Mutations in human homologue of chicken talpid3 gene (KIAA0586) cause a hybrid ciliopathy with overlapping features of Jeune and Joubert syndromes

Abstract

Background: In chicken, loss of TALPID3 results in non-functional cilia and short-rib polydactyly syndrome. This phenotype is caused by a frameshift mutation in the chicken ortholog of the human KIAA0586 gene, which encodes a novel coiled-coil domain protein essential for primary ciliogenesis, suggesting that KIAA0586 can be associated with ciliopathy in human beings.

Methods: In our patients with ciliopathy (http://www.clinicaltrials.gov: NCT00068224), we have collected extensive clinical and neuroimaging data from affected individuals, and performed whole exome sequencing on DNA from affected individuals and their parents. We analysed gene expression on fibroblast cell line, and determined the effect of gene mutation on ciliogenesis in cells derived from patients.

Results: We identified biallelic mutations in the human TALPID3 ortholog, KIAA0586, in six children with findings of overlapping Jeune and Joubert syndromes. Fibroblasts cultured from one of the patients with Jeune-Joubert syndrome exhibited more severe cilia defects than fibroblasts from patients with only Joubert syndrome; this difference was reflected in KIAA0586 RNA expression levels. Rescue of the cilia defect with full-length wild type KIAA0586 indicated a causal link between cilia formation and KIAA0586 function.

Conclusions: Our results show that biallelic deleterious mutations in KIAA0586 lead to Joubert syndrome with or without Jeune asphyxiating thoracic dystrophy. Furthermore, our results confirm that KIAA0586/TALPID3 is essential in cilia formation in human beings, expand the KIAA0586 phenotype to include features of Jeune syndrome and provide a pathogenetic connection between Joubert and Jeune syndromes, based on aberrant ciliogenesis.

Keywords: Clinical genetics; Developmental; ciliopathy; small thorax; whole exome sequencing.

Figure 1

Clinical photographs and imaging of subjects with Jeune–Joubert syndrome and Joubert syndrome. (A) Upper panel shows photograph of patient 1 (P1) with small thorax, relatively long trunk and short extremities, and craniofacial dysmorphism, including low set incompletely rotated ears, hypoplastic midface, epicanthal folds and lower lip scars due to biting. Brain MRI performed on the third day of life showing severe cerebellar vermis hypoplasia and dysplasia, marked retrocerebellar fluid (circle) and enlarged fourth ventricle with abnormal shape (asterisk) in (1); thickened superior cerebellar peduncles with abnormally oblique orientation resulting in the ’molar tooth sign‘ (circle) in (2) and cerebral cortex showing bilateral diffuse polymicrogyria (arrows) in (3). Middle panel shows photograph of patient 2 (P2) with normalised chest size at age 4.4 years; his chest was small and bell-shaped during the first year of life. Brain MRI at 18 months showing a mildly hypoplastic and dysplastic vermis (circle) in (4) and molar tooth sign (circle) in (5) and normal cerebral cortex (6). Lower panels show normal brain MRI images for comparison (7, 8 and 9). (B) First panel shows photograph of patient 3 (P3) displaying epicanthal folds, low set ears and normal chest size. Brain MRI at 2 years 4 months displaying a hypoplastic and dysplastic vermis (circle) in (1), molar tooth sign (circle) in (2) and normal cerebellar cortex (3). Second panel shows photograph of patient 4 (P4) showing normal chest size. Brain MRI of P4 at age 3 years 3 months displaying severely hypoplastic and dysplastic vermis (circle) and enlarged and abnormally shaped fourth ventricle (asterisk) in (4), molar tooth sign (5) and normal cerebral cortex (6). Third panel shows photograph of patient 5 (P5) showing a chubby 26-month-old with normal chest size. Brain MRI of P5 at age 14 months showing mildly hypoplastic and dysplastic cerebellar vermis (circle) and enlarged fourth ventricle (7), molar tooth sign (circle) in (8) and normal cerebral cortex (9). Fourth panel shows patient 6 (P6), whose chest size was normal. Brain MRI of P6 shows mildly hypoplastic and dysplastic cerebellar vermis (10, circle), enlarged fourth ventricle images, molar tooth sign (circle, in 11) and normal cerebral cortex (12).

Figure 2

Pedigree of families and molecular data. (A) Family pedigrees of patient 1 and patient 2 and representative chromatograms showing variants in KIAA0586. Affected individuals are shown in black, while arrows point to the probands in each family. Note that in patient 1, the c.990C>T mutation appears homozygous; patient 1 is hemizygous for this variant, as the other allele on this region is deleted due to the mutation that he inherited from his mother (c.745-350_1288+1117del). (B) Schema showing the main isoforms of KIAA0586. Mutations affect most, but not all, isoforms. Black triangle refers to mutations that are predicted to produce a premature stop; black circle refers to the synonymous variant that leads to deletion of exon 9 (exon highlighted in red). Probe 1 and probe 2 refers the exons amplified for expression analysis. (C) Protein schema showing the location of mutations. All mutations are found before amino acids 535-622, a highly conserved domain required for centrosome localisation (**, refers to the findings by Yin et al). # refers to the synonymous variant that leads to the deletion of exon 9; ## refers to the variant that deletes exon 8, 9 and 10.

Figure 3

Expression of KIAA0586 in multiple tissue panel and cells. (A) Tissue expression of KIAA0586 in multiple tissue panels from normal, unaffected individuals using two probes that amplify the first two and the last few exons of the gene, respectively (See figure 2B for probe sites). KIAA0586 appears ubiquitously expressed, but the levels of expression vary with the two probes. (B) Analysis of common coding isoforms of KIAA0586 in affected individuals compared with unaffected control samples. mRNA was extracted from the fibroblasts of control individual (control), patient 1 (P1) and patient 3 (P3) and analysed by QPCR using primer pairs that specifically amplify the known coding isoforms of KIAA0586 (NM_014749.3, isoform 1; NM_001244189.1, isoform 2; and NM_001244190.1, isoform 3). Expression of all three isoforms was markedly reduced in P1 and reduced by half in P3. (C) Overexpression of the full length human KIAA0586 (NM_014749.3) in fibroblasts by lentiviral-mediated transduction led to recovery of expression in both P1 and P3. KIAA0586 isoform expression was normalised to that of POLR2A.

Figure 4

Analysis of cilia in patient 1 and patient 3 with mutations in KIAA0586. (A) Fibroblasts from control, patient 1 (P1) and patient 3 (P3) were grown to near confluence and serum starved to allow formation of cilia. Forty-eight hours after serum starvation, cells were fixed in methanol and probed with anti-ARL13B antibody (green) that marks cilia and mouse antiacetylated tubulin (red) that stains the basal bodies. Insets show magnified images of cells (arrows). (B) Approximately 80% of control cells have cilia, while only 30% and 50% of cells are ciliated in P1 and P3, respectively. Overexpression of the full length human KIAA0586 (NM_014749.3) in fibroblasts by lentiviral-mediated transduction increased the number of ciliated P1 and P3 cells to normal. Scale bars represent 20 μm. Asterisks indicate p value <0.001 between control and patient (P1 or P3) cells using non-parametric t test. (C) Cilia length in cells was measured using Zen 2009 software (Zeiss). In control cells, cilia were ~3.5–7 μ in length. In P3, the cilia length ranged from 0.05 to 2.5 μ. In P3, cells had longer cilia than P1 (without pLenti-KIAA0586), measuring 2.5–5 μ, but still shorter than control. The length of cilia in P1 and P3 cells returned to normal after transduction with full length human KIAA0586 (with pLenti-KIAA0586). Asterisks indicate p value <0.001 between control and patient (P1 or P3) cells using non-parametric t test.