Genetic Studies on a Rare Mendelian Disease: Shwachman-Diamond Syndrome

The Shwachman-Diamond syndrome (OMIM: #260400, SDS) is a rare, autosomal recessive ribosomopathy. It is characterized by bone marrow failure, exocrine pancreatic insufficiency and skeletal alterations, with an increased risk to develop haematological malignancies. The majority of patients bears biallelic pathogenic variants in SBDS gene. During my internship I worked on two projects concerning SDS. The first one was about the hypothesis of an Italian founder effect in four families (three from Sicily and one from Lazio) for the pathogenic variant [c.258+533_459+403del] in SBDS gene. The fact that, among the rare mutations usually observed in single cases, the exon 3 deletion has been detected in four index cases, prompted us to hypothesize the presence of a founder effect. The study has been carried out by doing a microsatellite analysis, to define the haplotype surrounding the SBDS locus. Results showed that the three families from Sicily shared a common haplotype of the mutation, while the one from Lazio carried a completely different haplotype. Data were elaborated to predict the age of the variant, which resulted to be 171 years. Therefore, we demonstrated that [c.258+533_459+403del] originated in Italy in an independent way twice, in Sicily and Lazio. The second Project was suggested by the results obtained during the bioinformatic work applied to the analysis of exome files of sixteen SDS patients, with biallelic SBDS mutations. While I was attending my internship, bioinformatic analysis identified a single case carrying a heterozygous germline variant in EIF6 gene (c.100T>C, p.Phe34Leu), which product must be evicted from 60S ribosomal subunit by SBDS and EFL1 to complete 80S ribosome. Structural analysis suggested that the variant could obstacle EIF6 folding and reduce the binding affinity between EIF6 and 60S ribosomal subunit, rescuing the ribosomal dysfunction caused by SBDS germline mutations. Therefore, it was hypothesized that a lower dose of EIF6 could lead the patient to experience a genetic rescue at constitutional level. My work involved EIF6 sequencing in a group of SDS patients without available WES (Whole Exome Sequencing) data. In this group we planned to check for the presence of germinal pathogenic variants, which could be relevant to produce a genetic rescue effect. I found a total of twenty-three variants, seventeen were intronic and six exonic. Bioinformatic evaluation was carried out through several in silico prediction tools and all exonic variants and seven intronic variants were evaluated to have a possible significant effect on EIF6 protein functionality. About the single heterozygous nucleotide substitutions, four intronic had overlapped peaks while, in the remaining ones, peaks had different heights, with the substituted base being the shortest. Since sequences were well-defined and easily interpretable, we excluded the hypothesis that these substitutions could be due to background noise. The fact that peaks had different heights suggested that variants could be less represented compared to the wild type sequence, at a quantitative level. The next step of this project will be to analyse, in patients with significant variants, additional samples of DNA extracted from bone marrow, to check another tissue different from peripheral blood. Analysis of bone marrow may allow to confirm the variants found or identify additional somatic EIF6 variants. For some of these patients it could be suggested a lower risk for the development of haematological complications, as it has already been demonstrated for the cytogenetic abnormality del(20)(q) but also, more recently, for EIF6 somatic point mutations.