A novel hypomorphic allele of Spag17 causes primary ciliary dyskinesia phenotypes in mice

Abstract

Primary ciliary dyskinesia (PCD) is a human condition of dysfunctional motile cilia characterized by recurrent lung infection, infertility, organ laterality defects, and partially penetrant hydrocephalus. We recovered a mouse mutant from a forward genetic screen that developed many of the hallmark phenotypes of PCD. Whole exome sequencing identified this primary ciliary dyskinesia only (Pcdo) allele to be a nonsense mutation (c.5236A>T) in the Spag17 coding sequence creating a premature stop codon (K1746*). The Pcdo variant abolished several isoforms of SPAG17 in the Pcdo mutant testis but not in the brain. Our data indicate differential requirements for SPAG17 in different types of motile cilia. SPAG17 is essential for proper development of the sperm flagellum and is required for either development or stability of the C1 microtubule structure within the central pair apparatus of the respiratory motile cilia, but not the brain ependymal cilia. We identified changes in ependymal cilia beating frequency, but these did not apparently alter lateral ventricle cerebrospinal fluid (CSF) flow. Aqueductal (Aq) stenosis resulted in significantly slower and abnormally directed CSF flow and we suggest this is the root cause of the hydrocephalus. The Spag17Pcdo homozygous mutant mice are generally viable to adulthood, but have a significantly shortened life span with chronic morbidity. Our data indicate that the c.5236A>T Pcdo variant is a hypomorphic allele of Spag17 gene that causes phenotypes related to motile, but not primary, cilia. Spag17Pcdo is a novel useful model for elucidating the molecular mechanisms underlying central pair PCD pathogenesis in the mouse.

Quantifying cilia beat frequency using high‐speed video microscopy: Assessing frame rate requirements when imaging different ciliated tissues

“…The easiest way to assess motile cilia function is by quantifying CBF via highspeed video microscopy (Francis & Lo, 2013;O'Callaghan et al, 2012;Sisson et al, 2003) and this forms a sizable chunk of the cilia literature. However, no standardized frame rate exists when imaging cilia motility for subsequent CBF calculation, this has resulted in a large range of frames rates used to image cilia motility within the published literature (Abdelhamed et al, 2018;Abdelhamed et al, 2020;Bustamante-Marin et al, 2019;Chen et al, 2016;Hagiwara et al, 2020;Hennessy et al, 1986;Liu et al, 2021; F I G U R E 3 Assessing how movie framerate influences CBF measurement accuracy for cilia beating between 0-15 Hz, as quantified by either manual counting or FFT-based algorithm (MATLAB). Cilia data from all tissues and temperatures that displayed reference CBFs between 0-15 Hz was pooled (n = 73) (reference CBF was calculated from 300 fps movies).…”

“…The easiest way to assess motile cilia function is by quantifying CBF via highspeed video microscopy (Francis & Lo, 2013 ; O'Callaghan et al, 2012 ; Sisson et al, 2003 ) and this forms a sizable chunk of the cilia literature. However, no standardized frame rate exists when imaging cilia motility for subsequent CBF calculation, this has resulted in a large range of frames rates used to image cilia motility within the published literature (Abdelhamed et al, 2018 ; Abdelhamed et al, 2020 ; Bustamante‐Marin et al, 2019 ; Chen et al, 2016 ; Hagiwara et al, 2020 ; Hennessy et al, 1986 ; Liu et al, 2021 ; Mateos‐Quiros et al, 2021 ; Mikhailik et al, 2021 ; Reula et al, 2021 ; Smith et al, 2012 ; Zahid et al, 2020 ). The lack of a standardized fps for imaging motile cilia is especially problematic because it hinders high‐speed video microscopy from being clinically adapted as a diagnostic tool to help uncover motile cilia diseases, such as primary ciliary dyskinesia (Bricmont et al, 2021 ; Shapiro et al, 2018 ).…”

“…The most used method to assess motile cilia function is to quantify cilia beat frequency (CBF) via high‐speed video microscopy (Reula et al, 2021 ; Rubbo et al, 2019 ; Shapiro et al, 2018 ). However, a large heterogeneity exists within the published literature as to what frame rate per second (fps) to use when imaging cilia motility for subsequent CBF calculation; with published frame rates varying between 28–500 fps (Abdelhamed et al, 2018 ; Abdelhamed et al, 2020 ; Bustamante‐Marin et al, 2019 ; Chen et al, 2016 ; Hagiwara et al, 2020 ; Liu et al, 2021 ; Mateos‐Quiros et al, 2021 ; Mikhailik et al, 2021 ; Reula et al, 2021 ; Smith et al, 2012 ; Zahid et al, 2020 ). The large variation in sampling rates suggests some studies may be under‐sampling or oversampling cilia motility, which could result in inaccurate quantification of CBF or generation of large volumes of oversampled data requiring analysis and storage.…”

Alterations of the Primary Cilia Gene SPAG17 and SOX9 Locus Noncoding RNAs Identified by RNA‐Sequencing Analysis in Patients With Systemic Sclerosis

“…The most common effects are in the ears (chronic ear infections, hearing loss), sinuses (chronic sinus congestion), and lungs (recurrent pneumonia, chronic cough). Rare SPAG17 variants can lead to a PCD-like phenotype in mice and have been shown to cause human PCD as well (44,45). The PCD phenotypes are largely driven by the altered ability of the cilia to beat.…”

RNA-Seq analysis identifies novel roles for the primary cilia gene SPAG17 and the SOX9 locus non-coding RNAs in systemic sclerosis

“…The most common effects are in the ears (chronic ear infections, hearing loss), sinuses (chronic sinus congestion), and lungs (recurrent pneumonia, chronic cough). SPAG17 rare variants can lead to a PCD-like phenotype in mice and have been shown to cause human PCD as well (Abdelhamed et al, 2020, Andjelkovic et al, 2018. The PCD phenotypes are largely driven by the altered ability of the cilia to beat.…”