Biallelic RSPH4A loss-of-function variants cause primary ciliary dyskinesia in a Chinese patient

Abstract

Background Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder characterized by defective motile cilia function, affecting approximately one in 7,500 to one in 10,000 live births. Pathogenic variants in radial spoke head genes, including RSPH4A, cause PCD with distinctive central-microtubular-pair defects. However, the functional consequences of novel RSPH4A variants remain poorly characterized, limiting genetic counseling and prenatal diagnostic capabilities. This study aims to reveal the genetic etiology of PCD in an affected family and the clinical significance of the two novel RSPH4A variants identified in this PCD-affected pedigree. Methods We recruited a five-member Chinese family including an 11-year-old female PCD proband presenting with chronic bronchiectasis and recurrent respiratory infections. Comprehensive clinical evaluations, whole exome sequencing (WES), and Sanger sequencing were performed to identify genetic variants. Bioinformatics analyses including protein sequence alignment and structural modeling were conducted. Experimental validation employed site-directed mutagenesis, quantitative real-time PCR, and Western blotting in HEK293T cells to characterize variant effects on mRNA stability and protein expression. Results WES identified compound heterozygous RSPH4A variants in the proband: RSPH4A (NM_001010892.3): c.2T>C (p.Met1Thr) inherited from the mother and RSPH4A (NM_001010892.3): c.854delA (p.Lys286Serfs*22) inherited from the father, showing strict co-segregation with the disease phenotype. The c.2T>C variant disrupted the translation initiation codon, while c.854delA introduced a premature termination codon within the radial spoke head domain. Cross-species analysis demonstrated high conservation of the affected region across 15 vertebrate species. Structural modeling predicted complete loss of the radial spoke head domain in the truncated protein. Functional studies revealed that c.2T>C severely impaired protein translation despite intact mRNA levels, whereas c.854delA triggered nonsense-mediated mRNA decay and produced unstable truncated protein. Both variants resulted in substantial reduction in functional RSPH4A protein expression. Conclusion This study identifies novel loss-of-function RSPH4A variants causing PCD through distinct molecular mechanisms, expanding the mutational spectrum of radial spoke head protein-related ciliopathies. These findings offer compelling proof in favor of a molecular diagnosis of PCD in this family and enable carrier screening for at-risk relatives. The experimental validation strategy establishes a framework for interpreting variants of uncertain significance in PCD genes, facilitating accurate prenatal diagnosis and preimplantation genetic testing to reduce the recurrence risk and birth defect incidence in PCD. Furthermore, understanding the precise functional consequences of RSPH4A variants informs genotype-phenotype correlations and may guide future development of targeted therapeutic interventions for this debilitating respiratory disorder.