To further analyze the ciliary defect resulting from DNAH9 loss of function, we performed TEM as previously described30 with samples of one DNAH9 mutant individual who was available for analysis (OP-2905 II1). A total of 153 ciliary axonemes were analyzed. Confirming our high-resolution IF microscopy findings, ultrastructural analyses found ODAs to be absent from the distal ciliary axoneme and only ODA docking complexes were visible by TEM analyses of ciliary cross-sections of individual OP-2905 II1 (Figure 6A). Consistent with the restricted localization of DNAH9 to the distal part of the axoneme, outer dynein arms in the proximal ciliary axonemes were still present (Figure 6B). These findings confirm that mutations in DNAH9 result in specific absence of ODAs type 2 and consequently result in an aberrant distal ciliary beating pattern. Usually, ODA defects documented by TEM or high-resolution IF are considered hallmark defects for PCD.34 Thus, the report of three individuals without a classical PCD phenotype and absence of distal ODAs documented by high-resolution IF (n = 3) and TEM (n = 1) might be of interest for diagnostic testing in PCD.
Figure 6 Distal Ciliary Axonemes of DNAH9-Deficient Respiratory Cells Display Subtle Ultrastructural Defects of the Outer Dynein Arms (ODAs)
(A and B) Loss-of-function mutations in DNAH9 cause distal absence of ODAs in the transmission-electron microscopy but do not affect the ODA docking complex (DC) (red arrow in A).
(C and D) Interestingly, ODAs in the proximal part of the ciliary axonemes (blue arrow in C) are not affected by DNAH9 deficiency. The green arrow in (D) marks microvilli extending from the apical side of the cell.
Scale bars represent 200 nm. A total of 153 ciliary cross-sections were analyzed.