DEVELOPMENT OF FUNCTIONAL ASSAYS TO DETERMINE THE PATHOGENIC VARIANTS OBTAINED BY NEXT GENERATION SEQUENCING IN INHERITED THROMBOCYTOPENIAS

Inherited thrombocytopenias (IT) are a heterogeneous group of diseases characterized by platelet count lower than 150x109/L. They are clinically and genetically heterogeneous diseases, with mutations in at least 30 causative. However, these genes account for approximately 50% of the IT patients, suggesting that novel forms are still to be characterized. For this reason, in collaboration with Medical Genetic Unit of Policlinico Sant\u2019Orsola Malpighi in Bologna and the department of General Medicine 3 , IRCCS San Matteo in Pavia, our laboratory is applying the Next Generation Sequencing (NGS) technologies to identify mutations and genes responsible for the disease in thombocytopenic families. However, understanding the effects of the thousands variants identified remains a major problem in diseases like ITs, which are mainly autosomal dominant diseases caused by private, often missense, mutations. Whereas the deleterious effect of nonsense or frameshift variants is clear, that of the amino acid substitutions, which are classified as variants of unknown significance (VUS) is not always obvious. Therefore, it is fundamental to develop assays to tackle the pathogenicity question with functional studies. My PhD work fits into this project and is focused on the development of functional assays to test the pathogenicity of the variants of the IT genes encoding for cytosckeleton components, such as ACTN1 and TUBB1. Thanks to Whole Exome Sequencing (WES) in a family suffering from an IT of unknown origin, we identified a single-base substitution located within exon 19 of the ACTN1 gene (c.2305G>A), which was predicted to result in a missense mutation, p.Glu769Lys. In order to identify ACTN1 mutations in our cohort of patients, we searched for ACTN1 mutations in 127 probands affected and identified nine different heterozygous missense variants in ten families. Three were known amino acid substitutions (p.Gly225Lys, p.Arg738Trp and p.Arg752Gln) that were previously reported (Kunishima, al. 2013). The other six were not present in either the dbSNP or the 1000 Genomes Project datasets and therefore regarded as novel variants of ACTN1. For each of these novel variants, we evaluated their potential effect on protein function using three different pathogenicity prediction tools: SIFT, PolyPhen2 and Mutation Taster. We also performed segregation analysis which was confirmed in all but one (p.Asp666Val) cases. To confirm the genetic analysis, we performed an immunofluorescence analysis of human fibroblasts overexpressing wild-type and mutated forms of ACTN1. Confocal microscopy analysis revealed a well organized cytoskeleton in which \u3b1-actinin1 colocalizes with actin along the filaments in cells transfected with the wild type construct. On the contrary, all but one ( p.Asp666Val) mutant constructs presented an abnormal distribution of actin, which was no longer able to form filaments causing an apparent disruption in the cytoskeletal structure. Determination of the pathogenicity of variants identified through screening of mutation has allowed clinicians to define ACTN1-Related Thrombocytopenia as a mild form of thrombocytopenia without platelet dysfunction. These data have been reported in a paper published on Blood (Bottega et al, 2015)