Mapping the Most Common Founder Variant in RSPH9 That Causes Primary Ciliary Dyskinesia in Multiple Consanguineous Families of Bedouin Arabs

Abstract

Introduction: Primary ciliary dyskinesia (PCD) is a congenital thoracic disorder caused by dysfunction of motile cilia, resulting in insufficient mucociliary clearance of the lungs. The overall aim of this study is to identify causative defective genes in PCD-affected individuals in the Kuwaiti population.

Methods: A cohort of multiple consanguineous PCD families was identified from Kuwaiti patients and genomic DNA from the family members was isolated using standard procedures. The DNA samples from all affected individuals were analyzed by whole exome sequencing (WES). Transmission electron microscopy (TEM) and immunofluorescent analysis (IF) were performed on samples obtained by nasal brushings to identify specific structural abnormalities within ciliated cells.

Results: Here, we present six multiplex families with 11 patients who all presented with typical PCD symptoms. Ten out of eleven patients inherited a 3 bp homozygous deletion of GAA in RSPH9, whereas the eleventh patients inherited this variant in trans with a frameshift deletion in RSPH9. Genetic results were confirmed by segregation analysis. The in-frame deletion of GAA in RSPH9 has previously been published as pathogenic in both annotated RSPH9 transcript variants (1 and 2). In contrast, the previously unpublished RSPH9 frameshift deletion identified in KU-15.IV2 impacts only RSPH9 transcript variant two. Regarding all 11 PCD individuals analyzed, IF results demonstrated absence of RSPH9 protein and TEM analysis showed the typical findings in RSPH9 mutant individuals.

Conclusions: We present the largest cohort of PCD individuals affected by the founder in-frame deletion GAA in RSPH9. This founder variant is the most common PCD-causing variant in Bedouin Arabs in Kuwait.

Keywords: RSPH9; consanguinity; genetics of ciliopathy; primary ciliary dyskinesia; pulmonary diseases.

Figure 1

Summary of the Agile Multi Ideograms showing linkage scans for the 22-autosomal chromosomes for the affected individuals in each family with hereditary founder variant in RSPH9. Each ideogram represents the linkage scan as a circular axis for the PCD individuals belonging to the same family in one group. In multiplex families: the exclusive regions of homozygosity (ROH) shared between the affected relatives are displayed as navy blue bars. The homozygous regions that are not shared among affected relatives are displayed as light blue bars. In singleton families, all the detected ROH are displayed as navy blue bars since there is no shared ROH to be prioritized by the software, as seen for families KU-20 and KU-24. The shared ROH across the RSPH9 locus at chr: 6 is indicated by a red arrow in each ideogram for families KU-3, KU-11, KU-24, and KU-28. Family KU-15 indicates that there are no detectable ROH regions for the IV-2 PCD individual, while the IV-4 patient has a minor shade at the gene locus (arrowhead) consistent with the genotype of the two PCD individuals. The linkage scan for KU-24, IV-3 shows a tremendous ROH for the PCD individual at several chromosomal segments and across RSPH9 locus. However, the linkage for KU-20, II-1, PCD individual shows only one exclusive ROH at chr: 3 and a minor ROH interval (thin navy blue line) at the RSPH9 locus at chr: 6 (arrowhead), which indicates that this founder variant has very high penetrance. Interestingly, the KU-28 family shows two small-scale ROH intervals shared among the three affected individuals IV-1, IV-4, and IV-5 (navy blue line) at chr: 6, across RSPH9 locus, and they also have a shared IBD at chr: 17. There are multiple ROH that are not shared among the three affected individuals (light blue). In each ideogram, the linkage scan for each PCD individual belonging to the same family is presented from the ideogram edge (first circle) towards the center (last circle). The alignment is solely controlled by the software.

Figure 2

Radiological chest X-ray images for eight PCD individuals covered in this study. Chest X-ray of the KU-3.IV-2 patient showing mild diffuse bronchiectasis and patchy consolidations in bilateral lower zones, predominantly the retrocardiac regions (arrows), suggestive involvement of the right middle and the left lingular lobes. Chest X-ray of the KU-3.IV-3 patient showing dilated bronchi with “tram-track” appearance in the bilateral perihilar regions radiating into the lower zones (arrows). Chest X-ray of the KU-3.IV-4 patient showing subtle bronchiectatic changes with ill-defined hazy opacification in the medial right lower zone, suggestive of partial right middle lobe collapse (arrows). Chest X-ray of the KU-11.III-3 patient shows diffuse bilateral bronchiectasis with associated patchy opacities in all lung zones (arrowheads), representing secondary inflammatory changes (arrows). Chest X-ray of the KU-11.IV-1 patient showing central right perihilar bronchiectatic changes with an associated ill-defined opacity in the medial right lower zone causing obscuration of the right heart border, suggestive of complete right middle lobe collapse (arrows). Chest X-ray of the KU-24.II-1 patient showing dilated bronchi with “tram-track” appearance in the bilateral perihilar regions radiating into the lower zones (arrows). Small patches of consolidation are also seen in the right perihilar region (arrowheads), denoting infective changes. Chest X-ray of the KU-28.IV-1 patient showing mild diffuse bronchiectasis and patchy consolidations in bilateral lower zones, predominantly the retrocardiac regions (arrows), suggestive of involvement of the right middle and the left lingular lobes. Chest X-ray of the KU-28.IV-5 patient showing mild diffuse bronchiectasis and patchy consolidations in bilateral lower and the right upper zones (arrows). Situs solitus is seen in all patients.

Figure 3

Radiological data for the KU-3.IV-3 patient shows the clinical morphology of the founder variant. Selected coronal (A), axial (B), and sagittal (C) recons of the high-resolution computed tomography (HRCT) chest study show mild diffuse bronchiectatic changes in both lungs, most notably in the lingular segments of the left upper lobe (arrows; blue), with extensive scattered nodular opacities showing “tree-in-bud” appearance (arrowheads; orange). These findings represent secondary infection in a patient with underlying chronic bronchiectatic changes. Selected axial (D) and coronal (E) images of a computerized tomography (CT) for paranasal sinus (PNS) study of the same patient show diffuse polypoid mucosal thickening opacifying both maxillary, ethmoid, and sphenoid sinuses, denoting chronic inflammatory PAN sinusitis changes.

Figure 4

The sequencing chromatographs for the ten PCD patients versus control. All the patients with IBD across the gene locus have homozygous variant (c.856_858delGAA; p.Glu286del) in RSPH9. The position of the (GAA) deletion is indicated by three red asterisks in the healthy subject and by the red arrows in the chromatograms of PCD individuals.

Figure 5

Immunofluorescence images of primary respiratory epithelial cells of PCD individuals versus healthy control sample. The IF was performed using monoclonal anti-acetylated α tubulin (panel a) and polyclonal anti-RSPH9 (panel b) antibodies. As seen in the healthy control sample, the merged images (panel c) show a yellow co-staining within the ciliary axoneme, which indicates that both proteins co-localized within respiratory cilia, compared with the images for PCD individuals that demonstrate an absence of anti-RSPH9 staining. (d) shows differential interference contrast figures (DIC) for analyzed cells, Scale bar is 10 μm.

Figure 6

TEM images showing ultrastructural abnormalities of the ciliary axoneme for PCD patients. TEM results for ciliary axoneme of the epithelial cells from the respiratory system indicate that the PCD individuals mainly have an absence of central pairs (CP), as highlighted in arrowheads (orange), and mislocalization of the peripheral microtubule doublets (MDs) in some sections, as highlighted in arrowheads (blue). The ultrastructural ciliary defects indicate that the causative mutations in these patients affect the stabilization of the MDs and CP due to radial spoke defects consistent with their genotypes.