A quantitative super-resolution imaging toolbox for diagnosis of motile ciliopathies

Abstract

Airway clearance of pathogens and particulates relies on motile cilia. Impaired cilia motility can lead to reduction in lung function, lung transplant, or death in some cases. More than 50 proteins regulating cilia motility are linked to primary ciliary dyskinesia (PCD), a heterogeneous, mainly recessive genetic lung disease. Accurate PCD molecular diagnosis is essential for identifying therapeutic targets and for initiating therapies that can stabilize lung function, thereby reducing socioeconomic impact of the disease. To date, PCD diagnosis has mainly relied on nonquantitative methods that have limited sensitivity or require a priori knowledge of the genes involved. Here, we developed a quantitative super-resolution microscopy workflow: (i) to increase sensitivity and throughput, (ii) to detect structural defects in PCD patients’ cells, and (iii) to quantify motility defects caused by yet to be found PCD genes. Toward these goals, we built a localization map of PCD proteins by three-dimensional structured illumination microscopy and implemented quantitative image analysis and machine learning to detect protein mislocalization, we analyzed axonemal structure by stochastic optical reconstruction microscopy, and we developed a high-throughput method for detecting motile cilia uncoordination by rotational polarity. Together, our data show that super-resolution methods are powerful tools for improving diagnosis of motile ciliopathies.

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

“…As new genes and proteins related to PCD are discovered, the IF antibody panels may need to be revised and expanded in the future for an accurate diagnosis [49]. In fact, antibodies against a high number of ciliary proteins are already commercially available, although most of them have not yet been tested and/or validated for immunofluorescence or in human respiratory tissue [11]. For this reason, the optimization of antibodies in nasal brushing samples is difficult and time-consuming.…”

“…Furthermore, from our experience, we have not even been able to properly optimize some commercially available antibodies, i.e., DNAH11. Further antibody optimization is necessary, and, as a matter of fact, Liu et al's extensive IF technical protocols may help with this [11]. Another pitfall is the lack of consensus regarding the performance of the IF technique and, more importantly, the agreement in the IF considerations when the analysis is performed.…”

“…Alternatively, centers with available TEM analysis might use a first step IF panel with DNAH11, SPEF2, GAS8 and RSPH9 antibodies, omitting DNAH5 and DNALI1. For the translation to clinical diagnosis, Liu et al also proposed a restricted 10-antibody panel (instead of 21) based on proteins which are non-detectable by TEM or those indirectly detecting mislocalization of other proteins (DNAH5, DNAH11, DNALI1, GAS8, CCDC65, RSPH4A, RSPH9, RPGR, OFD1 and SPEF2) [11]. Although a quantitative super-resolution imaging tool, such as the one proposed by Liu et al [11], gives much more information and may solve so-called difficult or unsolved cases, it would be hard to implement in our clinical setting due to its high costs in time and personnel.…”

“…In a recent study, Liu et al presented a quantitative super-resolution imaging workflow for the detection of cilia defects thanks to the validation of 21 commercially available IF antibodies. Molecular defects using this super-resolution imaging toolbox were described in 31 clinical PCD cases, including patients with negative TEM results and/or with genetic variants of uncertain significance (VUS) [11].…”

A Revised Protocol for Culture of Airway Epithelial Cells as a Diagnostic Tool for Primary Ciliary Dyskinesia

“…Transmission electron microscopy (TEM) is used to assess and quantify ultrastructural abnormalities of motile cilia [ 13 , 14 ]. Immunofluorescence labelling (IF) can demonstrate the absence or mis-localisation of ciliary proteins [ 15 , 16 ], particularly helpful in cases where no TEM abnormalities are detected such as with DNAH11 [ 17 ], DNAH9 [ 18 ] and HYDIN gene mutations [ 19 ]. Genotyping can detect pathogenic bi-allelic or X-linked hemizygous mutations in 50 PCD-related genes to confirm the diagnosis in approximately 70% of well characterized cases [ 1 , 3 , 20 ].…”