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. 2018 Nov 1;103(5):727-739.
doi: 10.1016/j.ajhg.2018.10.003.

Biallelic Mutations in LRRC56, Encoding a Protein Associated With Intraflagellar Transport, Cause Mucociliary Clearance and Laterality Defects

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Biallelic Mutations in LRRC56, Encoding a Protein Associated With Intraflagellar Transport, Cause Mucociliary Clearance and Laterality Defects

Serge Bonnefoy et al. Am J Hum Genet. .
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Abstract

Primary defects in motile cilia result in dysfunction of the apparatus responsible for generating fluid flows. Defects in these mechanisms underlie disorders characterized by poor mucus clearance, resulting in susceptibility to chronic recurrent respiratory infections, often associated with infertility; laterality defects occur in about 50% of such individuals. Here we report biallelic variants in LRRC56 (known as oda8 in Chlamydomonas) identified in three unrelated families. The phenotype comprises laterality defects and chronic pulmonary infections. High-speed video microscopy of cultured epithelial cells from an affected individual showed severely dyskinetic cilia but no obvious ultra-structural abnormalities on routine transmission electron microscopy (TEM). Further investigation revealed that LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88. The link with IFT was interrogated in Trypanosoma brucei. In this protist, LRRC56 is recruited to the cilium during axoneme construction, where it co-localizes with IFT trains and is required for the addition of dynein arms to the distal end of the flagellum. In T. brucei carrying LRRC56-null mutations, or a variant resulting in the p.Leu259Pro substitution corresponding to the p.Leu140Pro variant seen in one of the affected families, we observed abnormal ciliary beat patterns and an absence of outer dynein arms restricted to the distal portion of the axoneme. Together, our findings confirm that deleterious variants in LRRC56 result in a human disease and suggest that this protein has a likely role in dynein transport during cilia assembly that is evolutionarily important for cilia motility.

Keywords: cilia; ciliopathies; dynein arms; flagella; intraflagellar transport; left-right asymmetry; leucine-rich repeat protein; trypanosome.

Figures

Figure 1
Figure 1
LRRC56 Mutations Cause Chronic Infective Lung Disease and Laterality Defects (A) The homozygous splice-site mutation (c.423+1G>A, GenBank: NM_198075.3) disrupts an invariant splice site in family 1 individual IV:1. The unaffected sibling IV:2 is heterozygous for the mutation. The homozygous missense mutation (c.419T>C, GenBank: NM_198075.3) was identified in the two affected siblings IV:3 and IV:4 from family 2. Consistent with autosomal-recessive inheritance, the mutations described were detected in a heterozygous state in the unaffected parents (data not shown). The third individual (family 3 II:1) was a compound heterozygote for the variants c.760G>T and c.326+1G>A (GenBank: NM_198075.3). (B) The family 1 proband (IV:1) had dextrocardia, documented by chest X-ray (left). High-resolution axial computed tomography of the thorax in the same individual demonstrates mild bronchiectasis (red arrows) with adjacent inflammatory consolidation in the right lower lobe. Dextrocardia is also visible (CT scan; right). (C) Cross section through the axoneme from cultured respiratory cells from family 1 individual IV:1. The position of the section is indicated, bar = 100 nm. Normal axonemal structure is visible, with intact dynein arms. (D) Recombinant human LRRC56 and intraflagellar transport protein IFT88 interact in vitro. HEK293 cells were co-transfected with plasmids encoding human LRRC56 and IFT88 tagged with V5 and YFP, respectively (1.5 μg of each). After 48 hr, immunoprecipitation (IP) was performed with transfected and untransfected cells using Cell-TRAP magnetic beads bound to an anti-GFP antibody fragment. Protein from input whole-cell extracts (WCE, left) and immunoprecipitated proteins (IP, right) were blotted using anti-V5 or anti-GFP. The IFT88-GFP fusion is a fairly large protein and in our conditions, slight differences in migration are frequently observed. This does not impact on the underlying hypothesis being tested. A β-actin control is also shown.
Figure 2
Figure 2
LRRC56 Associates with IFT Trains and Not with the Axoneme (A) An YFP::LRRC56-expressing cell that assembles its new flagellum (yellow arrow) shows staining (anti-GFP, green on merged images) in the new flagellum (blue arrowheads) that co-localizes with the anti-IFT172 (red on merged images) but not with the anti-axoneme marker (mAb25, blue on merged images). No YFP staining is visible in the old flagellum (white arrow) whereas IFT172-positive trains are clearly present. IFT trains are predominantly found on microtubules doublets 4 and 7, so the visual aspect of two separate tracks around the axoneme. DNA is stained with DAPI (cyan) showing the presence of two kinetoplasts (mitochondrial DNA) and two nuclei typical of cells at late stage of their cycle. (B) Cytokinesis results in two daughter cells each containing a unique flagellum. The one inheriting the new flagellum remains positive for YFP::LRRC56 that still shows association with IFT trains (same staining as in A). (C) The same immunofluorescence assay was performed on IFT140RNAi cells expressing YFP::LRRC56 after 24 hr in RNAi conditions. DNA staining shows that the top cell is mitotic and assembles a new flagellum. The IFT172 staining reveals the presence of a stalled IFT train that contains a considerable quantity of YFP::LRRC56 (arrowhead). The bottom cell is at a further stage of its cell cycle, yet its new flagellum is much shorter, indicating that RNAi knockdown occurred at a very early phase of construction (star). In these conditions, IFT172 staining shows that the very short flagellum contains a large amount of accumulated IFT material, yet no signal is visible for YFP::LRRC56 (star).
Figure 3
Figure 3
Absence of LRRC56 or Expression of the p.Leu259Pro Variant Reduces Flagellum Beating and Cell Motility (A–C) Tracking analysis showing the movement of individual trypanosomes in wild-type control (A), in cells expressing only YFP::LRRC56L259P (B) and in lrrc56−/− cells (C). Sustained motility is observed only in control conditions. (D) Quantification of the straight-line movement confirms the visual impression that motility was reduced in a statistically significant manner in YFP::LRRC56L259P cells and almost abolished in lrrc56−/−. Total number of cells, mean, and standard deviation are indicated on the figure. Statistical analysis was performed using t test.
Figure 4
Figure 4
LRRC56 Is Required for Assembly of ODA in the Distal Portion of the Axoneme (A–E) Sections are shown through the flagella of detergent-extracted cytoskeletons from various cell lines. Stripping the flagellum membrane and matrix facilitates the analysis of structures., , Sections through control YFP::LRRC56-expressing cells (A) possess all nine ODA, whereas a mixture of sections with normal profiles or with several missing ODA (orange arrowheads) is encountered in YFP::LRRC56L259P-expressing cells (B and C) or lrrc56−/− cells (D and E). (F) Sections were grouped in three categories: defects in two ODA or less (blue), in five ODA or less (green), and in six ODA or more (red). The total counted number of sections is 50 for each sample. Full details are given in Table S3. (G and H) IFA with the anti-DNAI1 antibody stains the whole axoneme of wild-type cells (G) as expected. However, the staining was limited to the proximal portion in lrrc56−/− cells in both growing (missing portion shown by yellow arrowheads) and mature (white arrowheads) flagella (H).

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