Development of new genetic tests: atypical cystic fibrosis and HCV genotyping

Recent revolutionary progress in human genomics is reshaping the approach to therapy and diagnosis. Nucleic acid–based testing is the major diagnostic tool not only in the setting of inherited genetic disease but in a wide variety of neoplastic and infectious processes. The “Reverse Line Blot” (RLB) is a robust method for the molecular characterization of several disorders. It is a simple technique that can be used in diagnostic routine also by small laboratories, because the request instruments are few and not expensive. The aim of our project is to exploit the potential of this method for assessment of two different disorders: a rare hereditary disease (atypical cystic fibrosis) and a hepatic pathology caused by the Hepatitis C Virus (HCV). In the first part of the study we explore the potentiality of RLB in detecting known genetic mutations in the human genome and our interest was focused on atypical CF. This pathology is characterized by cystic fibrosis (CF) mild symptoms, involvement of few organs, and only one or none mutations in the CFTR gene. This phenotypic heterogeneity could be explained by modifier genes of the CFTR gene, such as the genes coding for the Epithelial Sodium Channel (ENaC). During this project, we developed a new RLB molecular assay for detection of nine already described variations in the ENaC genes, involved in CF-like disorders. The performance of the method were evaluated analyzing 208 patient samples, with respiratory or pancreatic manifestations, and 169 true healthy individuals as control group. Among the atypical CF patients, with one or none CFTR mutations and showing respiratory symptoms, we found seven ENaC different variants: the mutations p.S82C-SCNN1B, p.P267L-SCNN1B, p.G294S-SCNN1B, p.E539K-SCNN1B, p.1670-2A>G-SCNN1B, and the polymorphisms p.W493R-SCNN1A and p.R181W-SCNN1A. In particular, p.P267L-SCNN1B, p.G294S-SCNN1B, p.E539K-SCNN1B, p.1670-2A>G-SCNN1B showed a frequency significantly higher in patients rather in controls (P<0.05), while for the other variants there was no significantly differences between the two groups. Instead, in the group of patients with pancreatitis, we found only 3 variants (p.W493R-SCNN1A, p.R181W-SCNN1A, p.S82C-SCNN1B), but without significantly differences between patient group and controls. In the second part of the study, we explore the potentiality of RLB in the identification of the genotypic strand of Hepatitis C virus. HCV is a single-strand positive RNA virus, which strains can be classified into 7 genotypes. Knowing HCV genotype is essential for the therapeutic management and it is related to antiviral therapy outcome. The 5’ untranslated region (5’ UTR) of the HCV genome is used as target region in most genotyping test. However it is not enough informative for subtyping genotype 1, distinguishing 1a and 1b, and for discrimination of genotypes 1 from 6 (subtypes c-l). During this project, we developed a molecular assay for HCV genotyping, which allows reliable distinction between genotypes and subtypes. The method is based on reverse-transcription of two viral regions (5’UTR and core), followed by RLB genotyping. For the performance evaluation of the developed assay, 283 serum samples were collected: 212 HCV positive, 71 HCV negative and 8 synthetic samples (representative of less common genotypes 5, 6 and 7a). Another CE-IVD genotyping assay and the sequencing of NS5b viral region were used as reference methods. Our results showed that the use of two viral regions for genotyping allows a better discrimination of subtypes 1a and 1b. Among 145 genotype 1 samples, 22 samples (whose subtyping 1a or 1b was not solved by the use of the sole 5’UTR) were correctly typed as 1a or 1b. The genotyping of other 8 samples, previously incorrectly subtyped, were solved. Moreover, the new assay has successfully genotyped 5 samples with genotype 6 that were misdiagnosed as genotype 1 with the analysis of 5’UTR. Our assay showed a diagnostic specificity of 100% and a sensibility of 98.6% both at genotype and subtype levels, in a range of viral loads between 1 x 10^3 and 6 x 10^7 IU/mL. Therefore, combined analysis of both 5’UTR and core regions could be very useful for a better discrimination between subtypes 1a and 1b, and between genotypes 1 and 6, leading to a more powerful instrument for the application of appropriate therapeutic regimen. In conclusion, this work has demonstrated that RLB is a robust method for the management of several diseases, requiring a genetic characterization. However, genetic knowledge about target disorders must to be well defined.I recenti progressi nel campo della genomica umana hanno fortemente influenzato l’approccio diagnostico e terapeutico alle patologie. La biologia molecolare si è affermata come strumento di elezione per la diagnosi non solo di malattie ereditarie ma anche di una grande varietà di processi neoplastici ed infettivi. Tra le metodologie di questa disciplina, il “Reverse Line Blot” (RLB) occupa un posto di rilievo, grazie alla sua versatilità. E’ una tecnologia molto semplice e può essere utilizzata per la routine diagnostica anche da piccoli laboratori, in quanto la strumentazione necessaria è limitata e non eccessivamente costosa. L'obiettivo del nostro progetto è stato quello di sfruttare le potenzialità di questa tecnologia applicandola allo studio di due diverse patologie: una malattia ereditaria (fibrosi cistica atipica) ed una patologia epatica causata dall’infezione del virus dell'epatite C (HCV). Nella prima parte dello studio, abbiamo esplorato le potenzialità della tecnologia RLB per l’identificazione di mutazioni genetiche note, concentrandoci sullo studio della fibrosi cistica atipica. Si tratta di una patologia caratterizzata dagli stessi sintomi della fibrosi cistica (FC), che si presentano però in forma più lieve, con il coinvolgimento solamente di alcuni organi, ed una o nessuna mutazione nel gene CFTR. Questa eterogeneità fenotipica può essere dovuta all’azione di geni modificatori di CFTR, come, per esempio, quelli che codificano il canale epiteliale del sodio (ENaC). Nel corso di questo progetto è stato sviluppato un nuovo test molecolare basato sulla metodologia RLB, per la rilevazione di 9 varianti nei geni ENaC, coinvolte nella FC atipica e precedentemente descritte da altri gruppi. Le performance del metodo sono state valutate analizzando 208 campioni di pazienti con sintomi respiratori o manifestazioni pancreatiche, e 169 individui sani del gruppo di controllo. Tra i pazienti con sintomi respiratori e affetti da FC atipica, recanti una o nessuna mutazione CFTR, abbiamo trovato 7 diverse varianti ENaC: le mutazioni p.S82C-SCNN1B, p.P267L-SCNN1B, p.G294S-SCNN1B, p.E539K-SCNN1B, p.1670-2A>G-SCNN1B, ed i polimorfismi p.W493R-SCNN1A e p.R181W-SCNN1A. In particolare, p.P267L-SCNN1B, p.G294S-SCNN1B, p.E539K-SCNN1B, p.1670-2A>G-SCNN1B mostravano una frequenza significativamente più alta nei pazienti rispetto ai controlli (p