AML is the most common form of sporadic leukemia in adults 1
while MDS is a clonal disorder of hematopoietic stem cells characterized by ineffective hematopoiesis, with a tendency to progress to AML2
. The study of families predisposed to particular malignancies is a successful strategy for discovering causative oncogenes and tumour suppressor genes (TSG). While rare, dozens of families developing non-syndromic forms of MDS and AML (i.e.
lacking other systemic manifestations) have been described. To date, only two MDS/AML predisposition genes have been recognized: Runt-related transcription factor 1 (RUNX1
) and CCAAT-enhancer binding protein α (CEBPA
) (reviewed in3
Here we report a highly specific p.Thr354Met heritable mutation in GATA2 co-segregating with with early onset MDS/AML in three families. We also report a family with MDS with a 3 bp heritable deletion in the GATA2 gene (p.Thr355del) deleting the second threonine in this sequence.
We determined the genomic DNA sequence of all RefSeq exons in 50 candidate genes (Supplementary Table 1
) from patients representing five pedigrees with predisposition to MDS/AML, prescreened for absence of RUNX1
germline mutations. In three families, there was an identical heritable heterozygous variation in the transcription factor GATA2
, c.1061C>T (p.Thr354Met) (, Supplementary Fig. 1
). In all three families p.Thr354Met segregated with the disease for the samples tested, and no family members had AML or MDS who did not also carry p.Thr354Met (, Supplementary table 2
). There were also members in each family who carried this variant but were unaffected (Pedigree 1: III-5 and III-8; Pedigree 2: II-6; Pedigree 3: III-9).
Identification of novel germline p.Thr354Met and p.Thr355del variants in the highly conserved zinc finger 2 domain of GATA2 that is associated with MDS-AML
We recently identified a fourth family in which a father and son, both affected by MDS, shared a heterozygous heritable deletion of 3 bp in GATA2
(c.1063-1065delACA) resulting in p.Thr355del (, Supplementary Fig. 1
). This codon is adjacent to the codon mutated in the first three families, and encodes the second of the five consecutive threonines.
p.Thr354 and p.Thr355del are the first two of five consecutive threonine residues in a highly conserved region of the GATA2 protein (Supplementary Fig. 1b
) encoding zinc finger 2 (ZF2), which is involved in DNA binding, homodimerization and interaction with transcription factor PU.14,5
. PolyPhen-2 predicts that p.Thr354Met and p.Thr355del are likely to affect GATA2 function. Somatic mutations in ZF2 of GATA2 have also been reported during chronic myeloid leukaemia (CML) blast crisis (BC) (p.Leu359Val, p.Ala341_Gly346del)6
and recently in ZF1 and ZF2 in AML-M57
(). Somatic mutations in the corresponding ZF2 of the related protein family member GATA3 are found in breast cancer8
High resolution melt (HRM) analysis did not detect p.Thr354Met, p.Thr355del or other variants in exon 5 of GATA2
in 695 non-leukemic, ethnically-matched normal controls (i.e.
1390 chromosomes) (Supplementary Fig. 2a
, Supplementary Note
). Thus it is improbable that these variants represent rare polymorphisms. These variants were also not present in dbSNP132 or 1000 Genomes Project (January, 2011; URLs). Together with the disease segregation data, these results indicate that the GATA2 p.Thr354Met and p.Thr355del variants are the predisposing mutation in these families with familial MDS/AML.
A distinguishing feature of our families with GATA2
mutation was a lack of apparent “accessory” phenotype inside or outside the hematopoietic system, akin to the thrombocytopenia and eosinophilia seen in AML-predisposed families due to RUNX1
. In all 4 families, the GATA2
mutations were associated with early-onset MDS and/or AML displaying highly penetrant autosomal dominant inheritance and resulted in a poor outcome unless successfully transplanted (e.g.
Pedigree 1: age of death from AML – 10 – 50 years; 2 individuals of age 58 and 62 years have mutation but no disease; all other siblings without the mutation are alive or have lived beyond 53 years) (Supplementary Table 2 and 3
, Supplementary Note
). For Pedigrees 1 and 311,12
, the presentation varied with some displaying protracted MDS and others acute onset; the FAB subtype and karyotypic features of AML varied. In Pedigree 4, MDS was first diagnosed at age 13 in the son, who was treated with allogeneic bone marrow transplant at age 15; MDS was later diagnosed at age 53 in the father, who underwent allogeneic bone marrow transplantation.
coding variations were not found in samples from another 8 families with multiple cases of AML, or in another 27 families with multiple occurrences of various lymphoid malignancies (11 NHL, 5 HL, 3 ALL, 7 CLL, 1 Multiple myeloma families). Also, no mutations were detected in GATA2
in 15 hematopoietic cell lines (Supplementary Table 4
). No sequence variations were detected in the entire GATA2
coding region of 268 sporadic AML patient sample DNAs except a single c.182C>T (p.Ala61Val) variant in exon 2 (Supplementary Fig. 2c
), which was assessed to be benign using PolyPhen-2 (URLs). Together, this suggests that point mutations and small indels in the GATA2
coding sequence are not frequent in sporadic AML.
Haplotype mapping using 8 informative single nucleotide polymorphisms (SNPs) within and surrounding the GATA2
gene demonstrated that the c.1061C>T (p.Thr354Met) mutation segregated within two distinct haplotypes (Supplementary Table 5
) indicating that this mutation has arisen at least twice among the three families in which it is found.
GATA2 is a DNA-binding transcription factor which localizes predominantly to the nucleus. We generated cDNAs for the p.Thr354Met and p.Thr355del mutant GATA2 proteins and the acquired CML BC p.Leu359Val mutant6
. Wildtype (WT) and mutant proteins expressed at comparable levels when transiently expressed in HEK293 fibroblasts () and when induced to express from a 4HT-responsive dual vector lentivirus system in stably transduced HL-60 promyelocytes (Supplementary Fig. 3a
). Mutant proteins were expressed at comparable levels to wildtype (WT) GATA2 when transiently introduced into WT and mutant proteins appropriately localized to the nucleus (, Supplementary Fig. 3b
). However, the p.Thr354Met mutation dramatically reduced the ability of GATA2 to bind its consensus WGATAR DNA motif while p.Thr355del almost completely ablated DNA binding (, Supplementary Fig. 3c
Subcellular localisation and DNA binding properties of GATA2 WT and mutants
Molecular modeling of GATA2 ZF2 (Supplementary Fig. 4
, Supplementary Note
) demonstrated that the p.Thr354 residue does not contact DNA, but rather makes polar contact with adjacent threonines, and via its amino group with p.Cys349 which coordinates the zinc atom. Replacement of p.Thr354 with the bulky methionine moiety is predicted to alter the overall structure of this zinc finger by affecting zinc contacts. This may explain reduced binding of p.Thr354Met to DNA (, Supplementary Fig. 3c
). In contrast, p.Thr355del shortens the conserved threonine string, likely impacting the orientation and position of p.Leu359, which directly contacts DNA. These observations likely explain the almost-complete ablation of DNA binding.
Luciferase reporter assay experiments show that GATA2 p.Thr354Met and p.Thr355del had significantly reduced transactivation ability compared to WT on known GATA2
responsive enhancers (RUNX1
) and the LYL1
promoter (). Experiments mixing WT with p.Thr354Met or p.Thr355del at a 1:1 ratio, mimicking heterozygosity, demonstrated a dominant negative effect of the mutants over WT transcription activation in multiple systems (, Supplementary Fig. 5
). Interestingly, WT and PU.1 transactivated the CSF1R
) promoter 2.4 and 2.5-fold, respectively, but together synergized to induce 18-fold (). While p.Leu359Val was similar to WT GATA2, p.Thr354Met and p.Thr355del gave dramatically reduced induction alone (1.5- and 0.9-fold) or with PU.1 (7- and 9-fold, respectively, compared to 18-fold with WT). p.Thr354Met and p.Thr355del also displayed dominant negative activity with transactivation by WT GATA2 reduced to 9- and 10-fold, respectively in the presence of these mutants (only marginally above the 7- and 9-fold with mutants alone). Hence, p.Thr354Met and p.Thr355del perturb the transactivation ability of GATA2, presumably by disrupting association with PU.1 or other interacting transcription factors, and are likely impact expression of downstream targets. Interestingly, while WT GATA2 displayed different responses in HEK293 versus Cos-7 cells on the RUNX1
enhancer (activating versus repressing, respectively), p.Thr354Met displayed loss-of-function activity in both cell types (Supplementary Fig. 6
). Thus, on multiple GATA responsive elements, p.Thr354Met and p.Thr355del show loss-of-function and also dominant negative effects.
p.Thr354Met and p.Thr355del cause altered transactivation via target GATA2 response elements
HL-60 promyelocytes differentiate into granulocytes upon exposure to all-trans
retinoic acid (ATRA), resulting in upregulation of CD11b, cessation of proliferation and subsequent promotion of apoptosis (). When expressed at equivalent levels under non-differentiating conditions, unlike WT and p.Leu359Val which inhibited proliferation and promoted apoptosis, p.Thr354Met and p.Thr355del acted as loss-of-function mutants (). However, in the presence of ATRA, p.Thr354Met alone enabled cell proliferation/survival (), while simultaneously inhibiting differentiation and apoptosis (, Supplementary Fig. 7
). p.Thr355del appeared to be a null mutant under these conditions.
p.Thr354Met inhibits differentiation and apoptosis while allowing accumulation of cells in the presence of ATRA-induced differentiation
In order to better understand the effects of the GATA2 mutants on gene expression, microarray analysis was performed to compare global gene expression in HL-60 cells expressing WT GATA2 and the three GATA2 mutants (Supplementary Table 6
and Supplementary Fig. 8
). The data clearly showed that p.Thr355del and p.Thr354Met are almost total loss-of-function mutants (Supplementary Fig. 8
). Note that, p.Leu359Val exhibits gain-of-function (1,253 newly regulated genes compared to WT GATA2) and partial loss-of-function (457 genes no longer regulated) while retaining 786 genes commonly regulated. These results are consistent with EMSA-western blot and transactivation assays. Further bioinformatics analysis indicated that MYC
may be among key target which is repressed by GATA2 WT but not p.Thr354Met and p.Thr355del (Supplementary Table 6, Supplementary Note
Interestingly, recurrent p.Leu359Val mutation in ZF2 of GATA2 was reported in 8/85 cases of CML BC6
, a disease often phenotypically indistinguishable from AML. As shown in .Thr354Met is situated between the deleted residues (p.Ala341_Gly346del), also observed in CML BC, and the p.Leu359 residue. p.Leu359 contacts DNA at the guanine residue of the WGATAR consensus motif and based on in vitro
DNA binding and transactivation assays, p.Leu359Val has previously been reported to be a gain-of-function mutation while p.Ala341_Gly346del appears to be a partial loss-of-function mutation. p.Met354 or deletion of p.Thr355 may affect overall ZF structure (Supplementary Fig. 4
) although we cannot exclude disrupted heterodimerization with GATA2’s interacting partners of GATA2 (Supplementary Table 7
). We speculate that aberrant protein partnerships may explain dominant negative activity and adversely influences expression of genes critical to myelopoiesis.
The MDS/AML observed within these families is clinically heterogeneous, and demonstrate a variety of somatic chromosomal abnormalities, including monosomy 7, trisomy 8, and trisomy 21 (Supplementary Table 2
). As such it is similar to familial MDS/AML with monosomy 73,13
. Moreover, GATA2
mutations were not detected in 8 MDS/AML families in which RUNX1
mutations were excluded and 27 families presenting with lymphoid malignancy. Mutations at this position within ZF2 are likely to initiate an exclusively myeloid pathway of oncogenesis in which subsequent gene-specific, somatically acquired mutations probably define the particular type of disease that ultimately arises.
The mechanism by which GATA2 p.Thr354Met and p.Thr355del mutations function is distinct to that generally described for RUNX1
, which commonly act as classical TSG with a wide-range of mutations and requiring functional disruption of both alleles. Transcription factors are well characterized as targets of dominant negative or constitutively active mutations in cancer14,15
, with RUNX1
mutations leading to a spectrum of outcomes including AML and ALL consistent with both TSG and dominant oncogene models16,17
. While we have only been able to detect single allele GATA2
germline mutations in affected samples, we cannot rule out the possibility of acquired mutations in the “normal” allele.
is indispensable for hematopoiesis17, 18,19
. It associates with, regulates or is regulated by transcription factors implicated in myeloid malignancy (Supplementary Table 7
). Many of these interactions involve ZF2 in which the p.Thr354Met and p.Thr355del mutations reside, and it is likely that changes in the nature of these interactions play an important role in predisposition to MDS/AML. Indeed, our co-transfection studies are consistent with altered transactivation by p.Thr354Met and p.Thr355del with PU.1 ().
p.Thr354Met, p.Thr355del or any other mutations in GATA2
were absent in our heterogeneous cohort of sporadic AML patients, although we cannot rule out possible mutations in samples with low percentage blasts. This is consistent with other recent studies, however, suggesting that somatic GATA2
mutations in both ZF1 and ZF2 could be acquired only in specific AML subtypes such as AML-M57,20,21
is, however, overexpressed in many cases of sporadic MDS22
and AML, particularly in FLT3-ITD+
, suggesting that alterations to GATA2
expression, rather than direct mutation, may occur more commonly.. Further, chromosomal aberrations at the 3q21 breakpoint cluster encompassing a presumptive GATA2
regulatory region resulted in upregulated GATA2
expression in MDS and AML22,24–26
. In addition, retroviral insertional mutagenesis in NUP98-HOXD13
mice, a model for MDS/AML, identified Gata2
as a common insertion site in induced AML, all of which overexpressed Gata227
. Hence, accumulating evidence suggests that aberrant activation or overexpression of GATA2
contributes to AML.
In this study, we show that GATA2 is a new predisposition gene for familial MDS/AML and demonstrate functional changes due to mutations within a highly conserved threonine repeat located in the second zinc finger. Our findings highlight the power of approaches investigating familial predispositions to cancer, and have implications for diagnostic genetic testing. The poor outcome associated with these mutations may suggest that an aggressive treatment strategy is appropriate for individuals carrying GATA2 mutations.