1
Title
Germline loss of MBD4 predisposes to leukaemia due to a mutagenic cascade
driven by 5mC
Authors
Mathijs A. Sanders
1,8
, Edward Chew
2,3,4,5,8
, Christoffer Flensburg
2,4
, Annelieke
Zeilemaker
1
, Sarah E. Miller
2
, Adil S. al Hinai
1,6
, Ashish Bajel
3,5
, Bram Luiken
1
,
Melissa Rijken
1
, Tamara Mclennan
7
, Remco M. Hoogenboezem
1
, François G.
Kavelaars
1
, Marnie E. Blewitt
4,7
, Eric M. Bindels
1
, Warren S. Alexander
2,4
, Bob
Löwenberg
1
, Andrew W. Roberts
2,3,4,5
, Peter J.M. Valk
1,9
*, Ian J. Majewski
2,4,9
*
Affiliation
1
Department of Hematology, Erasmus University Medical Center, Rotterdam, The
Netherlands
2
Division of Cancer and Haematology, The Walter and Eliza Hall Institute of Medical
Research, Parkville, Australia
3
Department of Clinical Haematology and Bone Marrow Transplantation, Royal
Melbourne Hospital, Parkville, Australia
4
Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne,
Parkville, Australia
5
Victorian Comprehensive Cancer Centre, Parkville, Australia
6
National Genetic Center, Royal Hospital, Ministry of Health, Sultanate of Oman
7
Division of Molecular Medicine, The Walter and Eliza Hall Institute of Medical
Research, Parkville, Australia
8
These authors contributed equally to this work
9
These authors jointly directed this work
* Correspondence
Peter J.M. Valk
Department of Hematology
Erasmus University Medical Center
Em: p.valk@erasmusmc.nl
Ian J. Majewski
Cancer and Haematology Division
The Walter and Eliza Hall Institute of Medical Research
Em: majewski@wehi.edu.au
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2
Cytosine methylation is essential for normal mammalian development, yet also
provides a major mutagenic stimulus. Methylcytosine (5mC) is prone to spontaneous
deamination, which introduces cytosine to thymine transition mutations (C>T) upon
replication
1
. Cells endure hundreds of 5mC deamination events each day and an
intricate repair network is engaged to restrict this damage. Central to this network
are the DNA glycosylases MBD4
2
and TDG
3,4
, which recognise T:G mispairing and
initiate base excision repair (BER). Here we describe a novel cancer predisposition
syndrome resulting from germline biallelic inactivation of MBD4 that leads to the
development of acute myeloid leukaemia (AML). These leukaemias have an
extremely high burden of C>T mutations, specifically in the context of methylated CG
dinucleotides (CG>TG). This dependence on 5mC as a source of mutations may
explain the remarkable observation that MBD4-deficient AMLs share a common set
of driver mutations, including biallelic mutations in DNMT3A and hotspot mutations in
IDH1/IDH2. By assessing serial samples taken over the course of treatment, we
highlight a critical interaction with somatic mutations in DNMT3A that accelerates
leukaemogenesis and accounts for the conserved path to AML. MBD4-deficiency
was also detected, rarely, in sporadic cancers, which display the same mutational
signature. Collectively these cancers provide a model of 5mC-dependent
hypermutation and reveal factors that shape its mutagenic influence.
We identified three patients with AML, including two siblings, that were distinctive
because of their early age of onset (all <35 years old) and an extremely high
mutational burden (~33-fold above what is typical for AML) (Fig. 1a, Clinical
Synopsis). Virtually all of the somatic mutations identified were C>T in the context of
a CG dinucleotide (>95% of SNVs) (Fig. 1b, Extended Data Fig. 1). This differs
markedly from the distribution of C>T mutations in AML generally and is more
refined than the mutational signature ascribed to ageing, which includes a strong
contribution from 5mC deamination
5
. All three cases carried rare germline loss-of-
function variants in the gene encoding the DNA glycosylase MBD4
2
(Fig. 1c,
Extended Data Table 1). Case EMC-AML-1 carried a homozygous MBD4 in-frame
deletion of Histidine 567 (His567) in the glycosylase domain. An in vitro glycosylase
assay confirmed that loss of His567 results in a catalytically inactive MBD4 protein
(Fig. 1c). The siblings (WEHI-AML-1, WEHI-AML-2) were compound heterozygotes
with a frameshift in exon 3 and a variant that disrupts the splice acceptor of exon 7
(Fig. 1c, Extended Data Table 1). Analysis of the MBD4 mRNA allowed for phasing
of the variants to distinct alleles and confirmed aberrant splicing that excludes exon 7
and disrupts the glycosylase domain (Extended Data Fig. 2). MBD4 has not
previously been associated with haematological malignancy, but somatic mutations
have been detected in sporadic colon cancers with mismatch repair (MMR)
deficiency
6,7
. Two patients (EMC-AML-1, WEHI-AML-2) also had colorectal polyps, a
common manifestation of DNA repair defects, including those associated with loss of
BER components MUTYH
8-10
and NTHL1
11
.
We accessed large cancer databases to explore the link between MBD4-deficiency
and the distinctive CG>TG signature. Analysis of the Cancer Genome Atlas (TCGA)
identified nine cases, from 10,683 total, that carried germline loss-of-function
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variants in MBD4 (Fig. 1c, Extended Data Table 1). In two cases, a uveal
melanoma (TCGA-UVM-1) and a glioblastoma multiforme (TCGA-GBM-1), splice
site mutations were accompanied by loss of the wildtype MBD4 allele due to somatic
copy number alterations (Extended Data Fig. 3a). Analysis of RNA sequencing from
both tumours confirmed aberrant splicing of MBD4, predicted to result in protein
truncation and loss of function (Extended Data Fig. 3b). Both cases exhibited an
elevated mutation rate and strong enrichment for CG>TG mutations (Fig. 1d,
Extended Data Fig. 1a). This signature was also observed in a glioma cell line,
SW1783, that carries a homozygous truncating variant in MBD4 at Leu563
(Extended Data Fig. 1a). The cancers that retained a wildtype allele did not display
a prominent CG>TG signature (Fig. 1d). These results suggest both alleles of MBD4
must be inactivated to block its repair activity, which is consistent with other BER-
associated cancer syndromes
8,11
. Analysis of a larger cohort will be required to
determine whether heterozygous loss of MBD4 predisposes to cancer.
Whole genome sequencing and methylation profiling were performed to refine the
mutational signature associated with MBD4-deficiency in AML. While MBD4 is
known to interact with the MMR pathway
12
, MBD4-deficienct leukaemias were
largely devoid of small insertions and deletions, suggesting MMR remains intact.
Overall, >15,000 substitution mutations were identified in each AML genome, of
which >90% were CG>TG (Fig. 2a, Extended Data Fig. 1b). The proportion of
mutations was higher in the context of the ACG triplet and lower in the context of
TCG, with CCG and GCG being intermediate. This difference remained after
correction for trimer abundance and methylation status (Fig. 2b), and was found to
be significant in the exome data from the five MBD4-deficient cancers (p= 0.007937,
Mann-Whitney U test) (Extended Data Fig. 1). The ACA trimer was the most
commonly mutated site outside of a CG context, and this matches the most common
site of non-CG methylation
13
. The mutation rate for a given region was linked to 5mC
abundance. Sparsely methylated regions, such as promoters and CG islands, were
rarely mutated (Fig. 2c). Correcting for 5mC abundance revealed a consistent
mutation rate across different genomic features (Fig. 2c). Reduced representation
bisulfite sequencing (RRBS) confirmed that >95% of CG sites mutated in the AML
were fully methylated in matched normal bone marrow available for two cases (Fig.
2d). Assessment of the mutated sites in each AML directly revealed ~50%
methylation, indicating the non-mutated CG site on the alternate allele was
methylated (Fig. 2d). Similar results were obtained when we assessed sites mutated
in the MBD4-deficient glioblastoma (Extended Data Fig. 4). We next assessed the
influence of genetic and epigenetic features known to influence mutation rate
14
.
Extending the analysis of sequence context to include one base either side of the
CG identified higher mutation rates in the context of a 3’ cytosine (NCGC), with the
highest rate at ACGC (Fig. 2e). The relative mutation rate was not influenced by the
transcriptional strand (Extended Data Fig. 5a), but was higher in late replicating
regions (Fig. 2f) and at lowly expressed genes (Extended Data Fig. 5b).
Collectively these results suggest that while 5mC is the dominant factor contributing
to the mutation rate, the local sequence context, replication timing and expression
status also contribute. The differences between tetramers and enrichment in late
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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replicating regions were also evident in rare germline CG>TG SNPs from the
gnomAD database
15
, indicating this phenomenon is not restricted to cancer
(Extended Data Fig. 5c).
The three cases with germline MBD4-deficiency shared a common path to AML.
They acquired biallelic DNMT3A mutations and IDH1/IDH2 hotspot mutations, all of
which were CG>TG (Fig. 3). Biallelic DNMT3A mutations are uncommon in AML,
affecting ~3% of patients in TCGA-AML, and when considering they also have
coincident IDH1/IDH2 mutations, it is highly unlikely that these three individuals
share this pattern of driver mutations by chance. These mutations impact 5mC at
multiple levels – deposition (DNMT3A), removal (IDH1/IDH2) and repair (MBD4) –
and this convergence suggests that modulating DNA methylation is central to AML
pathogenesis in MBD4-deficient cases. Analysis of sequential bone marrow biopsies
taken during treatment and single cell genotyping allowed us to refine the order of
somatic mutation acquisition in two cases (EMC-AML-1, WEHI-AML-1) (Fig. 3a-b,
Extended Data Fig. 6). DNMT3A mutations present in the AML at diagnosis were
also detected in non-malignant bone marrow populations in both cases, indicating
that these mutations are among the first acquired. Mutations in DNMT3A enhance
the self-renewal capacity of haematopoietic stem cells (HSCs) and are associated
with age-related clonal haematopoiesis
16-19
. In both patients, marked expansion of
clones carrying DNMT3A mutations occurred with treatment (Fig. 3a-b), suggesting
a strong advantage over normal HSCs. EMC-AML-1 experienced multiple clonal
outgrowths, with nine distinct DNMT3A mutations, and repeated selection of clones
with biallelic mutations. This shift in functional activity – the expansion of DNMT3A-
mutant clones – increases the likelihood that cells with biallelic DNMT3A mutations
will emerge, which appears to be key for initiating AML in MBD4-deficient patients.
There is a marked discrepancy between the substantial mutation burden in MBD4-
deficient AMLs and the modest 2-3 fold increase in mutation rate in MBD4-deficient
mice
20,21
. It is unclear whether this difference is a reflection of longer disease latency
in humans, as compared to mice, or whether somatic mutations in the AML further
compromise DNA repair. Mutations in DNMT3A and IDH1/IDH2 have been
associated with altered DNA repair in model systems
22,23
. It also remains unclear
why TDG, a glycosylase with overlapping substrate specificity, does not compensate
for MBD4 loss. One possible explanation stems from the observation that
DNMT3A/B can directly stimulate TDG glycosylase activity
24,25
. We confirmed that
recombinant DNMT3A enhances TDG glycosylase activity in vitro (Fig. 4a), but had
no impact on MBD4 glycosylase activity (Extended Data Fig. 7). Mutant forms of
DNMT3A showed weaker stimulation, and even inhibit TDG at higher concentrations
(Fig. 4a). We propose a model for AML pathogenesis whereby inhibition of DNMT3A
contributes in two ways: loss of one allele enables expansion of a premalignant
clone, then acquisition of a second DNMT3A mutation increases the CG>TG
mutation rate due to impaired TDG activity (Fig. 4b). Supporting this model, the
premalignant clone identified in WEHI-AML-1, which had a monoallelic DNMT3A
mutation, did not carry additional mutations that would suggest an elevated mutation
rate. The sporadic cancers that became MBD4-deficient (TCGA-UVM-1 and TCGA-
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GBM-1) did not acquire mutations in DNMT3A or IDH1/IDH2, which may indicate
that this interaction is specific to the haematopoietic compartment.
The last five years have seen a concerted effort to define mutational processes that
shape the cancer genome
5
. Deamination of 5mC is the most common source of
somatic mutations and this damage continues to accumulate with age
26
. Our results
highlight the important role for MBD4 in safeguarding against damage wrought by
5mC deamination. One manifestation of this damage is clonal haematopoiesis, a
phenomenon typically observed in people >70 years of age. Individuals with biallelic
loss of MBD4 in the germline sustain high levels of damage from 5mC deamination
and experience clonal expansions decades earlier, which eventually progress to
AML. There are more than 40 million 5mC residues in the genome, yet these
individuals develop the same type of cancer – AML – with a common set of driver
mutations. Our results indicate this convergence results from the combination of a
highly restricted mutational signature, which accesses a select set of driver genes,
and the dual role of DNMT3A, which regulates HSC function and directly contributes
to DNA repair. This interaction between mutational process, driver landscape and
stem cell biology has broad implications, and may explain the tissue restricted
pattern of disease in this and other cancer predisposition syndromes.
Acknowledgements
The authors would like to thank Simon He, Anita Rijneveld, Kirsten van Lom and
Kirsten Gussinklo for providing clinical information and reviewing samples; Meaghan
Wall for assistance with cytogenetics; Naomi Sprigg for assistance with sample
collection; Elwin Rombouts for assistance with single cell sorting; Hideharu
Hashimoto and Xiaodong Cheng for the TDG expression vector; Sari van Rossum
and Joyce Lebbink for assistance with recombinant protein isolation; the
Australasian Leukaemia and Lymphoma Group for access to clinical samples; and
Stephen Wilcox for technical assistance with sequencing. Additional sequencing was
performed at The Australian Genome Research Facility (Melbourne, Australia) and
the Kinghorn Centre for Clinical Genomics (Sydney, Australia).! Sean Grimmond,
Jason Wong, Oliver Sieber, Alicia Oshlack and Stephen Nutt provided valuable
feedback on the manuscript.
!
This work was made possible through support from the Australian National Health
and Medical Research Council (NHMRC) (Program Grant 1113577, to W.S.A and
A.W.R), an Independent Research Institutes Infrastructure Support Scheme Grant
(9000220), a Victorian State Government Operational Infrastructure Support Grant,
the Netherlands Organisation for Scientific Research (NWO) and the Center for
Translational Molecular Medicine (CTMM). M.A.S is supported by a grant from
CTMM (GR03O-102) and a Rubicon fellowship from NWO (019.153LW.038), E.C. is
a recipient of a PhD scholarship from the Leukaemia Foundation of Australia, A.H. is
a recipient of a PhD scholarship from the Ministry of Health - Sultanate of Oman,
M.E.B is supported by the Bellberry-Viertel fellowship, W.S.A and A.W.R are
supported by fellowships from NHMRC (1058344 and 1079560, respectively), and
I.J.M. is supported by the Victorian Cancer Agency.! We wish to acknowledge the
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted November 1, 2017. ; https://doi.org/10.1101/180588doi: bioRxiv preprint