A homozygous frameshift variant in the CILK1 gene causes cranioectodermal dysplasia

Abstract

Cranioectodermal dysplasia (CED) is a ciliopathy characterized by skeletal and ectodermal abnormalities, renal failure, and liver fibrosis. Pathogenic variants in genes that encode the intraflagellar transport (IFT) complex components, particularly IFT-A, are responsible for approximately two-thirds of the CED cases. However, the cause of the remaining cases remains unknown. Ciliogenesis-associated kinase 1 (CILK1) is a highly conserved ciliary serine/threonine kinase with an N-terminal catalytic domain responsible for kinase activity and a C-terminal non-catalytic domain that interacts with the IFT-B complex. Biallelic variants in the catalytic domain are associated with lethal skeletal dysplasia, endocrine cerebroosteodysplasia, and short-rib polydactyly syndrome. No human disease has been linked to biallelic variants in the non-catalytic domain. We present a homozygous frameshift variant in the CILK1 gene that affects the distal part of the non-catalytic domain, causing CED in five patients from two pedigrees. All the patients survived into childhood and had disproportionately short stature, skeletal abnormalities, ectodermal dysplasia, renal issues, and liver complications. Functional data from patient-derived cells and the C. elegans model indicate that the variant reduces cilia number, increases cilia length, and disrupts the localization of IFT components. In contrast, the ciliary localization of CILK1 bearing the variant itself remains unaffected. Notably, we rescued the majority of these abnormalities by reintroducing CILK1 into patient-derived cells. Finally, our study describes CILK1 as a novel causal gene and the first non-IFT protein-encoding gene in the etiology of CED, thus expanding the known genotypic, mechanistic, and phenotypic spectrum of CED.

Fig. 1. Pedigrees, clinical images, radiographs, and an illustration of the clinical features of the affected individuals.

A Pedigrees of the patients. The pedigree of Family 1 includes four molecularly confirmed patients with CED (Patients 1–4) and 11 unconfirmed patients suspected of having CED. The pedigree of Family 2 includes Patient 5. M: Mutant, WT: Wild-type, N/A: Not available. B Clinical images of the patients. All patients exhibited dolichocephaly, a depressed nasal bridge, full cheeks, ectodermal dysplasia, brachydactyly, and a sandal gap. Written informed consent was obtained from the parents of patients concerning publishing the clinical report along with their clinical pictures. C X-ray radiographs of Patients 1, 2, and 5 show a narrow thorax and brachydactyly. Pelvic bones were normal (middle panel). Patient 5 presented with shortening of both proximal and distal parts of the limbs. D Brain MRI of Patient 1 at 3 years of age, revealing external hydrocephalus, ventriculomegaly, and cerebral and cerebellar atrophy. E An illustration depicting the clinical findings of the patients.

Fig. 2. Identification of the causal variant in CILK1 in individuals with CED phenotype.

A Homozygosity stretch identification showed only a single locus shared among the patients, chr6:51732807-71011831. The colored bars indicate regions of homozygosity found in each patient. The shaded box indicates the homozygosity stretch region. B Schematic of the localization of genotypes associated with CILK1-related phenotypes on the transcript and protein. The variant reported in this study is located on a quite distant part of the protein, in contrast to ECO and SRPS-related variants. C The diagram shows the frequency of cardinal features in CILK1-related ciliopathies, CED, and the patients in this study. Note that the novel CILK1-related phenotype closely resembles CED. The patients in this report are labeled as CILK1-CED and represented in red.

Fig. 3. The disease-causing variant in CILK1 leads to longer cilia in patient-derived cells.

A Immunofluorescence images of control, patient-derived, and rescue of patient-derived cells. Staining includes DAPI (blue), acetylated tubulin (red), and CILK1 (green). An asterisk marks the ciliary tip. Scale bar: 3 μm. B, C Bar and scatter plots show the percentage of ciliated and non-ciliated cells and ciliary lengths after serum deprivation in control, patient-derived, and rescued patient-derived cells. D Immunofluorescence images of patient-derived cells displaying curly cilia. Staining includes DAPI (blue) and acetylated tubulin (red). Scale bar: 3 μm.

Fig. 4. IFT distribution is altered in patient-derived cells.

A, B Immunofluorescence images of control and patient-derived cells showing staining for acetylated tubulin (red), IFT88 (green), IFT70 (green), and DAPI (blue). Scale bar: 5 μm. C Density plot illustrating the distribution of acetylated tubulin (red), IFT88 (green), and IFT70 (green) along the ciliary length.