Acquired ASXL1 mutations are common in patients with inherited GATA2 mutations and correlate with myeloid transformation

Abstract

© F e r r a t a S t o r t i F o u n d a t i o n (Online Supplementary Table S2). The PCR were done with AccuPrime Taq DNA Polymerase High Fidelity (Life Technologies, Grand Island, NY, USA) using the manufacturer's recommended conditions with 30 ng of substrate DNA and 35 amplification cycles, with the following amplification parameters: 94°C for 20 seconds; 58°C for 30 seconds, and 68°C for 60 seconds/kilobase of amplified product. The p.G646Wfs*12insG mutation was verified first by repeating the PCR using two different primer sets (Online Supplementary Table S1: primers 1/4, primers 2/3), then by repeating the reactions with different polymerase enzymes [AccuPrime Pfx (Life Technologies, Grand Island, NY, USA), DreamTaq (Thermo Scientific, Pittsburgh, PA, USA)] using the manufacturers' recommended conditions. PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN Sciences, Germantown, MD, USA) and sequenced using ABI 3130XL and 3730 fluorescence-based sequencers. Sequences were analyzed with MacVector, version 12.0 (MacVector, Inc, Cary, NC, USA). Mutations were confirmed with independent PCR with separate primer sets. Statistical analyses were done with GraphPad Prism, version 5.0 (GraphPad Software, Inc., La Jolla CA, USA). The clinical protocols under which these studies were undertaken were reviewed and approved by the Institutional Review Board of the National Institutes of Health, Clinical Research Center. Patients in this study were enrolled in National Institutes of Health/National Institute of Allergy and Infectious Diseases ClinicalTrials.gov Identifier: NCT00018044, NCT00404560, NCT00001467, and National Cancer Institute ClinicalTrials.gov Identifier: NCT00923364. Results and DiscussionWe screened 48 patients with inherited GATA2 mutations to determine the incidence of somatic ASXL1 mutations. A schematic of the ASXL1 region that was sequenced, which contains ~90% of known ASXL1 mutations (COSMIC, v66 14 ), and the position of the mutations found in this study, are shown in Figure 1A. Sequence variations found in the Database of Single Nucleotide Polymorphisms (dbSNP) were not included as mutations Supplementary Table S3). Somatic ASXL1 mutations were detected in 14/48 (29%) patients with GATA2 deficiency (Table 1). All of these mutations were heterozygous and located within exon 13. The ASXL1 mutations found among GATA2 deficiency patients were similar to mutations previously reported in MDS and AML patients,15 including five independent cases of the most frequently described ASXL1 mutation (p.G646Wfs*12insG). The p.G646Wfs*12insG mutation was previously reported in two cousins with a GATA2 mutation who had developed MDS. 12 However, there has been concern over the validity of this particular mutation, with suggestions that it is a PCR artifact since it occurs immediately 3' to an eight base poly G sequence. 16 We confirmed this mutation by © F e r r a t a S t o r t i F o u n d a t i o nrepeating the PCR at least three times for each positive sample (for patie...

GATA Transcription Factors: Basic Principles and Related Human Disorders

“…Considering the important role of GATA-2 in HSC proliferation (Tsai et al 1994;Tsai and Orkin 1997;Ezoe et al 2002;Rodrigues et al 2005), additional genetic events might explain the progression of patients with GATA-2 deficiency to MDS/ AML. In this regard, the most commonly associated cytogenetic finding is monosomy 7 as well as additional acquired mutations such as those in ASXL1 (Hahn et al 2011;Bödör et al 2012;West et al 2014;Churpek et al 2015). However, the molecular basis for the evolution of GATA-2 deficiency into MDS/AML has not been elucidated, which impacts early detection and treatment of the disease.…”

Haematopoietic and immune defects associated with GATA2 mutation

“…Recently, acquired mutation of ASXL1 (chr 20q11) has been demonstrated in approximately 30% of individuals with GATA2 mutation evolving to MDS. Acquired ASXL1 mutation is strongly associated with the presence of monosomy 7, BM hypercellularity and chronic monomyelocytic leukaemia (Bodor et al, 2012;West et al, 2013;Micol & Abdel-Wahab, 2014). Whole genome sequencing in one patient has also identified mutations in EZH2, HECW2 and GATA1 (Fujiwara et al, 2014); the spectrum of somatic mutations that are known to occur with germline GATA2 mutation is summarized in Tables IV and SI.…”

“…Constitutive genetic background may have an influence on the risk of leukaemic transformation and susceptibility to infection although it is notable that a range of clinical phenotypes can be seen in different individuals within one pedigree (Holme et al, 2012;Mutsaers et al, 2013;Spinner et al, 2014). The acquisition of additional genetic abnormalities in the transformation of GATA2 mutation to multilineage dysplasia is clearly presaged by the high incidence of monosomy 7 and trisomy 8 in familial cases of MDS/AML (Hahn et al, 2011;Ostergaard et al, 2011;Bodor et al, 2012;West et al, 2013;Dickinson et al, 2014;Micol & Abdel-Wahab, 2014;Spinner et al, 2014). Recently, acquired mutation of ASXL1 (chr 20q11) has been demonstrated in approximately 30% of individuals with GATA2 mutation evolving to MDS.…”

“…Whole genome sequencing in one patient has also identified mutations in EZH2, HECW2 and GATA1 (Fujiwara et al, 2014); the spectrum of somatic mutations that are known to occur with germline GATA2 mutation is summarized in Tables IV and SI. The presence of monosomy 7, ASXL1 mutation and trilineage dysplasia are all high risk features in the biogenesis of AML (West et al, 2013). A number of patients with GATA2 mutation have received successful haematopoietic stem cell transplantation, precisely because a high risk AML was detected, according to standard criteria.…”

Heterogeneity of GATA2-related myeloid neoplasms

“…However, some patients with GATA2-related MDS can present with monocytosis rather than monocytopenia [6,34]. Somatic ASXL1 mutations are associated with the presence of monosomy 7, BM hypercellularity and CMML [35]. Furthermore, several (rare) cases of adult CMML disease were reported in germline GATA2 mutation carriers.…”