Identificazione e caratterizzazione dei nuovi pathway molecolari in malattie non diagnosticate

ABSTRACT The clinical application of Next Generation Sequencing (NGS) technologies has significantly improved the diagnosis of inherited human diseases, highlighting the extensive variability of the human genome and the complexity of the molecular mechanisms underlying pathological conditions. This thesis focuses on the tertiary analysis of NGS data, with the aim of exploring its potential in several areas: the identification of novel disease genes and molecular pathways in rare and undiagnosed conditions, the in-depth phenotypic characterization to define new genotype–phenotype correlations, and the implementation of the Italian reference genome. In the first section, Schuurs-Hoeijmakers syndrome is described in 2 patients from the Abruzzo region. This ultra-rare condition, also known as PACS1-related neurodevelopmental disorder, is characterized by intellectual disability, distinctive craniofacial anomalies, and congenital malformations, and has been associated with variants in the PACS1 gene in 63 patients, all carrying the recurrent de novo variant p.(Arg203Trp). In the second section, a novel de novo variant in the KCNT2 gene is reported in a patient of Moroccan origin affected by developmental and epileptic encephalopathy, characterized by dysmorphic facial features. To date, this condition has been described in 25 patients; the present case is undergoing further investigation aimed at a systematic clinical characterization and detailed description of the dysmorphic gestalt, suggesting the possible existence of a distinct syndromic entity. The third section describes the study of a 12-year-old patient, born to consanguineous parents (first cousins), in whom a novel candidate gene associated with a phenotype overlapping cartilage-hair hypoplasia was identified. The analysis highlighted the involvement of the RNase MRP complex, which includes the RMRP gene, already known to be responsible for cartilage-hair hypoplasia, a condition characterized by skeletal abnormalities and clinical manifestations significantly overlapping with those observed in the patient described. In the fourth section, a systematic review of variants and associated phenotypes in the ATP2B3 gene is presented, following the identification by WES of a novel ATP2B3 variant in a 27-year-old patient. The aim is to systematically define the observed clinical phenotype and provide a detailed characterization, with the hypothesis of contributing to the identification of a novel genetic syndrome. The fifth section presents a cohort of ultra-undiagnosed patients for whom no genetic cause has been identified, highlighting that the lack of a molecular diagnosis remains a not infrequent outcome in genomic investigations. Finally, the mutational spectrum at specific genes or whole genome sequencing was studied in the Abruzzo population. Both control and patient samples were analysed and the distribution of these variants in the Abruzzo provinces have been registered shedding lights on their cluster distribution. Bioinformatic analysis of recurrent pathogenic variants, conducted in collaboration with the DISIM Department, allowed the identification of potential high-risk areas, improved the interpretation of variants of uncertain significance, and enhanced variant classification. Overall, the results confirm the central role of medical genetics and NGS in molecular and personalized medicine and emphasize the need for further technological advancements, such as long-reads sequencing, to elucidate the molecular bases of still undiagnosed genetic disorders.