Value of transmission electron microscopy for primary ciliary dyskinesia diagnosis in the era of molecular medicine: Genetic defects with normal and non-diagnostic ciliary ultrastructure

Abstract

Primary ciliary dyskinesia (PCD) is a genetic disorder causing chronic oto-sino-pulmonary disease. No single diagnostic test will detect all PCD cases. Transmission electron microscopy (TEM) of respiratory cilia was previously considered the gold standard diagnostic test for PCD, but 30% of all PCD cases have either normal ciliary ultrastructure or subtle changes which are non-diagnostic. These cases are identified through alternate diagnostic tests, including nasal nitric oxide measurement, high-speed videomicroscopy analysis, immunofluorescent staining of axonemal proteins, and/or mutation analysis of various PCD causing genes. Autosomal recessive mutations in DNAH11 and HYDIN produce normal TEM ciliary ultrastructure, while mutations in genes encoding for radial spoke head proteins result in some cross-sections with non-diagnostic alterations in the central apparatus interspersed with normal ciliary cross-sections. Mutations in nexin link and dynein regulatory complex genes lead to a collection of different ciliary ultrastructures; mutations in CCDC65, CCDC164, and GAS8 produce normal ciliary ultrastructure, while mutations in CCDC39 and CCDC40 cause absent inner dynein arms and microtubule disorganization in some ciliary cross-sections. Mutations in CCNO and MCIDAS cause near complete absence of respiratory cilia due to defects in generation of multiple cellular basal bodies; however, the scant cilia generated may have normal ultrastructure. Lastly, a syndromic form of PCD with retinal degeneration results in normal ciliary ultrastructure through mutations in the RPGR gene. Clinicians must be aware of these genetic causes of PCD resulting in non-diagnostic TEM ciliary ultrastructure and refrain from using TEM of respiratory cilia as a test to rule out PCD.

Keywords: Electron microscopy; PCD; genetic testing; primary ciliary dyskinesia.

Figure 1

Illustration and TEM image of normal ciliary ultrastructure Normal 9+2 ciliary arrangement, with 9 outer doublet microtubules surrounding a central pair and normal outer and inner dynein arms. Even in this normal sample, electron dense material inside the ciliary membrane makes the inner dynein arms, radial spokes, and nexin links appear indistinct, as is often the case in clinical samples. Not that only one to two radial spokes are visible in this normal sample. Reproduced with permission from .

Figure 2

TEM and electron tomography images with disease causing HYDIN mutations TEM cross sections of HYDIN-mutant individual, showing normal ciliary ultrastructure (A & B). Rarely, ciliary transposition defects can be seen (C). Healthy control TEM images (D-F) demonstrate normal ciliary ultrastructure. Composite image averages of six affected (G) and 6 control (J) TEM images show loss of the C2b appendage from the central apparatus (arrow). A detailed analysis of the CP apparatus by electron microscopic tomography (H and I) identified the absence of the C2b projection in HYDIN-mutant cilia from one individual compared to control samples (K and L). Each rendered image (H and K) represents six cross-sections averaged from a dual-axis tomogram made up of 140 images at 2 degree tilts. The scale bars represent 10 nm (rendered picture) and 200 nm (TEM pictures). Reproduced with permission from .

Figure 3

TEM images in PCD caused by CCNO mutations TEM photographs of respiratory epithelial cells of individuals with CCNO mutations show severe reduction in the numbers of motile cilia and basal bodies at the apical cell region and mislocalization of basal bodies and rootlets within the cytoplasm. (a) Respiratory epithelial cell from a healthy control individual showing several basal bodies (BB) on the apical membrane nucleating multiple motile cilia (C) and microvilli (MV) at the apical cell region. (b–e) Respiratory epithelial cells of individuals with CCNO mutations. Occasionally, one can see basal bodies abnormally located inside the cytoplasm, with ciliary rootlets (R). Scale bars, 2 μm. Mutations in MCIDAS produce similar TEM images. Reproduced with permission from .

Figure 4

Illustration of the proposed ciliary structure for the inner dynein arm and dynein regulatory complex Reproduced with permission from .

Figure 5

TEM images of cilia with disease causing CCDC65 mutations (B) TEM of a patient with PCD from CCDC65 mutations, showing normal dynein arms and central pairs. Note that upon close examination, N-DRC links are missing (arrowheads). Microtubule disorganization (B, inset) was observed in 5%–15% of PCD individual cilia sections with CCDC65 mutations. (C) TEM from a healthy control, showing normal dynein inner arms and nexin links (arrowheads). Reproduced with permission from .

Figure 6

TEM images of cilia with disease causing CCDC39 mutations Transmission electron microscopy of respiratory cilia from an individual who is homozygous for CCDC39 mutations, showing absence of inner dynein arms in all ciliary sections, associated with a range of other, heterogeneous defects: isolated absence of the nine inner dynein arms (1), axonemal disorganization with mislocalized peripheral doublet associated with either a displacement of the central pair (2), an absence of the central pair (3), or supernumerary central pairs (4). Magnification of the axoneme from a normal cilium is shown in the upper right panel with presence of inner dynein arms (black arrow), nexin links (white arrow) and radial spokes (short arrow). The axonemal disorganization found in cases is associated with defects of inner dynein arms (black flash), nexin links (white flash) and radial spokes (star) that are better seen after magnification (lower right panel). Scale bar, 0.2 μm. Reproduced with permission from .

Figure 7

Ciliary ultrastructural defects seen on TEM in various radial spoke head gene mutations * Other changes consists of the following: 8+0 arrangement, 7 outer doublets with central pair and 2 single microtubules in the center, 8 outer doublets with 2 central pairs, 8 outer doublets with 4 central single microtubules, 8 outer doublets without a central pair and 1 doublet outside of the outer ring. ** Only accounting for 8+1 transposition defects Reprinted and adapted with permission of the American Thoracic Society. Copyright © 2017 American Thoracic Society. The American Journal of Respiratory and Critical Care Medicine is an official journal of the American Thoracic Society.

Figure 8

TEM images of cilia with transposition defects The TEM ultrastructural arrangement of the axoneme as seen in cilia with transposition defects. (A) A transverse view showing a normal appearing 9+2 ciliary arrangement that changes distally to (B) a 9+0 arrangement where the central pair disappears, and towards the tip of the cilium (C) where one of the microtubule doublets moves into the center to give an 8+1 arrangement. This particular pattern indicates a transposition defect. The change in arrangement can be seen in the longitudinal section (D). White arrows indicate microtubule doublets (MTD), radial spokes (RS) and the central pair (CP). Scale bar 100 nm. Reproduced with permission from .