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Structural insights into the cause of human RSPH4A primary ciliary dyskinesia.

ABSTRACT: Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Component 4A (RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A^-/- PCD remains elusive. Here, we present the native three-dimensional structure of RSPH4A^-/- human respiratory cilia using samples collected noninvasively from a PCD patient. Using cryo-electron tomography (cryo-ET) and subtomogram averaging, we compared the structures of control and RSPH4A^-/- cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1^-/- cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A^-/- cilia. However, RSPH4A^-/- cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A^-/- PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC8351563 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: The radial spoke head component 4A (RSPH4A) is involved in the assembly of radial spokes, which is essential for motile cilia function. Asthenoteratozoospermia in primary ciliary dyskinesia (PCD) related to RSPH4A variants has not been reported. Materials and Methods: RSPH4A variants were identified and validated using whole-exome and Sanger sequencing in three unrelated Chinese families. High-speed video microscopy analysis (HSVA) was performed to measure the beating frequency and pattern of nasal cilia of the patients and healthy control. Papanicolaou staining and computer-aided sperm analysis were performed to analyze the morphology and motility of the sperm in patient 1. Immunofluorescence was adopted to confirm the structure deficiency of sperm and nasal cilia. Results: Patient 1 from family 1 is a 22-year-old unmarried male presented with bronchiectasis. Semen analysis and sperm Papanicolaou staining confirmed asthenoteratozoospermia. Novel compound heterozygous RSPH4A variants c.2T>C, p.(Met1Thr) and c.1774_1775del, p.(Leu592Aspfs*5) were detected in this patient. Patients 2 and 3 are from two unrelated consanguineous families; they are both females and exhibited bronchiectasis and infertility. Two homozygous RSPH4A variants c.2T>C, p.(Met1Thr) and c.351dupT, p.(Pro118Serfs*2) were detected, respectively. HSVA showed that most of the cilia in patients 1 and 3 were with abnormal rotational movement. The absence of RSPH4A and RSPH1 in patient 1's sperm and patient 3's respiratory cilia was indicated by immunofluorescence. Patient 2 died of pulmonary infection and respiratory failure at the age of 35 during follow-up. Conclusion: Dysfunctional sperm flagellum and motile cilia in the respiratory tract and the fallopian tube were found in patients with RSPH4A variants. Our study enriches the genetic spectrum and clinical phenotypes of RSPH4A variants in PCD, and c.2T>C, p.(Met1Thr) detected in our patients may be a hotspot RSPH4A variant in Chinese.

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Structural insights into the cause of human RSPH4A primary ciliary dyskinesia.

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