A novel splice-specific GFI1B mutation in a family with recessive macrothrombocytopenia

H. Schulze1, A. Schlagenhauf2, G. Manukjan1, O. Andres1, E. Klopocki1, S. Panzer3, C. Beham-Schmid2, K. Althaus4, A. Greinacher4, T. Bakchoul4, M. G. Seidel2 (1Würzburg, Germany, 2Graz, Austria, 3Vienna, Austria, 4Greifswald, Germany)

Date: 18.02.2017,
Time: 10:00 - 11:15

Objective: Growth Factor Independent 1B (GFI1B) is a transcription factor, essential for the erythroid and megakaryocytic lineage. Alternative splicing leads to a shorter p32 isoform which lacks the two out of 6 zinc fingers. Germline mutations have been reported to cause autosomal-dominant macrothrombocytopenia with a grey-platelet syndrome phenotype. We report on a Chechen family whose affected family members present with severe, life-threatening bleedings. The index patient had recurrent hematoma and multiple petechiae since childhood. Both her children (age 9 and 7) have platelet counts less than 45/nL and a similar cutaneous bleeding pattern. The brother also had low platelet counts and died with age 33 due to a spontaneous cerebral hemorrhage. The index patient's husband, her parents and the children of the deceased brother were clinically unaffected.

Methods: We analyzed blood smears with May-Grünwald-Giemsa and immunofluorescence microscopy. Platelet function was tested by aggregometry and flow cytometry. DNA was subjected to targeted next generation sequencing to screen for variants in 59 genes reported to be relevant for platelet formation or function.

Results: Platelets showed reduced staining for alpha-granule markers. White and red blood parametets were unaffected. Aggregometry with ADP, TRAP-6, or arachidonic acid was impaired. We found a novel homozygous single nucleotide insertion in GFI1B (NM_004188.5; c.551insG), expeced to cause a premature stop-codon and which segregated with the phenotype. The unaffected mother, the husband and two unaffected nephews were heterozygous, confirming an autosomal-recessive trait. Dysplastic micromegakaryocytes and peripheral platelets were CD34-positive. Quantitative PCR of platelet RNA showed residual homozygous c.551_G insertion in the p37 transcript and an unexpected expression of p32. The p37 transcript was markedly reduced, with an increased p32/p37 ratio compared to controls.

Conclusion: Our findings imply that the first two zinc fingers of GFI1B are dispensable for human erythropoiesis, but essential for normal megakaryopoiesis and functional platelets. While previous mutations affect both isoforms, this insertion variant results in a premature stop-codon and affects only the p37 isoform. We conclude that the transcriptional regulation of erythropoiesis is uncoupled from that of megakaryopoiesis through alternative splicing of GFI1B.