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Although
familial myelodysplasia and acute myeloid leukemia (MDS/AML) is rare, its
true prevalence is likely underestimated due to wide variations in the age of
onset, disease latency and outcome between and within families, with some
mutation carriers remaining asymptomatic into late adulthood. Reduced
penetrance is a notable feature of germline GATA2 p.Thr354Met
pedigrees. In this study, we demonstrate that silencing of the wildtype (WT) GATA2
allele discriminates between symptomatic and asymptomatic carriers and is
linked with allele-specific differences in DNA methylation and H3K4me3
promotor deposition, providing a molecular explanation for the clinical
heterogeneity observed within a GATA2-mutated AML family.
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Follicular lymphoma is an
incurable B cell malignancy1 characterized by the t(14;18) translocation and
mutations affecting the epigenome2,3. Although frequent gene mutations in key
signaling pathways, including JAK-STAT, NOTCH and NF-κB, have also been defined2,3,4,5,6,7,
the spectrum of these mutations typically overlaps with that in the closely
related diffuse large B cell lymphoma (DLBCL)6,7,8,9,10,11,12,13. Using a
combination of discovery exome and extended targeted sequencing, we
identified recurrent somatic mutations in RRAGC uniquely enriched in patients
with follicular lymphoma (17%). More than half of the mutations
preferentially co-occurred with mutations in ATP6V1B2 and ATP6AP1, which
encode components of the vacuolar H+-ATP ATPase (V-ATPase) known to be
necessary for amino acid−induced activation of mTORC1. The RagC variants
increased raptor binding while rendering mTORC1 signaling resistant to amino
acid deprivation. The activating nature of the RRAGC mutations, their
existence in the dominant clone and their stability during disease
progression support their potential as an excellent candidate for therapeutic
targeting.
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ملخص المشاركة:
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Background: While
myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are
considered sporadic hematopoietic stem cell clonal disorders, there are rare
occurrences of familial cases (<5%) where two or more individuals within
the same family are affected. These high-risk examples are characterised by
wide variations in the age of onset, disease latency and outcome between and
within families, making their investigation, follow-up and treatment all the
more challenging. To date,
germline mutations in 11 disease genes have been described, with mutations in the myeloid transcription factor GATA2 representing one of the
best-characterised genetic loci predisposing to inherited hematological
malignancies. We have noted that within GATA2
families, particularly those segregating a germline p.Thr354Met mutation, there is striking evidence of reduced
penetrance. In our example, two
first-degree cousins developed high-risk MDS with monosomy 7 with a third
cousin presented with significant leukopenia (monocytopenia [0.1×109/L]
and neutropenia [0.8×109/L]). This contrasts with the parental
generation who all remain hematologically normal and symptom free into their
mid-late 60s. We therefore set out to understand these differences in
clinical presentation between mutation carriers.
Aim: To
investigate the molecular mechanisms underlying the variable penetrance and
clinical heterogeneity observed in a GATA2-mutated family.
Results:
Targeted deep-sequencing of 33 genes
frequently mutated in MDS/AML revealed a low overall burden of acquired
mutations in the symptomatic carriers with no mutations detected in
asymptomatic family members. It was
noteworthy that an acquired ASXL1 mutation
(p.Gly646TrpfsTer12) was identical in all affected individuals although the variant allele frequency was lower (12%) in
the symptomatic third cousin and remained stable (range 12-6%) over a 4 year
monitoring period. GATA2 expression
was lower in this symptomatic carrier as assessed by quantitative RT-PCR and
strikingly this was associated with monoallelic expression of the mutated GATA2 allele with complete loss of the
wild-type (WT) allele expression. Temporal analysis at yearly intervals
demonstrated reactivation of the WT allele 2 years later, coinciding with a
marked improvement in hematological parameters (normal monocyte count,
neutrophils >1×109/L). These changes in GATA2 expression were not linked to gross changes in methylation,
as assessed by methylation specific PCR and bisulphite sequencing, nor
acquisition of additional mutations in the WT promoter. Instead, we believe
that allele-specific fluctuations in expression are accompanied by changes in
chromatin structure at the promoter. Using a SNP (rs1806462 [C/A])
located in the 5’UTR of GATA2, we assessed allele-specific enrichment of H3K4me3 and H3K27me3
chromatin marks by chromatin immunoprecipitation. Sanger sequencing revealed a significant
enhancement in the deposition of H3K4me3 activating chromatin mark on the mutated allele compared to the WT
allele at diagnosis and this was reversed at later follow-up, correlating
with reactivation of the WT allele expression. There were no discernible
allele-specific differences in the H3K27me3 mark across the phenotypes at
different time-points.
Conclusion: Reduced penetrance amongst germline
mutation carriers is a feature of many families with inherited forms of
MDS/AML and this may be related to the nature of secondary genetic events
acquired in at-risk individuals. In this study, however, we show that changes
in the WT:mutant allele expression ratio as a result of local and
allele-specific changes in chromatin deposition may also influence the
penetrance of the inherited mutation.
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