to bepresent in cDNA.

WES was performed on 24 patients. These were selected based on theirphenotype, the availability of family members and the results of previous
investigations completed as part of this study. Putatively pathogenic variants werevalidated with Sanger sequencing.

4.2

Targeted Exome Screening and Sequencing

Targeted exome sequencing used the Haloplex Assay for gene capture. The assay
was designed in 2014, and was based on one of the commercially available CRC

127

gene panels available at the time, produced by AmbryGen: the Colonext NGS panel(http://www.ambrygen.com/tests/colonext accessed 20/4/16). 14 of the genes
included on the study plate were present on the Colonext NGS plate (BMPR1A,
CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, PMS2, POLD1, POLE, PTEN,
SMAD4, STK11, TP53). AXIN2 was added based on current evidence that it may
have a role in the pathogenesis of colorectal neoplasia (Lammi et al 2004; Marvin etal 2011; Rivera et al 2014). The rationale for all of the genes investigated as part ofthis chapter is described below.

4.2.1.1

NTHL1

NTHL1 is mutated in NTHL1-associated polyposis/ CRC as described in Chapter 1(1.5.1.4).

4.2.1.2

BMPR1A and SMAD4

Bone morphogenetic proteins (BMPs) are part of the TGFβ family. They bind totheir receptors, triggering receptor activation, which proceeds to phosphorylate a
receptor-associated SMAD protein. This then complexes with SMAD4, whichtranslocates to the nucleus to regulate gene transcription (reviewed in Hardwick et
al 2008) and plays a role in growth inhibition (reviewed in Fleming et al 2013). The
BMPR1A gene encodes the BMP-receptor 1A, and SMAD4 encodes the SMAD4
protein described above. Germline mutations in both genes are associated with
impaired growth inhibition and are found in patients with Juvenile Polyposis
Syndrome (JPS), in which patients develop intestinal hamartomas and have an
increased risk of CRC (reviewed in Hardwick et al 2008). Germline BMPR1A
mutations have also been identified in a 58-year old male patient with ‘multiple’ (8)colorectal adenomas (Lipton et al 2003b) and in families with microsatellite stable
hereditary non-polyposis CRC (Nieminen et al 2011).

4.2.1.3

CDH1

The CDH1 gene codes for E-cadherin, a member of the cadherin family of cellsurface glycoproteins (reviewed in Kim et al 2000). It has roles in embryogenesis,
polarisation, differentiation and cell migration in inflamed tissue (reviewed in Kim et128

al 2000). Importantly, it is also known to be involved in intestinal cancer: loss of Ecadherin function is associated with invasiveness, lymph node metastasis anddistant metastasis (reviewed in Kim et al 2000). It is well-established that germline
mutations in CDH1 result in an increased risk of developing hereditary diffuse
gastric cancer (HDGC) and breast cancer (Pharoah et al 2001). It has also beensuggested that inherited CDH1 mutations might increase the risk of an individual
developing CRC (Richards et al 1999; Kim et al 2000).

4.2.1.4

CHEK2

The protein product of the CHEK2 gene acts as a checkpoint kinase (reviewed in
Cybulski et al 2004). Activation of CHEK2 in response to DNA damage prevents
the cell from undergoing mitosis (reviewed in Cybulski et al 2004). It is clear that
CHEK2 variants can play a role in the pathogenesis of breast cancer (Walsh et al
2006; Cybulski et al 2004), and there is evidence that CHEK2 variants may also be
associated with malignancies in multiple other organ systems, including the thyroid
gland, prostate, colorectum and kidney (Cybulski et al 2004). In 2007, Cybulski et al(Cybulski et al 2007) reported that carriers of a CHEK2 missense variant,p.Ile157Thr, had an increased risk of CRC compared to a control cohort (OR = 1.5,
p = 0.002), and that this specific variant might be responsible for 3% of all CRC in
Poland. Very similar results regarding increased CRC risk have also been reported
in more recent papers investigating Russian populations (Yanus et al 2018), Polishpopulations (Suchy et al 2010) and in Finnish populations (Kilpivaara et al 2006).

4.2.1.5

EPCAM and the MMR Genes: MLH1, MSH2, MSH6,

PMS2
The MMR system involves 7 key genes: MSH2, MSH6, MSH3, MLH1, PMS1, PMS2
and MLH3. The role of five of these genes in polyposis, CRC and Lynch Syndrome
has been described in Chapter 1 (1.3 and 1.5.1.5 ).
EPCAM is not a MMR gene, rather it is a cell adhesion molecule. However, it too
plays an important role in the pathogenesis of Lynch Syndrome. Patients with
germline deletions at the 3’ end of the EPCAM gene experience epigenetic silencingof MSH2, which is 17kb downstream of EPCAM (reviewed in Tutlewska et al 2013).

129

4.2.1.6

POLE and POLD1

POLE and POLD1 are mutated in PPAP and have been described in Chapter 1(1.5.1.3).

4.2.1.7

PTEN

PTEN is a tumour suppressor gene: its product is a phosphatase which negatively
regulates the phosphatidylinositol 3-kinase-AKT and mammalian target of
rapamycin (mTOR) signalling pathways, which are involved in cell proliferation, cell
cycle progression and apoptosis (reviewed in Lam-Himlin et al 2014). PTEN
mutations are involved in the pathogenesis of several carcinomas, including breast,
endometrial, thyroid, large bowel and kidney (reviewed in Lam-Himlin et al 2014).
PTEN mutations are identified in 80% of patients with Cowden’s Syndrome(reviewed in Omunsden and Lam 2012), described in Chapter 1 (1.5.3.3).

4.2.1.8

STK11

The LKB1/ STK11 gene is a nuclear serine threonine kinase which regulates cell
polarisation, growth and metabolism. Mutations in the gene account for
approximately 50% of cases of PJS (reviewed in Omunsden and Lam 2012),
described in Chapter 1 (1.5.3.1).

4.2.1.9

TP53

The p53 protein is a transcription factor which has a vital role in maintaining
genomic stability (reviewed in Sarasqueta et al 2013). Following DNA damage, p53
activation causes arrest of the cell cycle to allow DNA repair (reviewed inSarasqueta et al 2013). If the damage is too extensive, p53 can drive a cell towards
senescence or apoptosis (reviewed in Sarasqueta et al 2013; reviewed in
Vogelstein and Kinzler 2004). Somatic p53 mutations are a key event in thedevelopment of CRC (reviewed in Iacopetta 2003; Vogelstein et al 1988). Germline
p53 mutations give rise to Li Fraumeni Syndrome, a highly penetrant cancer
predisposition syndrome, in which patients develop a variety of early onset tumours,
including sarcomas, carcinomas, haematological malignancies and brain tumours130(reviewed in Gonzalez et al 2009). CRC is observed in 2.8% of patients with
germline p53 variants (Gonzalez et al 2009).

4.2.1.10

AXIN2

It is well established that the majority of CRC are characterised by activation of theWnt signalling pathway. As described in the previous chapters, nuclear β-catenincomplexes with DNA-binding proteins of the TCF/ LEF family, which leads toenhanced expression of oncogenes involved in tumour progression (reviewed in Wu
et al 2012). AXIN2 is a scaffold protein which is involved in regulating Wnt
signalling. It forms part of the ‘destruction complex’ which targets β-catenin for
degradation: AXIN2 supports the GSK3β-dependent phosphorylation of β-catenin,
which marks the protein for ubiquitination and subsequent proteosomal degradation(reviewed in Wu et al 2012). AXIN2 is a transcriptional target of β-catenindependent Wnt signalling, and AXIN2 levels are elevated in cancers with Wnt
activating mutations, therefore potentially negatively regulating Wnt signalling(reviewed in Mazzoni and Fearon 2014). There is some evidence that AXIN2
mutations may have a role in colorectal neoplasia: this will be described in detail in
Chapter 5.

4.2.2 Aims and Objectives: Targeted Exome Sequencing
The aim of the first part of this chapter was to identify novel genetic variants ingenes thought to be/ known to be associated with intestinal neoplasia in NMI
patients with multiple colorectal polyps. This involved screening for the known
pathogenic mutation in NTHL1 using Sanger sequencing and examining the exonsof 15 genes using NGS technologies.

The polyposis patients included in this study are the same 60 patients that
underwent UDS of APC and MUTYH described in Chapter 3, as are the 11
unaffected controls (3.2.5).

4.2.3 Methods

131

4.2.3.1

Screening NTHL1

DNA was extracted from whole blood of 60 NMI patients using standard protocols(2.3.2). PCR and Sanger sequencing was performed using standard reagents andreaction conditions (2.3.8.1, 2.3.8.5, 2.3.9-2.3.13) to screen for the recurrent
polyposis-associated mutation in NTHL1, c.268A>T, p.Gln90*. The primers were
designed by Dr. Laura Thomas (Appendix 4.1) who carried out the PCR reactions
and sequencing.

4.2.3.2

Haloplex Assay and Sequencing

4.2.3.2.1

Targeted Exome Capture, Sequencing and Data

AnalysisTargeted exome capture was achieved using the Haloplex assay (Agilent). The
probes for the Haloplex assay were designed using Agilent’s Sure Design software(https://earray.chem.agilent.com/suredesign/home.htm). DNA extracted from blood
samples underwent UDS using the HiSeq 2500, as described in Chapter 2 (2.4.2).
Bioinformatic analysis followed the protocol detailed in Chapter 3 (2.4.3).

The data was filtered to select for variants which were:

rare, with an allele frequency </= 1% based on data from The 1000
Genomes Project

not synonymous

not known to be benign when investigated using 4 public databases(dbSNP, HGMD, InSiGHT and LOVD)present in all affected relatives

not present in unaffected relatives (where family members were available
for analysis)/ not present in healthy controls (the pattern of inheritance wastaken into account. For example, when modelling dominant traits, variants
which were present in a healthy control were excluded; when modelling
autosomal recessive traits, variants identified in polyposis patients were only
excluded if they were also homozygous in a healthy control)

present in </= 3 samples

132

Variants were then analysed using CADD software(http://cadd.gs.washington.edu/home). Variants which had a CADD score >/= 15
were validated with Sanger sequencing provided they appeared real when observed
using Integrative Genomics Viewer (IGV) (https://www.broadinstitute.org/igv/).

4.2.3.3

Validation of Identified Variants

Variants identified were validated with Sanger Sequencing, using standard reagentsand reaction conditions (2.3.8.1, 2.3.8.5, 2.3.9-2.3.13). Variants present at a low
frequency (<20%) were validated with COLD-PCR, using standard protocols(2.3.8.3, 2.3.8.4, 2.3.8.6, 2.3.8.7).
The primers were designed by Marc Naven (Cardiff University) using a combinationof his own scripts, samtools and primer3 software, and by myself using primer3
software. They were supplied by Eurofins (Appendix 4.2).

4.2.3.4

Confirmation of the Variants in cDNA

RNA was converted to cDNA following standard protocols (2.3.3.2). DNA
underwent PCR and Sanger sequencing as per the standard protocol (2.3.8.1,
2.3.8.5, 2.3.9-2.3.13). The primers used are listed in Appendix 4.3.

4.2.4 Results

4.2.4.1

Screening for the common mutations in NTHL1

60 patients were screened for the known pathogenic NTHL1 mutation, c.268A>T,
p.Gln90*. This was not identified in any of the patient samples.

4.2.4.2

Haloplex Assay and Sequencing

1334.2.4.2.1

Metrics of UDS

The mean depth of coverage was 2900 reads across all 15 genes. The mean ontarget coverage across all the genes was 98.1%. The details for each gene arelisted in Table 4.1:
Gene

Mean Depth of Coverage

Mean On-Target Coverage(number of reads)

(%)

2928

100

BMPR1A 3010

99.6

CDH1

3627

99.7

CHEK2

2232

89.3

EPCAM

2343

100

MLH1

2876

94.8

MSH2

2290

97.2

MHS6

3523

100

PMS2

2605

98.1

POLD1

3526

99.9

POLE

3910

99.8

PTEN

1834

97.6

SMAD4

2649

99.9

STK11

3112

99.8

TP53

3039

95.5

AXIN2

Table 4.1 Coverage of the 15 genes on the Haloplex assay

1344.2.4.2.2

Variants Selected for Validation

60 NMI patients underwent targeted exome capture and UDS. A total of 33 different
variants were selected for validation across 32 patients (Table 4.2). The validation
sequencing traces are in Appendix 4.4.

135

Sample Gene

Location

Variant Frequency

Halo05

CHEK2

22: 29091740

C>G

POLD1

19: 50918229

G>A

MSH2

2:47639637

T>A

Halo07

SMAD4

18:48603114

C>A

Halo14

CHEK2

22:29107974

C>T

Halo15POLE

12:133249812 T>C

Halo17TP53

17:7577117

Halo18POLE

12:133202816 C>THalo20MSH62:48026861

1335/3023(44%)
1716/3706(46.4%)

CADD
Score

Amino Acid Change

26.9

p.Arg406Pro

22.4

p.Arg849His

24.5

p.Leu244Met

24.7

p.Pro472Gln

24.8

p.Glu239Lys

24.4

p.Met471Val

29.8

p.Val274Asp

23.8

p.Glu2140Lys

32

p.Ser580Leu

Genomic
Annotation
nonsynonymous
SNV
nonsynonymous
SNV

Validated

Yes

Yes

Halo06

A>T

C>T

3/3 (100%)
9/890(1.01%)
277/632(43.8%)
22813/45964(49.6%)
55/3547(1.56%)
310/609(50.9%)
7/669(1.05%)

nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV

No

No

Yes

Yes

No

Yes

No

136

Sample Gene

Location

Variant Frequency

Halo25

CDH1

16:68867388

G>A

Halo26MSH2

2:47630458

A>G

Halo27

POLD1

19:50919693

C>T

AXIN2

17:63534353

T>C

MSH2

2:47639633

C>A

Halo29PTEN

10:89720870

T>G

Halo31

POLE

12:133245032 A>T

Halo35

CHEK2

22:29121042

G>A

Halo43PTEN

10:89720678

A>T

Halo45MSH6

2:48026228

C>T

1856/3910(47.5%)
439/858(51.2%)
1081/2232(48.5%)
1147/3499(32.8%)

CADD
Score

Amino Acid Change

27.8

p.Gly879Ser

26.2

p.Tyr43Cys

34

p.Thr954Met

15.42

p.Ser390Gly

25

p.Asn242Lys

27.9

p.Phe341Val

23

p.Phe695Ile

16.85

p.Thr215Ile

25.1

p.Thr277Ser

29.5

p.Thr369Ile

Genomic
Annotation
nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV

Validated

Yes

Yes

Yes

Yes

Halo288/767(1.04%)
264/2436(10.84%)2428/4862(49.9%)
152/3145(4.83%)
12/848(1.41%)
580/1532(37.9%)

nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV

No

No

Yes

No

No

Yes

137

Sample Gene

Location

Variant Frequency

Halo46MLH1

3:37067140

G>T

Halo47

AXIN2

17:63533512

C>A

Halo49MSH62:48010592

G>T

Halo51

PMS2

7:6045634

T>C

Halo54

CHEK2

22:29090061

G>A

Halo58MSH62:48030603

C>T

Halo61

POLE

12:133245452 T>C

CDH1

16:68855966

G>A

EPCAM

2:47612347

G>A

BMPR1A 10:88677029

T>G

7/619
(1.13%)
357/742(48.2%)
8/734 (1%)
936/2788(34%)
71/4620(1.5%)
44/4148(1%)
3617/6785(53%)
609/1192
(51%)

CADD
Score

Amino Acid ChanGlye

Genomic
Annotation

Validated

39

p.Gly351*

stopgainNo

37

p.Gly548*

stopgainYes

36

p.Gly74*

stopgainNo

25.6

p.Ile18Val

34

p.Arg474Cys

23.8

p.Pro1073Ser

26.0

p.Tyr623Cys

23.9

p.Ala592Thr

29.1

p.Glu301Lys

29.7

p.Phe272Val

nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV

No

Yes

No

Yes

Yes

Halo62

Halo64

267/922(29%)
19/1240(1.5%)

nonsynonymous
SNV
nonsynonymous
SNV

Yes

No

138

Sample Gene

Location

Variant Frequency

Halo68AXIN2

17:63533512

C>A

Halo69POLE

12:133253974 C>T

Halo70MSH62:48030669

C>T

Halo72TP53

17:7578245

G>A

Halo80TP53

17:7578388

C>T

Halo81

POLE

12:133253974 C>T1176/2351(50%)
1611/3091(52%)
398/997(40%)
763/1656(46%)
56/162(35%)
1851/3784(48.9%)

CADD
Score

Amino Acid ChanGlye

37

p.Glylu548*

24.4

p.Arg259His

35

p.Arg1095Cys

16.71

p.Arg202Cys

28.5

p.Arg181His

24.4

p.Arg259His

Table 4.2 Variants selected for validation following targeted exome capture

Genomic
Annotation
stopgainnonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV
nonsynonymous
SNV

Validated

Yes

Yes

Yes

Yes

Yes

Yes

139

4.2.4.2.3

Confirmation of the Variants in cDNA

At the time of the experiments, RNA was available for 14 patients with variants
validated in DNA. All the variants which were identified in DNA were confirmed to
be present in cDNA (Appendix 4.5).

4.2.5 Further Assessment of Validated Variants
Validated variants were checked in dbSNP(https://www.ncbi.nlm.nih.gov/projects/SNP/), HGMD (https://portal.biobaseinternational.com/hgmd/pro/start.php), InSiGHT (https://www.InSiGHTgroup.org/variants/databases/) and LOVD (http://lovd.nl/3.0/home). International
experts who specialise in specific genes were contacted to determine whether they
had any further information regarding the clinical significance of the confirmed
variants. I also liaised with AmbryGen, a company which provides a clinicalgenetics testing and diagnostic service in the United States of America.

Several of the variants identified using the Haloplex assay followed by UDS havebeen deemed to be pathogenic, but in clinical situations other than colorectal
polyposis. For example, the CHEK2 mutation found in Halo14(c.715G>A:p.Gln239Lys) is assigned to be DM (pathological) in prostate cancer(CM030421: inherited variant) on HGMD and the TP53 mutation in Halo072(c.604C>T:p.Arg202Cys) is reported as DM in adrenocortical carcinoma(CM121764: inherited variant).
With regards to the CDH1 variants, I contacted Professor Seruca’s research groupin Portugal. One of the CDH1 variants which had been found in Halo62,c.1774G>A:p.Ala592Thr, has been classified as non-pathogenic. The group had
already evaluated its significance in vivo and demonstrated that cells expressing thevariant are not invasive (Keller et al 2004). The second variant identified in Halo25,
c.2635G>A:p.Gly879Ser, had been reported in an 81-year old Caucasian woman
with lobular breast cancer, but its functional relevance remains to be evaluated.
Professor Seruca noted that although CRC can occur as part of the tumour
spectrum in Hereditary Diffuse Gastric Cancer, which can develop as aconsequence of germline mutations in CDH1, it is specifically of signet ring cell type.
She reported that there is no evidence to suggest that CDH1 mutations increase therisk of CRC associated with colorectal polyposis (personal communication140

10/10/2016). In light of this, the CDH1 mutations were not pursued as part of thisproject.

AmbryGen provided their classification, if available, of the validated variants (VLB:
variant likely benign, Poly: polymorphism, VUS: variant of unknown significance,
VLP: variant likely pathogenic). This is listed in Table 4.3:

141

Based on this information, the following conclusions were drawn:

4.2.5.1

CHEK2

It is possible that CHEK2 variants may have a role in colorectal neoplasia. Threedifferent variants were validated in this cohort of NMI patients:
c.1217G>C:p.Arg406Pro in Halo05, c.715G>A:p.Glu239Lys in Halo14 andc.1420C>T:p.Arg474Cys in Halo54. However, during the time course of this project
it was not possible to recruit additional family members for de novo/ segregation
analysis. Therefore these variants have not been pursued in the short-term.

1424.2.5.2

POLE and POLD1

The VUS/ unreported POLE variants were selected for additional studies in Chapter
5 of this thesis. One of the POLD1 variants, c.2546G>A:p.Arg849His, is recorded
as a polymorphism by AmbryGen, and has recently been classified as benign in
dbSNP (https://www.ncbi.nlm.nih.gov/clinvar/variation/220865/ accessed
08/05/2018) so was not pursued. The second POLD1 variant,c.2861C>T:p.Thr954Met, is not previously reported. It was taken forward for further
work described in Chapter 5.

4.2.5.3

CDH1

These variants were not investigated following Professor Seruca’s advice that they
are unlikely to be pathogenic in the context of polyposis. This was also consistent
with AmbryGen’s classification of the variants identified as VLB/ Poly.

4.2.5.4

MSH2, MSH6, MSH6, MLH1, EPCAM

Variants in the MMR genes were selected for further work. Although the AmbryGenclassification was taken into consideration, the gold standard classification is thatprovided by the InSiGHT consortium (https://www.InSiGHTgroup.org/syndromes/lynch-syndrome/), so this was given the greatest weight. The
only variant which wasn’t pursued was the EPCAM missense variant. Only
deletions of EPCAM that inactivate the adjacent MSH2 gene in Lynch syndrome areconsidered pathogenic, (Tutlewska et al 2013) rather than missense changes.

4.2.5.5

AXIN2Neither of the variants identified had been reported by AmbryGen. We were unable
to recruit further relatives for Halo28, and the variant was a missense change, so isless likely to be pathogenic. Therefore, the c.1168A>G:p.Ser390Gly variant was nottaken forward. The variant found in Halo47 and Halo68, c.1642G>T:p.Glu548*, wasextensively investigated as reported in Chapter 5 of this thesis.

1434.2.5.6

TP53

One of the variants has been discussed above. TP53 c.604C>T:p.Arg202Cys was
identified in Halo72, and is thought to have a role in adrenocortical carcinoma.
Halo72 had an interesting family history, including several family members in thepreceding generation having CRC/ lung cancer/ cancer of unknown origin.
However, we were unable to recruit any further family members, so the variant has
not been pursued at the present time. The second variant, c.542G>A:p.Arg181His,
is recorded as likely pathogenic by AmbryGen, who have 12 cases of patients
carrying this variant. 11/12 had breast cancer, and the remaining patient had afamily history of breast cancer. Although most of the families had histories of further
malignancies, they did not fulfil Li Fraumeni diagnostic criteria, and there were nocases of polyposis. In light of this, the variant was not investigated further.

The variants which were selected for further investigations were therefore those
whose significance was unknown/ which were unreported and for which basic
functional/ in silico work could be carried out and/ or those which were identified inpatients in whom relatives were available to be recruited for genetic analysis todetermine de novo status or segregation.

The genes I selected to focus on were the MMR genes, POLE, POLD1 and AXIN2.
This work is described in Chapter 5 of this thesis.

144

4.3

Whole Exome Sequencing

The next phase of this project involved whole exome sequencing (WES) of 24
patients, plus 5 relatives. The relatives were included to assess whether variants
identified in NMI patients were occurring de novo and to allow variants to be
assigned as likely non-pathogenic if they were found in a healthy control, once themode of inheritance was taken into consideration. Although most of the patients
included in this thesis will ultimately undergo WES and/ or whole genome
sequencing, this cohort was selected based on a phenotype of early onset or
profuse polyposis and/ or the availability of relatives and/ or those patients in whoma novel putative polyposis-causing variant had already been identified, to help
exclude the presence of an additional previously undetected polyposis-associatedvariant.

The patients and relatives included are recorded in Table 4.4 below:

UniqueDemographic

Clinical

Identifier

Details

Phenotype

Relative Available

Aged 39 had multiple

Halo0546-year-old
femaleadenomas (at least19): one polypcontained Duke's pT1
CRC

Halo08

Halo1570-year-old
female84-year-old male

629 adenomas

12 adenomas
25 mixed polyps:
adenomas andhyperplastic lesions

Halo1766-year-old male(exact numbers ofeach is not clear). 2
CRC aged 63(pT2,pT3)

145

Referred from
Halo1872-year-old malescreening programme.
27 polyps: adenomas
with 4 HPPs
>10 adenomas andHalo1962-year-old

CRC (no further

femaleinformation available
about the CRC)

Halo2767-year-old
female17 adenomas

1990: sigmoid
colectomy for aprobable diverticularperforation. Multiple'benign metaplastic
polyps' were noted
Halo2861-year-old male?number. 2012:
subtotal colectomy for>30 polyps. The
majority of the polyps
were TA; 2 wereserrated adenomas
with LG dysplasia.

Halo40

Halo45

Halo4670-year-old
female77-year-old man

44-year-old
female18 adenomas, 3 HPPs

27 adenomas, 7 HPPClinical FAP – has had
a colectomy andproctectomy
Colonoscopy found in

Halo4751-year-old
femaleexcess of 50 sessile

Halo68 (Sister) 3 TVA with

polyps throughout the

LGD, 10 TA with LGD and 7colon. 2 of these were

HPPs.

biopsied: TVA. Px had

146

a subtotal colectomy ?4 polyps were
sampled: 2 TA, 2’serrated adenomas’.
The patient
subsequently had acompletion colectomy– she was found tohave a rectal
carcinoma (pT4N2Mx)
Sister of Halo68
Referred fromscreening. 30 polyps: 4
Halo4871-year-old malehave been biopsied:
all adenoma.
Subsequent colectomy.
Rectaladenocarcinoma age

Halo4957-year-old
female32 (?stage),ameloblastoma age 38.
22 polyps: mixture of
adenomas andhyperplastic lesions
Halo77 (mother):Halo5143-year-old
female208 adenomas

unaffected
Halo78 (father):unaffected

Halo5259-year-old maleOver 1000 adenomas

Approximately 400
Halo5351-year-old maleadenomas, carcinoma
age 27 (Duke’s C)

18 polyps: 9
Halo5554-year-old maleadenomas, 9 serrated
lesions

Halo75 (mother):unaffected
Halo76 (father):unaffected

F_Halo55 (father):unaffected147

60-year-old female with
Halo6160-year-old

11 polyps: 2 serrated

femaleadenomas, 7 TA, 1
TVA, and 1 HPP
At least 24 polyps. 5

Halo6368-year-old malebiopsies show 4
adenomas and 1 HPP
Thousands ofcolorectal polyps,

Halo6454-year-old
femalethose which have been
biopsied showedadenomas. Caecal
carcinoma aged 23(?stage)

Halo6533-year-old male

117 adenomas

Halo6670-year-old male

23 adenomas, 4 HPPs

Table 4.4: Patients and relatives undergoing WES

4.3.1 Methods
DNA was extracted from whole blood by the AWMGS (2.3.2). The concentration of
the DNA samples to be examined were determined using the Invitrogen Qubit kit
following the manufacturer’s protocol. The DNA was diluted to a concentration of
5ng/μl.

4.3.1.1

Sequencing

Sequencing was carried out by Sarah Edkins, Wales Gene Park, using the Nextera
Rapid Capture Enrichment Library Preparation and Sequencing Protocols (2.4.4).

148

4.3.1.2

Sequence Analysis

Bioinformatic analysis was performed by Dr. Kevin Ashelford (Cardiff University)(2.4.5).

4.3.1.3

Variant Analysis and Selection

A candidate gene approach was initially adopted, and the data was filtered forgenes which were:
1) present in an in-house list of 1177 candidate CRC genes (Smith et al Appendix
4.6) or2) were established cancer predisposition genes (Rahman 2014) or
3) were genes which are differentially expressed in solid tumours (Digital Differential
Display (DDD) genes, Scheurle et al 2000)

Novel genes outside of this list were being investigated by Beth Bradford as her
professional training year (PTY) project (Bradford, B (2017) Use of Whole Exome
Sequencing for the Identification of Novel Genetic Mechanisms in Colorectal
Polyposis). Any patient not investigated by Bradford was subject to interrogation by
myself, to ensure that all patient samples had been examined in the same way. I
therefore examined samples Halo05, Halo15, Halo27, Halo28, Halo47, Halo61 andHalo68.

The complete protocol for shortlisting variants of interest is outlined in Figure 4.1.

149

Candidate Gene
Approach
Variants are shortlisted to inclue thosewhich are present in an in-house list of
candidate CRC genes or which areestablished cancer predispostion genes
or which are differentially expressed insolid tumours (4.3.1.3)

Variants are further filtered, selecting
for heterozygous/ homozygous/compound heterozygous variants which:pass the bioinformatic quality control,
have an allele frequency <0.5%(according to data from The 1000
Genomes Project), are present in >20%of reads, are present in <50% of
samples (to exclude sequencing
artefacts), are frameshift/ nonsynonymous/ are at a splice site/ stop
gain, which are not present in a healthy
control (taking into consideration themode of inheritance). Non-coding
variants are excluded unless they are at
a splice site

CADD Score >20

Perform a literature review for eachvariant and select those for validation

Novel Gene Approach
Variants are filtered, selecting forheterozygous/ homozygous/ compound
heterozygous variants which: pass thebioinformatic quality control, have anallele frequency <0.5% (according todata from The 1000 Genomes Project),are present in >20% of reads, arepresent in <50% of samples (to exclude
sequencing artefacts), are frameshift/non-synonymous/ are at a splice site/
stop gain/ non frameshift deletions/non frameshift insertions/ stoploss,
which are not present in a healthy
control (taking into consideration themode of inheritance)

Exclude variants which are present in an
in-house list of candidate CRC genes or
which are established cancer
predispostion genes or which are
differentially expressed in solid tumours(4.3.1.3)
CADD Score >20
Prioritise variants using ToppGene
software(https://toppgene.cchmc.org/prioritizati
on.jsp accessed August 2017). Smith’s
CRC candidate gene list was used as thetraining set. Patients’ filtered genes
were compared according to thefollowing rules: similar molecularfunction/ involvement in similar
biological processes/ similar cellularcomponent/ involvement in similar
human and mouse phenotypes/
involvement in the same molecularpathways/ PubMed similarity/ similarinteractions/ similar transcription factor
binding sites/ involvement in similardiseases
Perform a literature review for variants
with a CADD score >/= 30 and the top 10
hits in ToppGene and any truncating
variants. Select those for validation

Figure 4.1 WES variant analysis and selection150

4.3.1.4

Validation of Identified Variants

Variants were validated with Sanger sequencing, using standard reagents andreaction conditions (2.3.8.1, 2.3.8.5, 2.3.9-2.3.13). Primers were designed by Dr.
Marc Naven (Cardiff University) using a combination of his own scripts, samtools
and primer3 software, and by myself using primer3 software. They are listed inAppendix 4.7.

4.3.2 Results

4.3.2.1

Coverage

The mean depth of coverage across the 29 patients/ relatives was 61.99 reads.
The details for each patient, including the percentage of target region covered at
different depths, are recorded in Table 4.5:

Percentage of target region covered at the following
coverage depths:
SampleMean Reads

1x

4x

10x

20x

30x

40x

50x

Halo05

88.82

97.20

95.24

92.51

87.21

81.18

74.76

68.09

Halo08

50.36

97.26

92.08

81.69

67.66

56.34

46.60

38.23

Halo15123.42

98.47

95.99

91.60

84.90

79.14

74.13

69.61

Halo17

49.62

97.88

93.33

83.02

67.78

55.27

44.74

36.01

Halo18

49.11

97.95

93.48

83.17

69.90

55.46

44.85

36.02

Halo19

44.73

97.36

91.86

80.32

64.60

52.03

41.55

33.01

Halo27

85.95

97.97

95.01

89.39

80.82

73.54

67.13

61.17

Halo28

64.59

97.26

94.97

91.20

83.39

74.30

64.67

55.17

Halo40

60.54

98.01

97.73

86.87

73.69

62.23

52.22

43.53

151

Halo45

40.69

97.42

91.46

79.15

64.40

49.11

38.20

29.52

Halo46

76.93

98.24

95.63

89.59

79.02

69.69

61.43

53.92

Halo47

73.33

97.93

95.68

92.77

86.91

79.50

71.04

62.15

Halo61139.76

98.66

96.73

93.43

87.76

82.43

77.62

73.23

Halo68

74.90

97.25

95.11

91.90

85.40

77.84

69.70

61.41

Halo48

36.24

98.07

93.33

79.35

57.19

41.45

30.65

22.99

Halo49

64.64

98.26

95.19

87.78

75.11

64.14

54.52

46.03

Halo51

52.73

98.06

94.47

85.40

70.66

58.30

47.70

38.79

Halo77

51.57

97.70

93.77

84.16

69.62

57.62

47.27

38.49

Halo78

61.28

98.04

93.93

84.21

69.45

57.28

46.81

37.98

Halo52

62.20

98.26

95.27

87.78

74.86

63.69

53.94

45.40

Halo53

62.15

98.22

95.10

87.52

74.69

63.58

53.85

45.30

Halo75

38.16

96.85

90.30

77.47

60.55

46.94

35.84

27.21

Halo76

62.76

97.56

92.81

83.67

71.53

61.88

53.57

46.27

Halo55

45.34

97.56

92.19

80.85

65.29

52.89

42.50

33.87

56.64

98.90

94.08

85.05

71.50

60.30

50.63

42.21

Halo63

54.32

98.14

94.38

85.26

70.92

59.00

48.77

40.03

Halo64

58.35

98.05

94.42

85.95

72.61

61.50

51.83

43.31

Halo65

54.59

97.95

94.01

84.97

71.07

59.38

49.24

40.47

Halo66

14.02

93.60

76.76

50.51

24.75

12.15

5.83

2.71

Halo55
FatherTable 4.5: Patient-specific depth of coverage for WES

152

4.3.2.2

Variants Selected for Validation

106 variants were selected for validation across the 24 patients. The details andresults of validation are in Table 4.6. The validation sequencing traces are in
Appendix 4.8.

153

CADD

Notes

Gene

Chr.

Location

Variant

Halo05LZTS1

820107358

G>A

34

Yes

Missense

Halo05ERCC61050690821

G>A

34

No

Missense

Halo05LRP4

1146918516

G>A

29.3

Yes

Missense

Halo05ATM

11108114679

G>T

24.4

No

Halo05ATM

11108114684

G>T

23.3

No

Missense

Only present in 2/7 reads (NGS)

Halo05ATM

11108114689

C>T

23.4

No

Missense

Only present in 2/10 reads (NGS)

Halo05ATM

11108196797

G>A

34

Yes

Missense

Halo05PALB2

1623635370

C>T

26.6

Yes

Missense

Halo05LIG1

1948620943

C>A

29

Yes

Missense

Score

Validated

Genomic

Patient ID

Annotation

Splice site

There was a synonymous variant
adjacent to the called variant

Only present in 2/5 reads (NGS)

acceptor

154

CADD

Gene

Chr.

Location

Variant

Halo05FZD52208633009

C>T

22.5

Yes

Missense

Halo05ATR

3142272170

A>G

20.5

Yes

Missense

Halo05MCPH1

86479113

C>T

24.6

Yes

Missense

Halo08EYA4

6133789765

C>T

24.8

Yes

Missense

Halo08

BCLAF1

6136599544

G>A

36

Yes

Stopgain

Halo08BMP1

822049596

G>A

33

Yes

Missense

Halo15SFN

127190196

A>T

26.1

Yes

Missense

Halo15

CELSR2

1109812092

G>A

22

Yes

Missense

Halo15CAPN91230907799

C>T

35

Yes

Missense

Halo15ATR

3142272170

A>G

20.5

Yes

Missense

Score

Validated

Genomic

Patient ID

Annotation

Notes

155

Patient ID

Halo15Gene

ZKSCAN
4

Chr.

6

Location

28219686

Variant

CGGTC
A>C

CADD
Score

Validated

Genomic

Notes

Annotation
There was low coverage at this locus.

24.4

No

Frameshift

The variant was called in 2/3 reads(NGS)

Halo15DAAM2

639864686

C>T

34

Yes

Missense

Halo15EGFR

755273086

G>A

35

Yes

Missense

Halo15ST18

853030923

G>T

31

Yes

Missense

Halo15NCOR2

12124824869

G>A

24.3

Yes

Missense

Halo15NCOR2

12124911260

C>T

24.6

Yes

Missense

Halo15HDAC51742171169

G>A

25.6

Yes

Missense

Halo17BMPR2

2203407059

G>A

23.1

Yes

Missense

Halo17BMPER

734125622

C>T

34

Yes

Missense

156

CADD

Gene

Chr.

Location

Variant

Halo17TTI2

833361016

C>T

24.6

Yes

Missense

Halo17CBL

11119169085

G>A

21.6

Yes

Missense

Halo17DSC31828605748

C>T

35

Yes

Missense

Halo17DSC2

1828647999

T>TTC

35

Yes

Frameshift

Halo18CNKSR1

126515380

G>A

33

Yes

Missense

Halo18POLQ

3121207509

34

Yes

Frameshift

Halo18CBL

11119148958

T>C

23.9

Yes

Missense

Halo18RAD51B

1468353893

A>G

27.2

Yes

Missense

Halo19THRAP3136757052

G>A

24.3

Yes

Missense

CAATAG
TA>C

Score

Validated

Genomic

Patient ID

Annotation

Notes

157

CADD

Gene

Chr.

Location

Variant

Halo19RB1

1349047524

G>A

28

Yes

Missense

Halo19MGA

1542028820

A>G

22.2

Yes

Missense

Halo19WNT9B

1744953675

G>A

31

Yes

Missense

Halo19MED12

X

70342603

G>A

33

Yes

Missense

Halo27PLK3

145267346

G>T

33

Yes

Missense

Score

Validated

Genomic

Patient ID

Notes

Annotation

There was relatively low coverage at thisHalo27RBM5350155887

TGA>T

35

No

Frameshift

locus and the variant was only called in
5/22 reads (NGS)Halo27TRRAP

798591187

G>C

21.6

Yes

Missense

Halo27KAT5

1165482096

G>A

28.1

Yes

Missense

Halo27PTPRH

1955697712

G>A

36

Yes

Stopgain

158

CADD

Gene

Chr.

Location

Variant

Halo28PIK3C2A

11

17140810

A>G

23.5

Yes

Missense

Halo28CD82

1144626709

G>A

33

Yes

Missense

Halo28RBMS2

1256982155

C>T

21.3

Yes

Missense

14104205127

T>C

23.1

Yes

Missense

Halo28

PPP1R1
3B

Score

Validated

Genomic

Patient ID

Annotation

Halo28MGA

1542058553

G>C

22

Yes

Missense

Halo28DCC

1850961517

G>A

28.2

Yes

Missense

Halo28BIRC6

232706513

G>T

32

Yes

Missense

Halo28EYA4

6133789765

C>T

24.8

Yes

Missense

Halo28AKAP9

791623985

G>C

26

Yes

Missense

Notes

159

CADD

Gene

Chr.

Location

Variant

Halo28MET

7116381047

A>G

23.1

Yes

Missense

Halo40GDF7

220870532

C>A

23.6

Yes

Missense

333883492

G>A

30

Yes

Missense

Halo40

PDCD6IP

Score

Validated

Genomic

Patient ID

Annotation

Halo40LRP5

1168183958

G>A

23.9

Yes

Missense

Halo40DSC2

1828672114

C>T

21.7

Yes

Missense

Halo45EIF3I

132688188

A>G

28.2

Yes

Missense

Halo45HELQ

484374567

C>T

25.5

Yes

Missense

Halo45RPLP2

11810305

A>G

22.6

Yes

Missense

Halo46MSH4176345823

A>G

28.6

Yes

Missense

Notes

160

Patient ID

Halo47Gene

PPP1R1
3B

CADD

Location

Variant

14104245134

C>T

23.5

Yes

Missense

Score

Validated

Genomic

Chr.

Annotation

Halo47XAB2

197694391

G>C

23.5

Yes

Missense

Halo47ENG

9130588091

C>T

23.9

Yes

Missense

Halo48ADAM1729633092

C>T

34

Yes

Missense

Halo48FZD3828420428

G>A

23

Yes

Missense

Halo49BMPR1B

496070060

G>C

33

Yes

Missense

Halo49MSH3

580063899

G>C

23.7

Yes

Missense

Halo49EPHB4

7100421340

C>T

25.2

Yes

Missense

Halo49ID1

2030193351

C>T

29

Yes

Missense

Notes

161

CADD

Gene

Chr.

Location

Variant

Halo52EP400

12132471141

C>T

15.34

Yes

Missense

1345008887

G>T

24.6

Yes

Missense

Halo52

TSC22D1

Score

Validated

Genomic

Patient ID

Annotation

Halo52FOXC21686602293

G>C

28.3

Yes

Missense

Halo52PHF121727239701

T>G

18.87

Yes

Missense

Halo55DVL1

11273404

G>A

22.8

Yes

Missense

Halo55BMP8B

140228846

G>T

39

Yes

Stopgain

Halo55TLE1

984228372

G>A

29.5

Yes

Missense

Halo61PARP1

1226564855

G>A

26.2

Yes

Missense

Halo61ANAPC29140069828

C>T

31

Yes

Missense

Notes

162

CADD

Gene

Chr.

Location

Variant

Halo61RECQL

1221623219

G>C

38

Yes

Stopgain

Halo61INHBE

1257850383

G>A

33

Yes

Missense

Halo61POSTN

1338156538

C>T

34

Yes

Missense

Halo61EEF2K

1622269048

C>T

26.8

Yes

Missense

Halo61ZFP14

1936831616

T>A

25.9

Yes

Missense

Halo63BIRC6

232726929

A>C

22.3

Yes

Missense

Halo63XRCC52217026733

G>A

24.1

Yes

Missense

Halo63

WNT10A

2

219754822

G>A

24

Yes

Missense

Halo63CHD7

861734439

G>A

25.7

Yes

Missense

Halo63WISP1

8134232908

C>T

24.3

Yes

Missense

Score

Validated

Genomic

Patient ID

Annotation

Notes

163

CADD

Gene

Chr.

Location

Variant

Halo63ARID2

1246233172

C>T

26.4

Yes

Missense

Halo64

MTHFR

111854085

T>A

23.3

Yes

Missense

Halo64WAPAL

1088259879

T>C

25

Yes

Missense

Halo65POLQ

3121207520

G>A

24.2

Yes

Missense

Halo65ERCC8560194107

G>T

24.4

Yes

Missense

Halo65NUDT1

72284301

G>A

23.7

Yes

Missense

Halo65ATM

11108186610

G>A

31

Yes

Missense

Halo65

TMBIM61250146761

C>T

24.7

Yes

Missense

Halo66MSH3

580063896

C>T

22.8

Yes

Missense

Halo66MMS19

1099218456

C>T

32

Yes

Missense

Score

Validated

Genomic

Patient ID

Annotation

Notes

164

CADD

Gene

Chr.

Location

Variant

Halo66XAB2

197694391

G>C

23.5

Yes

Missense

14104245134

C>T

23.5

Yes

Missense

Halo68

PPP1R1
3B

Score

Validated

Genomic

Patient ID

Annotation

Halo68XAB2

197694391

G>C

23.5

Yes

Missense

Halo68ENG

9130588091

C>T

23.9

Yes

Missense

Table 4.6: WES validation

Notes

165

All of the validated variants were subsequently investigated using multiple
databases to determine whether there was any additional information available
about their likely pathogenicity: dbSNP, Exac, Ensembl, HGMD, LOVD, COSMIC,
CBioPortal, ClinVar, CanVar (Databases accessed May/ June 2017,
http://exac.broadinstitute.org/, http://grch37.ensembl.org/index.html,
https://portal.biobase-international.com/hgmd/pro/start.php,
http://www.lovd.nl/3.0/home, http://grch37-cancer.sanger.ac.uk/cosmic,
http://www.cbioportal.org/index.do, https://www.ncbi.nlm.nih.gov/clinvar/,https://canvar.icr.ac.uk/, https://genome.ucsc.edu/cgibin/hgTracks?db=hg19&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=chr11%3A108186595108186625&hgsid=594778521_y7cxR9MaVPipen9AAAExDrahzrKP).
The results are summarised in Appendix 4.9. Particular attention was paid to theclinical situations in which specific inherited variants had been identified in, andwhether or not the variants had been described as a somatic change in different
cancer types.

Relatives were available for Halo51, Halo53 and Halo55. For both Halo51 andHalo53, no putative pathogenic de novo variants were identified. For Halo55 only 2
were found: missense changes in TLE1 and BRIC6. A third variant identified in
Halo55 was a stopgain in BMP8B. At a relatively late stage in the project, DNA
became available for the patient’s unaffected mother, in whom the BMP8B variant
was also identified, so it is unlikely to be clinically significant.

166

4.4

Discussion

4.4.1 Targeted Exome Sequencing
The Haloplex assay was used for targeted exome capture of 15 candidate polyposis
genes, followed by UDS using the HiSeq 2500 (Illumina). 33 different variants
across 32 patients were selected for validation (a total of 35 variants as 2 different
variants were identified in 2 sets of different patients). 21 variants which appeared
to be heterozygous changes were validated. A possible MHS2 homozygous variant
observed in patient Halo06 was not validated. There were only 3 reads with UDS,
so this is highly likely to be an artefact. A PMS2 variant identified in Halo51 in 34%
of reads was not validated. There are known to be multiple pseudogenes of PMS2
(Vaughn et al 2010), so it is likely that UDS had identified a variant in such a
pseudogene.

Twelve low frequency variants underwent COLD-PCR protocols followed by Sanger
sequencing. In 2 samples, Halo17 and Halo49, the COLD-PCR protocols used did
not successfully amplify the desired amplicon, and the required melting temperature
was 95⁰C as in standard PCR. For sample Halo64 the full COLD PCR protocol
appeared to work at denaturation temperatures as low as 68°C. However, thiswould be extremely unlikely, if not impossible. It is most probable that the
hybridisation step did not work, therefore the effective denaturation temperature
would have been 95°C.

11/12 low frequency variants were not validated, so the UDS results are likely to beartefacts. However, it is possible that the COLD-PCR was not sufficiently sensitive
to detect them. COLD-PCR has been discussed in Chapter 3 (3.4.3.1). The one low
frequency variant which was validated was identified in sample Halo54: CHEK2
22:29090061 G>A. The UDS results indicated a variant fraction of 1.5% (71/4620
reads). However, the trace obtained with the Sanger sequencing validation
revealed that the patient was approximately heterozygous rather than a very-low
frequency mosaic (Appendix 4.4), suggesting that there had been preferential
amplification of the wild-type allele with UDS.

167

The aim of this part of the project was to identify novel/ rare variants in genes
already known to have/ potentially have a role in colorectal neoplasia. A key issue
encountered was the lack of availability of relatives for many of the probands fordetermination of the variant’s de novo status or assessment of whether it wassegregating with the disease. Without such evidence, it is often not sensible tocommence functional studies. The variants that have been selected for further workin this thesis are a novel truncating AXIN2 mutation (c.1642G>T:p.Glu548*), which
has been identified in 2 affected siblings, and variants in POLE, POLD1 and the
MMR genes. Although relatives were not always available for the patients found tocarry variants in the latter group of genes, their established roles in inherited
predisposition to colorectal tumours and availability of basic functional studies madeit reasonable to investigate the pathogenicity of the variants.

The work in this part of the project required database interrogation to aid inappraising identified variants, and also communication with international experts.
This highlights the importance of communication and collaboration in clinicalresearch. It is apparent that genetic variants identified through clinical research
projects are often not recorded in publicly accessible databases. This is a great
shame. It would be excellent if all variants were recorded in a centralised
repository, such as LOVD, as this would allow improved understanding of disease
pathogenesis and therefore improved patient care. With the advent of the 100 000
Genomes Project it is hoped that there will be improved understanding of the role of
the genome in health and disease. The project aims to sequence 100 000 genomesof individuals with cancer/ rare diseases/ infectious diseases. Genomes will besequenced, annotated and analysed in the context of the individual’s medical
history. The project seeks to enhance the clinical interpretation of the data andderive new findings from the data (https://www.genomicsengland.co.uk/faqs-aboutgecip/ accessed 10/5/2018). The Human Variome Project has a similar ethos. It is
an international non-governmental organisation which functions to ensure that all
information on genetic variation and its effects on human health can be collected,
crated, interpreted and shared freely and openly(http://www.humanvariomeproject.org/about/about-the-human-variome-project.html
accessed 10/05/18). The project itself doesn’t physically store the data, but it
develops standards and approaches so that data from different sources can beshared in an appropriate manner. LOVD is one example of a database which now
functions under the auspices of the Human Variome Project.

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4.4.2 Whole Exome Sequencing
24 patients underwent WES to try and identify further genes which may be
implicated in colorectal neoplasia. Two approaches were taken: a candidate geneapproach and a novel gene approach (Bradford 2017). Using the candidate geneapproach, 106 variants were selected for validation: only 6 of these were notvalidated. In one of these cases, ERCC6 in Halo05, there was a synonymouschange at the base adjacent to that called, so it is likely that the bioinformatic
analysis was miscalling the actual change. In 3 cases also occurring in Halo05, in
the ATM gene, there was poor coverage at the loci, so the variants called are likely
to be artefactual. In Halo15, a ZKSCAN4 variant was not validated, but again there
was low coverage at this locus, and the variant was called in only 2/3 reads. The
final variant which was not validated was an RBM5 frameshift in Halo27. The
variant was only called in 5/22 reads, and it was at a region where there werenumerous AC dinucleotide repeats, so the call was likely to be an artefact.

There are several important points to note about the WES results using thecandidate gene approach:There were no obvious mutations which could explain the reduced APC
expression for samples Halo52, Halo53 and Halo64.

There was not a ‘second hit’ in a gene involved in BER for Halo40, who is amonoallelic carrier of a pathogenic MUTYH mutation.



There were no mutations for Halo46 which were more likely to bepathogenic than her previously-identified APC mutation.

In Halo47 and Halo68 there were no mutations in genes involved in thepathogenesis of oligodontia/ ectodermal dysplasia (CXORF5, DLX1, DLX2,
EDA, EDAR, EDARADD, FGFR1, GLI2, GLI3, LEF1, LTBP3, MSX1,
NEMO, PAX9PITX2, P68, Wnt10a. Chhabra et al 2014; Deshmukh et al2012) (See Chapter 5).



Of the truncating mutations, which are generally more likely to be clinicallysignificant than missense changes:o

The BCLAF1 variant identified in Halo08 was not present in her
affected son, whose DNA became available at a late stage in the
project.

o

The DSC2 variant in Halo17 may be implicated in a clinicalphenotype other than colorectal neoplasia (cardiomyopathy) and no169

relatives were available for screening, so it was not followed up in
the short term as part of this project.
o

A frameshift POLQ variant was identified in Halo18. I liaised with
AmbryGen about this variant, and they commented that ‘thesubpopulation frequencies in 1K Genomes for
POLQ c.4262_4268delTACTATT are high indicating that this would
probably be likely benign based on our current classification scheme’(Felicia Hernandez personal communication 09/03/2017).
Furthermore, relatives were not available for screening as the patient
sadly passed away during the course of the project, so familial follow
up was not possible.

o

The PTPRH variant found in Halo27 may be involved in thepathogenesis of Parkinson’s Disease (Appendix 4.9) and colorectal
neoplasia is not typically part of the phenotypic spectrum, so it was
not followed up in the short term as part of this project.

o

The RBMS2 variant in Halo28 had been mis-described in thebioinformatic analysis of the exome sequencing: it was actually asynonymous change rather than a stop-gain.

o

The BMP8B variant in Halo55 was subsequently identified in his
unaffected mother, as described above.

o

The RECQL variant in Halo61 is interesting as RECQL is involved in
DNA repair. Unfortunately, during the time course of this project it
was not possible to recruit any relatives, but this variant will be
pursued by the Inherited Tumour Syndromes Research Group,
Cardiff University. Halo61 also carries a POLE mutation, which is
being followed up in Chapter 5 of this thesis.



The remaining variants, although all potentially interesting, were notfollowed up in the short term due to at least one of the following reasons:oNo relatives available.

oVariant more likely to be involved in a phenotype other thancolorectal neoplasia.

oToo common.

oInsufficient evidence of a role in colorectal neoplasia to prioritise
further study.

The second strategy in data analysis involved searching for novel polyposis genes(Bradford 2017). Bradford found one interesting variant. In Halo53, a novel de170

novo stopgain in MAP3K11 (p.Arg561*) was identified. MAP3K11 is a mitogenactivated protein kinase that is involved in the regulation of the c-Jun N-terminal
kinase (JNK) pathway (Mishra et al 2007). There is some evidence that it may havea role in tumourigenesis: in HER+ breast cancer tissue, there is reduced function of
MAP3K11 kinase activity (Das et al 2015). Furthermore, HER2-directed drugs suchas trastuzumab and lapatinib, as well as depletion of HER2/ HER3, stimulate
MAP3K11 kinase activity in HER2+ breast cancer cell lines (Das et al 2015).
MAP3K11 has known pro-apoptotic effects and stable knockdown of MAP3K11 in
the HER2+ cell line blunted the pro-apoptotic effects of trastuzumab and lapatinib.
These findings suggest that HER2 activation inhibits the pro-apoptotic function of
MAP3K11, which plays a mechanistic role in mediating anti-tumour activities of
HER2-directed therapies (Das et al 2015). If the apoptotic effects of MAP3K11 are
reduced, as may be the case with the p.Arg561* variant identified in Halo53, there
may be enhanced tumour cell survival (Bradford 2017). Interestingly, there may beinteractions between MAP3K11 and the Wnt-pathway. Although MAP3K11 canstabilise -catenin, it has also been shown to inhibit conventional -catenin/ TCF
transcriptional activation (Thylur et al 2011). Therefore, MAP3K11 mutations might
be expected to lead to enhanced expression of Wnt-target genes. The MAP3K11
variant is being further investigated by the Inherited Tumour Syndromes ResearchGroup.

In this study, the most important factors limiting progress in the search for novel
variants associated with polyposis were the lack of additional family members andthe size of the study cohort. Other studies have been more successful because
they were larger or prioritised the study of familial cases (Adam et al 2016; Weren etal 2015) with availability of samples from multiple affected relatives.

Although the mean depth of coverage across the 29 patients/ relatives who
underwent WES was 61.99 reads, the percentage of the target region covered at
50x depth was only 43.9%. The coverage was also variable between patients,
ranging from a mean of only 14.02 reads (Halo66) to a mean of 139.76 reads(Halo61). When apparent variants were not validated with Sanger sequencing,these tended to have occurred in regions of low coverage.

WES generates enormous amounts of data. Although a predetermined pipeline
was used to analyse the data, the potential for human error remained. A literaturesearch was performed for all the shortlisted variants to try and inform decisions

171

about which ones to pursue further, but the process remained subjective. This
inevitably introduces bias into the process of data analysis. All the data is in theprocess of being re-analysed by a second, independent reviewer, Dr. Hannah West,but the scope for missing potentially significant variants will always remain. When
analysing the results using the novel-gene approach, the volume of data potentiallyfor literature review was significantly larger than when using the candidate-geneapproach. In view of this, even more stringent criteria had to be applied before
carrying out the literature search. Again, this introduces another layer of bias into
data analysis. It is important to be aware that all pipelines used for WES data
analysis included the criteria that the variant must have an allele frequency <0.5% in
the general population. This would allow the identification of rare, dominantvariants, but it means that recessive/ compound heterozygous diseases would only
have been identified if they were very rare. This threshold was employed as it iscomparable to the minor allele frequencies of pathogenic mutations identified in the
recently identified autosomal recessive polyposis syndromes. In NTHL1-associatedpolyposis the mutant NTHL1 variant has an allele frequency of 0.0036 in the control
population (Weren et al 2015). In Adam’s paper describing MSH3-associatedpolyposis the group had used a minor allele frequency of 1% as part of their filtering
criteria for recessive variants, but the actual allele frequencies of the pathogenic
mutations identified were 0.008%/ 0.0016%/ unreported (Adams et al 2016).

As anticipated, exome sequencing using both targeted and WES approaches
identified many variants. These had to be prioritised in the short term, so variants
were selected in genes which have an established role in inherited predisposition tocolorectal tumours (POLE, POLD1 and the MMR genes) or variants which wereidentified in families in which multiple family members were available for analysis(AXIN2). Assessment of variants using knowledge from databases is improving
continuously. For example, in the 100 000 Genomes Project, different contributing
centres may identify just 1 variant in a gene possibly associated with a particular
phenotype. Once the data from all centres is collated, these variants can be linked
so that an informative pattern emerges. Therefore review of the variants I haveidentified will be possible in the future, and may be helpful in highlighting further
variants implicated in inherited polyposis.

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4.5

SummaryThis chapter utilised exome sequencing to try and identify novel genetic variants
responsible for tumourigenesis in the cohort of NMI polyposis patients. Initially atargeted approach was used: 60 patients underwent deep sequencing of 15 genes
known to be/ possibly involved in the pathogenesis of colorectal neoplasia. A
subset of the cohort then had WES performed on germline DNA. A large number ofvariants were identified, from which variants in AXIN2, POLE, POLD1 and the MMR
genes were selected for further follow up in Chapter 5 of this thesis.

173

174

Chapter 5 Functional
Characterisation of Variants
Identified in the Mismatch Repair
Genes, POLE, POLD1 and AXIN2
5.1

Introduction

The final part of this thesis involved the genetic and functional characterisation ofvariants identified in Chapter 4. Mutations of interest were found in 3 genes/ groupsof functionally similar genes: the MMR genes, two of the pol genes: POLE and
POLD1, and AXIN2. This chapter is therefore divided into 3 sections, eachaddressing one of these genes/ groups.

5.2

The Mismatch Repair (MMR) Genes

5.2.1 Introduction
The human MMR system involves 7 key genes: MSH2, MSH6, MSH3, MLH1,
PMS1, PMS2 and MLH3. The role of five of these genes in CRC and Lynch
Syndrome has been described in Chapter 1 (1.3 and 1.5.1.5 ).
As a result of targeted exome sequencing, three patients were found to carrypotentially pathogenic variants in the MMR genes. They are summarised in Table
5.1:

175

All three variants were investigated using a combination of database interrogation,
MMR IHC and microsatellite instability testing.

5.2.2 Database Interrogation
The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) is aninternational organisation which seeks to improve the quality of care for thoseindividuals with any hereditary condition resulting in gastrointestinal tumours(https://www.InSiGHT-group.org/ accessed 31/08/2017). Part of their work includes
the curation of a database which records variants in the MMR genes, along with aclassification of the likelihood of pathogenicity. This database is recognised byclinicians as the ‘gold standard’ for the interpretation of such variants and is widelyused in the clinical genetic diagnostic and research settings. There is also a French
database, the Universal Mutation Database, UMD-MMR, which records variants in
MLH1, MSH2 and MSH6 which have been reported in French laboratories inpatients with CRC/ endometrial carcinoma (http://www.umd.be/ accessed
31/08/2017). It is also possible to calculate the prior probability of pathogenicity of176

MMR gene variants, based on sequence conservation and position (Thompson et al2013).
All these tools were employed in the investigation of the MMR variants which had
been identified in NMI patients.

5.2.3 MMR Immunohistochemistry (IHC)
It is well established that mutations in the MMR genes, MLH1, MSH2, MSH6 and
PMS2 lead to Lynch Syndrome (LS), which accounts for around 5% of cases of
CRC (reviewed in Mishra and Hall 2012). LS-related cancers are characterised by
the absence of MMR protein expression (reviewed in South et al 2009), and MMR
IHC is a fundamental component of the molecular diagnosis of LS. Previous
authors have reported that colorectal polyps which develop in carriers of known LS
mutations show loss of MMR staining: this was observed in 79% of adenomas and
27% of serrated lesions (Walsh et al 2012).

I hypothesised that if the MMR mutations identified in NMI patients were pathogenic,
the majority of adenomas in the patients would show loss of MMR protein staining.

5.2.4 Microsatellite Instability (MSI) Testing
Microsatellite instability (MSI) refers to a hypermutable phenotype caused by
defective MMR activity (Boland and Goel 2010). It is the characteristic genetic
signature identified in cancers associated with LS. It has previously been shownthat 80% of adenomas from patients with LS show evidence of MSI (Iino et al 2000).

I hypothesised that if the MMR variants identified in the NMI patients were
pathogenic, their adenomas would show evidence of MSI.

5.2.5 Methods

1775.2.5.1

Database Interrogation

All variants were investigated to determine what their InSiGHT and UMD
classifications were, and what their prior probability of pathogenicity was (Thompson
et al 2013). The databases accessed are available at:

1. http://www.InSiGHT-database.org/genes
2. http://www.umd.be/MSH2/
3. http://www.umd.be/MSH6/
4. http://hcilovd.hci.utah.edu/variants.php?select_db=MSH2_priors&action=search_all&search_Variant%2FDNA=c.128A%3EG, http://hcilovd.hci.utah.edu/variants.php?select_db=MSH6_priors&action=search_uniq
ue&order=Variant%2FDNA%2CASC&hide_col=&show_col=&limit=100&sear
ch_Variant%2FExon=&search_Variant%2FDNA=&search_Variant%2FRNA=&search_Variant%2FProtein=p.T369I&search_Variant%2FCustom_PP2_sc
ore=&search_Variant%2FMAPP_score=&search_Variant%2FMAPP%2FPP
2_Prior=&search_Variant%2FReference=&search_Variant%2FDetection%2
FTemplate=&search_Variant%2FDetection%2FTechnique=&search_Variant%2FDBID=
5. http://hcilovd.hci.utah.edu/variants.php?select_db=MSH6_priors&action=search_uniq
ue&order=Variant%2FDNA%2CASC&hide_col=&show_col=&limit=100&sear
ch_Variant%2FExon=&search_Variant%2FDNA=&search_Variant%2FRNA=&search_Variant%2FProtein=p.R1095C&search_Variant%2FCustom_PP2_
score=&search_Variant%2FMAPP_score=&search_Variant%2FMAPP%2FP
P2_Prior=&search_Variant%2FReference=&search_Variant%2FDetection%
2FTemplate=&search_Variant%2FDetection%2FTechnique=&search_Varia
nt%2FDBID=(accessed 26/06/2017)

5.2.5.2

MMR IHCFFPE tissue (multiple adenomas and HPPs) was available for all 3 patients in whom
we had identified MMR gene variants: Halo26 (6 x TVA LGD, 7 x TA LGD, 1 x VA
LGD), Halo45 (13 x TVA LGD, 2 x HPP) and Halo70 (2 x TVA LGD, 13 x TA LGD, 1

178

x HPP). All FFPE tissue blocks underwent IHC analysis of MLH1, MSH2, MSH6
and PMS2 at the Cellular Pathology Department, University Hospital of Wales. The
EnVision FLEX System (Agilent) was used, following the manufacturer’s protocol,and the primary antibodies MLH1 Flex RTU, MSH2 FE11, MSH6 EP49 and DAKOPMS2 1/80. I reviewed the slides using an Olympus BX43 light microscope.

5.2.5.3

MSI Testing

DNA was extracted from FFPE polyps by the AWMGS using the Maxwell Promega
LEV FFPE kit. MSI testing was carried out by the AWMGS following the protocol
given in Appendix 5.1.

5.2.6 Results

5.2.6.1

Database Interrogation

The results from the database interrogation are summarised in Table 5.2 (InSiGHT
class 3: uncertain; UV: uncertain variant):

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The MSH2 variant, c.128A>G:p.Tyr43Cys, has an InSiGHT classification of 3,
meaning that its pathogenicity is uncertain. The prior probability of pathogenicity is
0.8980, in concordance with its InSiGHT classification. The MSH6 variant,c.1106C>T:p.Thr369Ile, has not previously been recorded in the InSiGHT/ UMD
databases. However, it has a low prior probability of pathogenicity score: 0.0462.
This would put it in the InSiGHT class 2 category (https://www.InSiGHTgroup.org/criteria/) i.e. likely not pathogenic. The variant in Halo70, MSH6
c.3283C>T:p.Arg1095Cys, had not previously been reported in the InSiGHT
database. There were 5 reports on UMD, and it was recorded as a UV. It had arelatively high prior probability of pathogenicity score: 0.9342. This would place it in
the InSiGHT class 3 category (https://www.InSiGHT-group.org/criteria/).

5.2.6.2

MMR IHC

Attempted IHC staining of the MMR proteins was performed on all tumours from the
3 patients. One TA LGD from Halo45 had cut out, so there was no tissue available

180

for MMR IHC, and there was insufficient tissue available for complete MMR IHC for
one HPP from Halo45, so results for MLH1 were not available.

For the tumours with successful MMR IHC, there was no evidence of loss of MMR
staining in any of the lesions and the staining pattern was normal.
A representative image of the MMR IHC is shown in Figure 5.1:

A

B

D

E

C

Figure 5.1: Halo70 TA LGD. A: H&E, B: MLH1 IHC, C: MSH2 IHC, D: MSH6, E: PMS2

5.2.6.3

MSI TestingA subset of 20 tumours underwent MSI testing (Appendix 5.2). The remaining
tumour samples did not undergo screening due to significant diathermy artefact/ thelesion cutting out/ a small number of dysplastic glands being present amongst
normal glands/ the specimen being too small.

There was no evidence of MSI in any of the tumours tested.

5.2.7 Conclusions of MMR Gene Investigations
Database interrogation, MMR IHC and MSI testing was performed for the patients inwhom variants in the MMR genes had been identified through targeted exome
sequencing. Database interrogation showed that all three variants were likely to be181

of uncertain pathogenicity (MSH2 c.128A>G:p.Tyr43Cys and MSH6
c.3283C>T:p.Arg1095Cys) or were likely not pathogenic (c.1106C>T:p.Thr369Ile).
Tumour samples from the carriers of these variants showed normal MMR IHC andno evidence of microsatellite instability. Although relatively small numbers oftumours underwent testing, it is unlikely that any of the identified mutations areclinically significant.

There were no relatives available for any of the patients to be screened.

In summary, 3 patients were identified as carrying variants in the MMR genes.
Although polyposis is not the typical phenotype seen in heterozygous carriers of
MMR gene mutations, these variants were investigated to gain a greater
understanding of whether they might have significant functional effects. It is unlikelythat any of the variants are clinically significant, in view of the normal MMR IHC andmicrosatellite stability of the tumours.

182

5.3

POLE and POLD1

5.3.1 Introduction
Mutations in the proofreading domains of the human polymerase enzymes, POLE
and POLD1, have been shown to have a role in the pathogenesis of colorectal
neoplasia, as described in Chapter 1 (1.5.1.3). As a result of the targeted exome
sequencing, four patients were found to carry missense variants in POLE/ POLD1.
They are summarised in Table 5.3, along with the patients’ phenotypes and thelocation of the resultant amino acid change in the protein:

183

Patient
ID

Gene

Halo15POLE

Halo18POLE

Halo27

POLD1

Halo61

POLE

Posn.

12:133249812
12:133202816
19:50919693

12:1332
45452

CADD

Amino Acid

Score

ChangeT>C

24.4

p.Met471Val

C>T

23.8

p.Glu2140Lys

C>T

34

p.Thr954Met

Variant

T>C

26

p.Tyr623Cys

Location of Amino Acid Change

AmbryGen Class

Patient Phenotype

In ribonuclease H-like domain, DNA directedNot84-year-old male with 13

DNA polymerase Family B exonuclease domain

reported

TA LGDNo domain

VUS

In DNA directed DNA polymerase family B

Not67-year-old female with 17

multifunctional domain

reportedadenomas

In DNA directed DNA polymerase family B
multifunctional domain

polyps, majority TAs

60-year-old female with 11
VUS

Table 5.3: Variants in the pol genes identified through targeted exome sequencing. The amino acid location was determined using
http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=NP_006222.2 (accessed 09/03/16)

72-year-old male with 27

polyps: 2 SAs, 7 TA, 1
TVA, 1 HPP

184

Only one of the variants was located in the exonuclease domain of the protein,
POLE p.Met471Val in Halo15. All of the pathogenic variants that have beenreported in the literature thus far are also located within the exonuclease domain of
POLE/ POLD1 (Palles et al 2013). However, I hypothesised that a variant may bepathogenic, even if it lies outside of the exonuclease domain, if it can alter the abilityof the exonuclease domain to elicit its functional effects, for example throughaltering the movement of DNA through the active site of the polymerase.
In Palles’ paper (Palles et al 2013), the group examined 39 tumours from
11 POLE p.Leu424Val carriers for somatic mutations. Second hits by LOH involving
the germline wildtype allele were found in some tumours and all tumours weremicrosatellite stable. Most tumours were screened for KRAS and BRAF driver
mutations, and a sub-set of tumours were screened for known pathogenic mutationsin APC, CTNNB1, PIK3CA and FBXW7. Mutations were all base substitutions.
This was especially interesting for APC, as ~60% of mutations seen in sporadic
tumours are frameshifts (reviewed in Palles et al 2013). In addition to this, certain
sites not commonly mutated in sporadic colorectal tumours seemed to be mutation
hotspots, for example codons 1114 and 1338 of APC and codon 146 of KRAS. In
view of this, I hypothesised that the same ‘hot spot’ locations may be mutated intumours obtained from the patients we identified as harbouring germline POLE
variants, if the variants were pathogenic. I also hypothesised that the tumours
should be microsatellite stable, in keeping with Palle’s results.
Palle’s group is currently carrying out work to determine whether tumours
developing in patients carrying pathogenic mutations in the pol genes have acharacteristic mutational signature. We collaborated with their group to examine thesomatic mutations in tumours occurring in the patients we identified as carrying pol
gene variants.

The genetic variants identified were therefore investigated through a combination of:

Modelling the mutant proteins to investigate whether the amino acidchanges could theoretically affect movement of DNA through theexonuclease domains of the polymerases. This work was carried out incollaboration with Dr. Pierre Rizkallah, Cardiff UniversitySomatic mutation screening, to identify ‘hotspot’ mutations as described byPalles et al (2013)

185



MMR IHC and MSI testing to determine whether tumours were microsatellite
stable

Segregation/ de novo analysis in families, where family members wereavailable for testing

Investigation of the mutation signature of the tumours. This work was
carried in collaboration with Professor Ian Tomlinson and Dr. Claire Palles(Oxford University/ Birmingham University)

5.3.2 Methods

5.3.2.1

In Silico Modelling of Pol Variants

Thank you to Dr. Pierre Rizkallah for his expert help with this part of the project.
The Protein Databank (PDB) (http://www.rcsb.org) server’s BLAST based sequencealignment algorithm was used to match the human POLD1 sequence with thesequence of many POLD1 entries represented in the structural database. The top
hit was 3IAY (Swan et al 2009), with the alignment shown in Appendix 5.3a. The
sequence of the human POLE had the top hit 4M8O (http://www.rcsb.org, Hogg et
al 2014). The alignment is shown in Appendix 5.3b. These models were then used
to analyse the effect of variants on a per residue basis. The program
Crystallographic Object-Oriented Toolkit (COOT) (Emsley and Cowtan 2004)
introduced the identified mutations and regularised the geometry in the immediateneighbourhood. PyMOL (Delano 2002) was used to produce graphical images tovisualise the results of the predictions.

5.3.2.2

Somatic ‘Hot Spot’ Mutation Screening in APC andKRAS
At the time of the experiments, a total of 15 FFPE tumours were available for
Halo15 and Halo61 (Halo15: 11 x TA LGD; Halo61: 4 x TA LGD) (Appendix 5.4). A
further 45 tumours were obtained from patients with variants in POLE that wereassumed to be non-pathogenic, due to their AmbryGen classification (4.1.4.2.4)

186

(Halo31: 10 x TA LGD, 2 x HPP; Halo69: 6 x TA LGD, 10 x HPP, 1 x CRC; Halo81:
3 x TA LGD, 3 x TVA LGD, 9 x HPP, 1 x TVA arising in a SSL) (Appendix 5.4).
These were included in the study protocol to ensure that somatic ‘hotspot’ mutations
were not present: if such mutations had been identified it would suggest that thedesignation of the variants as benign may not be correct. DNA was extracted fromthe tumours using the GeneRead DNA FFPE Kit (Qiagen) following the standard
protocol (2.3.4). DNA underwent PCR and Sanger sequencing using standard
reagents and reaction conditions for DNA extracted from FFPE tissue (2.3.8.2,
2.3.8.5, 2.3.9-2.3.13). Tumours were screened at the ‘hotspot’ mutation locations in
APC and KRAS described by Palles et al (2013): APC codons 1114 and 1338 andKRAS codon 146. Primers were designed using primer3 software and were
supplied by Eurofins (Appendix 5.5).

5.3.2.3

MMR IHC and Microsatellite Stability of Tumours

in Carriers of Pol Gene Variants
34 FFPE tumours from Halo15, Halo27 and Halo61 were available (Halo15: 11 x
TA LGD; Halo27: 17 x TA LGD, 1 x SSL; Halo61: 5 x TA LGD (Appendix 5.6).
All FFPE tumours underwent IHC analysis of MLH1, MSH2, MSH6 and PMS2 at the
Cellular Pathology Department, University Hospital of Wales. The EnVision FLEX
System (Agilent) was used, following the manufacturer’s protocol, and the primary
antibodies MLH1 Flex RTU, MSH2 FE11, MSH6 EP49 and DAKO PMS2 1/80.
A subset of 28 tumours underwent DNA extraction. This was done by the AWMGS
service using the Maxwell Promega LEV FFPE kit. MSI testing was carried out
following the protocol given in Appendix 5.1. The remaining tumours did notundergo MSI testing due to the dysplastic glands being too scanty to warrant DNA
extraction or there being significant diathermy artefact.

5.3.2.4

Segregation Analysis

I sought to recruit relatives of the patients in whom we had identified germline
variants in POLE and POLD1 to try and determine whether the variants were
occurring de novo in the index cases or whether they were segregating with disease
if multiple family members were affected.

187

Halo15 was an only child and there was no family history of colorectal neoplasia.
Halo18’s mother and maternal aunt had a possible history of CRC, reported by thefamily. Halo18 has two siblings who are thought to be unaffected. Halo27 has 3
siblings with colorectal polyposis and her father had CRC. Halo61 has one siblingwith colorectal polyposis and 2 unaffected siblings. She had a son who died of CRC
at the age of 23. The son’s tumour IHC was suggestive of a MMR defect, but there
was insufficient DNA for MSI testing. Halo61 also has a daughter who had
developed a single colorectal polyp at the age of 28. There was, in addition, ahistory of CRC on her husband’s side of the family.

The only relatives that were successfully recruited to the study were both children of
Halo15. The daughter of Halo15 was a 43-year old female. She had a colonoscopy
at age 30 for rectal bleeding: it showed colonic endometriosis but no polyps. The
son was a 42-year old male who has never had a colonoscopy. Germline DNA wasextracted from whole blood by the AWMGS (2.3.2). RNA was extracted from whole
blood following the standard protocol (2.3.3). RNA was reverse transcribed to
cDNA (2.3.3.2). DNA and RNA underwent PCR and Sanger sequencing to screen
for the POLE mutation, c.1411A>G:p.Met471Val. Standard reagents and reaction
conditions were used (2.3.8.1, 2.3.8.5, 2.3.9-2.3.13). DNA primers were thoseused for Haloplex validation (Appendix 4.2). cDNA primers are listed in Appendix
5.7.

5.3.2.5

Investigation into the Mutation Signature of

Tumours
This work was carried out with Professor Ian Tomlinson’s group at the Wellcome
Trust Centre for Human Genetics, Oxford University. I am extremely grateful for
their help and involvement.

The mutation signature of tumours occurring in patients with germline POLE/
POLD1 variants was determined by examining 30 genes. For some genes, the fullcoding regions, with 10 bp flanking region were interrogated. For others, hotspots
or domains were selected. The genes interrogated were: ACVR2A, APC, ARID1A,
ATM, B2M, BCL9L, BMPR2, BRAF, CTNNB1, ELF3, FBXW7, GNAS, KRAS, MLH1,
MSH2, MSH6, NRAS, PIK3CA, POLD1, POLE, PTEN, RNF43, RPL22, SMAD2,
SMAD4, SOX9, TCF7L2, TGIF1, TP53, ZFP36L2.

188

The genes which didn’t have the full coding region examined were: APC (codons 11600), POLD1 (chr19:50905938-50910304), POLE (chr12:133249809-133253238),
GNAS (codon 200), BRAF (exons 11 and 15: chr7: 140453074-140453193,
chr7:140481375-140481493), CTNNB1 (hotspots chr3:41266444-41266698,
chr3:41267150-41267352, chr3:41274831-41274935, chr3:41275019-41275358,
chr3:41277214-41277334, chr3:41266016-41266244), PIK3CA (hotspots
chr3:178916613-178916965, chr3:178935997-178936122, chr3:178951881178952152).

Gene capture was achieved using single molecule molecular inversion probes(smMIPs), which were designed by Dr. Palles using MIPgen software and were
supplied by Integrated DNA Technologies. A total of 300 smMIPs were used totarget 63.1kb. The smMIPs capture ~1800 sites which are commonly mutatedwhen there is impaired POLD1/ POLE proofreading due to a functional mutation in
the exonuclease domain (Palles personal communication).

I travelled to Oxford to assist with the smMIP gene capture. The protocol for thecapture and subsequent sequencing is described in Chapter 2 (2.4.6).

5.3.3 Results

5.3.3.1

Modelling of Pol Variants

The 3-dimensional location of the variants is described and shown, along with theirpotential functional effects, in Table 5.4:

189

190

5.3.3.2

Somatic ‘Hot Spot’ Mutation Screening in APC andKRAS
60 tumours from patients carrying presumed benign or putatively pathogenic Pol
gene variants were screened for mutations at APC codons 1114 and 1338, andKRAS codon 146. No mutations were identified.

5.3.3.3

MMR IHC and Microsatellite Stability of Tumours

in Carriers of Pol Gene Variants
34 tumours from patients carrying putatively pathogenic variants in POLE/ POLD1
were screened using MMR IHC +/- MSI testing. There was no evidence of loss of
MMR protein staining or MSI in any of the samples (Appendix 5.8).

5.3.3.4

Segregation Analysis

The son and daughter of Halo15 both underwent screening for the POLE variant,c.1411A>G:p.Met471Val. The variant was present in the daughter only, in both
DNA and cDNA (sequencing traces in Appendix 5.9).

5.3.3.5

Investigation into the Mutation Signature of

Tumours
A total of 2 tumour samples from Halo15 have thus far undergone successful
smMIP gene capture and sequencing. Both lesions were TA LGD. Tumour samples
from Halo27 are currently being analysed: DNA from 7 x TA LGD has been
submitted. The tumour DNA (one TA LGD) from Halo61 failed quality control andno results are available.

At the time of writing, the high quality raw results for somatic mutation sequencing
for Halo15 were available (Appendix 5.10). However, these were being further
analysed by the bioinformatics team at Birmingham University, to determine
whether they are consistent with a hypermutated signature observed in tumours
occurring in carriers of known pathogenic mutations in the pol genes.

191

5.3.4 Conclusions of POLE/ POLD1 Gene Interrogation
Four patients were found to carry potentially pathogenic germline mutations in
POLD1 or POLE. All variants were missense changes. The variants were
investigated using a combination of database interrogation, modelling the variants
using in silico structural modelling, MMR IHC and MSI testing, somatic mutation
analysis and family segregation studies.

Based on in silico modelling, two of the variants were thought most likely to bedisease-causing. One was a POLD1 variant in Halo27, p.Thr954Met, which waspredicted to distort the structure of the active site of the enzyme. It had a very high
CADD score of 34. The second variant was identified in Halo15, p.Met471Val. Thisis a novel variant which is located in the exonuclease domain of the protein.

The remaining two variants included a POLE variant in Halo18, p.Glu2140Lys, anda POLE variant in Halo61, p.Thr623Cys. POLE p.Glu2140Lys is reported as benign
on dbSNP. It does not lie within any of the POLE functional domains and was not in
the protein model used as part of this project. The variant is considered very
unlikely to be clinically significant.

POLE p.Thr623Cys was suggested to possibly have pathogenic effects that could
be mediated through an alteration in the protein structure in the proximity of DNA as
it moves through the active site of the enzyme (Dr. Pierre Rizkallah, personalcommunication). It has a CADD score of 26 and is classified as a VUS by
AmbryGen. Tumour DNA from Halo61 did not have any evidence of ‘hot spot’
mutations in APC or KRAS, although only 4 lesions were available for analysis.
Unfortunately, tumour DNA was not successfully sequenced following smMIP gene
capture, so its mutation signature remains uncertain. Therefore, at the present time,
there is insufficient evidence to support or refute a pathogenic effect of this mutationand it remains a VUS.
Tumours from Halo15 and Halo61 underwent testing for somatic ‘hotspot mutations’described by Palles et al (2013). No such mutations were identified, however thisis not surprising: in Palles’ paper, the mutations were present at the following
frequencies:

192

Prevalence in

Prevalence in

Adenomas

Carcinomas

APC p.Arg1114* (C>T)

1/36 (3%)

1/3 (33%)

APC p.Gln1338* (C>T)

3/36 (8%)

1/3 (33%)

1/36 (3%)

1/3 (33%)

Variant

KRAS p.Ala146Thr(A>C)

There were 11 tumours available for Halo15/ p.Met471Val and 4 for Halo61/
p.Tyr623Cys. This study was therefore underpowered to identify mutations at the
hotspot locations, as we had insufficient numbers of tumours. Additionally thedysplastic epithelial component of the lesions was highly variable – ranging from
10%-100%. For the lesions with a small dysplastic component, even if a hotspot
mutation was present it may not have been observed with Sanger sequencing.

Tumour material for Halo27 only became available at a late stage of the project.
Tumour DNA did not undergo screening for the ‘hot spot’ variants but is undergoing
mutation signature analysis, which potentially has a greater power to support orrefute the pathogenicity of the POLD1 variant.

All of the tumours from Halo15, Halo27 and Halo61 which underwent MSI testing
were found to be microsatellite stable.

The only relatives available for family studies were the son and daughter of Halo15.
The daughter was found to carry the same variant as her affected father, and thiswas confirmed to be present in both DNA and cDNA. However, her phenotype is
not accurately known, as she has not had a recent colonoscopy. The genetic
finding has been fed back to her clinician, who has arranged surveillancecolonoscopy.

SmMIP gene capture is being performed on tumour DNA from Halo15 and Halo27,
in collaboration with Professor Ian Tomlinson’s group at Oxford University/
Birmingham University. The aim of this work is to determine whether the tumoursfrom these patients have a hypermutated genetic signature, in keeping with thatobserved in the tumours which occur in carriers of known pathogenic mutations in193the pol genes. Although the raw results are available for 2 TA LGD from Halo15,
bioinformatic analysis is still pending. There was a seven-month delay between
carrying out the sample preparation and obtaining the raw data, due to factors
outside of our control. It is anticipated that the complete analysis will be finalised
within the next several months. Therefore, at present, the variants identified in
Halo15 and Halo27, c.1411A>G:p.Met471Val and c.2861C>T:p.Thr954Met
respectively, remain as VUS.

194

5.4

AXIN2 and AXIN2-Associated Polyposis (AxAP)

5.4.1 Introduction
The role of the Wnt-pathway in colorectal neoplasia has been described in theprevious chapters of this thesis. AXIN2 is a scaffold protein which is involved inregulating Wnt signalling. It forms part of the ‘destruction complex’ which targets βcatenin for degradation: AXIN2 supports the GSK3β-dependent phosphorylation of
β-catenin, which marks the protein for ubiquitination and subsequent proteosomal
degradation (reviewed in Wu et al 2012). The AXIN2 protein has several domains,
including a Tankyrase binding domain which regulates protein stability, an RGS
domain which mediates binding to APC, a β-catenin binding domain and a DIX
dimerization domain (reviewed in Mazzoni and Fearon 2014). AXIN2 is atranscriptional target of β-catenin dependent Wnt signalling, and its levels areelevated in cancers with Wnt activating mutations, therefore potentially negatively
regulating Wnt signalling (reviewed in Mazzoni and Fearon 2014).

In view of its effects on Wnt signalling, it is possible that germline AXIN2 mutations
might be associated with an increased risk of colorectal neoplasia. In 2004, Lammi
et al described a Finnish family with oligodontia (Lammi et al 2004). The family
exhibited oligodontia segregating as an AD trait across 4-generations. Two patientsfrom the oldest generation were known to have a history of colorectal neoplasia(one had metastatic CRC and the other had 68 adenomas) and 10 further family
members underwent a colonoscopy/ sigmoidoscopy: 7 with oligodontia and 3
healthy individuals. Colorectal neoplasia was identified in 6 individuals witholigodontia, but not in the unaffected individuals. The colorectal phenotype was
highly variable, ranging from a single HPP to 10-20 HPP plus 2 adenomas. Family
members were screened for AXIN2 mutations in coding regions/ flanking intronic
sequences, and a c.1966C>T transition in exon 7 of was identified. This results in ap.Arg656* codon and premature termination of translation. The mutation waspresent in 11 individuals with oligodontia, but in none of 6 healthy family members.
The group also identified a further AXIN2 truncating mutation in a 13-year-old boy
with oligodontia. They describe a 1bp G insertion after nucleotide 1994 in exon 7,
which results in re-coding of the amino acids starting at p.Asn666 and a stop codon,
40 codons later. The same mutation had previously been identified as a somatic
mutation in CRC (reviewed in Lammi et al 2004).

195

Lammi et al concluded that their results provide strong evidence that familial
colorectal cancer can be caused by mutations in AXIN2.

In 2011, Marvin et al reported the identification of a novel AXIN2 mutation,
c.1989G>A, in a family with a history of oligodontia, absent eyebrows, sparse hair,colonic polyps, early onset CRC and early onset breast cancer (Marvin et al 2011).
The colorectal phenotype included an individual with >100 adenomas and an
individual with 2 metanchronous CRC plus 5 adenomas. The mutation introduces astop codon at amino acid 663, p.Tyr663*, in exon 7 (Marvin et al 2011). It was
identified in 3 individuals with a phenotype of oligodontia and colorectal neoplasia,
and was absent in 2 unaffected family members. Subsequent in vitro transcription
and translation of a c.1989G>A construct and expression of the construct in
HEK293T cells produced a truncated AXIN2 product (Marvin et al 2011). The groupcommented that their findings provided further evidence of an AD multisystem
ectodermal and neoplastic phenotype associated with a germline AXIN2 mutation.
They noted that the truncated protein would lack its DIX dimerization domain, which
they predicted would impair the inhibitory action of AXIN2 on WNT signalling.

Functional studies subsequently carried out on the mutation identified by Marvin etal (2011), AXIN2 c.1989G>A, revealed that the mutation did not result in nonsense
mediated decay (NMD), and that it could cause activation of the Wnt pathway,
although this was context dependent (Mazzoni et al 2015). The group transiently
transfected HEK293T cells with wild-type and mutant-containing plasmids. They
showed that truncated AXIN2 protein was more abundant than wild-type (WT)
protein, despite equivalent transcript levels. Interestingly, despite lacking thedimerization domain, truncated AXIN2 was still found to interact with both WTAXIN2 and truncated AXIN2. It was suggested that such interactions may not bedirect but might be mediated by another protein in the destruction complex, such as
APC, which contains AXIN binding sites. Although truncated AXIN2 was able tointeract with AXIN2, its ability to bind to AXIN1 was impaired. An important point tonote is that when overexpressed, truncated AXIN2 was able to inhibit SW480 CRC
cell colony formation and was able to inhibit β-catenin/ T cell factor- dependent
reporter gene activity, as would be expected with WT protein. However, whenstably expressed in rat intestinal IEC-6 cells, truncated AXIN2 did not match WT
AXIN2’s ability to inhibit Wnt-mediated expression of Wnt-regulated target genes(Mazzoni et al 2015). It was suggested that when AXIN2 levels are low, such as in
the absence of Wnt-stimulation, the mutation may have a loss-of-function effect.

196

When there is elevated AXIN2 expression, the truncated protein may have gain-offunction effects.

In 2014, Rivera et al identified a further novel variant in AXIN2 in a 3-generation
family with a phenotype of multiple colorectal adenomas and/ or CRC, but without
any signs of ectodermal dysplasia/ oligodontia. The missense variant, c.1387C>T,p.Arg463Cys is located in exon 5 within the β-catenin binding domain. However, its
pathogenicity is questionable, as the mutant allele was apparently lost in polyps
from the proband and his sister (Mazzoni and Fearon 2014), and the mutation was
found to be present in an unaffected relative (Rivera et al 2014).

Although these three papers have identified potentially pathogenic germline AXIN2
mutations in patients with colorectal polyposis/ CRC, there are also several studies
which have failed to identify pathogenic variants, although the study cohorts were
small (Mongin et al 2012; Lejeune et al 2006). Mongin et al (2012) did not identify
any mutations in the coding sequence of AXIN2 in a cohort of 38 NMI patients with
>40 adenomas or >20 polyps, and Lejeune et al (2006) failed to identify pathogenic
variants in AXIN2 in a cohort of 31 patients with multiple colorectal adenomas (18individuals), one of whom also had tooth agenesis, or microsatellite stable CRC (13individuals). However, in the latter paper, the patients had only undergone APC
screening to define their NMI status, and when MUTYH was analysed as part of thestudy protocol, 32% of the patients were found to have MAP.

Chapter 4 described the targeted exome capture and UDS of genes which might be
implicated in polyposis/ CRC. A sibling pair was found to carry a truncating
mutation in AXIN2: c.1642G>T:p.Glu548* (Halo47 and Halo68, Family A). Thissection of Chapter 5 will focus on this mutation and approaches to investigate its
possible pathogenicity.

5.4.2 Halo47 and Halo68: Family History and Clinicalinformation
Halo47 was a 51-year-old female when she was recruited to the study. In 2013, atthe age of 50, she had removal of 2 TVAs with LGD and 3 TAs with LGD. A
subsequent colonoscopy found >50 sessile polyps, up to 10mm, throughout thecolon. She underwent a subtotal colectomy, and approximately 40 sessile polyps

197were identified macroscopically. Four blocks were taken of polyps: the majority
were TA LGD, but 2 were serrated adenomas (it is not clear exactly how many
polyps were sampled). The patient was due to have a completion colectomy, but at
the time of surgery she was found to have a large rectal cancer. She underwent
sigmoidectomy/ proctectomy/ abdominoperineal resection and the cancer was
reported as being pT4bN2MX, Duke's C1. Unfortunately, she developed liver andlung metastases and she passed away during the course of this project.

Her sister, Halo68, had a colectomy for colorectal polyposis at the age of 50. She
had 14 TA with LGD and 6 HPPs.

Both Halo47 and Halo68 had been diagnosed with colorectal neoplasia following
symptomatic presentation.

There is a family history of intestinal neoplasia. Their mother (1.2) had CRC(moderately differentiated Duke’s A adenocarcinoma), and her medical records
described a VA with LGD and ‘three small metaplastic polyps’ identified
macroscopically, one of which was confirmed microscopically to be a HPP. When
all available specimens were reviewed for this research, she was found to have had
at least one VA LGD, CRC arising in a VA, one HPP and one TA LGD.

The sisters Halo47 and Halo68 had a maternal aunt (1.1) with pT3N2MX CRC and
8 polyps (5 were sampled and were found to be TA with LGD) and a maternal aunt
with ‘stomach cancer’ reported by the family (it was not possible to access her
medical records).

The family tree is illustrated in Figure 5.2 and the family is hereafter referred to as
Family A:

198

Key
CRC/ Polyposis
1.1

1.2

Halo68

Halo47

Figure 5.2: Family tree of Family A

Both sisters and their mother allegedly lacked all adult teeth. This was confirmed in
Halo68 by a formal dental assessment at the University Hospital of Wales dental
department. Halo68 was reviewed in clinic and was found to have sparse hair on
the outer third of her eyebrows, thin hair on her head, and she was born with amalformation of the nail on her left index finger.

One of the daughters of Halo68 also has sparse hair on the outer third of her
eyebrows, but her head hair and teeth are normal. Family photos of Halo47 andIndividual 1.2 are suggestive of scanty eyebrow hair. See Figure 5.3.

199

A

B

C

D

E

F

G

200

H

I

J

K

L

M

N

O

Figure 5.3: Phenotype of Halo68 (A-G) and her daughters (H-M). Halo68: note sparse
eyebrows and thin hair (A-D), malformation of finger nail (E, F), and absence of teeth on an
orthopantogram (G). One daughter has sparse eyebrows (H, I) but her hair (J), nails andteeth are normal. The second daughter is phenotypically normal (K-M). N and O: Halo47
wearing false teeth (N) and Individual 1.2 (O): both appear to have scanty eyebrow hair201

5.4.3 AXIN2: c.1642G>T:p.Glu548*
The germline mutation identified in the affected siblings Halo47 and Halo68 is
AXIN2 c.1642G>T:p.Glu548* in exon 5. The mutation was confirmed to be present
in the cDNA of Halo47 (4.1.4.2.3). If a truncated protein results from this mutation itis predicted to lose its DIX dimerization domain(http://www.uniprot.org/uniprot/Q9Y2T1#showFeaturesViewer accessed
04/05/2017).

5.4.4 Studies to Investigate the Mutation
The further studies performed to determine the pathogenicity of AXIN2c.1642G>T:p.Glu548* are described below.

5.4.4.1

Family Segregation Studies

There is a family history of colorectal neoplasia (Figure 5.2). It is important toascertain whether the mutation is segregating with the disease phenotype. It is also
important to try to determine which of the additional clinical features observed in thefamily, i.e. sparse eyebrows/ thin hair/ nail abnormalities/ oligodontia, are a part ofthe disease spectrum.

5.4.4.2

Protein Analysis to Confirm that the AXIN2

Mutation Produces a Truncated Protein
It has already been shown that the mutation is present in DNA and is transcribed toRNA. This is interesting, as the location of the mutation is predicted to trigger NMD.
NMD refers to the process in which mRNA harbouring premature termination
codons (PTCs) is destroyed (Wen and Brogna 2008). It is a surveillancemechanism which prevents the accumulation of aberrant mRNA, and therefore
potentially toxic truncated peptides (Wen and Brogna 2008). AXIN2 c.1642G>T ispredicted to result in NMD (http://www.mutationtaster.org/cgibin/MutationTaster/MutationTaster69.cgi accessed 31/08/2016) and yet it remains
present in RNA, similar to the findings of Marvin et al (2011) for the reportedly202pathogenic mutation p.Tyr663*. The next logical step is to confirm that the mutation
produces a truncated protein.

5.4.4.3β-catenin IHC on FFPE Tumours to Assess

Somatic Activation of the Wnt Pathway
APC mutations allow the accumulation and nuclear translocation of β-catenin.
Histologically normal colorectal epithelia exhibit a membranous location of βcatenin, as observed with IHC (Wong et al 2004; Iwamoto et al 2000; Hao et al
1997; Valizadeh et al 1997). Colorectal adenomas, which are typicallycharacterised by APC mutations, may display membranous and nuclear staining(Wong et al 2004; Iwamoto et al 2000; Hao et al 1997; Valizadeh et al 1997), but
nuclear staining is not always observed (Kobayashi et al 2000). It is likely that
nuclear staining increases with increasing degrees of dysplasia (Wong et al 2004;
Iwamoto et al 2000; Kobayashi et al 2000; Hao et al 1997; Valizadeh et al 1997). βcatenin nuclear staining seems to occur in the majority of CRC (Wong et al 2004;
Iwamoto et al 2000; Hao et al 1997), although not all papers report the same
finding: Kobayashi et al (2000) observed that 55% of sporadic CRC exhibited
negative/ scattered nuclear β-catenin expression. Different results could be
attributed to differences in the protocols used for immunostaining, and differences inhistological interpretation of neoplastic lesions (Wong et al 2004).
AXIN2 is a further component of the β-catenin ‘destruction complex’. I therefore
hypothesised that pathogenic AXIN2 mutations might result in a similar pattern of βcatenin immunostaining as is observed in adenomas/ CRC with APC mutations. I
sought to determine the subcellular location of β-catenin in adenomas/ CRC frompatients with inherited variants in AXIN2 using IHC. The results from this work are
to be interpreted alongside the results from the somatic APC gene mutation
screening in the same tumours.

5.4.4.4

Somatic APC Screening

The APC mutations which play a role in the pathogenesis of colorectal neoplasia
are not random: ‘typical’ patterns are identified. Approximately a third of germline
mutations in FAP patients occur at codons 1061 and 1309, and the reminder occur

203largely uniformly between codons 1200 and 1600 (Fearnhead et al 2001). As
described in the introduction chapter (1.5.1.1) of this thesis, in FAP the nature of thefirst mutation determines the type of second hit to APC. If the germline mutation
occurs between codons 1194 and 1392, there is selection for allelic loss of APC as
the second hit, whereas if the germline mutation lies outside of this region, thesecond hit is most likely to be a truncating mutation in the MCR (Fearnhead et al2001). In sporadic tumours, over 60% of somatic mutations occur between codons
1286 and 1513, the MCR, which accounts for <10% of the coding sequence of APC(Fearnhead et al 2001). Within the MCR, there are further hotspots for mutations atcodons 1309 and 1450 (reviewed in Fearnhead et al 2001) and codon 1554 (Rowanet al 2000).

APC mutations partially mediate their effects through enhanced Wnt-signalling.
AXIN2 mutations are hypothesised to have a similar effect. If this is true, then thetumours which occur in patients with inherited AXIN2 mutations might not need toacquire APC mutations, so the ‘typical’ APC mutations observed in colorectal
tumourigenesis might be absent. However, if typical APC mutations are identified inpatients with germline AXIN2 variants this could suggest that AXIN2 variants are notpathogenic or alternatively that they are exerting tumourigenic effects through amechanism other than through the Wnt-pathway.

To further elucidate the mechanism(s) by which AXIN2 mutations may exert
pathological effects, DNA was extracted from FFPE tumours originating in patientswith inherited AXIN2 variants. DNA was screened to determine whether ‘typical’
APC mutations were present.

5.4.4.5

AXIN2 Loss of Heterozygosity (LOH) Analysis

LOH describes the situation whereby one allele of a gene is ‘lost’ somatically. It is acommon genetic event in many cancers. It may develop through copy neutralmechanisms, in which there is a homologous recombination event or because aretained chromosome was duplicated, or it may be observed with copy loss, inwhich all/ part of the chromosome is lost (Ryland et al 2015). LOH is strongly
associated with the loss of the wild-type allele in the tumours of individuals with
inherited cancer predisposition syndromes who carry a germline mutation in atumour suppressor gene (Ryland et al 2015).

204

Of the three papers which suggest that germline AXIN2 mutations may predispose
an individual to the development of polyposis +/- CRC (Lammi et al 2004; Marvin etal 2011; Rivera et al 2014), only one sought to investigate AXIN2 LOH (Rivera et al2014). Paradoxically it was the mutant allele which was lost in the tumours whichwere sampled. Two adenomas from the proband and four polyps (two adenomas,
one HPP and one mixed polyp) from a sister were examined: LOH of the mutant
allele was identified in one polyp from the proband, and in an adenoma and themixed polyp from the sister. The authors also noted that the ‘other adenoma(with low-grade dysplasia) had reduced amplification of one allele, and therefore
incipient LOH, but not sufficiently so to confirm LOH per se’.

If AXIN2 is acting as a typical tumour suppressor gene, and inherited mutations
result in an increased risk of neoplasia, it would be expected that there might beloss of the wild type allele in tumours. This was investigated in DNA extracted fromFFPE tumours.

5.4.4.6

Functional Characterisation of AXIN2c.1642G>T:p.Glu548*
Pathogenic mutations in AXIN2 are proposed to result in increased activation of the
Wnt-pathway, in a similar manner to APC mutations. To determine whether thevariant identified in this study impacts Wnt-signalling, HEK293 TCF-Luc reporter
cells were transfected with WT-AXIN2, the AXIN2 mutation identified by Marvin et al(2011) (c.1989G>A, p.Tyr663* referred to hereafter as Fearon-AXIN2. See
5.3.2.7.1) or with the variant identified during this study (c.1642G>T:p.Glu548*,
Short-AXIN2). Wnt-pathway activation was assessed using luciferase assays.

5.4.5 Methods

5.4.5.1

Family Segregation Studies

In addition to Halo47 and Halo68, a further 6 family members were recruited to thestudy. The details of these individuals are in Figure 5.4 and Table 5.5 below:

205

1.2

1.1

Halo68

3.1

Halo47

3.2

2.1

3.3

Figure 5.4: Family tree of Family A illustrating recruited individuals. The yellow circles
indicate recruited family members.

FamilyDemographic

MemberDetails

Material
Phenotype

Analysis
Moderately differentiated CRC

Individual
1.1

pT3N2Mx. 8 polyps identified
Deceased

macroscopically: 5 sampled: TA

femalewith LGD. Hemicolectomy
performed when the patient was
79-years-old

Moderately differentiated Duke’s
Individual
1.2

DeceasedfemaleAvailable for

A CRC, a VA with LGD and‘three small metaplastic polyps’,
one of which was confirmed
microscopically to be a HPP.

FFPE material:
tumour andnormal mucosa(4 x TA LGD, 1
x CRC)

FFPE material:
tumour andnormal mucosa(1 x TA LGD, 1
x VA LGD, 1 x
VA LGD that206

CRC diagnosed when the patient

CRC has arisen

was 59-years-old.

in, 1 x CRC, 1 x

Allegedly did not develop adult

HPP)

teeth
Individual

48-year-old

Clear colonoscopy in 2016

DNA from

2.1

male

Normal teeth

whole blood

Individual
3.1

Clear colonoscopy as a
35-year-old

teenager, no recent colorectal

DNA from

femaleexamination

whole blood

Normal teeth
Individual
3.2

Individual
3.3

27-year-old

Never had a colonoscopy

DNA from

femaleNormal teeth

whole blood

24-year-old

Never had a colonoscopy

DNA from

femaleAllegedly lacks 2 adult teeth

whole blood

Table 5.5: Demographic details and clinical phenotypes of recruited family members

For the individuals in whom blood was available, DNA was extracted from whole
blood by the AWMGS (2.3.2). DNA was screened for the familial AXIN2 mutation
with PCR and Sanger sequencing using standard reagents and reaction conditions(2.3.8.1, 2.3.8.5, 2.3.9-2.3.14). The primers were those used for Haloplex validation(Appendix 4.2).

For the individuals in whom FFPE tissue was available, DNA was extracted fromnormal mucosa by the AWMGS using the Maxwell Promega LEV FFPE kit. DNA
underwent PCR using reagents as per the protocol for PCR for DNA extracted fromFFPE tissue (2.3.8.2, 2.3.8.5, 2.3.10). The primers were those used for Haloplex
validation (Appendix 4.2), and the products were sequenced from reactions using
Tas of 55.1 and 63.4⁰C, which were the Tas resulting in successful PCR
amplification. 6.25 µl of PCR product was used in the Big Dye reaction, which207

followed standard conditions (2.3.11). Subsequent steps in the sequencing protocol
followed the standard conditions (2.3.12, 2.3.13).

For any individual not found to carry the AXIN2 mutation, the PCR and sequencing
reactions were repeated with a second set of primers (Appendix 5.11).

5.4.5.2

Protein Analysis: Confirmation of a TruncatedProtein
The familial AXIN2 mutation, c.1642G>T:p.Glu548*, had been shown to be present
in cDNA. To confirm that it produces a truncated protein, HEK293 TCF-Luc cells
were transfected with WT-AXIN2 plasmid, Short-AXIN2 plasmid or empty plasmid.
The Short-AXIN2 plasmid had been generated through SDM of WT-AXIN2(5.3.2.6.1, 5.3.2.6.2). The same protocols and plasmid dilutions were followed as for
the Luciferase assays (5.3.2.7, 2.5.7-2.5.10). A 24-well plate was used so thereaction volumes were adjusted accordingly. 48-hours after transfection, the cells
were lysed and protein extracted (2.5.13). Protein was quantified using a BCA
assay (2.5.14), and equivalent amounts of protein underwent Western Blotting,using anti-myc primary antibodies, and anti-mouse secondary antibodies (2.5.15).
To ensure that equal amounts of protein had been loaded onto the electrophoresis
gel, the membrane was stripped using Restore Plus Stripping Buffer (2.5.16) andwas re-probed using primary antibodies against -actin and anti-mouse secondary
antibodies (2.5.15).

5.4.5.3β-Catenin Immunohistochemistry (IHC)FFPE tumours were available for Halo47 (CRC, TA LGD x 3, SA x 2) and Halo68(TA LGD x 14, HPP x 6), their mother (Individual 1.2. CRC, the VA LGD that the
CRC had arisen from, TA LGD x 1, VA LGD x 1, HPP x 1) and their aunt (Individual
1.1. CRC, TA LGD x 4).

IHC was also performed on a known FAP patient (germline mutation APC
c.2940_2941 delA) as a positive control (CRC, TA LGD x 8, TA HGD x 1, mixed
TVA/ HPP x1, biopsy of HGD/ CRC).

208

The Cellular Pathology Department, Cardiff and Vale NHS Trust, carried out βcatenin IHC using the EnVision FLEX System (Agilent) following the manufacturer’s
protocol and primary antibody Beta-Catenin Flex RTU.

5.4.5.4

Somatic APC Mutations in Colorectal Neoplasms

FFPE tumours were available for Halo47, Halo68, Individual 1.1 and Individual 1.2
as described in 5.4.5.3. DNA was extracted by the AWMGS using the Maxwell
Promega LEV FFPE kit.
Due to a limited supply of poor quality DNA, APC MCR screening was performed,
rather than the whole of the coding sequence. Codons 1181-1648 were sequenced.
Work was carried out by the AWMGS following the protocol described in Appendix
5.12.

5.4.5.5

AXIN2 Loss of heterozygosity Analysis (LOH)

FFPE tumours were available for Halo47, Halo68, Individual 1.1 and Individual 1.2
as described in 5.4.5.3. DNA was extracted by the AWMGS using the Maxwell
Promega LEV FFPE kit.
LOH analysis was carried out by testing for heterozygosity at the site of the germline
mutation. Standard reagents and reaction conditions were employed (2.3.8.2,
2.3.8.5, 2.3.9-2.3.13). The primers used are listed in Appendices 4.2 and 5.11.
LOH analysis was performed for all the DNA samples which remained after somatic
APC screening: several of the samples had been used to exhaustion.

5.4.5.6

Functional Characterisation of the AXIN2 Variant

5.4.5.6.1

AXIN2-Containing Plasmids

Plasmids containing the human AXIN2 coding sequence were very kindly supplied
by Professor Eric Fearon, University of Michigan Medical School. The plasmid usedduring this study was N-terminal 6xmyc-tagged AXIN2 in pCS2+MT. Both wild-type
AXIN2 was provided, and mutant AXIN2 containing the c.1989G>A variant
described by Marvin et al (Marvin et al 2011), Fearon-AXIN2.

209

Plasmids were retrieved from filter paper (2.5.1) and used to transform XL-1 Blue
Competent cells (2.5.4). Plasmid DNA was extracted using the MiniPrep kit(Qiagen) (2.5.5.) and the identity of the AXIN2 coding sequence was confirmed withsequencing (2.5.5).

5.4.5.6.2

Site Directed Mutagenesis (SDM)

Site directed mutagenesis and bacterial transformation were employed to generate
plasmids containing the AXIN2 mutation identified as part of this study,c.1642G>T:p.Glu548* (2.5.6), Short-AXIN2. Primers are described in Appendix
5.13. Plasmid DNA was extracted using the MiniPrep kit (Qiagen) (2.5.5).
To confirm the presence of the desired mutation, sequencing was carried out byDundee Sequencing. Primers are described in Appendix 5.14.

5.4.5.6.3

The Luciferase Reporter Assay in HEK293 TCF-

Luc Cells
HEK293 TCF-Luc cells were kindly supplied by Professor Jeremy Nathans, John
Hopkins University. They were kept at -80˚C. Once defrosted (2.5.7) they wereseeded onto a 96-well Assay plate (2.5.9) prior to transfection with varying amountsof WT-AXIN2, Fearon-AXIN2, Short-AXIN2 or WT/ Short-AXIN2 plasmids (2.5.10).
Following transfection, cells were stimulated with exogenous Human Wnt3a andHuman R-Spondin-1 (2.5.11). WIST was subsequently added, before measuring
WIST and luciferase activity (2.5.12).

5.4.6 Results

5.4.6.1

Family Segregation Studies

Six further family members were screened for the familial AXIN2 mutation,c.1642G>T:p.Glu548*. The mutation was identified in Individuals 1.1 and 1.2, who

210

had a history of colorectal neoplasia. It was not present in the family members who
were unaffected, Individuals 2.1, 3.1, 3.2, 3.3 (sequencing traces in Appendix 5.15)

5.4.6.2

Protein Analysis

HEK293 TCF-Luc cells were transfected with a series of dilutions of WT-AXIN2,
Short-AXIN2 or empty plasmid. Protein was extracted and analysed using Western
Blotting.

Results showed that Myc-tagged-WT-AXIN2 was detected in the cells transfectedwith WT-AXIN2 plasmid, and a truncated AXIN2 protein, approximately 30 KDa
smaller than the WT protein, was detected in the cells transfected with Short-AXIN2
plasmid. Protein was only detectable at the highest two concentrations of plasmid
used for transfection. No AXIN2 protein was detected in the cells transfected withempty vector.
-actin protein was detected with approximately equal intensity in all samples ofprotein lysate, confirming that the same amounts of protein had been loaded in eachwell.
The mutant AXIN2 was detected at greater intensity than WT protein, suggestingthat it may possibly be a more stable protein. The protein ran at a larger size than
predicted, by approximately 30 KDa, but this is commonly observed with AXIN2 (Dr.
Anika Offergeld, personal communication). Possible reasons include it interactingwith other proteins or the way in which it was interacting with the gel. See Figure
5.5.

211

L

L

L

L

Figure 5.5. A: Western blot probing for AXIN2. L: ladder. Decreasing concentrations of
AXIN2 plasmid used for cell transfection (1. 90 ng/25 µl, 2. 30 ng/25 µl, 3. 10 ng/25 µl, 4.
3.33 ng/25 µl, 5. 1.11 ng/25 µl, 6. 0.37 ng/25 µl, 7. 0.12 ng/25 µl, 8. ng/25 µl). B: Western
blot probing for β –actin. L: ladder.

5.4.6.3β-Catenin Immunohistochemistry (IHC)

Immunostaining was interpreted by myself and Professor Geraint Williams, and I am
extremely grateful for his help and advice.
Results from the FAP positive control patient showed that β-catenin IHC in the
adenomas exhibited largely membranous staining, but in most of the lesions, there
were occasional cells with nuclear positivity. However, this did not seem tocorrelate with the degree of dysplasia, i.e. membranous staining was observed inboth low-grade and high-grade regions, and the focal nuclear positivity present
occurred in regions of low-grade dysplasia. However, LGD was by far the
predominant morphology, so it is feasible that there wasn’t sufficient HGD present toobserve nuclear staining. The CRC exhibited mainly membranous staining of β-212catenin. There were occasional cells with nuclear positivity, and these tended tooccur at the invasive margin of the tumour. See Figure 5.6.

213

A1

A2

A3

B1

B2

C1

C2

C3

Figure 5.6: -catenin IHC of colorectal adenomas and CRC from a patient with confirmed
FAP, germline mutation APC c.2940_2941delA. A1: TA LGD H&E. A2: -catenin IHC islargely membranous but there are scattered cells with nuclear staining as seen in A3 (redarrows). B1: Region of HGD H&E. Note membranous staining in B2. C1: CRC H&E. catenin IHC is predominantly membranous, C2, but there are occasional cells with nuclear
positivity, C3 (red arrows). Scale bars in the bottom right corner of each image.

214

There was no evidence of nuclear β-catenin in any of the serrated/ hyperplastic
lesions from patients carrying the AXIN2 mutation (Figure 5.7):

A1

A2

B1

B2

C1

C2

Figure 5.7: A1: Halo47 SA LGD H&E, A2. Halo47 SA LGD β-catenin IHC; B1: Halo68
HPP H&E, B2: Halo68 HPP β-catenin IHC; C1: Individual 1.2 HPP H&E, C2: Individual 1.2
HPP β-catenin IHC

215

All of the tubular adenomas from patients carrying the AXIN2 mutation were low
grade lesions. The prominent staining pattern was membranous. The only patient
to exhibit any β-catenin nuclear positivity was Individual 1.1: in two adenomas there
was an occasional cell with nuclear staining (Figure 5.8):

A1

A2

B1

B2

C1

C2

D1

D2

E1

E2

Figure 5.8: A1: Halo47 TA LGD H&E, A2 Halo47 TA LGD β-catenin IHC; B1: Halo68 TA
LGD H&E, B2: Halo68 TA LGD β-catenin IHC; C1: 1.2 TA LGD H&E, C2: 1.2 TA LGD βcatenin IHC; D1: 1.1 TA LGD H&E, D2 TA LGD β-catenin IHC. Note focal nuclear staining(red arrow); E1: 1.1 TA LGD H&E, E2: TA LGD β-catenin IHC. Note focal nuclear staining(red arrow)

216

The only villous adenomata present were in Individual 1.2. Both the solitary VA andthe VA which had progressed to carcinoma showed focal evidence of nuclear βcatenin staining. There was weak membranous staining of the carcinoma, but noevidence of nuclear β-catenin, although overall the staining pattern was very weak(Figure 5.9):
A1

A2

B1

B2

C1

C2

C3

Figure 5.9: Individual 1.2 A1: Solitary VA H&E. A2: Solitary VA β-catenin IHC. Note focal
nuclear staining (red arrows). B1: VA that has progressed to CRC H&E, B2: VA that has
progressed to CRC β-catenin IHC. Note focal nuclear staining (red arrows). C1: CRC H&E.
C2: CRC β-catenin IHC. C3: Contrast between the intensity of the staining of β-catenin
between the villous adenoma (blue arrow) and the CRC (green arrow)

217

For Halo47, IHC staining of the adenocarcinoma showed different patterns: most of
the tumour was moderately differentiated, but there were regions of poordifferentiation. In the moderately differentiated regions, there were cells withprominent nuclear β-catenin staining, but this was not universal. In the poorly
differentiated area, nuclear β-catenin was ubiquitous (Figure 5.10):

A1

A2

B1

B2

Figure 5.10: Halo47: A1: Moderately differentiated CRC H&E. A2: Moderately differentiatedCRC β-catenin IHC. Note nuclear staining (red arrows) B1: Poorly differentiated CRC H&E.
B2: Poorly differentiated CRC β-catenin IHC

218

The CRC in Individual 1.1 again showed predominantly membranous staining of catenin, but there were occasional cells with nuclear positivity (Figure 5.11).

A1

A2

A3

A4

Figure 5.11: Individual 1.1: A1: Moderately differentiated CRC H&E; A2-A4: Moderately
differentiated CRC β-catenin IHC. Focal nuclear staining is present (red arrows)

5.4.6.4

Somatic APC mutations in colorectal neoplasms

Several DNA samples extracted from FFPE tissue were of very low concentration,
and PCR amplification followed by sequencing was not always successful. The
successful PCR and sequencing reactions are described in Table 5.6. A pathogenic
APC mutation refers to stop gains and frameshift mutations, it does not include
silent or missense changes.

219

220

221

It was therefore seen that only a minority of tumours were identified as having asomatic APC mutation present.

5.4.6.4.1

Correlation between β-Catenin

Immunohistochemistry (IHC) and Somatic APC Mutations
Only a small proportion of lesions exhibited evidence of nuclear -catenin or wereidentified as having a somatic APC mutation. The correlation between thesefindings is described in Table 5.7.

SampleNuclear -Morphology

CateninHalo47

CRC

Yes

1.1

TA LGDYes

1.1

TA LGD

Yes

1.1

CRC

Yes

1.2

VA

Yes

Patient

1.2

VA progressed
to CRC

Yes

Somatic APC Mutation
Uncertain (11/12 fragments
sequenced)
Uncertain (7/12 fragments
sequenced)
Yesc.3870_3877delTCAGACGAYes c.3667delT
Uncertain (3/12 fragments
sequenced)
Yes c.3856 G>T

Table 5.7: Correlation between nuclear -catenin IHC and the presence of a somatic APC
mutation

5.4.6.5

AXIN2 Loss of Heterozygosity Analysis (LOH)

Successful LOH screening was achieved for a total of 12 tumours/ tumour regions.
There was no evidence of LOH of the WT AXIN2 allele in any of the tumours
sampled (sequencing traces in Appendix 5.16).

222

5.4.6.6

Functional Characterisation of the AXIN2 Variant

In order to further characterise the effects of the AXIN2 c.1642G>T:p.Glu548*
variant, studies were performed in HEK293 TCF-Luc cell lines. Luciferase assays
were used to determine the effects of the mutation on Wnt-pathway activation.

Cultured cells transfected with empty plasmids were stimulated with exogenous
Wnt3a and R-Spondin. This lead to an approximately 67-fold increase in thenormalised luciferase score, compared to unstimulated cells.

Cells were transfected with a series of dilutions of AXIN2 plasmid: WT-AXIN2,
Fearon-AXIN2, Short-AXIN2 or a WT/ Short mix. At all concentrations of plasmid,
mutant AXIN2, both Fearon and Short, was less able to inhibit reporter gene activity
than WT-AXIN2 (Figure 5.12A). Interestingly, it was the WT/ Short mix which had
the least effect on Wnt-pathway inhibition.

223

WT

Luciferase /(WST450/WST590)
score compared tostimulated cells
transfected withempty plasmid vector

B
1.5
1
0.5
0
0.01

0.1

1

10

100

Amount of AXIN2 plasmid used for transfection (ng)Luciferase /(WST450/WST590)
score compared tostimulated cells
transfected withempty plasmid vector

Fearon
1.5
1
0.5
0
0.01

0.1

1

10

100

Amount of AXIN2 plasmid used for transfection (ng)Luciferase /(WST450/WST590)
score compared tostimulated cells
transfected withempty plasmid vector

Short
1.5
1
0.5
0
0.01

0.1

1

10

100

Amount of AXIN2 plasmid used for transfection (ng)Luciferase /(WST450/WST590)
score compared tostimulated cells
transfected withempty plasmid vector

WT/ Short
1.5
1
0.5
0
0.01

0.1

1

10

100

Amount of AXIN2 plasmid used for transfection (ng)Figure 5.12A: Graph to show the different effects of AXIN2 mutations on Wnt-pathway
inhibition. Mutant AXIN2 is less able to inhibit Wnt-pathway activation than WT-AXIN2;
5.12B: Standard deviations shown for each separate plasmid used for transfection.

224

Statistical analysis was carried out with the help of Dr. Matthew Mort. A 2-way
ANOVA analysis and Tukey multiple pairwise analysis was performed to determine
whether the difference in Wnt-pathway inhibition was statistically significant between
the WT-AXIN2 and the mutant plasmids. Statistical significance was reached when
WT-AXIN2 was compared to the Short/ WT- AXIN2 mix, with a p-value of 0.00155.
Statistical significance was not reached for Fearon-AXIN2 (p=0.1021872) or ShortAXIN2 (p=0.1274879) compared to WT-AXIN2. This could be due to the small
sample size.

5.4.7 Conclusions of Studies into AXIN2 c.1642G>T:p.Glu548*
The AXIN2 c.1642G>T:p.Glu548* mutation was initially identified in the sibling pair
Halo47 and Halo68, both of whom had been recruited to the study due to theirphenotype of colorectal polyposis. The same variant was subsequently identified intheir mother and maternal aunt. Both had colorectal neoplasia: they had a historyof CRC, but their polyp counts were below 10. The variant was not present in abrother, who had recently had a clear colonoscopy, and in the daughters and nieceof Halo68.

Both sisters and their mother allegedly lacked all adult teeth. This was confirmed bydental X-rays in Halo68. We were unable to get any information about the dental
history of the aunt. Halo68 also exhibited clinical signs of ectodermal dysplasia –she had sparse hair on the outer half of her eyebrows, thin hair on her head and anail malformation. We were unable to do a clinical assessment of the remaining
variant-carriers in the family as they had all passed away, but family photos of
Halo47 and her mother were suggestive of sparse eyebrow hair. Whether thesefeatures are part of the phenotypic spectrum associated with AXIN2 mutations
remains to be determined. In both Lammi’s (Lammi et al 2014) and Marvin’s (Marvin
et al 2011) papers, AXIN2 mutant-carriers had oligodontia, and in the family
reported in Marvin’s paper, signs of ectodermal dysplasia were also present. In thisstudy, a daughter of Halo68 had sparse eyebrows and no other signs of ectodermal
dysplasia, but did not carry the familial mutation. It seems that oligodontia
segregates with the colorectal phenotype, but further studies in AXIN2-mutant
families are required to better characterise the clinical features.

225

Western blotting confirmed that cell lines transfected with Short-AXIN2 plasmids
produce a protein approximately 30 KDa smaller than cells transfected with WTAXIN2 plasmids. The truncated protein may possibly be more stable than WT
protein. This would need to be confirmed by a technique such as a pulse chaseexperiment. Increased stability of mutant AXIN2 had previously been reported forthe truncated protein investigated by Mazzoni et al and may be due to the loss of adestabilising phosphorylation site (Mazzoni et al 2015).

It is possible that the mutation may have further effects in addition to being a
nonsense change. Analysis using MutPred Splice(http://www.mutdb.org/mutpredsplice/about.htm accessed 06/03/2018) suggeststhat the mutation is a splice neutral variant with a score of 0.37. However, in silico
analysis using Human Splicing Finder (http://www.umd.be/HSF3/ accessed
06/03/2018) suggests that the mutation could possibly add an exon splicing silencer
and remove an exonic splicing enhancer. In theory, this could lead to skipping ofExon 5. Analysis with Mutalyzer (https://mutalyzer.nl accessed 06/03/2018)confirmed that the loss of Exon 5 would produce a truncated protein, but that thetranscript would be in-frame. The truncated product would be approximately 88
KDa. Exon 5 is partially responsible for encoding the domain of AXIN2 whichinteracts with GSK3β (http://www.uniprot.org/uniprot/Q9Y2T1#showFeaturesViewer
accessed 06/03/018) so if it was absent from the protein, this could have functional
consequences. To further investigate this, techniques such as mass spectrometry
could be used.
-catenin IHC was initially performed on tumour samples from a known FAP patient.
Throughout all adenomas and CRC, the staining pattern was predominantly
membranous, with occasional cells exhibiting nuclear positivity. For the patients
carrying the familial AXIN2 mutation, Halo47, Halo68, Individual 1.1 and Individual
1.2, there was no nuclear staining in any of the hyperplastic lesions. This is as
expected, as such lesions do not typically exhibit activation of the Wnt-pathway. Of
all the tubular adenomas present, only two had any focal nuclear -catenin staining.
It was anticipated that if the adenomas had similar levels of Wnt-pathway activation
as do APC-mutant tumours, there should have been more evidence of nuclear
positivity. Interestingly, the villous adenomata, which are more likely to progress tocancer than tubular lesions did show evidence of nuclear β-catenin staining.

226

There were three CRC present – in Halo47, Individual 1.1 and Individual 1.2. The
cancer in Halo47 showed different patterns of -catenin staining: most of thetumour was moderately differentiated, but there were regions of poor differentiation.
In the moderately differentiated regions, there were cells with prominent nuclear βcatenin, but this was not universal. In the poorly differentiated area, nuclear βcatenin was ubiquitous. In Individual 1.1, the cancer again showed predominantly
membranous staining of -catenin, but there were occasional cells with nuclear
positivity. In Individual 1.2, the cancer had arisen in a VA. There was weakmembranous staining of the carcinoma, but no evidence of nuclear β-catenin.
However, the staining of the cancer was repeatedly very weak. The reason for thisis not clear. One possibility is that -catenin had acquired a mutation which
rendered it relatively resistant to IHC staining. Regardless of the aetiology, the poor
staining makes interpretation difficult.
Nuclear positivity for β-catenin in the VA and CRC, but not in most of the TA, implies
that Wnt pathway activation may be occurring at a later stage of tumourigenesis
than in FAP adenomas.
Somatic APC analysis was performed to determine whether ‘typical’ APC mutations
were present in tumours from patients carrying germline AXIN2 mutations.
Although DNA was extracted from 36 lesions, less than half of the desired PCR
fragments were successfully amplified, making interpretation difficult. Only 3
truncating mutations were identified: 1 of these was present in cancer and anotherwas present in the VA which had developed into CRC. This raises the possibility
that adenomas arising in AXIN2 mutation carriers might need to acquire mutation(s)in a gene such as APC in order to acquire sufficient Wnt pathway activation toprogress to malignancy.

We attempted to determine whether we had identified statistically significantly fewer
APC mutations than would have been predicted in adenomas from AXIN2 mutation
carriers. However, it was not possible to perform such an analysis due to the lack ofa robust control set to compare our results to. Whilst there is a wealth of data in theliterature documenting somatic mutations in sporadic CRC and in FAP-associatedadenomas and CRC, there are scanty papers addressing somatic changes insporadic adenomas. Of the papers that do exist, different experimental approaches
have been taken. Some studies considered the whole of the coding region of APC,
some look at exon 15 only and others address specific nucleotide sequences; some227

of the studies sequence fresh tissue, some use frozen tissue and others use FFPE
material; some studies investigate sporadic lesions only, others include some FAPassociated tumours (Van Wyk et al 1999; Olschwang et al 1998; De Benedetti et al
1994; Miyoshi et al 1992b; Powell et al 1992).
The correlation between a somatic APC mutation with the presence of nuclear catenin staining was addressed: in both of the cancers and the TA LGD which had
identified somatic APC mutations (Individual 1.2 and Individual 1.1) nuclear staining
was present. There were tumours with nuclear -catenin staining which did notseem to have a somatic APC mutation. However, in all of these samples, the MCR
was not successfully completely sequenced, so the actual somatic APC MCR
mutation status is uncertain.

It appears that tumours arising in individuals carrying the germline AXIN2c.1642G>T:p.Glu548* mutation may not exhibit the ‘typical’ APC mutations seen inpatients with FAP or sporadic adenomas, although the poor coverage of the MCR
makes it difficult to say this with certainty. If this is true, it implies that lesions follow
a different genetic tumourigenic pathway. This exciting possibility is going to befurther investigated by carrying out WES on tumour DNA, but this was not possible
during the timescale of this project.

There was no evidence of LOH in any of the tumours sampled. If AXIN2 was a
typical tumour suppressor gene, and if the variants identified are pathogenic, itwould be anticipated that there would be loss of the wild-type allele in the tumours.
This was not observed. It is possible that the mutation is exhibiting dominantnegative effects. It is also possible that there is a second hit to the WT allele by amechanism other than LOH, for example epigenetic silencing or a second
mutational event.

The effects of the Fearon and Short AXIN2 mutations on Wnt-pathway activation
were examined through the use of luciferase assays in HEK293 TCF-Luc cell lines.
Fearon-AXIN2 and Short-AXIN2 both demonstrated possibly impaired Wnt pathway
inhibition, but this did not reach statistical significance. Interestingly, the plasmids
least able to inhibit the Wnt-pathway were the Short/ WT mix. This would beconsistent with the notion that inherited AXIN2 mutations can act via a dominantnegative mechanism. Previous papers have shown that mutant AXIN2 is still able228

to dimerise with itself and with WT-AXIN2 (Mazzoni et al 2015). If the same is true
for the mutation we have identified, it is possible that a complex composed of WT
and mutant AXIN2 is less efficient at promoting -catenin degradation than mutant
AXIN2 alone.

Halo47 and Halo68 have also undergone WES of blood DNA (4.3). This did not
identify any inherited variant that was considered more likely to be clinicallysignificant than the confirmed AXIN2 mutation. In particular no likely pathogenic
variants were identified in known adenomatous polyposis or ectodermal dysplasia
genes.

Overall the results from the studies performed would be consistent with someinherited AXIN2 mutations having a role in the pathogenesis of a colorectal
polyposis syndrome in which individuals also display signs of ectodermal dysplasia,
in particular oligodontia. If confirmed, this apparently very rare syndrome could be
named AxAP: AXIN2-Associated Polyposis. The effects of mutant AXIN2 are likely
to be at least partially mediated through activation of the Wnt-signalling pathway. It
is important that further studies are performed to confirm and characterise thesyndrome, so that optimal clinical management can be determined. Such further
studies would include an improved genetic characterisation of the tumours arising inpatients with inherited AXIN2 mutations to better understand the molecularpathways of tumourigenesis in these individuals. Currently the WGP is developing
a platform to allow WES on DNA extracted from FFPE tissue, which will be
performed later this year. It is also important to gain a greater knowledge about anyother effects which mutant AXIN2 may have, for example on the numbers of stem
cells in tissue, its effects on cell proliferation and its effects on other signalling
pathways.

The AXIN2 mutation identified in this study, c.1642G>T:p.Glu548*, is not present in
Exac, HGMD or in data from the 1000 Genomes Project. However, somatic AXIN2
mutations are well described in a variety of cancer types. The COSMIC databasereports 22 somatic nonsense mutations of AXIN2 in 46376 cancer samples tested:
10 of these were large intestine adenocarcinoma(https://cancer.sanger.ac.uk/cosmic/gene/samples?all_data=&coords=AA%3AAA&d
r=&end=844&gd=&id=4988&ln=AXIN2&mut=substitution_nonsense&seqlen=844&s
rc=gene&start=1 accessed 15/05/2018). CBioPortal reports that 1.4% of cancers
screened had a somatic mutation of AXIN2. 243 truncating mutations are229

described: the vast majority of these were cancers in the gastrointestinal tract(http://www.cbioportal.org/index.do?session_id=5aec70b3498eb8b3d565fab9
accessed 15/05/2018). In the Exac database, there are 9 entries of loss of function
germline mutations in AXIN2. They are all rare – the commonest is a frameshift,p.Asn666Glnfs*41, which has an allele frequency of 0.0005561 and was identified in
65/116894 alleles, being overrepresented in African populations. Although thephenotype of these patients is not known, Lammi’s paper (2004) describes aframeshift mutation in a 13-year old boy with oligodontia, which truncates AXIN2 atalmost the same location: the group reported a heterozygous 1 bp insertion after
nucleotide 1994 in exon 7, which expands one of the several mononucleotide
repeats. It recodes the amino acids starting at Asn666 and incorporates a stopcodon 40 codons later. On CBioportal there are 6 reports of a stop codon being
inserted 41 codons after p.Asn666 in CRC(http://www.cbioportal.org/index.do?session_id=5aec70b3498eb8b3d565fab9
accessed 15/05/2018). The significance of the frameshift reported in Exac istherefore not determined. It is possible that it is associated with a phenotype that
we are not aware of, or alternatively it may be well-tolerated, possibly because it
occurs relatively close to the C terminus of the protein.

It is interesting that the colorectal phenotype in patients carrying reported specificAXIN2 mutations seems to be very variable, and both adenomas and HPPs seem toform part of the clinical spectrum. The consistent observation in this and previous
reports is that patients with polyposis and truncating AXIN2 mutations also haveoligodontia. Oral abnormalities are common in FAP patients, with a prevalence of
17% (reviewed in Groen et al 2008). Such abnormalities include supernumeraryteeth, dentigerous cysts and secondary retention of teeth (reviewed in Groen et al2008), likely due to aberrations in Wnt signalling. The canonical Wnt pathway plays
an essential role in tooth initiation and morphogenesis, and also in dental celldifferentiation (reviewed in Järvinen et al 2018). In AXIN2 mutant patients,
individuals appear to grow their primary dentition (baby teeth) but do not develop a
full complement of secondary teeth. Järvinen et al (2018) sought to investigate theeffects of the Wnt pathway on the formation of molars in mouse models and in exvivo cultures. They showed that increasing Wnt/ β-catenin activity in dental
mesenchyme inhibited the development of posterior molars, whereas reducing
activity was associated with continued tooth development (Järvinen et al 2018).
The authors concluded that the dental abnormalities observed in AXIN2 mutant

230patients results from the modulation of Wnt/ β-catenin signalling in the dental
mesenchyme (Järvinen et al 2018).

In mice studies of AXIN2, homozygous knockout of AXIN2 leads to reduced head
growth, due to premature fusion of cranial sutures. This resembles craniosyntosis
in humans (Yu et al 1995). The dental and colorectal phenotype in such models is
not described.

5.5

Chapter Conclusions

The aim of this chapter was to carry out genetic, in silico and functional assessmentof mutations identified through targeted exome sequencing (Chapter 4). Variants
were selected in the MMR genes, POLE, POLD1 and AXIN2.

Tumours from patients carrying MMR gene variants underwent MMR IHC and MSI
testing. There was no evidence of loss of MMR proteins or microsatellite instability
in any of the lesions. This leads to the conclusion that the MMR gene variants
identified are unlikely to be clinically significant.

Four patients were found to have germline variants in POLE/ POLD1. These were
investigated through a combination of protein modelling, somatic ‘hotspot’ mutation
screening and mutation signature analysis. One of the variants, POLE
p.Glu2140Lys in Halo18, is reported as benign on dbSNP. It does not lie within anyof the POLE functional domains and was not in the protein model used as part ofthis project. The variant is presumed not to be clinically significant. A POLE
variant, p.Tyr623Cys in Halo61, remains a VUS. Although mutation signature
analysis was attempted, this was not successful. Somatic mutation analysis is stillin progress for tumours from Halo15 and Halo27. Although this should be completein the near future, at the present time the mutations identified in Halo15 and Halo27,
c.1411A>G:p.Met471Val and c.2861C>T:p.Thr954Met, remain as VUS.

A previously unreported truncating AXIN2 mutation, c.1642G>T:p.Glu548*, wasidentified in a family with a 2-generation history of oligodontia and colorectal
neoplasia. The mutation segregates with the disease phenotype in an AD manner.
It is present in DNA and cDNA and produces a truncated protein. P.Glu548* mutant
AXIN2 appears less able to inhibit the Wnt pathway than WT AXIN2 and could havedominant negative effects.

231

The study of Family A has added to evidence from other previously reported familes
with AXIN2 mutations that there may exist a rare AXIN2-Associated Polyposis
Syndrome, in which individuals have signs of ectodermal dysplasia and a variable
colorectal phenotype. The effects of the mutant protein could be at least partiallymediated through activation of the Wnt signalling pathway. Further studies are
required to confirm and better characterise this syndrome. Research programmes,
including the UK 100 000 Genomes Project that is recruiting patients with colorectal
polyposis, may identify further affected patients with germline AXIN2 mutations.

232

Chapter 6

Thesis Discussion

Colorectal carcinoma (CRC) is the fourth most common cancer in the United
Kingdom (UK) and the second most common cause of cancer death (Cancer
Research UK 2015). The majority of colorectal carcinomas occur sporadically, but
in 15-35% of patients, hereditary factors are important (reviewed in Mishra and Hall
2012; Burt 2007). These factors include the inherited polyposis syndromes, FAP,
MAP, PPAP, NTHL1-Associated Polyposis and MSH3-Associated Polyposis. In allof these syndromes, affected individuals develop multiple colorectal polyps andhave an increased risk of developing CRC. In order to assess whether an inherited
disorder is present in patients with multiple polyps, it has been traditional practice torefer individuals with >10 polyps to a medical genetics service for genetic
counselling and consideration of genetic testing. In the NHS, APC and MUTYH are
routinely screened, with increasing numbers of laboratories also screening for therecurrent mutations in POLE and POLD1. Of the cohort of individuals who undergo
genetic diagnostic testing, up to 50% do not have a mutation identified. It is thisgroup of patients that this thesis has addressed.
The key hypotheses thought to underlie the patients’ phenotypes were that
mutations may be present outside of the ORF of APC/ MUTYH, that the affectedindividuals may exhibit somatic mosaicism of APC or that further polyposis genes
may play a role. This thesis has not considered epigenetic effects or oligogenic
inheritance.

To address these hypotheses, firstly APC and MUTYH were interrogated. UDS ofthe entire genomic loci was performed, qPCR analysis was utilised to study genetranscription, and patient-derived RNA samples underwent sequencing and AI
analysis.
The next stage was to carry out targeted exome sequencing of 15 genes either
known to be, or possibly, involved in the pathogenesis of colorectal neoplasia.
Following on from this, a subset of patients underwent WES. The final phase of the
project was to carry out further genetic, in silico and functional studies on thecandidate variants most likely to be clinically significant, to obtain further evidence tosupport or refute their role in colorectal polyposis.

233

6.1

APC and MUTYH Studies

The most interesting results from the examination of APC and MUTYH were thosefrom the gene expression studies. Four patients appeared to have evidence of
reduced APC transcription. These individuals all had phenotypes in keeping with aclinical diagnosis of FAP. Their gene transcription results were consistent with their
AI results, which either showed apparent loss of transcription of one APC allele(Halo52 and Halo53) or were uninformative (Halo46 and Halo64). The cause of the
reduced APC expression was determined in Halo46. UDS identified a 5’UTR
variant (G>A at 5:112043225), at position -c190. Subsequent testing of family
members confirmed that the mutation was segregating with the phenotype, andpreviously described functional studies reported the variant to be pathogenic
through impaired binding of a transcription factor (Li et al 2016). Predictive testingfor this variant is now being established in the clinical diagnostic setting forextended family members.

I sought to determine the cause of the reduced gene transcription in the remaining 3
samples. However, in addition to apparently normal UDS of the entire genomic
APC locus, including the promoter regions and UTRs, karyotype analysis andpromoter methylation studies also gave normal results. We are planning that thesepatients and appropriate members of their families undergo whole genome
sequencing with the aim of searching for variants in the regions of the genome
which have not yet been examined.
UDS only identified one pathogenic mutation - the APC 5’UTR variant in Halo46
described above. There was no evidence for any likely pathogenic variants outsideof the ORF or for APC mosaicism in the other patients. With regards to APC
mosaicism, the average depth of coverage of APC using the HiSeq 2500 was
1665.5x, so if there were any cases with significant levels of mutant alleles in blood
DNA, they were unlikely to have been missed. However, others have publishedevidence indicating that somatic mosaicism restricted to the gut or a section of thebowel may be relatively common (Jansen et al 2017; Spier et al 2016), limiting theusefulness of studying DNA derived from blood.

234

6.1.1 Strengths and Weaknesses of the Study of APC and
MUTYH
There were several strengths to this study. DNA was obtained for all study
participants, and RNA from the majority of individuals. FFPE tissue was available
for most cases when it was required for IHC analysis or for somatic DNA analysis.
Furthermore, it was possible to access patients and their medical notes when they
were registered with the AWMGS. We worked closely with the WGP, which allowed
access to NGS technologies, making it possible to examine whole genomic loci andto perform exome sequencing.

One of the key problems encountered throughout this project was the lack ofadequate clinical information about the patients. Often the phenotype,
histopathological assessment of lesions and family history were not complete, so it
was difficult to determine whether cases were familial or sporadic, and what the true
polyp counts/ morphology were. We found that despite acquiring written patientconsent to access medical records under an ethically approved study, it was often
difficult to access clinical details for patients who were not under follow up with theregional genetics service in Wales. Busy hospital services have priorities other than
supporting research studies and the dispersion of clinical data in the NHS makes its
retrieval time consuming. The gradual move to digitalised records will likely benefit
clinical research in the longer term. The optimal information would have included acomplete family history, the age of onset of polyposis, the cumulative numbers ofpolyps, the size and location of polyps and their microscopic morphology.
It also often proved difficult to recruit family members of the proband. This madeanalysis difficult as it was often not possible to ascertain whether identified variants
were occurring de novo or were segregating with disease if multiple family members
were affected.

Part of the study involved using NGS technologies to carry out UDS. NGS
generates large amounts of data, and a challenge is to identify what is clinicallysignificant. In this thesis, CADD scoring was used as a tool to indicate the likelihoodof pathogenicity and to therefore prioritise variants for further study. Although the
CADD score gives a measure of predicted deleteriousness, this is not the same asits likelihood of causing a clinical phenotype. Furthermore, CADD scoring was used
to appraise all variants. Recent evidence suggests that ‘CADD scores may be
useful for identification of pathogenic intronic or nonsynonymous variants in235

targeted testing situations when used in combination with other data. However
our data suggest that CADD scores are unlikely to be useful for identifying
disease-causing mutations in other noncoding regions in cancer-risk genes’(Mather C. et al 2016), so the utility of this method in scoring variants in regions
such as promoters/ UTRs is questionable. The authors acknowledge that there isa distinction between variants that are functionally deleterious and clinicallypathogenic (Mather C. et al 2016). They note that there are many situations inwhich a deleterious variant does not cause clinical phenotype (Mather C. et al2016).

6.1.2 Clinically Translatable Outcomes
The key clinically translatable outcomes from this stage of the project are detailed
below:
1. APC promoter sequencing and sequencing of Exon 1/ 5’UTR. In theclinical diagnostic setting, APC and MUTYH undergo sequence and dosage
analysis through sequencing and MLPA respectively. Standard protocols
involve sequencing of the coding exons (APC and MUTYH) and dosage
analysis of coding exons (APC and MUTYH) and promoter (APC only)(2.2.3.1). It is recommended that diagnostic laboratories consider including
the APC promoters 1A and 1B and Exon 1 in protocols for patients who
remain NMI after sequencing of coding exons. This would allow theidentification of variants which could result in abnormalities of gene
expression.

2. RNA studies. For NMI patients, diagnostic laboratories could consider RNA
studies as part of their investigative tool kit. cDNA sequencing of APC is arelatively simple procedure to undertake. If abnormalities were identified,sequencing of the entire genomic locus of APC could be undertaken to
determine the underlying cause. Quantitative studies of APC transcription
could also be considered. However, this would first necessitate a largescale study to determine the normal range of APC transcription in a healthy
cohort and to compare this to expression levels in known APC mutation
carriers. qPCR was used in this study to identify reduced APC transcription
in 4 of 45 NMI patients when compared to a healthy control cohort of 40
individuals, but for clinical applications the control cohort would need to be236

much larger, and multiple Taqman probes would need to be used. Again, if
an individual was found to have reduced APC transcription, sequencing of
the entire genomic locus could be performed to try to determine its cause.
However, as was seen in this work, the cause isn’t always apparent.
Through research such as the 100 000 Genomes Project, which includes acohort of patients with multiple bowel polyps who were NMI following testingin the NHS, further clinically significant variants in gene enhancers/
upstream regions may be characterised.

6.2

Targeted and Whole Exome Sequencing

Targeted exome sequencing was initially carried out to interrogate 15 genes known
to be, or possibly, involved in the pathogenesis of colorectal neoplasia. A total of 33
variants across 32 patients were selected for further investigation: 22 of these werevalidated. The variants which were not validated were largely those occurring at a
low frequency or in regions with low NGS coverage. Whole exome sequencing(WES) was undertaken on a subset of 24 patients, based on their phenotype, theavailability of relatives and those patients in whom a novel and possibly causative
mutation had already been identified, to confirm that missed mutations in the known
genes were not involved. 106 variants were selected for validation, and 100 ofthese were validated. Again, the variants which were not validated were largely
present in regions of low NGS coverage.

The genes/ variants which were selected in the short term for functional
characterisation were those identified as part of the targeted exome testing:variants in AXIN2, POLE, POLD1 and the MMR genes. Although many othervalidated variants were potentially interesting, they were not taken forward forfurther studies. The reasons for not selecting other variants included 1) lack ofavailability of relatives, 2) the variant was in a gene already associated with adisease phenotype other than colorectal neoplasia, 3) it was too frequent to be areasonable candidate for the relatively uncommon phenotype of polyposis or 4)
there was not sufficient evidence of a likely role in colorectal neoplasia to prioritise
the gene concerned. As mentioned previously, some variants were identified whichwere known to be clinically significant in contexts other than colorectal neoplasia.
Where these were clinically actionable, they were fed back to the patient’s clinician,according to the study protocol.

237

6.2.1 Strengths and Weaknesses of Targeted and Whole
Exome Sequencing in NMI Polyposis Patients
As described above (6.1.1) the strengths to this study included the availability of
DNA and RNA and the access to NGS technologies. A further strength is that wewere fortunate to have had input from international experts, Professor Seruca, andindustry, AmbryGen, to assist in variant appraisal.

The most important factors limiting progress in the search for novel variants
associated with polyposis were the lack of additional family members and the size ofthe study cohort. Other studies have been more successful because they werelarger or prioritised the study of familial cases (Adam et al 2016; Weren et al 2015)
with availability of samples from multiple affected relatives.

Another issue faced was variable coverage with WES. The mean depth ofcoverage across the 29 patients/ relatives was 61.99 reads, but this varied from14.02 reads (Halo66) to 139.76 reads (Halo61). Furthermore, the percentage of thetarget region covered at 50x depth was only 43.9%. When variants were notvalidated following Sanger sequencing, these tended to have occurred in regions of
low coverage.

Exome sequencing generates large amounts of data, including many novel variants,
leading to a challenge in the selection of variants for further study. CADD scores
were one tool used for variant prioritisation in the current work, and their limitations
have already been discussed (6.1.1). Ultimately choices have to be made based
upon a variant’s predicted impact on gene function, previous literature on thefunctions of the gene involved and genetic data, if available, on de novo occurrenceor segregation with the disease phenotype.

The work in this part of the project required database interrogation to aid inprioritising identified variants. As described in Chapter 4 (4.4.1) it is apparent thatgenetic variants identified through clinical research projects are often not recordedin publicly accessible databases. It would be excellent if all variants were recorded
in a centralised repository, such as LOVD, as this would allow improved
understanding of disease pathogenesis and therefore improved patient care. With
the advent of the 100 000 Genomes Project it is hoped that there will be improved
understanding of the role of the genome in health and disease. The project seeks238to enhance the clinical interpretation of the data and derive new findings from thedata (https://www.genomicsengland.co.uk/faqs-about-gecip/ accessed 10/5/2018).

6.2.2 Recommendations Resulting from Exome Sequencing
1. Ensure attempts are made to recruit family members at the onset of thestudy. Affected and/ or unaffected family members are required in order tomeaningfully evaluate variants identified in probands. I would recommendthat for future genetic research similar to this, family members should be
recruited to studies at their onset, if at all possible.

2. Record all variants in a pubic database. The aim of all clinical researchshould be to improve patient care. We were very fortunate throughout thisstudy to have had input from world experts and from Industry, for example
AmbryGen. The optimal situation would be to record all variants identified
through genetic research in a publicly-available repository, as described in
sections 4.4.1 and 6.2.1.

6.3

Functional Characterisation of Variants Identified in the

Mismatch Repair Genes, POLE, POLD1 and AXIN2
The final phase of this project sought to further characterise variants in the MMR
genes, POLE and POLD1 and AXIN2 to gain evidence either for or against theirclinical significance.

6.3.1 The MMR Genes
Three patients were found to carry germline variants in one of the MMR genes(Halo26, Halo45 and Halo70). Tumour samples were available for all three patients.
The pathogenic effects of the mutations were examined through IHC analysis of the
MMR proteins and MSI testing of tumour DNA. In all cases, MMR IHC was normal,
and all lesions tested were microsatellite stable. This lead to the conclusion that thevariants are unlikely to be pathogenic. This in keeping with their prior probability ofpathogenicity scores (5.2.6.1) which were all <0.95.

239

6.3.2 POLE and POLD1
Four patients were found to carry germline mutations in POLE or POLD1 (Halo15,
Halo18, Halo27 and Halo61). Basic in silico and functional studies were initially
carried out. These included structural modelling to try and predict whether the
mutations could theoretically impair the functioning of the proteins and studies ontumour samples to assess their MSI status and to test for the somatic ‘hot spot’
mutations which have been reported in tumours from carriers of pathogenic POLE
mutations (Palles et al 2013). Based on structural modelling, the p.Glu2140Lys
mutation in Halo18 was deemed unlikely to have functional consequences so was
not taken further forward.
The ‘gold standard’ of variant assessment was to determine the ‘mutation signature’of the tumours arising in carriers of mutations in the pol genes. Known pathogenic
mutations impair the proofreading activity of the polymerases, and they areassociated with a hypermutated genetic signature in tumours. Somatic genetic
analysis was performed in collaboration with Professor Ian Tomlinson and Dr. Claire
Palles (Oxford University/ Birmingham University). SmMIPs were used to capture
30 genes. Unfortunately, sequencing was not successful for Halo61, so
p.Tyr623Cys remains a VUS. There was a seven-month delay between preparing
the samples for sequencing and obtaining the initial results from sequencing. Thiswas due to factors beyond our control (the relocation of the Tomlinson group from
Oxford to Birmingham). However, it has meant that we currently only have the raw
sequencing results for Halo15, for which bioinformatic analysis is awaited. The
tumour sequencing results for Halo27 are pending. Therefore, at the present time,the variants identified in Halo15 and Halo27, c.1411A>G:p.Met471Val andc.2861C>T:p.Thr954Met still remain as VUS.

6.3.3 AXIN2 c.1642G>T:p.Glu548*
Two sisters participating in this study were found to carry a germline mutation in
AXIN2, c.1642G>T:p.Glu548*. When further family members were recruited, thiswas confirmed to segregate with occurrence of colorectal neoplasia in an AD
manner. All individuals affected by colorectal neoplasia also had a history ofoligodontia, and there were variable signs of ectodermal dysplasia. These findings
are in keeping with previous reports in the literature describing AXIN2 mutations in
the context of colorectal polyposis (Marvin et al 2011; Lammi et al 2004). Further

240

work investigating this mutation confirmed that it was present in patient-derived
RNA samples and that it was capable of producing a protein approximately 30k DA
smaller than the wild-type protein. The protein appeared to be more stable thenwild-type protein, again in keeping with the previous literature (Mazzoni et al 2015).
Assessment of the mutation’s impact on Wnt-pathway activation was investigatedusing the luciferase assay in HEK293 TCF-Luc cell lines. Results showed that
Short-AXIN2, WT/ Short-AXIN2 and Fearon-AXIN2 all had impaired ability to inhibit
Wnt-pathway activation, although this was only statistically significant for the WT/
Short-AXIN2 mix.
To further investigate the mutation, β-catenin IHC was performed on tumours arisingin carriers of germline AXIN2 mutations. There was evidence of nuclear β-catenin,
but only in a very small proportion of tumours, and it was more overt in higher-grade
lesions. This suggested that there is Wnt-pathway activation in colorectal tumoursfrom the AXIN2 mutation carriers investigated, but that it may be occurring at a laterstage of tumourigenesis compared to sporadic or FAP-associated lesions and/ or it
may be a subtler effect.

I attempted to screen the APC MCR to determine whether somatic mutations in
APC were present, as this is a common finding in FAP-associated and sporadic
colorectal neoplasia. Unfortunately, the quality and quantity of DNA extracted fromtumours was poor, and MCR sequencing was incomplete. Nonetheless, there
appeared to be fewer somatic APC mutations than would have been expected in the
adenomas, although this was not confirmed statistically due to the lack of a robust
control set. This raises the possibility that there may be a novel genetic pathway oftumourigenesis in carriers of germline AXIN2-mutations, such that APC mutations
are not required for the initial stages of tumourigenesis. This will be further
investigated through WES of tumour DNA.

To summarise, we have identified a family in which a truncating AXIN2 mutation issegregating with colorectal neoplasia and oligodontia in an AD manner. It is notclear whether the additional signs of ectodermal dysplasia, thin hair and nailabnormalities, are part of the phenotype or are incidental findings. The results
obtained from in vitro studies are in keeping with the effects of the mutation at least
being partially mediated through activation of the canonical Wnt-signalling pathway.
Interestingly, a key extra-intestinal finding appears to be oligodontia. The role of the
Wnt-pathway in tooth formation has been discussed in Chapter 5 (5.4.1).

241

The results of our study are consistent with previous reports in the literature –notably that truncating AXIN2 mutations have been found to segregate withcolorectal neoplasia and oligodontia in an AD manner (Marvin et al 2011; Lammi etal 2004). The colorectal phenotype was variable in our family, like that reported inother families. The polyp count is variable, the presence or absence of CRC isvariable, and the morphology of the lesions present is variable: both adenomas and
HPPs/ serrated lesions have been described. In this study, colorectal tumours
arising in the context of a germline AXIN2 mutation were characterised by IHC andmolecular genetic analysis. This pointed to potential differences from sporadic or
FAP-related tumours. However, further characterisation is required to confirm or
refute these differences. It is possible that adenomas may result from activation of
the Wnt-signalling pathway, whereas HPPs/ serrated lesions may be the result ofeffects on other signalling pathways impacted by AXIN2, for example through Ras/
ERK signalling pathways or SAPK/ JNK pathways (Mazzoni and Fearon 2014).

There is mounting evidence for the existence of an AXIN2-Associated Polyposis
Syndrome. Although it is likely to be rare, its true prevalence may beunderestimated due to the variable GI phenotype. It is possible that AXIN2
mutations are highly penetrant with regards to the dental phenotype but are ofvariable penetrance in terms of the colorectal phenotype. To further characterise
the syndrome, it might be sensible to aim to identify patients through dental clinics
rather than through gastroenterology/ colorectal surgery.

6.4

Thesis Conclusions

The aim of this study was to identify novel genes/ mechanisms of disease in acohort of patients with colorectal polyposis who had not had a mutation identified ina clinical genetics diagnostic setting.

A total of 60 NMI polyposis patients were included in this project. Putative
pathogenic mutations/ disease mechanisms have been identified in 8 of thesepatients: 4 with evidence of reduced APC expression, in one of whom theunderlying mutation has been confirmed (Halo46); 2 with variants in POLE/ POLD1
in whom further analysis of adenomas is in progress, and 2 with mutations in
AXIN2.

242

The phenotype of the patients recruited to this study was highly variable, in terms of
the age of patients, the family histories, the polyp counts and morphologies. There
are several possible explanations as to why we did not identify mutations in more of
the patients: variants may be in genomic regions not yet interrogated; they may be
restricted to a tissue not examined, for example the gut epithelium; epigenetic
factors may have a role; some patients may have their phenotype as a result ofoligogenic inheritance and some patients may have numerous sporadic lesions andrepresent one end of a normal distribution of sporadic lesions in the nonpredisposed population.

There are clinical recommendations that result from this project, namely that APC
promoter/ Exon 1 screening is carried out for patients who remain NMI after
standard genetic testing protocols and that RNA studies are considered for NMI
patients. With further characterisation it is possible that a case for AXIN2 to be
included on polyposis/ CRC gene testing panels will be made.

Further work which will be done following on from this project will be to carry out
whole genome sequencing for those patients with evidence of reduced APC
transcription and to examine the genetic signature of tumours developing in carriersof AXIN2 mutations.

The ultimate aim of this work is to optimise patient care and to prevent thedevelopment of CRC. Once causative mutations are identified, family members canbe screened, and appropriate clinical management instituted. The work is alreadyhaving an impact on the family of Halo46 and will hopefully have a positive impact
on other families and future generations. A relative of Halo46 has written:‘I would also like to thank you for the information you passed to … This means myson will be able to be checked for FAP without the initial endoscopy. Just this small
advance has lifted a world of anxiety from my shoulders, thank you so very muchindeed.’

243

244

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