C70-04 Structural 3D Modeling of the Puerto Rican RSPH4A P.Tyr230glnfs*8 Founder Genetic Variant

Background Primary Ciliary Dyskinesia (PCD) is a rare genetic disease that causes defects in ciliary structure and function. Among over 55 genes, the RSPH4A [c.921 + 3_6delAAGT] variant is a Puerto Rican founder genetic variant that leads to the majority of PCD without laterality defects on the island. This four-nucleotide intronic deletion disrupts normal splicing, resulting in a frameshift (r.687_921del) and premature termination of translation, producing a truncated protein: RSPH4A p.Tyr230Glnfs*8. Immunofluorescence studies have shown cytoplasmic mislocalization of this truncated protein, implicating it in the disease mechanism. However, the structural consequences of this truncation at the protein level remain poorly characterized, and there is limited understanding of how this specific genetic variation affects the integrity of the radial spoke head-like family sequence. To address this gap, we employed in-silico modeling to compare the wild-type (WT) and truncated RSPH4A proteins, aiming to elucidate structural disruptions and provide mechanistic insight into the pathogenicity of the Puerto Rican founder variant. Methods The gene (NG_012934.1), mRNA (NM_001010892.3), and protein (NP_001010892.1) sequences for WT RSPH4A were obtained from NCBI RefSeq. Sequence alignments were performed using MEGA12 to characterize the variant’s splicing impact. The resulting mis-spliced mRNA and corresponding amino acid sequence were modeled based on Daniels et al. (2013). Three-dimensional (3D) structural models of WT and p.Tyr230Glnfs*8 RSPH4A proteins were generated using SWISS-MODEL and evaluated via SWISS-MODEL Structure Assessment. The WT UniProt model (Q5TD94) served as a reference for comparative analysis. Results To avoid a disordered region in the N-terminal, both models were generated from residue E201 onward. The WT model achieved a QMEANDisCo Global score of 0.74 ± 0.05, a MolProbity Score of 1.11, and 95.62% Ramachandran favored residues. In contrast, the p.Tyr230Glnfs*8 model scored 0.64 ± 0.12 for QMEANDisCo, had a MolProbity Score of 0.50, and 100% Ramachandran favored residues. Structural comparison revealed a near-complete loss of the radial spoke head-like family sequence, which spans residues 214-687 in the WT protein according to Pfam (PF04712). Conclusions In-silico modeling of the RSPH4A p.Tyr230Glnfs*8 variant demonstrates substantial structural disruption due to truncation, including loss of the radial spoke head-like family sequence, critical for ciliary assembly and function. This structural loss supports observed mislocalization and highlights the molecular basis for PCD in individuals with the [c.921 + 3_6delAAGT] variant. These findings underscore the utility of computational modeling in elucidating disease mechanisms when experimental structural data are limited. Further studies, such as cryo-EM and functional assays, are needed to validate predictions.