Implementation of NGS protocols for the diagnosis of rare genetic diseases and development of models for the validation of mutations

Background. DNA sequencing is a field of evolving molecular biology. In the last decade, new generation sequencing technologies - known as Next Generation Sequencing (NGS) - have become increasingly common in clinical practice. These new technologies, by their characteristics, are completely revolutionized to the analysis of the human genome. Aim of the thesis. The aim of the thesis is to verify the applicability of the new generation sequencing methods (NGS). In this regard, the Illumina and Agilent different massively-parallel sequencing strategies were compared, which were adopted for the study of patients analyzed at the Genetics Unit of the Hospital of Padua. The research activity is also focused on the development of models for the pathogenicity validation and the characterization of the genetic variants identified by NGS sequencing. A new variant in the COQ4 gene was identified in a patient with coenzyme Q deficiency syndrome and its potential ability to alter the splicing mechanism was studied by hybrid minigene system. A functional complementation in S. cerevisiae yeast was performed in order to examine and validate the pathogenicity of some missense mutations in the ASS1 gene, identified in patients with citrullinemia type 1, with the intent to extending the analysis to the potential new variants identifiable in the future. Results and conclusions. The comparative analysis of the NGS technologies TruSeq Custom Amplicon Low Input Library Prep Kit (Illumina) and HaloPlex HS Target Enrichment System (Agilent Technologies) showed how the second one is more efficient in diagnostic, considering the followed parameters: coverage, results variability, pseudogenes and false positive discrimination, number of amplicons generated for the analysis. For these reasons, from 2017 we use only the HaloPlex HS Target Enrichment System (Agilent Technologies). In the study, 85 patients positive for neonatal metabolic screening for Mucopolysaccharidosis type I (MPS I) or for Biotinidase Deficiency or Phenylketonuria (PKU), have been analyzed by Next Generation Sequencing: molecular analysis made it possible to distinguish true positive patients from false positives to neonatal metabolic screening (it would be important to review the cutoff of this screening), allowing to avoid unnecessary therapies and significantly reduce the cost of the investigation. The hybrid minigene system used for the validation of the new IVS4+1G>A variant in COQ4 gene was considered an effective and simple method to demonstrate the pathogenicity of the mutation: it behaves like a hypomorph allele causing the activation of a cryptic splicing site at exon 4 of the COQ4 gene producing both the wild-type transcript and a smaller one less of about 100 bp. Finally, the functional complementation in S. cerevisiae yeast system developed to test the pathogenicity of missense mutations in the ASS1 gene, allowed to classify the mutants in the two classes: class I mutations that completely abolish yeast growth, class II mutations that use a residual growth. This system was useful to validate the pathogenicity of the mutations studied but not to made correlation between the genotype and the phenotype of the patients.