Primary Ciliary Dyskinesia (PCD) is a rare disorder of the motile cilia for which diagnosis comprises a combination of clinical investigation, analysis of cilia and identification of bi-allelic variants in one of at least 50 PCD-related genes, including HYDIN.

Analysis of HYDIN is complicated by the presence of a pseudogene, HYDIN2, with 98% sequence homology to 79 of the 86 exons of HYDIN. This presents a significant challenge for short read Next Generation Sequencing (SR-NGS) and analysis; consequently many diagnostic PCD panels do not include HYDIN, and affected patients do not receive a genetic diagnosis. To facilitate analysis of HYDIN by SR-NGS, we bioinformatically masked HYDIN2, forcing all reads from both genes to align to HYDIN. This ensured that all HYDIN sequence variants can be assessed and facilitated detection of bi-allelic HYDIN variants in 15 of 437 families referred for PCD genetic testing. However, masking significantly compromises detection of copy number variants. Supplementing testing with long read NGS (LR-NGS) allowed us to identify exonic HYDIN deletions in 2 families missed by SR-NGS. In addition, we used LR-NGS to confirm that identified disease-causing variants were homozygous in two families where parental testing was not possible.

Using our combined diagnostic approach, we detected bi-allelic HYDIN variants in 17 families from 242 genetically-confirmed PCD cases, comprising 7% of our cohort. This is the largest reported HYDIN cohort and supports the inclusion of HYDIN in PCD panels. Additionally, we provide further evidence for the utility of LR-NGS in diagnostic testing, in particular for regions of high genomic complexity.