Kozyra, Emilia J.
[VerfasserIn]
;
Wlodarski, Marcin W.
[AkademischeR BetreuerIn];
Grosschedl, Rudolf
[AkademischeR BetreuerIn]Albert-Ludwigs-Universität Freiburg Fakultät für Biologie
Functional studies of GATA2 germline mutations in GATA2-deficiency disorders
Beschreibung:
Abstract: GATA2 deficiency has been described for the first time in 2011 by several groups reporting germline heterozygous mutations in the GATA2 gene as an underlying cause of various hematological and systemic disorders, which eventually were recognized as manifestations of a single disease. Since then, the number of genetic variants and related phenotypes has been increasing; however, the complete spectrum is yet to be defined. Therefore, the aim of my project was to identify and characterize novel genetic and biological aspects of GATA2 deficiency.<br><br>My first study characterizes a new mechanism leading to GATA2 haploinsufficiency. By studying a cohort of 911 patients with cytopenias and pediatric myelodysplastic syndrome (MDS), I identified 9 cases with 5 distinct synonymous GATA2 variants previously considered as ‘silent’. Since the phenotypes of patients were consistent with GATA2 deficiency, I decided to elucidate the effect of detected variants. Unbalanced expression of GATA2 alleles with partial or complete loss of the mutated one, leading to GATA2 haploinsufficiency, was the main finding of this study. Modification of a splicing pattern, resulting in destabilization of the mutated transcript and its loss, was experimentally confirmed for one variant. Obtained results suggest that synonymous mutations should be incorporated into the diagnostic pipeline of individuals manifesting with GATA2-deficient phenotype but broadly also for other Mendelian disorders, and emphasize the importance of RNA analysis in establishing molecular diagnosis.<br><br>The second part of my thesis describes a novel clinical presentation of GATA2 deficiency observed in 2 unrelated infants with missense GATA2 variants (p.P385L and p.R398W). While various cytopenias constitute a hallmark of this disorder, cases described here presented with high leukocyte and monocyte count at birth. I performed a series of in vitro experiments to determine the impact of both mutations on the transcription factor activity and ascribed them loss-of-function (LOF) effect, manifested by impaired ability to bind DNA and induce transcription. The mutations failed to restore in vivo hematopoietic system in hematopoietic stem and progenitor cell-depleted zebrafish, corroborating their LOF character. In addition, ectopic expression of GATA2 WT and the mutants in murine and human progenitor cells confirmed previously reported inhibitory effect of supra-physiological level of GATA2 WT on normal hematopoiesis. Such an influence on the activity of hematopoietic cells was not observed upon overexpression of both mutants. Identification of gene pathways dysregulated by the mutations may enable to understand their contribution to the atypical representation of GATA2 deficiency, which I approached by performing transcriptome sequencing and chromatin immunoprecipitation-sequencing.<br><br>The last part of my thesis contains systematic characterization of somatic genetic changes acquired by GATA2 deficient patients and associated with malignant progression of a hematologic phenotype. Here, I was involved in the project delineating driver mutations in 50 pediatric primary MDS cases, 15 of whom carried germline GATA2 variants. We found higher mutational load within the group of advanced MDS in comparison to low-grade MDS. In the context of GATA2 deficiency (N=15), secondary alterations were detected in 47% of cases (N=7), predominantly in SETBP1, RUNX1 and ASXL1 genes. Finally, we discovered that acquired mutations are related to the underlying karyotype with monosomy 7 and occur in MDS with germline GATA2 variants as well as in MDS patients without known genetic predisposition.<br><br>Taking together, my work fills in the gaps about genetic and clinical aspects of GATA2 deficiency in children and adolescents. It compels us to take a broader look at this disease, beyond commonly recognized genotypes and phenotypes, and search for novel associations. Understanding the mechanism of underlying mutations, their contribution to the disease development and manifestation as well as establishment of reliable genotype-phenotype correlations will improve patient management and open new therapeutic possibilities