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

Keywords: PacBio; RPGR; TTLL5; long-read sequencing; molecular docking; molecular dynamics.

Figure 1

NGS sequencing coverage of RPGR ORF15 exon in the analyzed samples.

Figure 2

Graphical representation of RPGR^ORF15 and TTLL5 interaction. The basic domain (BD) of RPGR^ORF15 recruits TTLL5 and binds to its noncatalytic CID domain. The in-frame deletion is indicated in red.

Figure 3

Surface and cartoon view of TTLL5 active site. The peptide (in pink, shown as sticks) fits into the crevice centered at the active site, with Glu737 directed towards ADP. Glutamates align to form electrostatic interactions with the positively charged residues (shown as lines in the cartoon view), stabilizing the peptide into the pocket. The structural components surrounding the peptide chain are shown in light orange (loops α1-β1, α2-β3, β6-β7, and helix α6), with the rest of the protein shown in green; the donor glutamate is shown in cyan; magnesium ions and ADP are shown in gray and orange, respectively.

Figure 4

Loop configurations around the binding site. (A) Comparison of the binding cavity between the average structure of the bound (light green) and free (light red) TTLL5. The β6-β7 loop is slightly displaced to accommodate the peptide chain. (B) Comparison of the binding cavity between the average structure of the TTLL5 bound to the GEEEHGE737GEEEE (light green) and TTLL6 (light cyan). The open configuration is evident in TTLL5, with a notable displacement of the β6-β7 loop. Loops are marked with higher opacity.

Figure 5

RMSD of each peptide chain bound to TTLL5. While the variant (shown in light green) is characterized by large movements for the first 100 ns, the two wildtype segments (shown in dark green and yellow) are subject to fewer fluctuations, especially the one with Glu737 as the glutamylation site.