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Ellis-van Creveld syndrome (EvC) is caused by mutations in EVC and EVC2, genes in a divergent orientation separated by only 2.6 kb. We systematically sought mutations in both genes in a panel of 65 affected individuals to assess the proportion of cases resulting from mutations in each gene. We PCR amplified and sequenced the coding exons of both genes. We investigated mutations that could affect splicing by in vitro splicing assays and cDNA analysis. We have identified EVC mutations in 20 cases (31%); in all of these we have detected the mutation on each allele. We have identified EVC2 mutations in 25 cases (38%); in 22 of these we have isolated a mutation on each allele. The majority of the mutations introduce a premature termination codon. We sequenced the region between the two genes in 10 of the 20 cases in which we had not identified a mutation in either gene, revealing only one SNP that was not a common polymorphism. As we have not identified mutations in either gene in 20 cases (31%) it is possible that there is further genetic heterogeneity.

Acrodysostosis is a dominantly-inherited, multisystem disorder characterized by skeletal, endocrine, and neurological abnormalities. To identify the molecular basis of acrodysostosis, we performed exome sequencing on five genetically independent cases. Three different missense mutations in PDE4D, which encodes cyclic AMP (cAMP)-specific phosphodiesterase 4D, were found to be heterozygous in three of the cases. Two of the mutations were demonstrated to have occurred de novo, providing strong genetic evidence of causation. Two additional cases were heterozygous for de novo missense mutations in PRKAR1A, which encodes the cAMP-dependent regulatory subunit of protein kinase A and which has been recently reported to be the cause of a form of acrodysostosis resistant to multiple hormones. These findings demonstrate that acrodysostosis is genetically heterogeneous and underscore the exquisite sensitivity of many tissues to alterations in cAMP homeostasis.

Analysis of a nuclear family with three affected offspring identified an autosomal-recessive form of spondyloepimetaphyseal dysplasia characterized by severe short stature and a unique constellation of radiographic findings. Homozygosity for a haplotype that was identical by descent between two of the affected individuals identified a locus for the disease gene within a 17.4 Mb interval on chromosome 15, a region containing 296 genes. These genes were assessed and ranked by cartilage selectivity with whole-genome microarray data, revealing only two genes, encoding aggrecan and chondroitin sulfate proteoglycan 4, that were selectively expressed in cartilage. Sequence analysis of aggrecan complementary DNA from an affected individual revealed homozygosity for a missense mutation (c.6799G --> A) that predicts a p.D2267N amino acid substitution in the C-type lectin domain within the G3 domain of aggrecan. The D2267 residue is predicted to coordinate binding of a calcium ion, which influences the conformational binding loops of the C-type lectin domain that mediate interactions with tenascins and other extracellular-matrix proteins. Expression of the normal and mutant G3 domains in mammalian cells showed that the mutation created a functional N-glycosylation site but did not adversely affect protein trafficking and secretion. Surface-plasmon-resonance studies showed that the mutation influenced the binding and kinetics of the interactions between the aggrecan G3 domain and tenascin-C. These findings identify an autosomal-recessive skeletal dysplasia and a significant role for the aggrecan C-type lectin domain in regulating endochondral ossification and, thereby, height.

Ellis-van Creveld syndrome (EvC) is an autosomal recessive skeletal dysplasia. Elsewhere, we described mutations in EVC in patients with this condition (Ruiz-Perez et al. 2000). We now report that mutations in EVC2 also cause EvC. These two genes lie in a head-to-head configuration that is conserved from fish to man. Affected individuals with mutations in EVC and EVC2 have the typical spectrum of features and are phenotypically indistinguishable.

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BackgroundEvc is essential for Indian Hedgehog (Hh) signalling in the cartilage growth plate. The gene encoding Evc2 is in close proximity in divergent orientation to Evc and mutations in both human genes lead to the chondrodysplasia Ellis-van Creveld syndrome.ResultsBioinformatic analysis reveals that the Evc and Evc2 genes arose through a duplication event early in metazoan evolution and were subsequently lost in arthropods and nematodes. Here we demonstrate that Evc2 is essential for Hh pathway activation in response to the Smo agonist purmorphamine. A yeast two-hybrid screen using Evc as bait identified Evc2 as an Evc binding partner and we confirmed the interaction by immunoprecipitation. We developed anti-Evc2 antibodies and show that Evc2 and Evc co-localize at the basal body and also on primary cilia. In transfected cells, basal body and cilia localization is observed when Evc and Evc2 constructs are co-transfected but not when either construct is transfected individually. We show that Evc and Evc2 are cilia transmembrane proteins, the C-terminus for both being intracellular and Evc2, but not Evc, having an extracellular portion. Furthermore, Evc is absent at the basal body in Evc2 null cells. Using Western blots of cytoplasmic and nuclear protein, we also demonstrate that full length Evc2 but not Evc, is located in the nucleus.ConclusionsWe demonstrate for the first time that Evc2 is a positive regulator of the Hh signalling pathway and that it is located at the basal body of primary cilia. We show that the presence of Evc and Evc2 at the basal body and cilia membrane is co-dependent. In addition, Evc2, but not Evc, is present in the cell nucleus suggesting movement of Evc2 between the cilium and nucleus.

Fibrochondrogenesis is a severe, autosomal-recessive, short-limbed skeletal dysplasia. In a single case of fibrochondrogenesis, whole-genome SNP genotyping identified unknown ancestral consanguinity by detecting three autozygous regions. Because of the predominantly skeletal nature of the phenotype, the 389 genes localized to the autozygous intervals were prioritized for mutation analysis by correlation of their expression with known cartilage-selective genes via the UCLA Gene Expression Tool, UGET. The gene encoding the α1 chain of type XI collagen (COL11A1) was the only cartilage-selective gene among the three candidate intervals. Sequence analysis of COL11A1 in two genetically independent fibrochondrogenesis cases demonstrated that each was a compound heterozygote for a loss-of-function mutation on one allele and a mutation predicting substitution for a conserved triple-helical glycine residue on the other. The parents who were carriers of missense mutations had myopia. Early-onset hearing loss was noted in both parents who carried a loss-of-function allele, suggesting COL11A1 as a locus for mild, dominantly inherited hearing loss. These findings identify COL11A1 as a locus for fibrochondrogenesis and indicate that there might be phenotypic manifestations among carriers.