Germline loss of MBD4 predisposes to leukaemia due to a mutagenic cascade driven by 5mC
Summary (2 min read)
Affiliation
- These authors contributed equally to this work 9 MBD4-deficiency was also detected, rarely, in sporadic cancers, which display the same mutational signature.
- Both cases exhibited an elevated mutation rate and strong enrichment for CG>TG mutations (Fig. 1d, Extended Data Fig. 1a).
- This shift in functional activity – the expansion of DNMT3Amutant clones – increases the likelihood that cells with biallelic DNMT3A mutations will emerge, which appears to be key for initiating AML in MBD4-deficient patients.
- The authors confirmed that recombinant DNMT3A enhances TDG glycosylase activity in vitro (Fig. 4a), but had no impact on MBD4 glycosylase activity (Extended Data Fig. 7).
Contributions
- All authors discussed the results and agree with the conclusions presented.
- C, Relative mutation rate in different genomic features per Mb of CG dinucleotides (CG corrected), or corrected for methylation status in CD34+ cells (5mC corrected).
- Each coloured area is proportional to the representation of the clone and vertical lines indicate sampling points31.
- B, Schematic representation of the repair pathways governing T:G mismatch repair and the combined influence of germline mutations in MBD4 and somatic mutations in DNMT3A (at top) in AML.
Extended Data References – pg. 20-21
- Supplementary Information Somatic mutations detected in MBD4-deficient AML at diagnosis (hg19).
- A quality score is provided , variants with a score >0.5 were used for mutation signature analysis.
AML cases
- Sanger sequencing traces were generated from cloned PCR products after amplification from DNA (top).
- B, A schematic of the MBD4 gene is shown at top together with the position of two candidate loss-of-function variants that impact splice sites.
- Sites with mutations were typically fully methylated in the control sample.
- Individual values are plotted (n=2) and the bar shows the mean.
- The relative mutation rate was calculated per bin based on CG or 5mCG abundance (as in a).
Clinical synopsis
- The AML was negative for NPM1, FLT3 and CEBPa mutations.
- She had induction chemotherapy (high dose cytarabine, idarubicin and etoposide) and achieved complete morphologic and cytogenetic remission.
- Bone marrow examination 5 weeks post allogeneic HSCT showed complete morphologic and cytogenetic remission; and full donor chimerism.
- Relapsed AML (of WEHI-AML-1 origin) occurred 11 weeks post allogeneic HSCT.
Methods
- Patient characteristics and sample collection EMC-AML-1, WEHI-AML-1 and WEHI-AML-2 were diagnosed with AML and treated with combination chemotherapy as per the protocols at their respective institutions [see Clinical Synopsis].
- They gave informed consent according to the Declaration of Helsinki for participation in research and for collection of samples over the course of their treatment.
- DNA libraries were quantified and used for both whole genome sequencing and whole exome sequencing.
- Reduced representation bisulfite sequencing (RRBS) For WEHI-AML-1 and WEHI-AML-2, between 75 to 100 ng of DNA was used to construct RRBS libraries using the Ovation RRBS Methyl-Seq System (NuGEN, San Carlos, CA, USA).
- DNA was restriction enzyme digested using Mspl followed by ligation with indexed adaptors.
RNA sequencing
- For WEHI-AML-1 and WEHI-AML-2, total RNA was extracted using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) as per manufacturer’s instructions.
- As the mutations occurred almost exclusively in a CG context, the rate of CG>TG mutations per CG was calculated for each genomic feature.
- Transcriptional strand and expression level: Transcriptional strand bias analysis was performed by determining the template and non-template strands per gene as reported in Ensembl v7513.
- Libraries were generated as per manufacturer’s instructions and the sequencing was performed on a MiSeq.
Site-directed mutagenesis and cloning
- And anti-sense 5’- TTGTATTTCCAGGGCGGCACGACTGGGCTGGAGAGTCT-3’. QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) was used to generate the DNMT3A and MBD4 mutants.
- Proteins were verified by SDS-PAGE using a NuPage Novex 4-12% Bis-Tris Protein Gel run in a Bis-Tris XCell SureLock™ Mini-Cell system (Thermo Fisher Scientific, Waltham, MA, USA) with 1x MOPS at 200V for 90 minutes.
- MBD4 and TDG glycosylase activity assays MBD4 and TDG glycosylase activity assays were performed as described (Hashimoto et al., NAR, 2012).
Data availability
- Sequencing data from WEHI-AML-1 and WEHI-AML-2 have been deposited at the European Genome Phenome Archive (EGA) [EGAS00001002581].
- The data are available for ethically approved research into haematological malignancy upon completion of a data transfer agreement.
- Sequencing data from EMC-AML-1 were sourced from the dbGaP under accession phs001027.
- TCGA data were downloaded from the GDC Data Commons.
- Code to reproduce the figures and data are made available through GitHub (https://github.com/MathijsSanders/AML-RoaMeR).
Extended Data References
- Distinct evolution and dynamics of epigenetic and genetic heterogeneity in acute myeloid leukemia.
- The UCSC Genome Browser database: 2017 update.
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Citations
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23 citations
Cites background from "Germline loss of MBD4 predisposes t..."
...numbers of C > T mutations (associated with signature 1, following the deamination of methylated cytosines), researchers uncovered a germline mutation in the DNA glycosylase MBD4 that may predispose cells to subsequently developing certain driver mutations that accelerate oncogenesis (Sanders et al. 2017)....
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...…of C > T mutations (associated with signature 1, following the deamination of methylated cytosines), researchers uncovered a germline mutation in the DNA glycosylase MBD4 that may predispose cells to subsequently developing certain driver mutations that accelerate oncogenesis (Sanders et al. 2017)....
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22 citations
Cites background from "Germline loss of MBD4 predisposes t..."
...SuperFreq was designed to detect and track somatic mutations in exomes, and it has been applied to study breast cancer metastasis [2, 21], lung cancer xenografts [22], gastric cancer organoids [23], and myeloid leukaemia [24]....
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References
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