Unique pipeline for the assessment of novel genetic variants leads to confirmation of PCD diagnosis

Abstract

Background/Objectives: Primary ciliary dyskinesia (PCD) is a disease caused by impaired ciliary motility and mainly affects the lungs and reproductive organs. Inheritance is autosomal recessive and X-linked with more than 40 disease-causing genes, wherefore PCD patients have diverse clinical manifestations, thus making diagnosis difficult. The utility of next-generation sequencing (NGS) technology for diagnostic purposes allows a better understanding of the PCD genetic background. However, the identification of specific disease-causing variants is challenging. The objective of this study was to create a unique guideline that will enable the standardization of the assessment of novel variants within PCD associated genes. Methods: The study included designing a pipeline for the classification of the rare genetic variants detected using NGS. The pipeline included in silico (translation, 3D-model, protein-protein interactions, sequence conservation, posttranslational modifications) and functional analysis (expressional analysis, Western Blot) of the variants. Results: The designed pipeline consists of three steps: sequencing, detection, and identification of genes/variants; classification of variants according to their effect; and variant characterization using in silico structural and functional analysis. The pipeline was validated by the analysis of the variants detected in a disease-causing gene (DNAI1) and the novel candidate gene (SPAG16). Conclusion: The application of the pipeline resulted in the identification of disease-causing variants, as well as pathogenicity validation, through the analysis on transcriptional, translational, and posttranslational levels.The application of created pipeline leads to the confirmation of PCD diagnosis and enables a shift from candidate to disease-causing gene.