Functional assays to unravell the pathogenetic role of variants found in GFI1B in piastrinopenic patients

Objective GFI1B is an important transcription factor for megakaryopoiesis, known as causative of an Inherited thrombocytopenia (IT) form named platelet-type bleeding disorder 17. During screening analysis of IT patients, we have found five new missense variants and a splicing one. Unravelling the functional impact of non sense or frameshift variants is usually easy, while assessing the role of missense or splicing variants requires gene-specific functional studies.

Material and Methods Our patients have low platelet count and NGS analysis with our Ion Torrent panel showed no other variants in other ITs genes.

All the variants were analysed with bioinformatics tools and for the segregation in the family. We firstly analysed the splicing variant, which is predicted to cause skipping of exon 9. From patient RNA we performed RT-PCR to verify the presence of exon skipping and Real time PCR on genes involved in oncogenic pathways. Finally, we perform a luciferase reporter assay on some known target gene promoters.For the missense variant luciferase assays are in progress.

Results We have firstly confirmed the skipping of exon 9, and we found overexpressed a shorter form of the transcript, previously described in literature because is increased in chronic and acute leukemia. Then, we evaluated the transcriptional activity with a luciferase gene reporter assay: the wild type form act as a transcription repressor, while the variant loses the repression activity and acts as a dominant negative. We repeated this experiment with CD34 promoter, because the increased expression of CD34 on platelet surface is the major character in common between GFI1B patients. Also here, there is a loss of repression. To further confirm that the splicing variant has a role in malignancies development too, we performed Real time PCR on oncogenic genes, which we found dysregulated (overexpressed) in the patient when compared to healthy control.

Conclusion Our findings suggest that the splicing variant has probably a role in thrombocytopenia, because shows a dysregulation of the target genes. Moreover, the dysregulation of some oncogenic genes could be seen as a starting point for malignancies development, thus making patients more susceptible to develop these diseases. It is therefore clear how also try to evaluate the role of the missense variant found is very important for patient management and for a better understanding of the disease.

Publication History

Article published online:
18 June 2021