Specific variants in WDR35 cause a distinctive form of Ellis-van Creveld syndrome by disrupting the recruitment of the EvC complex and SMO into the cilium

Abstract

Most patients with Ellis-van Creveld syndrome (EvC) are identified with pathogenic changes in EVC or EVC2, however further genetic heterogeneity has been suggested. In this report we describe pathogenic splicing variants in WDR35, encoding retrograde intraflagellar transport protein 121 (IFT121), in three families with a clinical diagnosis of EvC but having a distinctive phenotype. To understand why WDR35 variants result in EvC, we analysed EVC, EVC2 and Smoothened (SMO) in IFT-A deficient cells. We found that the three proteins failed to localize to Wdr35(-/-) cilia, but not to the cilium of the IFT retrograde motor mutant Dync2h1(-/-), indicating that IFT121 is specifically required for their entry into the ciliary compartment. Furthermore expression of Wdr35 disease cDNAs in Wdr35(-/-) fibroblasts revealed that the newly identified variants lead to Hedgehog signalling defects resembling those of Evc(-/-) and Evc2(-/-) mutants. Together our data indicate that splicing variants in WDR35, and possibly in other IFT-A components, underlie a number of EvC cases by disrupting targeting of both the EvC complex and SMO to cilia.

Figure 1.

Clinical and molecular findings in patients with a new form of EvC. (A) Pedigree of family 1; the proband (case 1) is designated with an arrow. (B) Left foot of case 2 showing postaxial polydactyly and hypoplastic nails. (C) Full body view of case 1 demonstrating shortening of all four limbs and postaxial polydactyly. The photograph was taken soon after delivery which explains the abnormal shape of the head in this image. (D) RT-PCR analysis of WDR35 (5′-UTR to exon 7) in whole-blood from both parents of family 1 and an unrelated control individual (C1) revealing amplification of a smaller cDNA fragment corresponding to exon 3 exclusion (Δ3) in the parents. (E) Representative immunoblot showing response to SAG (+) or its vehicle (−) of Evc2^−/− MEFs in comparison to Wdr35^−/− MEFs retrotransduced with the empty vector (+pBabe) or with Wdr35Δ3:FLAG (+Wdr35Δ3), or full length wild-type Wdr35:FLAG (+Wdr35) cDNAs. Total cell extracts probed for anti-EVC2, anti-FLAG (WDR35) and anti-IFT43 are also shown. Asterisks denote non-specific bands. (F) Bar graphs representing GLI3R/GLI3FL ratio (left) and GLI1 levels (right) with respect to tubulin (TUB) from three independent experiments obtained by band densitometry. (G) Pedigree of family 2. The arrow designates the proband (case 3). (H) Full body view of case 3 showing narrow elongated chest, sloping shoulders, pectus excavatum and proximal shortening of limbs. (I and J). Clinical images demonstrating hypodontia, abnormally shaped teeth and enamel hypoplasia in case 3 (I) and multiple labiogingival frenulae in his affected sister (case 4; J). (K and L) RT-PCR analysis of WDR35 between exons 8–15 (K) and 26–28 (L) in whole-blood cDNA from family 2 members and an unrelated control (C1). Transcripts lacking exons 10 (Δ10), 9–10 (Δ9–10) and 27 (Δ27) are indicated. (M) Pedigree of family 3. The arrow designates the proband (case 5). (N) Full body view of case 5 showing dolichocephaly, sparse hair and eyebrows, short upper limbs and narrow chest with pectus excavatum. (O) Right hand of case 5 with brachydactyly postaxial polydactyly and dysplastic nails. (P) Feet of case 5 showing brachydactyly, bilateral postaxial polydactyly with hypoplastic nails and partial soft tissue syndactyly between 2nd and 3rd toes and between 5th and 6th toes bilateral. (Q) RT-PCR analysis of WDR35 from exons 8 to 17 in whole-blood from case 5 and an unrelated control (C1) revealing amplification of a longer transcript (ins 121 nt) in the patient. For D, K, L and Q, C2 is a negative control with no template and wt designates the normal transcript (NM_020779.3).

Figure 2.

Cilial localization of EVC, EVC2 and SMO requires WDR35. (A) Representative immunofluorescence image showing partial overlapping localization between EVC and IFT43 at the base of cilia (arrows) in normal MEFs. (B) Compartmentalization of Wdr35^−/− cilia was examined by immunofluorescence of the IFT-B component IFT88 as well as transition zone markers NPHP1 and CEP290 in MEFs. Cilia are oriented with the base to the left and tip to the right. Arrows point to the NPHP1 and CEP290 signals at the transition zone. (C) EVC and EVC2 immunobloting demonstrating unchanged total levels of EVC and EVC2 in Wdr35^−/−, Wdr35^+/− and Dync2h1^−/− MEFs. (D) Immunofluorescence images revealing absence of EVC, EVC2 and SMO signals in mutant axonemes of Wdr35^−/− MEFs indicating a defect in trafficking of these proteins into the ciliary compartment as opposed to cytoplasmic destabilization. SMO was visualized after activation of the Hh pathway with SAG. Shifted-overlay images are shown for IFT88, EVC, EVC2 and SMO. Nuclei were stained with DAPI. Scale bars: 10 µm.

Figure 3.

Primary cilia defects resulting from EvC versus CED WDR35 variants. (A) Representative images (shifted-overlay) corresponding to immunofluorescence analysis of EVC, EVC2, SMO, IFT43 and FLAG (WDR35) in primary cilia of Wdr35^−/−MEFs stably expressing the Wdr35:FLAG variants indicated on the left. Cilia are oriented with the base to the left and tip to the right. Arrows point to the specific staining of the pool of IFT43 and FLAG (WDR35) at the base of cilia. Acet TUB: Acetylated tubulin and Glu TUB: detyrosinated tubulin. (B) Frequency of ciliated cells in Wdr35^−/− cultures stably expressing Wdr35:FLAG variants or retrotransduced with the empty vector (+pBabe) or full length wild-type Wdr35:FLAG (+Wdr35) from three independent experiments. At least 500 cells were analysed per cell line. (C) Box and whiskers plot representing average primary cilia length. Three independent experiments were performed and a minimum number of 120 cilia were measured in each cell line. (D) Percentage of cilia positive for SMO staining after treatment with SAG (+) or its vehicle (−), n ≥ 120 cilia. (E) Percentage of cells with EVC- and EVC2-positive cilia (n ≥ 180 cilia). (F and G) Average fluorescence intensity in the cilium of EVC (F) and EVC2 (G) represented as a box and whiskers plot (n ≥ 50 cilia). Note that although EVC and EVC2 are detected in WDR35Δ3 cilia their corresponding fluorescence intensities are greatly diminished. (H) Frequency of cilia with positive staining for IFT43 (n ≥ 110 cilia) and FLAG (WDR35) (n ≥ 60 cilia) in the different Wdr35:FLAG cell lines. (I and J) Box and whiskers plots of the average fluorescence intensity at the ciliary base of IFT43 (I) and FLAG (WDR35) (J), n ≥ 40 cilia.

Figure 4.

Biochemical characterization of WDR35 variants causing EvC and CED. (A–D) Representative immunoblots (n = 3) showing levels of GLI3FL, GLI3R and GLI1 after incubation with SAG or its vehicle in cultures carrying Wdr35:FLAG variants corresponding to cases 3–4 (A) or CED variants (C) with respect to control cultures retrotransduced with empty vector (+pBabe) or full length Wdr35:FLAG (+Wdr35). Densitometry analysis of GLI3R/GLI3FL and relative levels of GLI1 corresponding to panels A and B are shown in B and D respectively. (E) Representative immunoblot demonstrating lower levels of IFT43 in Wdr35^−/− cells with respect to Wdr35^+/− control MEFs. (F and -G) Quantification of IFT43 (F) and WDR35:FLAG (G) total protein levels by densitometric analysis of Western blots corresponding to cell extracts of Wdr35^−/−cell lines retrotransduced with the indicated Wdr35:FLAG variants, n = 2. Generally the EvC-associated variants are more disruptive to the formation and stability of the IFT43-WDR35 complexes than the CED variants. (H) Representative immunoblot showing endogenous levels of IFT43 in the 20% input cellular extract and anti-FLAG immunoprecipitates of Wdr35^−/− cells retrotransduced with the indicated FLAG-tagged variants, n = 2. WDR35 was detected with anti-FLAG (left blot) or anti-WDR35 (right blot). Asterisks denote non-specific bands.