Loss-of-function germline GATA2 mutations in patients with MDS/AML or MonoMAC syndrome and primary lymphedema reveal a key role for GATA2 in the lymphatic vasculature

Abstract

Recent work has established that heterozygous germline GATA2 mutations predispose carriers to familial myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML), "MonoMAC" syndrome, and DCML deficiency. Here, we describe a previously unreported MDS family carrying a missense GATA2 mutation (p.Thr354Met), one patient with MDS/AML carrying a frameshift GATA2 mutation (p.Leu332Thrfs*53), another with MDS harboring a GATA2 splice site mutation, and 3 patients exhibiting MDS or MDS/AML who have large deletions encompassing the GATA2 locus. Intriguingly, 2 MDS/AML or "MonoMAC" syndrome patients with GATA2 deletions and one with a frameshift mutation also have primary lymphedema. Primary lymphedema occurs as a result of aberrations in the development and/or function of lymphatic vessels, spurring us to investigate whether GATA2 plays a role in the lymphatic vasculature. We demonstrate here that GATA2 protein is present at high levels in lymphatic vessel valves and that GATA2 controls the expression of genes important for programming lymphatic valve development. Our data expand the phenotypes associated with germline GATA2 mutations to include predisposition to primary lymphedema and suggest that complete haploinsufficiency or loss of function of GATA2, rather than missense mutations, is the key predisposing factor for lymphedema onset. Moreover, we reveal a crucial role for GATA2 in lymphatic vascular development.

Figure 1

Gata2 mRNA and protein levels are elevated in primary embryonic mouse LECs. (A) Quantification of Gata2 mRNA expression levels assessed by real-time RT-PCR in primary dermal LECs and BECs. Error bars represent mean ± SD of 3 replicates, and data are representative of 3 independent cell isolations from 20 to 30 embryos. (B) Primary dermal LECs and BECs cultured for 3 days on fibronectin and immunostained with CD31 (green), Prox1 (red), and Gata2 (cyan). Scale bar represents 30 μm. (C) Quantification of miR-451 expression levels assessed by real-time RT-PCR in primary dermal LECs and BECs. Error bars represent mean ± SD of 3 replicates, and data are representative of 3 independent cell isolations from 20 to 30 embryos. *P < .05 (Student paired t test).

Figure 2

Localization of Gata2 in the lymphatic vasculature. E16.5 (A-B) and adult (C-D) skin immunostained with anti-CD31 (green), Prox1 (red), and Gata2 (cyan) antibodies. Arrows indicate Gata2 localization in lymphatic vascular valves; and arrowheads, Gata2-positive hematopoietic cells. Boxed regions in panels A and C are shown at higher magnification in panels B and D, respectively. Scale bars represent 30 μm (A,C), 10 μm (B), and 15 μm (D).

Figure 3

Gata2 target genes are down-regulated after siRNA knockdown of Gata2. (A) Gata2 mRNA and protein levels are substantially reduced in primary embryonic LECs after transfection with Gata2 siRNA, compared with transfection with control siRNA. (B) Quantification of Kdr, Pecam1, Foxc2, Nfatc1, Angpt, and Itga9 mRNA expression levels by real-time RT-PCR in primary LECs transfected with control and Gata2 siRNA. (C) Prox1 mRNA and protein levels are substantially reduced in primary embryonic LECs after transfection with Gata2 siRNA, compared with transfection with control siRNA that has no established targets. Error bars represent mean ± SEM (n = 3), and data are representative of 3 independent cell isolations from 20 to 30 embryos. *P < .05 (Student paired t test). Quantification of protein levels was calculated after normalization with respect to β-actin and expressed as percentage change from controls. Each lane contains equal amounts of protein pooled from 3 separate transfections.

Figure 4

A model of Gata2 regulated transcription in LECs. Our data demonstrate that GATA2 regulates the expression of key transcription factors important for valve development. GATA2 lies upstream of Prox1, Foxc2, and Nfatc1. FoxC2 and NFATc1 have been demonstrated to interact together to coordinately regulate transcription in LECs. NFATc1 and Gata2 have been found to heterodimerize in myocytes, suggesting that these transcription factors might interact together in LECs. Gata2, Prox1, FoxC2, and NFATc1 have been demonstrated to bind the promoter and/or regulate the transcription of genes, including Pecam1, Kdr, Cdh5, Angpt2, and Itga9.