Immunofluorescence Analysis as a Diagnostic Tool in a Spanish Cohort of Patients with Suspected Primary Ciliary Dyskinesia

Abstract

Primary ciliary dyskinesia (PCD) is an autosomal recessive rare disease caused by an alteration of ciliary structure. Immunofluorescence, consisting in the detection of the presence and distribution of cilia proteins in human respiratory cells by fluorescence, has been recently proposed as a technique to improve understanding of disease-causing genes and diagnosis rate in PCD. The objective of this study is to determine the accuracy of a panel of four fluorescently labeled antibodies (DNAH5, DNALI1, GAS8 and RSPH4A or RSPH9) as a PCD diagnostic tool in the absence of transmission electron microscopy analysis. The panel was tested in nasal brushing samples of 74 patients with clinical suspicion of PCD. Sixty-eight (91.9%) patients were evaluable for all tested antibodies. Thirty-three cases (44.6%) presented an absence or mislocation of protein in the ciliary axoneme (15 absent and 3 proximal distribution of DNAH5 in the ciliary axoneme, 3 absent DNAH5 and DNALI1, 7 absent DNALI1 and cytoplasmatic localization of GAS8, 1 absent GAS8, 3 absent RSPH9 and 1 absent RSPH4A). Fifteen patients had confirmed or highly likely PCD but normal immunofluorescence results (68.8% sensitivity and 100% specificity). In conclusion, immunofluorescence analysis is a quick, available, low-cost and reliable diagnostic test for PCD, although it cannot be used as a standalone test.

Keywords: PCD; antibody; cilia; immunofluorescence; primary ciliary dyskinesia.

Figure 1

Ciliary axoneme in transverse section indicating the ultrastructural parts and the target proteins by immunofluorescence. Proteins are indicated in parentheses. DRC = dynein regulatory complex. A and B: outer microtubule doublets; C1 and C2: central pair.

Figure 2

Example results of immunofluorescence technique in control subjects and patients with primary ciliary dyskinesia. The first column shows cilia by acetylated α-tubulin (green); the second column shows the protein of interest (red); and the third column shows the final merged image with the nuclei stained with DAPI (blue). (a) Patient IF-024 exhibited absent DNAH5 and DNALI1. (b) Patient IF-056 had absent DNALI1 and cytoplasmatic localization of GAS8. (c) Patient IF-050 showed a normal axonemal localization of RSPH4A and absent RSPH9. (d) Patient IF-046 presented a proximal localization of DNAH5.