Molecular and Cellular Pathogenesis of Ellis-van Creveld Syndrome: Lessons from Targeted and Natural Mutations in Animal Models

Abstract

Ellis-van Creveld syndrome (EVC; MIM ID #225500) is a rare congenital disease with an occurrence of 1 in 60,000. It is characterized by remarkable skeletal dysplasia, such as short limbs, ribs and polydactyly, and orofacial anomalies. With two of three patients first noted as being offspring of consanguineous marriage, this autosomal recessive disease results from mutations in one of two causative genes: EVC or EVC2/LIMBIN. The recent identification and manipulation of genetic homologs in animals has deepened our understanding beyond human case studies and provided critical insight into disease pathogenesis. This review highlights the utility of animal-based studies of EVC by summarizing: (1) molecular biology of EVC and EVC2/LIMBIN, (2) human disease signs, (3) dysplastic limb development, (4) craniofacial anomalies, (5) tooth anomalies, (6) tracheal cartilage abnormalities, and (7) EVC-like disorders in non-human species.

Keywords: EVC2; Ellis-van Creveld syndrome; LIMBIN; ciliopathy; craniofacial.

Figure 1

Individuals affected by EVC, LIMBIN mutant cattle, and Evc2/Limbin mutant mice share similar dwarfism. (A) A 20-month-old infant affected by EVC syndrome (left) and a 20-month-old infant without EVC syndrome (right) © BMJ. The infant with EVC syndrome has un-proportional shortened legs. (B) A LIMBIN mutant cattle bears apparent short legs. (C) Evc2/Limbin mutant mice with littermate controls exhibit a smaller body size at 5 weeks old. (D) LIMBIN mutant cattle have decreased size of growth plate (marked in white arrow) in appendicular bones. (E) The growth plate of tibia from Evc2/Limbin mutant mice demonstrate shorter and disorganized structure at 5 weeks old compared with control mice. Bars indicate 200 um. (F) Tibiae from E18.5 mouse embryos were stained with alcian blue for cartilage and alizarin red for bone. Tibiae from Evc2/Limbin mutant embryos are shorter than those in control littermates, although body size in these two groups are similar at this stage.

Figure 2

Primary cilium, EVC and EVC2/LIMBIN proteins, and the mechanism of EVC-EVC2/LIMBIN in regulating Hedgehog signaling within the primary cilium. Diagrams of the motile cilium (A) and the primary cilium (B) are shown. CB, cilium base; CP, cilium pocket. (C). Structures of the EVC and the EVC2/LIMBIN are shown. The EVC and EVC2/LIMBIN are N terminal anchored proteins. Blue box in the EVC and orange box in the EVC2/LIMBIN indicate domains for interaction with each other. Green box indicates the domain for the ciliary localization of the EVC2/LIMBIN and the yellow box indicates the domain for localization of the EVC2/LIMBIN at the EVC zone. Numbers indicate the numbers of amino acids in each protein. (D) EVC-EVC2/LIMBIN complexes are localized at the bottom of cilia by tethering to EFCAB7 through the W domain in EVC2/LIMBIN. In the absence of Hedgehog ligand, PTCH1 resides within the primary cilium, and GLI proteins are processed to the repressor form (GLI-R) at the centrosome. (E) In the presence of Hedgehog ligand, binding of the ligands with PTCH1 leads to exclusion of PTCH1 out of the primary cilium, which allows SMO to enter the primary cilium. Within the primary cilium, SMO interacts with EVC-EVC2/LIMBIN at the bottom of the primary cilium, which allows GLI trafficking into the primary cilium and accumulation at the tip of the primary cilium. After entering the primary cilium, GLI are processed to the activator form (GLI-A). GLI activators exit the primary cilium and translocate into the nucleus to activate Hedgehog responsive genes. (F) EVC or EVC2/LIMBIN loss of function leads to absence of EVC-EVC2/LIMBIN complexes within the primary cilium. When Hedgehog signaling is activated, SMO still moves into the primary cilium, but without EVC-EVC2/LIMBIN complexes, SMO cannot lead to full activation of GLI. (G) In Weyers form of mutant cells, EVC-EVC2/LIMBIN complexes cannot be restricted at the bottom of primary cilium due to no interactions with EFCAB7 caused by loss of the W domain in EVC2/LIMBIN, thus EVC-EVC2/LIMBIN-SMO complex cannot lead to full activation of GLI.

Figure 3

Elevated FGF signaling is critical for the pathogenesis of the dwarfism developed in EVC. (A) In control growth plate, both Hedgehog signaling (yellow-green feedback loop) and FGF signaling (red) work on chondrocytes to ensure regulated proliferation and maturation. (B) In Evc2/Limbin mutant growth plate, moderately decreased Hedgehog signaling due to Evc2/Limbin loss of function within only chondrocytes moderately contributes to the pathogenesis of dwarfism, whereas elevated FGF signaling due to loss of Evc2/Limbin within perichondrium critically contributes the pathogenesis of the dwarfism. Green, resting chondrocytes; gold sand, proliferating chondrocytes; aqua, hypertrophic chondrocytes, IHH, Indian Hedgehog ligand.

Figure 4

A shortened skull base leads to mid-facial defects in Evc2/Limbin mutant mice. (A) Surface models of the mid-line regions were generated based on the micro-CT scans of controls (green) and Evc2/Limbin mutants (yellow). Two models were then superimposed at the occipital bones of the skull. Green and yellow lines are spanning the entire regions of the skull bases in control and mutant, respectively; black arrows indicate the intersphenoidal synchondrosis (ISS) and the spheno-occipital synchondrosis (SOS) in skull base. (B) Models were generated from the skull region containing nasal, frontal and parietal bones from micro-CT scans of control and mutant. No apparent defects were observed in Evc2/Limbin mutants in comparing to controls. (C) Models were generated from the skull base from micro-CT scans of controls and mutants. Apparent shortened anterior parts of skull bases from Evc2/Limbin mutants are observed in comparison to controls, whereas the posterior part of the mutant skull bases remains the same length with the controls. Black arrows indicate the ISS and the SOS.