Genetic and Clinical Heterogeneity in Shwachman-Diamond Syndrome (SDS): Bioinformatic Analysis on Exome Sequencing for 16 SDS patients

The clinical spectrum of patients affected with Shwachman-Diamond syndrome (SDS) is wide. Phenotypic variability has been noticed between unrelated patients, siblings, and even within the same patient over time, making clinical diagnosis challenging in some cases. The main research hypothesis has been addressed to explain this clinical variability by identifying any possible variants able to exert an additive or modifying effect to that produced by already known mutations in the SBDS. Through this thesis work, we aimed to understand if other germline variants, in addition to the SBDS mutations, could explain the large clinical variability observed among the patients using the data obtained by the WES project for 16 SDS patients, funded by the Italian Association of Shwachman Syndrome. The thesis work concerned several bioinformatic and molecular analysis on this type of data. These findings focused on the theory that the EIF6 germline variant mimics the effect of somatic deletions of chromosome 20, always including the EIF6’ s locus and that it may similarly be able to rescue the ribosomal stress and dysfunction caused by SBDS mutations. This rescue may likely contribute to the stable and not severe hematological status of the proband. The study of the distribution of variants in genes related to MDS and AML using a different type of bioinformatic filtering showed that UPN45, the only patient who developed MDS, had always the highest number of genetic variations that were distributed in various genetic pathways. Therefore, we postulated that in addition to the pre-existence of SBDS mutations, the presence of a greater number of variants in various genetic pathways could play a significant role in the development of MDS in UPN45. On the other hand, Pearson correlation testing showed a negative correlation between the absolute neutrophil count and the number of variants (R=-0.601, p=0.05) found in 16 SDS patients. We observed that the genes VPS13B and CSF3R were the most frequently mutated genes and their variants were found to be more common among patients with reduced neutrophil counts. Three out of four patients with severe neutropenia were found to be carrying biallelic rare variants in the CSF3R gene. PRF1 gene missense variants were identified in all patients with severe bone marrow hypocellularity. Additionally, VWF variants have been noticed only in patients with severe thrombocytopenia. Using eVAI and DIVAs methods, we explained some phenotypic variability observed in two siblings (UPN42 and UPN43) by analysing the WES data for both. For UPN42, the single variant analysis did not disclose any variation which could be related to the patient phenotype. Conversely, in UPN43, we found and confirmed, using Sanger sequencing, a novel de novo variant (c.10663G>A, p.Gly3555Ser) in the KMT2A gene that is associated with WTDS Syndrome. The variant is classified as pathogenic according to different in silico prediction tools. Interestingly, the de novo variants in the KMT2A gene were found to be associated with some of the HPO terms that only describe UPN43 including horseshoe kidney, developmental delay, bone abnormalities, expressive-language delay, minor facial dysmorphisms of hypertelorism and a wide nasal bridge and cryptorchidism. Moreover, the DIVAs method predicted the digenic combination between KMT2A and SBDS genes as pathogenic and subclassified it as a likely Dual Molecular Diagnosis. The findings of this thesis underline the need of performing a comprehensive genomic analysis on SDS patients who present unexpected or unusual features. Whole exome sequencing and bioinformatics tools based on artificial intelligence are now readily available, raising the possibility that these methods may contribute to the well-known clinical variability reported with Mendelian diseases. These new data are likely, in the future, to also be relevant for personalized medicine and therapy in selected cases or groups of patients.