Mutations in KIAA0753 cause Joubert syndrome associated with growth hormone deficiency

Abstract

Joubert syndrome and related disorders (JSRD) are a heterogeneous group of ciliopathies defined based on the mid-hindbrain abnormalities that result in the characteristic "molar tooth sign" on brain imaging. The core clinical findings of JSRD are hypotonia, developmental delay, abnormal eye movements and breathing abnormalities. To date, more than 30 JSRD genes that encode proteins important for structure and/or function of cilia have been identified. Here, we present 2 siblings with Joubert syndrome associated with growth hormone deficiency. Whole exome sequencing of the family identified compound heterozygous mutations in KIAA0753, i.e., a missense mutation (p.Arg257Gly) and an intronic mutation (c.2359-1G>C). The intronic mutation alters normal splicing by activating a cryptic acceptor splice site in exon 16. The novel acceptor site skips nine nucleotides, deleting three amino acids from the protein coding frame. KIAA0753 (OFIP) is a centrosome and pericentriolar satellite protein, previously not known to cause Joubert syndrome. We present comprehensive clinical descriptions of the Joubert syndrome patients as well as the cellular phenotype of defective ciliogenesis in the patients' fibroblasts.

Fig. 1

Clinical photographs of patients 1 and 2. a–c Patient 1, at age 10.5 years, displayed slight head tilt, bilateral epicanthal folds, downturned corners of the mouth and short stature. d–f Patient 2 at age 22 months had macrocephaly, frontal bossing, epicanthal folds, borderline low-set ears and esotropia

Fig. 2

Brain MRI images of patients 1 and 2 in comparison to normal. a Sagittal T1-weighted and b axial T2-weighted brain MRI images of healthy controls showing normal appearance of pituitary gland (small circle) and cerebellar vermis (large circle), and superior cerebellar peduncles (arrows). c–f Both patients had cerebellar vermis hypoplasia (large circles), dysplastic vermis (arrows) and thickened and horizontally oriented superior cerebellar peduncles resulting in the appearance of “molar tooth sign” on axial MRI images (red circles). c Patient 1 had an ectopic posterior pituitary gland (small circle). c Patient 2’s pituitary stalk appeared to be absent and the pituitary gland itself was very small (small circle). There was a suggestion of a pituitary bright spot immediately posterior to the chiasm, suggesting an ectopic posterior pituitary

Fig. 3

Molecular analysis of the patients’ mutations in KIAA0753. a Sequence chromatograms representing the c.769A>G missense mutation and the c.2359-1G>C splice site mutation. b Conservation of protein sequence across different species, encompassing both missense mutation (upper panel) and in-frame deletion (lower panel). c Agarose gel images of PCR products using cDNA specific primers flanking exon 16 showing absence of exon skipping/large deletion in both the patients, compared to wild type. d TA cloning of the PCR products followed by sequencing showing deletion of nine nucleotides encoding 3 amino acids (KYQ), at the beginning of exon 16. e Western blotting, performed in duplicate, for control and both patients showing no significant difference in the protein expression level of KIAA073. Housekeeping protein beta actin (ACTB) was used a loading control

Fig. 4

Mutations in KIAA0753 cause defects in ciliogenesis. Fibro-blasts were grown to ~70% confluence, starved for 48 h, methanol fixed. Cilia were stained with ARL13B (green) and centrosome with gamma tubulin (red). Representative cilia images of cells from control, Patient 1, and Patient 2. Z-stacked images were analyzed for percentage of cells with cilia. a Staining identified both reduced length ciliated cells (upper panel) and non-ciliated cells (lower panel) in both patients. b Graph representing the percentage of ciliated cells in patients, comparison to control. c Dot plot demonstrating the length of cilia (in micrometer) in both patients compared to control. A total of ~200 cells (3 replicates) were analyzed for cilia measurement. Error bars represent standard deviation. **p < 0.05 (nonparametric t test)