PIH1D2 Loss-of-Function Directs Ciliary and Cell Cycle Defects Resulting in Left Isomerism With or Without Biliary...

Laterality disorders are rare congenital conditions caused by disruption of left-right embryonic patterning, often associated with ciliary dysfunction. Despite the identification of numerous causative genes, the molecular basis remains unresolved in many patients. We aimed to identify the genetic cause of left isomerism heterotaxy in two unrelated patients, and investigate the functional consequences of the implicated gene.

Genome sequencing was performed in both patients. Candidate variants were validated by RNA and protein analyses in patient-derived fibroblasts. To study the functional consequences, we conducted RNA-seq, cell cycle assays (flow cytometry, EdU incorporation, Ki-67 staining), rescue experiments with the wild-type gene, and primary ciliogenesis studies.

Both patients harboured biallelic loss-of-function variants in PIH1D2, a gene not previously associated with human disease. Transcript analyses confirmed aberrant splicing, and immunofluorescence staining revealed loss of PIH1D2 expression in Patient 1's primary fibroblasts. RNA-seq demonstrated dysregulation of pathways related to cell cycle regulation and developmental signalling. Patient fibroblasts showed impaired cell cycle re-entry from G₀ following serum starvation and reduced S-phase progression. Wild type PIH1D2 overexpression partially rescued these defects. Furthermore, patient cells exhibited significantly reduced primary ciliogenesis compared to controls.

Our findings establish PIH1D2 as a novel disease gene for left isomerism heterotaxy, with biliary atresia observed in one of the patients. Loss of PIH1D2 impairs ciliogenesis and cell cycle progression, providing a mechanistic link between ciliary defects and abnormal left-right patterning. These results expand the spectrum of cilia-related laterality disorders and identify PIH1D2 as a critical regulator of human development.