The Genetics of Familial Leukaemia and Myelodysplasia

PhDBackground & 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

Germline heterozygous DDX41 variants in a subset of familial myelodysplasia and acute myeloid leukemia

The Brazilian National Council for Scientific and Technological Development), Bloodwise, Children with Cancer and MRC (Medical Research Council, UK)

Genome instability is a consequence of transcription deficiency in patients with bone marrow failure harboring biallelic ERCC6L2 variants

Bloodwise Program Grant (14032) and the Medical Research Council Research Grant (MR/P018440)

GATA2 monoallelic expression underlies reduced penetrance in inherited GATA2-mutated MDS/AML.

Saudi Arabian Ministry of Higher Education through a doctoral scholarship awarded to A.F.A.S. and a Bloodwise Programme grant (14032) awarded to J.F., T.V., and I.D

The complex genetic landscape of familial MDS and AML reveals pathogenic germline variants.

The inclusion of familial myeloid malignancies as a separate disease entity in the revised WHO classification has renewed efforts to improve the recognition and management of this group of at risk individuals. Here we report a cohort of 86 acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) families with 49 harboring germline variants in 16 previously defined loci (57%). Whole exome sequencing in a further 37 uncharacterized families (43%) allowed us to rationalize 65 new candidate loci, including genes mutated in rare hematological syndromes (ADA, GP6, IL17RA, PRF1 and SEC23B), reported in prior MDS/AML or inherited bone marrow failure series (DNAH9, NAPRT1 and SH2B3) or variants at novel loci (DHX34) that appear specific to inherited forms of myeloid malignancies. Altogether, our series of MDS/AML families offer novel insights into the etiology of myeloid malignancies and provide a framework to prioritize variants for inclusion into routine diagnostics and patient management

225: The addition of etoposide to Bu16/Cy200 in the conditioning of children with acute myeloid leukemia (AML) undergoing allogeneic stem cell transplantation (SCT) is associated with improved survival

Relapse remains the major concern for children with AML undergoing SCT, and we have previously shown that intensifying the conditioning by adding etoposide at 60 mg/kg and consequently reducing the busulfan (Bu) dose to 12 mg/kg and the cytoxan (Cy) to 90 mg/kg did not result in any significant improvement of the survival; we suggested then that the reduction of Bu/Cy doses necessitated by addition of the high dose of etoposide could have affected the outcome and we hypothesized that adding a lower dose of etoposide and keeping the Bu/Cy at the conventional doses may offer better survival to AML patients undergoing allogeneic SCT. We present here our results using such a protocol. Patients and Methods: From March 2003 until December 2006, 33 patients with AML (24 in CR1, 8 in CR 2) underwent allogeneic SCT and were conditioned with Bu 16 mg/kg po, Cy 200 mg/kg iv plus etoposide 900 mg/m2 iv, median age was 9.6 years. This cohort of patients (group B) was compared with 18 AML patients (17 in CR 1, 1 in CR 2) who underwent SCT from July 93 thru February 96, median age at SCT was 7.25 years, patients were conditioned with only Bu 16 mg/kg po and Cy 200 mg/kg iv (group A). Results: Median days to ANC ≥ 500 × 106/l was 21 days and 15 days in groups A and B respectively, median days to platelet count ≥ 20 × 109/l was 22 days and 26 days in groups A and B respectively (P= NS). The incidence of complications was similar, acute GVHD grade 2 or higher developed in 5% and 9% in groups A and B respectively (P= NS), hemorrhagic cystitis developed in 11% and 15% in groups A and B respectively, no VOD developed in either group. The 4 year overall survival for groups A and B respectively was 50% and 68.2% (P = 0.3) and the 4 year event-free survival for groups A and B respectively was 33% and 68.2% (P = 0.1). Conclusions: The addition of etoposide to Bu16/Cy200 was not associated with increased toxicity, and although it did not reach statistical significance, it does appear to be associated with a better overall and event-free survival. Larger scale studies are advised to further corroborate our findings