Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants

Autor: Gabriele Bonetti, William Cozza, Andrea Bernini, Jurgen Kaftalli, Chiara Mareso, Francesca Cristofoli, Maria Chiara Medori, Leonardo Colombo, Salvatore Martella, Giovanni Staurenghi, Anna Paola Salvetti, Benedetto Falsini, Giorgio Placidi, Marcella Attanasio, Grazia Pertile, Mario Bengala, Francesca Bosello, Antonio Petracca, Fabiana D’Esposito, Benedetta Toschi, Paolo Lanzetta, Federico Ricci, Francesco Viola, Giuseppe Marceddu, Matteo Bertelli
Jazyk: angličtina
Rok vydání: 2023
Předmět:
Zdroj: International Journal of Molecular Sciences, Vol 24, Iss 23, p 16881 (2023)
Druh dokumentu: article
ISSN: 1422-0067
1661-6596
DOI: 10.3390/ijms242316881
Popis: Sequencing of the low-complexity ORF15 exon of RPGR, a gene correlated with retinitis pigmentosa and cone dystrophy, is difficult to achieve with NGS and Sanger sequencing. False results could lead to the inaccurate annotation of genetic variants in dbSNP and ClinVar databases, tools on which HGMD and Ensembl rely, finally resulting in incorrect genetic variants interpretation. This paper aims to propose PacBio sequencing as a feasible method to correctly detect genetic variants in low-complexity regions, such as the ORF15 exon of RPGR, and interpret their pathogenicity by structural studies. Biological samples from 75 patients affected by retinitis pigmentosa or cone dystrophy were analyzed with NGS and repeated with PacBio. The results showed that NGS has a low coverage of the ORF15 region, while PacBio was able to sequence the region of interest and detect eight genetic variants, of which four are likely pathogenic. Furthermore, molecular modeling and dynamics of the RPGR Glu-Gly repeats binding to TTLL5 allowed for the structural evaluation of the variants, providing a way to predict their pathogenicity. Therefore, we propose PacBio sequencing as a standard procedure in diagnostic research for sequencing low-complexity regions such as RPGRORF15, aiding in the correct annotation of genetic variants in online databases.
Databáze: Directory of Open Access Journals
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