GATA2 haploinsufficiency caused by mutations in a conserved intronic element leads to MonoMAC syndrome

Abstract

Previous reports of GATA2 mutations have focused on the coding region of the gene or full gene deletions. We recently identified 2 patients with novel insertion/deletion mutations predicted to result in mRNA nonsense-mediated decay, suggesting haploinsufficiency as the mechanism of GATA2 deficient disease. We therefore screened patients without identified exonic lesions for mutations within conserved noncoding and intronic regions. We discovered 1 patient with an intronic deletion mutation, 4 patients with point mutations within a conserved intronic element, and 3 patients with reduced or absent transcription from 1 allele. All mutations affected GATA2 transcription. Full-length cDNA analysis provided evidence for decreased expression of the mutant alleles. The intronic deletion and point mutations considerably reduced the enhancer activity of the intron 5 enhancer. Analysis of 512 immune system genes revealed similar expression profiles in all clinically affected patients and reduced GATA2 transcript levels. These mutations strongly support the haploinsufficient nature of GATA2 deficiency and identify transcriptional mechanisms and targets that lead to MonoMAC syndrome.

Figure 1

Organization and conservation within the GATA2 locus. (A) GATA2 locus. The 3 identified isoforms of human GATA2 are shown with the associated GERP, DNaseI hypersensitive sites, and reported transcription factor binding sites.^, (B) The conserved region within intron 5 including the composite element encompassing an E-box and GATA motifs and the ETS motif. Bold text denotes motifs for transcription factor binding. Underline denotes deletion in patient 6.II.1; *recurrent ETS motif point mutation, c.1017+572C>T. Figure modified from UCSC browser.

Figure 2

Mutation of conserved ETS motif or deletion of the composite element reduces intron 5 enhancer activity. Luciferase activity from exon 1 preceded by the wild-type intron 5 enhancer set and exon 1 preceded by the intron 5 enhancer containing either the 28 base deletion seen in patient 6.II.1 or the ETS motif C>T point mutation seen in patients 4.II.1, 11.II.1, 25.I.1, and 28.I.1. ***P < .001.

Figure 3

Reduced expression of mutant allele in a patient with c.1017+572C>T. (A) Genomic sequences of GATA2 exons in patient 4.II.1 and her sister 4.II.5 indicate the phase of the SNPs and the mutation. M, K, and S refer to base calls of mixed nucleotide bases at a single site, A/C, G/T, and C/G, respectively. (B) GATA2 cDNA sequences from isolated granulocytes (Grans), CD3⁺ T-cells (CD3⁺), and CD3^– PBMCs (CD3^–) demonstrate reduced expression of c.1017+572C>T allele. (C) Quantitation of relative peak heights of patient 4.II.1 mutant allele as a percentage of the combined peak height at that site.

Figure 4

Reduced allelic expression of GATA2 in MonoMAC patients. (A) Genomic vs cDNA GATA2 sequence for patients 23.I.1, 7.I.1, 29.I.1, 28.I.1, and a healthy control. Shown is a portion of the transcript in which the patient is heterozygous at the genomic level with reduced or absent expression of 1 allele by full-length cDNA sequence. Y, S refer to base calls of mixed nucleotide bases at a single site, C/T and C/G, respectively. (B) Quantitation of relative peak heights of patient’s mutant allele as a percentage of the combined peak height at that site.

Figure 5

GATA2 patients have unique transcript profiles compared with healthy normals and diseased controls. Normalized transcript counts from nCounter analysis used for (A) 2-dimensional principal component analysis of patients with GATA2 i5C>T (patients 4.II.1, 11.II.1, 25.I.1), missense mutation (GATA2 Mis; patients 15.I.1, 19.II.1, 30.II.1), and reduced allelic expression without identified GATA2 mutation (GATA2 Unk; patients 7.I.1, 29.I.1) compared with healthy controls and diseased controls with similar infection types but wild-type GATA2. Fungal, disseminated coccidioidomycosis or histoplasmosis; Mycobacteria, disseminated mycobacteria; Cryptosporidiosis, disseminated cryptosporidium. (B) Hierarchical clustering of transcripts differentially regulated (P < .05) between healthy normals and GATA2 mutated patients irrespective of genotype. Samples and color labels correspond to mutations as in A.

Figure 6

Transcript counts of GATA2, IKBKG, FERMT3, and RUNX1 from patients segregated by mutation class. i5C>T (n = 5, patients 4.II.1, 4.II.5, 6.II.1, 11.I.1, 25.I.1); Haplo, identified mutations resulting in loss of expression of 1 allele (n = 2, patients 13.I.2, 41.I.1); Unk, patients without identified pathogenic mutations (n = 4, patients 7.I.1, 23.I.3, 29.I.1, 33.II.2); Mis, identified GATA2 mutations predicted to result in a single amino acid change or a late frameshift with demonstrated mRNA stability (n = 8, patients 1.II.5, 5.I.1, 9.II.1, 15.I.1, 30.II.1, 33.III.3, 37.I.1, 40.I.1); compared with healthy controls (HC) (n = 9) or patients with pNTM (n = 5) with wild-type GATA2. *P < .05; **P < .01; ***P < .001.