Genetic studies of hereditary thrombocythemia

Hereditary thrombocythemia (HT) is a familial myeloproliferative disorder characterized by an elevated platelet count in peripheral blood. Thrombocytosis is due to a genetic alteration that can be transmitted to the offspring. Recently, major progress has been made in understanding the biology of HT thanks to the discovery of mutations in two genes: thrombopoietin (THPO) and its receptor MPL. Interestingly, the analysis of these mutations has provided more insights in the physiological regulation of platelet homeostasis. However, not all the HT pedigrees carry mutations in the THPO or MPL genes. In 80-90% of these pedigrees the disease-causing gene remains unknown and it is likely that hereditary thrombocytosis can be caused by alterations in other genes, not yet identified.
The focus of my PhD studies was a large US family affected by HT. In this family, THPO and MPL were excluded as disease-causing genes. Therefore, genome-wide linkage analysis was performed to identify co-segregating regions shared by the affected family members as a target in search of possible candidate mutations responsible for the thrombocytosis phenotype. One region with significant logarithm of odds (LOD) score values has been located using microsatellites and SNP chip arrays. One novel candidate mutation was found in the gelsolin gene by next generation sequencing and confirmed by capillary sequencing in all the 12 affected family members. Gelsolin is a Ca2+ regulated actin filament severing, capping and nucleating protein abundant in platelets. It is involved in the regulation of cell structure and metabolism. Interestingly, it has a key-role in apoptosis regulation and modulation of platelets. Computational predictions showed that this alteration can probably affect protein function and the structural analysis indicated that the alteration is located at the interface with actin. The platelets-biogenesis in vitro assay showed that the candidate alteration can increase the release of platelets-like particles in DAMI cell line stably transfected with the mutant gelsolin. To study the in vivo role of the candidate mutation in the pathogenesis of HT, different mouse models have been established. In lethally irradiated recipient mice transplanted with BM cells transduced with retrovirus expressing the human mutant gelsolin, variations in platelet counts in peripheral blood have been observed. Transgenic mice expressing the human mutant gelsolin were generated to fully characterize the new discovered alteration. These mice developed a tendency to elevated platelet counts compared to their wild type littermates.
Taken together, these data illustrate the discovery of a new candidate mutation associated with the pathogenesis of HT. Until now, mutations in gelsolin gene were never described except for a mutant (D187N/Y) plasma gelsolin responsible for familial amyloidosis of Finnish type (FAF). My work contributed to further characterize this gene and to link it with the pathogenesis of HT.