Methyl‐CpG binding domain 4, DNA glycosylase (MBD4)‐associated neoplasia syndrome associated with a homozygous missense variant in MBD4: Expansion of an emerging phenotype

Germline biallelic loss-of-function variants in methyl-CpG binding domain 4, DNA glycosylase (MBD4) have recently been associated with a syndrome characterised by early onset haematological malignancy and colonic polyposis (provisionally termed MBD4-associated neoplasia syndrome [MANS]).1, 2 This clinical phenotype is caused by a base excision repair (BER) defect as a result of loss of MBD4 function and the subsequent accumulation of C>T transitions at CpG dinucleotides after spontaneous 5-methylcytosine deamination.2 To date, four patients (from three families) have been identified with MANS harbouring deleterious variants in MBD4 (NM_003925.2) including an in-frame deletion (p.(His567del)), canonical splice site (c.1562-1G>T) and truncating variants (p.(Glu314Argfs*13) and p.(Ser205Thrfs*9)).1, 2 Here, we describe a patient with MANS as a result of a homozygous missense germline variant in MBD4 providing novel insights into the clinicobiological features of this emerging phenotype. A 37-year-old man of Middle Eastern ethnicity and consanguineous parents presented with symptomatic anaemia and pancytopenia. A diagnosis of myelodysplastic syndrome with ring sideroblasts and multilineage dysplasia (MDS-RS-MLD) was made on bone marrow biopsy. His history was significant for the presence of multiple (75) colonic polyps, which had led to total colectomy 5 years previously. No variants were identified in mutY DNA glycosylase (MUTYH) or APC regulator of WNT signaling pathway (APC) at that time. He also had a right-sided vestibular schwannoma resected 3 years before diagnosis with MDS. There was no other relevant personal or family history. The patient was transfusion dependent as a result of MDS-RS-MLD and he underwent a matched unrelated donor allogeneic stem cell transplant (ASCT). Despite initial engraftment at day 28, he became transfusion dependent again at 3 months after ASCT and a bone marrow biopsy showed relapsed MDS (without blast excess) at that time. He was commenced on lenalidomide and azacytidine and had a further bone marrow biopsy (9 months after ASCT), which showed disease progression with a blast excess of 11% and karyotyping demonstrating a monosomy 7 and del(5q). He then progressed rapidly to acute myeloid leukaemia (AML) and despite the addition of venetoclax to azacytidine he had no disease response and care was redirected towards palliation. Whole genome sequencing (WGS) was first performed on DNA extracted from hair follicles in order to investigate possible germline causes for the patient's phenotype (Supplementary Methods). A homozygous missense variant in the catalytic domain of MBD4 was detected – c.1535G>A, p.(Arg512Gln). A different amino acid substitution (Arg512Trp) at this critical residue has been shown experimentally to result in reduced catalytic activity with the loss of an arginine predicted to disrupt protein structure.3 The Arg512Trp and Arg512Gln show a similar degree of predicted deleterious protein effect by commonly accepted in silico predictors (rare exome variant ensemble learner [REVEL] scores for the Trp and Gln substitution of 0.88 and 0.82 respectively). The Arg512Gln has an allele count of seven in the Genome Aggregation Database (gnomAD) (version 2.1.1) with no homozygotes present. The variant was classified as likely pathogenic using the American College of Medical Genetics guidelines for classification of germline variants.4 The patient's parents and sibling were heterozygous carriers of the Arg512Gln variant (Figure 1A). Paired WGS was then performed using DNA extracted from hair as a germline sample and from peripheral blood at time of diagnosis of MDS-RS-MLD as a tumour sample (Supplementary Methods). The global mutation profile from the haematopoietic compartment showed a markedly elevated tumour mutation burden (18 938 somatic single nucleotide variants, 16.88 mutations/Mb) and the vast majority of mutations being C>T transitions occurring at CpG dinucleotides (highest in the context of ACG triplet followed by CCG, GCG then TCG) (Figure 1B,C). Assessment of recurrently mutated genes in haematological malignancy was also performed using unique molecular identifier (UMI)-based targeted sequencing to detect variants in the MDS diagnostic blood sample (Supplementary Methods) and revealed mutations in tet methylcytosine dioxygenase 2 (TET2) and splicing factor 3B subunit 1 (SF3B1) at a variant allele frequency (VAF) of ~25% (Figure 2). In addition, low VAF mutations (0.5%–2%) in DNA methyltransferase 3 alpha (DNMT3A), isocitrate dehydrogenase (NADP(+)) 2 (IDH2) and tumor protein p53 (TP53) were detected at this time point. Sequencing of longitudinal samples from 3, 9 and 12 months post-ASCT revealed continued clonal evolution across all time points with emergence of a dominant clone containing TP53, isocitrate dehydrogenase (NADP(+)) 1 (IDH1), Cbl proto-oncogene (CBL) and DNMT3A mutations, which was then replaced by a tumour compartment dominated by TP53 mutations and wild-type for IDH1 and IDH2 (Figure 2). Three out of the four patients described to date have presented with AML and harboured mutations in DNMT3A and IDH1/IDH2. It is therefore notable that our patient presented in an MDS phase with dominant SF3B1 and TET2 mutations, particularly as variants in SF3B1 are central to the pathogenesis of MDS with ring sideroblasts and the specific variant in our patient (c.1873C>T; p.(Arg625Cys)) is the result of a C>T transition at a CpG dinucleotide. However, low level mutations in DNMT3A and IDH2 were detected at this diagnostic time point before ASCT, possibly representing an early transformation event. Interestingly, during overt transformation of his disease to a more aggressive phase characterised by blast excess post-ASCT, he acquired dominant mutations in DNMT3A and IDH1 (9 months after ASCT, Figure 2) further supporting the hypothesis that this may be a relatively stereotyped pathway to leukaemia in patients with MANS.2 Targeted sequencing (TSO500) was then performed on DNA extracted from the vestibular schwannoma (Supplementary Methods) that showed the same mutational signature that was observed in the haematopoietic compartment as well as two nonsense mutations in NF2, moesin-ezrin-radixin like (MERLIN) tumor suppressor (NF2; NM_000268.3) c.169C>T;p.(Arg57*) and c.1021C>T;p.(Arg341*), both C>T transitions at CpG dinucleotides. Biallelic loss-of-function mutations in NF2 are ubiquitous early molecular events in sporadic vestibular schwannoma.5 In summary, we have a described a patient with MANS as a result of a homozygous germline variant in MBD4 with multiple novel features including: (i) the first missense variant in MBD4 described in this condition, (ii) expansion of the phenotype to include vestibular schwannoma as part of the potential neoplastic spectrum and (iii) the observation of a DNMT3A/IDH1/IDH2-independent development of haematological malignancy prior to leukaemic transformation. Descriptions of more patients with this novel syndrome will contribute to understanding the full clinical spectrum of this emerging phenotype. The authors would like to thank Maddie Riewoldt's Vision (MRV0023), the Snowdome Foundation and the Wilson Centre for Blood Cancer Genomics for their funding support. The authors also thank the Genomics Core Facility and Genomics Platform Group (University of Melbourne Centre for Cancer Research) for whole genome sequencing and analysis support. Piers Blombery conceived of the project and designed the study and provided clinical care; Piers Blombery, Georgina L. Ryland, Ain Roesley, Ella R. Thompson, Zornitza Stark, Meg Wall, Sean M. Grimmond analysed genomic data; Piers Blombery, Anna Jarmolowicz, Lucy C. Fox, Shyam Panicker, Fiona Kwok provided clinical care to the patient; and all authors reviewed the data and contributed to critical revision of the manuscript. Data S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.