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Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGR^ORF15 Genetic Variants.

ABSTRACT: 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 RPGR^ORF15, aiding in the correct annotation of genetic variants in online databases.

SUBMITTER: Bonetti G

PROVIDER: S-EPMC10706286 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGR<sup>ORF15</sup> Genetic Variants.

Bonetti Gabriele G Cozza William W Bernini Andrea A Kaftalli Jurgen J Mareso Chiara C Cristofoli Francesca F Medori Maria Chiara MC Colombo Leonardo L Martella Salvatore S Staurenghi Giovanni G Salvetti Anna Paola AP Falsini Benedetto B Placidi Giorgio G Attanasio Marcella M Pertile Grazia G Bengala Mario M Bosello Francesca F Petracca Antonio A D'Esposito Fabiana F Toschi Benedetta B Lanzetta Paolo P Ricci Federico F Viola Francesco F Marceddu Giuseppe G Bertelli Matteo M

International journal of molecular sciences 20231128 23

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-complexit ...[more]

PMID: 38069202

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Project description:BackgroundThe clinical diagnosis of Fabry Disease (FD) can be challenging due to the clinical heterogeneity, especially in females. Patients with FD often experience a prolonged interval between the onset of symptoms and receiving a diagnosis. Genetic testing is the gold standard for precise diagnosis of FD, however conventional genetic testing could miss deep intronic variants and large deletions or duplications. Although next-generation sequencing, which analyzes numerous genes, has been successfully used for FD diagnosis and can detect complex variants, an effective and rapid tool for identifying a wide range of variants is imminent, contributing to decrease the diagnostic delay.MethodsThe comprehensive Analysis of FD (CAFD) assay was developed for FD genetic diagnosis, employing long-range PCR coupled with long-read sequencing to target the full-length GLA gene and its flanking regions. Its clinical performance was assessed through a comparative analysis with Sanger sequencing.ResultsGenetic testing was performed on 82 individuals, including 48 probands and 34 relatives. The CAFD assay additionally identified variants in two probands: one had a novel and de novo pathogenic variant with a 1715 bp insertion in intron 4, and the other carried two deep intronic VUS variants in cis-configuration also in intron 4. In total, CAFD identified 47 different variants among 48 probands. Of these, 42 (89.36%, 42/47) were pathogenic, while 5 (10.64%, 5/47) were VUS. Sixteen (34.04%, 16/47) of the variants were novel, including 15 SNV/Indels and one large intronic insertion. Pedigree analysis of 21 probands identified four de novo disease-causing variants. Hence, FD exhibits not only variable clinical presentations but also a wide spectrum of variants. Utilizing a comprehensive testing algorithm for diagnosing FD, which includes enzyme activity, clinical features, and genetic testing, the diagnostic yield of CAFD is 97.92% (47/48), which is higher than that of conventional Sanger sequencing, at 95.83% (46/48).ConclusionThe duration between initial clinical presentation and diagnosis remains long and winding. CAFD provides precise diagnosis for a wide spectrum of GLA variants, promoting timely diagnosis and appropriate treatment for FD patients.

Dataset Information

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGR^ORF15 Genetic Variants.

Publications

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGR<sup>ORF15</sup> Genetic Variants.

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Dataset Information

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

Publications

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGR<sup>ORF15</sup> Genetic Variants.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

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Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGR^ORF15 Genetic Variants.