A Novel Mutation Causing GATA2 Haploinsufficiency Activates a Cryptic Splice Site in Exon 3

A 48-year old woman presented with clinical symptoms consistent with GATA2 haploinsufficiency: recurrent human papilloma virus associated cervical intraepithelial neoplasia, extensive deep vein thrombosis of the lower extremity, a history of hepatitis C virus infection and recurrent pneumonias complicated by cryptogenic organizing pneumonia. Infectious agents isolated from the lungs included mycoplasma pneumonia, serratia marcescens, mycobacterium intracellulare and aspergillus nidulans . Laboratory findings revealed monocytopenia, B, NK and CD4 T cell lymphopenia. However, analysis of the bone marrow ruled out myelodysplastic syndrome and showed a normal karyotype. Sanger sequencing from genomic DNA isolated from peripheral blood did not reveal any published missense, nonsense or complex mutation associated with GATA2 haploinsufficiency, but showed a novel, synonymous mutation in exon 3 (c.351C>G, p.T117T). In order to elucidate the pathomechanism of the mutation, we performed an in depth in silico evaluation of the mutated sequence and analysed cDNA from the patient bone marrow and fibroblasts for aberrant splicing patterns by GATA2 specific RT-PCR and Sanger sequencing.

In silico studies of the mutation showed that the mutated cytosine at position 351 is conserved across most species (www.ensembl.org) and predicted aberrations of splicing, especially the activation of a crypticsplice site in exon 3 (www.fruitfly.org). GATA2 specific RT-PCR showed comparable results for the patient and controls and did not show any aberrant bands at visual inspection. Sanger sequencing of these RT-PCR amplified GATA2 specific transcripts from patient bone marrow and blood identified a clear "C" call for the wild type allele and a very weak "G" call for the mutated allele. In order to test if the mutated allele was expressed at minor frequency we cloned the transcripts into a topoisomerase I-activated pCR™4-TOPO® TA Vector. Sequencing of 60 clones showed an overrepresentation of the wild type allele (42/60 clones, 70%) in comparison to the mutated allele (8/70 clones, 13%). Further clones (10/60, 17%) showed an aberrantly spliced cDNA using the predicted cryptic splice donor site at cDNA position 351 and a cryptic splice acceptor site in exon 3 (cDNA position 487). The aberrant transcript showed a deletion of 136bp in exon 3 and introduction of a premature stop codon after 517bp. This presumably leads to a truncated GATA2 protein disrupting the DNA binding domains. The data support that GATA2 haploinsufficiency in our patient is caused by decreased expression of the mutated allele and abrogation of functional protein expression from the aberrantly spliced mRNA.

In summary, we report a novel mutation type leading to GATA2 haploinsufficiency. The described, synonymous mutation activates a cryptic splice donor site in GATA2 exon 3. This mutation type and mutations affecting splicing in general have so far not been described in GATA2 haploinsufficiency. Our finding underscores the need to thoroughly investigate also rare "silent" mutations in genetic counselling and diagnosis.