GATA2 mutations in patients with non-tuberculous mycobacterial or fungal infections with unknown immunodeficiency in Brazil

Background Non-tuberculous mycobacteria (NTM) and invasive fungal infections (IFI) may be considered opportunistic infections in immunocompromised patients with GATA2 haplodeficiency. Sporadic or familial GATA2 mutations are associated with infection susceptibility, autoimmunity, and myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). This work aimed to investigate GATA2 status in patients with NTM and/or IFI with unknown causes for immunodeficiency . Methods A series of incident patients with NTM and/or IFI from the Division of Hematology of the Institute of Infectious Diseases-FIOCRUZ at Rio de Janeiro, Brazil, from 2015 to 2018 were subject to GATA2 genotyping. Patients with HIV positivity or other immunodeficiencies were excluded. Results. Twenty-two patients and 9 of their relatives were enrolled. Seventeen patients had IFI, 4 NTM, and one patient present both infections. In 6 patients, the occurrence of malignant disease was found along with this infection, with MDS/AML (n =3) being the most frequent. The pathogenic T354M mutation was found in 4.5% (1/22) of patients and asymptomatic offspring (2/9). We also found the GATA2 polymorphisms rs2335052 and rs369850507 in 18.2% and 4.5%, respectively, and the rs11708606 intronic polymorphisms in 27.3% of cases. Conclusions GATA2 mutations are substantial findings in patients with NTM and/or IFI without known immunosuppression. As it can indicate a primary immunodeficiency and lead to cancers - particularly MDS and AML- the presentation with NTM or IFI should trigger GATA2 mutation testing. The carriers should receive genetic counseling, subsequent infection prevention measures , and surveillance for hematologic malignancies.


Introduction
Non-tuberculous mycobacteria (NTM) are infections caused by largely non-pathogenic organisms and, the immune host defects that may underly it were recently reviewed (1). Advances in imaging, microbiological, and molecular techniques have contributed to the recognition of these infections leading to an apparent increased incidence rate over time (2). Morbidity caused by NTM is clinically manifested by lymphadenitis, skin, and pulmonary diseases. The outcome of NTM infection depends upon a complex interaction between exposure, duration of host exposure, immune status, and genetic background (3). Overall, patients with immunodeficiencies, whether primary or acquired, are at risk of developing NTM or invasive fungal infections (IFI) (4). Nowadays, the recognition of the underlying genetic background for NTM and/or IFI understanding is essential for therapy and prognostic assessment. For instance, mutations in interleukin 12-interferon γ pathway genes (INGR1/2, IL12b, STAT1) are often associated with the onset of opportunistic infections in early childhood, while GATA2 mutations give rise to disseminated infection in late childhood or adulthood (1).
The GATA2 gene is located on chromosome 3q21 region, coding a 3,730 kb GATA2 protein. GATA2 has six exons and two zinc-finger domains, and it acts as a transcriptor factor in several tissues (5).
GATA2 is mostly expressed in hematopoietic progenitor, myeloid, and mast cells (6). GATA2 haplodeficiency in sporadic and familial mutations was found associated with MonoMAC syndrome that clinically presents decreases of monocytes, T, B and NK cell, viral, and bacterial infections autoimmunity and pulmonary alveolar proteinosis. Additionally, GATA2 haplodeficiency is associated with lymphedema, warts, and deafness, condition named Emberger syndrome. Both MonoMAC and Emberger syndromes predispose patients to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) (5). Because of the importance of identifying the underlying causes of overt immunodeficiency in patients with NTM and or IFI infections, this study aimed to identify GATA2 variants among such individuals and to establish the association with clinical-phenotypic features.
The heterogeneity of GATA2 aberrations might be reflected in the broad range of clinical features and deserve a distinctive approach to a multidisciplinary team.

Methods Subjects
A series of incident patients with pulmonary or disseminated NTM and/or IFI (older than 12 years old) were investigated between 2015 to 2018. Demographic, clinical, and laboratory features were extracted from the patients' charters and follow-up. A complete blood count was performed, from which the monocyte count was obtained, when the patients entered the study (Supplemental Table 1). Human immunodeficiency virus (HIV) serology was tested in all patients. To confirm the infectious diagnosis, direct microscopic examination, histopathological, and culture analysis was performed. Bone marrow puncture for a myelogram, immunophenotyping, cytogenetics, and histopathological examination was performed in patients who presented sustained unexplained cytopenias or organomegaly (Supplemental Table 2).
The exclusion criteria were patients with a prior diagnosis of other primary or secondary immunodeficiency such as acquired immunodeficiency syndrome (AIDS)/HIV infection, well established autoimmune diseases such as rheumatoid arthritis, solid organ transplantation, cystic fibrosis, and immunosuppressive or chemotherapeutic treatments.

GATA2 genotyping
Genomic DNA was extracted from peripheral blood samples using the commercial QIAamp DNA Blood Mini kit (Qiagen, CA, USA) according to the manufacturer's instructions. Sanger sequencing was performed throughout the five coding exons (2, 3, 4, 5, and 6) and intron 4 of the GATA2 using the oligo primers and reagents protocols according to Wang et al (7). GATA2 mutation found in the proband was investigated in the relatives. All PCR products were sequenced using the ABI3500xl automatic gene analyzer (Applied Biosystems, CA, USA). Sequences were analyzed in the Mutation Surveyer® program (Softgenetics, PA, USA). Genetic variants found in the GATA2 gene were compared to the reference databases available online at https://cancer.sanger.ac.uk/cosmic, https://www.ncbi.nlm.nih.gov/clinvar, and www.ensemble.org, to evaluate the population frequency and gene variant clinical association.

Statistical Analysis
We used a convenience patients sampling recruited during outpatient and inpatient hematology consults. The frequency analysis was calculated through IBM® SPSS Statistics version 22.0.

Results
Twenty-seven patients fulfilled the inclusion criteria in the study, but 5 patients declined the invitation to participate in the GATA2 genotyping investigation. Thirty-one samples from 22 patients and 9 healthy relatives from a single family were genotyped (shown in Fig. 1). There was a predominance of males and white patients (63.6%; 59.1%, respectively). The median age was 45.5 years old (range 5 19-75 years). The most frequent opportunistic infections were IFI (77.3%) followed by NTM (18.2%), and in one patient, both diseases concomitantly were found (4.5%). Fungal and mycobacterial infections found were: histoplasmosis, sporotrichosis, paracoccidiomycosis, aspergillosis, and candidosis (IFI) in 17 patients, and M. Kansassi, M.abscessus, M.fortuitum and M.intracellulare (NTM) in 5 patients. Viral infections didn't fit inclusion criteria, but we observed herpes simplex virus and varicella-zoster virus in two ascertained patients, as shown in Fig. 2.
The demography, infectious agent, diseases, and genotyping of GATA2 are shown in Table 1    T354M is one of the most prevalent mutations associated with MonoMAC syndrome and is located at the second zinc finger domain (8). We identified this mutation in one patient and his two healthy sons.
This identification was essential to drive his treatment as he was submitted received a hematopoietic stem cell transplant with his GATA2-wild type brother as his donor; in addition his his offspringwere able to receive genetic counseling for prevention and early complication recognition. Although it was not a pathogenic variant and does not affect the zinc finger regions, the rs2335052 is located in a coding and regulatory region and may contribute to the GATA2 protein expression. This polymorphism has been previously associated with worse prognosis in a colon cancer study (12). It is unknown if the presence of this SNP may have contributed to the poor outcome of the cases. The polymorphisms rs11708606 and rs369850507 observed in our patients do not lead to amino acid substitution, as they are located in a non-coding region and are not associated with clinical diseases.
With this study, we reinforce the need for multidisciplinary care of NTM and/or IFI infected patients with unknown causes for immunodeficiency, considering a hidden germline GATA2 haplodeficiency.
The identification of a pathogenic variant in one patient was not only useful for himself but also for his family. The identification of other non-pathogenic variants may be related to the clinical prognosis and can be better explored in future clinical association studies. Finally, we had limitations in this small and descriptive work; in particular we couldn´t rule out other causes of primary immunodeficiencies because we did not investigate such conditions.

Consent for publication
Written informed consent for publication of the clinical information and molecular data were obtained.
All this information is available for the journal.

Availability of data and materials
The clinical documentation is available to the corresponding author on reasonable request.

Competing interests
The authors declare no competing financial interests.

Funding
This study was financed in part by the

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