부산대학교 도서관

Background & Aim: While the majority of myelodysplasia and acute myeloid leukaemia (MDS/AML) cases are sporadic, rare familial predisposition syndromes have been delineated and are regarded a separate disease entity in the 2016 WHO classification system. Germline mutations in 14 disease genes have been uncovered thus far, with GATA2 representing one of the key transcriptional regulators commonly mutated in inherited leukaemias. The rarity of these familial cases opens the door to fundamental questions in biology, one of which is the phenomenon of reduced penetrance posing a clinical challenge particularly when identifying "silent" mutation carriers for genetic screening and exclusion as potential stem cell transplant donors. We have noted that this is indeed a feature within certain GATA2-mutated families, especially those carrying germline missense mutations such as (p.Thr354Met). In our example, two first-degree cousins developed MDS/AML with monosomy 7 while a third cousin presented with significant monocytopenia and neutropenia. This contrasted with the parental generation mutation carriers who all remain symptom free into their mid-late 60s. This thesis therefore sets out to investigate the molecular mechanisms underlying the reduced penetrance and clinical heterogeneity observed within a GATA2-mutated family with a view of identifying molecular features that distinguish between these two groups of mutation carriers. Results: Deep targeted sequencing of 33 genes frequently mutated in MDS/AML revealed acquisition of somatic ASXL1 mutation (p.Gly646TrpfsTer12) in all affected cousins with no mutations detected in asymptomatic family members. It was noteworthy that the variant allele frequency was lower (12%) in the third cousin symptomatic carrier and remained stable (range 12-6%) over a 6-year monitoring period. Total GATA2 expression was lower in the symptomatic compared with asymptomatic carriers as assessed by RT-qPCR and remarkably this was associated with monoallelic expression favouring the mutant GATA2 allele with loss of the wild-type (WT) allele expression. Temporal analysis of the symptomatic carrier over a 6-year disease period demonstrated a reactivation of the WT allele expression 3 years later, coinciding with a persistent improvement in haematological parameters. We believe these allele-specific changes in GATA2 expression are driven by dynamic epigenetic reprogramming that include changes in DNA methylation and chromatin mark deposition. Using a SNP (rs1806462 [C/A]) that generates/removes a CpG dinucleotide within GATA2 promoter region, we first assessed allele-specific differences in DNA methylation by bisulphite sequencing. This demonstrated a significant increase in promoter methylation in the WT allele that returned to normal levels at later time-points. We then assessed allele-specific deposition of H3K4me3 and H3K27me3 chromatin marks by chromatin immunoprecipitation (ChIP). Sanger sequencing revealed a significant enrichment in the deposition of H3K4me3 activating mark on the mutant allele at diagnosis that was reversed at later follow-up, correlating with reactivation of the WT allele expression. Conclusion: Reduced penetrance is a feature of many families with inherited forms of MDS/AML which may be governed by the acquisition of additional co-operating mutations (e.g. ASXL1). In this thesis, however, we show that changes in the WT:mutant GATA2 allele expression ratio as a result of local and allele-specific changes in DNA methylation and chromatin mark deposition may also influence the penetrance of the germline mutation, adding another layer of complexity to the (epi)genetic basis of familial MDS/AML.