Identification of disease gene variants that can lead to familial myelodysplasia and acute myeloid

319 Pages·2017·5.94 MB·English

Identification of disease gene variants that can
lead to familial myelodysplasia and acute
myeloid leukaemia

A thesis submitted for the degree of PhD

Shirleny Romualdo Cardoso

Supervisors: Professor Inderjeet Dokal, and
Professor Tom Vulliamy

Centre for Genomics and Child Health, Blizard Institute
Barts and The London School of Medicine & Dentistry,
Queen Mary University of London
In loving memory of my beloved sister Karla Romualdo Cardoso

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I, Shirleny Romualdo Cardoso, confirm that the research included within this thesis is
my own work or that where it has been carried out in collaboration with, or supported
by others, that this is duly acknowledged below and my contribution indicated.
Previously published material is also acknowledged below.

I attest that I have exercised reasonable care to ensure that the work is original, and
does not to the best of my knowledge break any UK law, infringe any third party’s
copyright or other Intellectual Property Right, or contain any confidential material.

I accept that the College has the right to use plagiarism detection software to check
the electronic version of the thesis.

I confirm that this thesis has not been previously submitted for the award of a degree
by this or any other university.

The copyright of this thesis rests with the author and no quotation from it or information
derived from it may be published without the prior written consent of the author.

Shirleny Romualdo Cardoso

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Abstract

Myelodysplasia (MDS) is characterised by inefficient haematopoiesis with dysplastic
features of blood and bone marrow, reduction of mature blood cells and continuous
bone marrow failure (BMF). Acute myeloid leukaemia (AML) is characterised by the
accumulation of immature myeloid blasts in the bone marrow. MDS and AML are
mostly sporadic clonal disorders affecting older patients. Familial occurrence of
MDS/AML is rare, and most of these cases occur in the setting of genetic syndromes.
However, it has also been reported to be caused by germline heterozygous mutations
in genes including RUNX1, CEBPA, TERC, TERT, GATA2, SRP72, and ANKRD26.

Our group has collected 115 families that have two or more individuals with BMF with
at least one of whom has MDS or AML. The aim of this project was to identify disease
causing gene variants that can lead to familial MDS/AML. Identification of
predisposing variants to familial MDS/AML is critical for effective management in
these families. This will also provide new insights into the biology of MDS/AML in
general.

Herein, we have characterised a subset of families with MDS/AML as well as identified
candidate disease genes using a range of genetic studies. Specifically, we have: i.
Identified new genetic variants in some of the known disease genes such as RUNX1
and GATA2. ii. Our studies have substantiated the discovery of DDX41 as a disease
gene as we have identified several families harbouring novel heterozygous loss of
function (LoF) DDX41 variants. iii. Identified germline heterozygous LoF RTEL1
variants in a subset of families with myelodysplasia and liver disease. This defines a
new disease group in this field, RTEL1 can now be added to the list of familial
MDS/AML disease genes. iv. We have identified nine new candidate disease genes
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which are involved in RNA splicing, transcription factor, DNA modification, cell
signalling and intracellular transport.

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Contents
List of figures 12
List of tables 15
List of abbreviations 17
Publications and presentations arising from this thesis 27
Acknowledgments 29
Introduction 31
1.1 General introduction 32
1.2 Myelodysplasia syndromes 32
1.2.1 Patterns of acquired genetic variants in MDS 37
1.3 Acute myeloid leukaemia 41
1.3.1 Patterns of acquired genetic variants in AML 45
1.4 Familial myelodysplasia and/or acute myeloid
leukaemia with germline predisposition syndromes 49
1.4.1 Familial platelet disorder with propensity to myeloid
malignancy (FPD/AML) 51
1.4.2 Thrombocytopenia 2 51
1.4.3 Thrombocytopenia 5 52
1.4.4 Familial AML with mutated CEBPA 53
1.4.5 Familial MDS/AML with mutated GATA2 53
1.4.6 Familial aplastic anaemia/MDS with SRP72 54
1.4.7 Bone marrow failure syndromes 54
1.4.7.1 Fanconi anaemia 55
1.4.7.2 Dyskeratosis congenita 56
1.5 Our cohort of families with familial MDS/AML 60
1.6 Aims of the project 63
Material and methods 64
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2.1 Introduction 65
2.2 Materials 65
2.2.1 Patient samples 65
2.2.2 Primers 65
2.2.3 Chemicals and reagents 66
2.3 Methods 67
2.3.1 DNA quantification 67
2.3.2 Polymerase chain reaction – PCR 68
2.3.3 Agarose gel electrophoresis 68
2.3.4 DNA restriction digestion 69
2.3.5 Rolling circle amplification reaction 69
2.3.6 Alkaline gel electrophoresis 71
2.3.7 Southern blotting 71
2.3.8 Hybridisation and chemiluminescence detection 73
2.3.9 Gel extraction and purification 74
2.3.10 Sanger sequencing 74
2.3.11 Analysis of sequencing traces 76
2.3.12 RNA extraction 77
2.3.13 Reverse Transcriptase PCR 77
2.3.14 Monochrome multiplex quantitative PCR 78
2.3.15 Denaturing high-performance liquid chromatography 79
2.3.16 Whole exome sequencing 81
2.3.17 Nextera library preparation (enrichment of exonic
fragments) 81
2.3.18 Tagmentation of genomic DNA and first PCR amplification 83
2.3.19 First hybridisation and first capture 84
2.3.20 Targeted resequencing – design of probes 86
2.3.21 Targeted resequencing library preparation 87
2.3.22 Sequencing of exome library 90
2.3.23 Variant calling 92
2.3.24 Mammalian cell culture 94
2.3.25 Polyacrylamide gel electrophoresis and Western blotting 95
2.3.26 In silico analyses 96
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Variants identified in known disease genes associated
with familial MDS/AML and related disorders 97
3.1 Introduction 98
3.1.1 Genetically uncharacterised familial MDS/AML patients 98
3.2 Results 99
3.2.1 Variants identified in previously known familial MDS/AML
causing genes 100
3.2.1.1 Variants identified in RUNX1 100
3.2.2 Variants identified in genes associated with complex
phenotypes 104
3.2.2.1 Variants identified in GATA2 104
3.2.2.2 Variant identified in TERT 109
3.2.2.3 TERT promoter region and ANKRD26 5’UTR screening in
our cohort of patients by dHPLC 113
3.2.3 Variants identified in genes associated with inherited
syndromes with predisposition to MDS/AML 118
3.2.3.1 Shwachman-Diamond syndrome 118
3.2.3.2 Fanconi anaemia 121
3.2.4 Variants identified in genes associated with other inherited
syndromes 124
3.2.4.1 Wiskott-Aldrich syndrome 124
3.3 Discussion 127
Germline heterozygous DDX41 variants in a subset of
familial myelodysplasia and acute myeloid leukaemia 131
4.1 Introduction 132
4.1.1 Familial myelodysplasia and acute myeloid leukaemia with
germline predisposing variants in DDX41 132
4.1.2 DDX41 – DEAD-box helicase 41 structure and function 133
4.1.3 The role of DExD/H box helicases and DDX41 in the innate
immunity response 136
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4.1.4 Association of DDX41 variants with myeloid neoplasms
and defects in mRNA splicing 141
4.1.5 The role of DDX41 in ribosome biogenesis and in post-
transcriptional regulation of protein translation in cell
growth 149
4.2 Results 153
4.2.1 DDX41 germline variants identified in our cohort 155
4.2.2 Telomere length analysis in patients harbouring DDX41
variants 158
4.3 Discussion 159
RTEL1 variants leading to myelodysplasia and liver
disease 163
5.1 Introduction 164
5.1.1 RTEL1 protein structure and function 164
5.1.2 RTEL1 in homologous recombination 167
5.1.3 RTEL1 in telomere maintenance 170
5.1.4 RTEL1 in human diseases 177
5.1.4.1 RTEL1 germline biallelic variants leading to dyskeratosis
congenita and Hoyeraal-Hreidarsson syndrome 177
5.1.4.2 RTEL1 germline heterozygous variants leading to
pulmonary fibrosis 179
5.2 Results 182
5.2.1 RTEL1 germline variants identified in our cohort 185
5.2.1.1 Patients with biallelic RTEL1 variants 185
5.2.1.2 Patients with heterozygous loss of function RTEL1 variants
189
5.2.1.3 Families with heterozygous variants of unknown
significance and heterozygous likely benign variants 193
5.2.2 Short telomeres and T-circles in distinguishing the
pathogenic status of RTEL1 variants 195
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5.3 Discussion 198
Variants identified in familial MDS/AML candidate
genes 202
6.1 Introduction 203
6.1.1 Genetically uncharacterised familial MDS/AML patients 203
6.2 Results 206
6.2.1 Data analysis 206
6.2.1.1 All MDS/AML families 208
6.2.1.2 Family FML012 217
6.2.1.3 Family FML003 223
6.2.1.4 All MDS/AML families – gene-level and variant-level
metrics combined to assess potential pathogenicity of a
variant 227
6.3 Discussion 235
Discussion 239
7.1 Variants identified in known disease genes associated
with familial MDS/AML and related disorders 240
7.2 Germline heterozygous LoF DDX41 variants in a
subset of familial myelodysplasia and acute myeloid
leukaemia 244
7.3 RTEL1 LoF variants leading to myelodysplasia and
liver disease 247
7.4 Variants identified in familial MDS/AML candidate
genes 250
7.5 Future work 259
7.6 Concluding remarks 261
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