Page 274 - 2022_01-Haematologica-web

M. Faleschini et al.
could be responsible for thrombocytopenia. In fact, consid- ering that p37 has a pivotal role in megakaryopoiesis and platelet production and that p32 is essential for erythroid lineage, distortion of the p32/p37 ratio is likely to dysregu- late hemopoiesis. On the contrary, the role of p39 and p34, which represent approximately 3% and 10%, respectively of the total GFI1B mRNA in both specimens of control samples, has not yet been defined.7
We further investigated the role of p37 and p32 in terms of cellular localization and ability to control the transcrip- tion of targeted genes. In overexpressed conditions, most of the two isoforms are in the nucleus at comparable level, though their transcriptional effect is opposite. Whereas p37 represses the activity of the luciferase that is under the con- trol of GFI1B target promoters, such as MEIS1, GFI1, and GFI1B itself, p32 increased its activity, reversing the tran- scriptional repression.
The same effect of c.648+5G>A on alternative splicing has previously been reported for c.648+1_648+8del GTGGGCAC, another mutation of GFI1B that was found in members of one family affected with MYH9- related disease.10 As in our family, in individuals carrying the GFI1B but not the MYH9 variant, the expression level of p32 was markedly increased, being in at least equal amounts to p37. However, these individuals were not thrombocytopenic (mean value of 228x109/L).
Another GFI1B variant, associated with skipping of exon 9 and significant reduction of the p37 expression level, is the synonymous substitution c.576C>T(p.Phe192=) in exon 9, which is reported as rs150813342 with a MAF of 0.0041.28 Indeed, in individuals heterozygous for c.576C>T the platelet count is reduced with an average of 25– 30x109/L in comparison with controls.
Finally, unbalance between p32 and p37 was identified in association with another mutation (c.551insG/p.Ser185Leufs*3).29 In patients homozygous for this mutation, while the expression level of p37 was significantly decreased, likely due to degradation by non- sense-mediated decay, the expression of p32 was the
same level as healthy controls. Therefore, in these patients the active form of GFI1B is mainly p32. Of note, whereas individuals homozygous for c.551insG had a severe phenotype, heterozygous individulas were clini- cally unaffected. Of note, in these patients, as well as in our affected individuals, erythropoiesis was not defec- tive, except for mild anisocytosis, suggesting that red cell production is rather independent of the p32/p37 ratio.29
Taken together all these observations suggested that the expressivity is extremely variable even when the variants exert the same effect. Indeed, bleeding and platelet count vary significantly, from individuals without defects (c.648+1_648+8delGTGGGCAC) or slight reduction of platelet count (c.576C>T) to those with an intermediate phenotype characterized by mild thrombocytopenia with mild bleeding tendency as in our family (c.648+5G>A) or with severe disease when the c.551insG mutation is homozygous.29 Of note, abnormalities in megakaryocyte maturation were observed in isogenic cell clones homozy- gous for rs150813342 and in hematopoietic stem cell pro- genitors in which GFI1B p37 was selectively silenced,28 sug- gesting that both expressivity and penetrance of the platelet phenotype might depend - at least in the cases enlisted above - on a specific threshold of the p32/p37 ratio, a value that cannot be estimated from the literature or our own data.
One feature that is likely to be pathognomonic of the GFI1B-associated bleeding disorder is the abnormal expres- sion of CD34 on the platelet surface.10 Indeed, platelets of affected individual III-2, as well as those mentioned above with an altered p32/p37 ratio, express the CD34 antigen. Strong expression has also been reported in association with the Cys168Phe, Arg184Pro, and His181Tyr mutations, all affecting ZNF 1, or H294fsX307, Gln287* and G272fsX2 removing ZNF 5 and 6.3,10–12
Changes in proportion between p37 and p32 significantly increasing p32 levels has been reported in acute and chronic leukemia, which could be interpreted as a triggering event for carcinogenesis.26 This hypothesis is supported by the
Figure 5. Quantitative polymerase chain reaction of GFI1B transcriptional target genes. The GFI, BCL2L1, TGFbR3, MYC, GATA1, and GATA3 mRNA expression levels were significantly increased in patients (II-2 and III-1) compared to controls (two unrelated healthy individuals [HC]) (fold change from 1.5 to >3). No significant dif- ference was observed for GATA2. Error bars represent the standard deviation of three independent experiments. Statistical analysis was performed using t-test.
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