Variants in members of the cadherin-catenin complex, CDH1 and CTNND1, cause blepharocheilodontic syndrome

Abstract

Blepharocheilodontic syndrome (BCDS) consists of lagophthalmia, ectropion of the lower eyelids, distichiasis, euryblepharon, cleft lip/palate and dental anomalies and has autosomal dominant inheritance with variable expression. We identified heterozygous variants in two genes of the cadherin-catenin complex, CDH1, encoding E-cadherin, and CTNND1, encoding p120 catenin delta1 in 15 of 17 BCDS index patients, as was recently described in a different publication. CDH1 plays an essential role in epithelial cell adherence; CTNND1 binds to CDH1 and controls the stability of the complex. Functional experiments in zebrafish and human cells showed that the CDH1 variants impair the cell adhesion function of the cadherin-catenin complex in a dominant-negative manner. Variants in CDH1 have been linked to familial hereditary diffuse gastric cancer and invasive lobular breast cancer; however, no cases of gastric or breast cancer have been reported in our BCDS cases. Functional experiments reported here indicated the BCDS variants comprise a distinct class of CDH1 variants. Altogether, we identified the genetic cause of BCDS enabling DNA diagnostics and counseling, in addition we describe a novel class of dominant negative CDH1 variants.

Fig. 1

Facial features of BCDS subjects. Pictures of 24 BCD patients, showing the variability of the phenotype at different ages. The patient identification below the picture corresponds with the patient identification in the tables and pedigrees

Fig. 2

BCDS variants in CDH1 and CTNND1. a Schematic overview of protein alterations in CDH1 identified in BCDS subjects, combined the number of observations in independent index cases. b Protein structures of CDH1 highlighting the fact that all missense variants alter amino acid that directly interact with a Ca²⁺ ion. The wild-type residues are depicted in green, the mutated form in red. c Splicing analysis on by PCR analysis of cDNA from a subject with c.1320 + 1 G > T; p.Tyr380_Lys440del, an exon 9 splice donor mutation using primers on exons 7 and 11. The BCDS subject (U2) shows a lower band on agarose gel (indicated by the arrow) compared to the control cDNA (C). Sanger sequencing of this band showed absence of the entire exon 9, predicted to result in an in-frame deletion of 61 amino acids (p.Tyr380_Lys440del). d Schematic overview of protein alterations in CTNND1 identified in BCDS subjects. e Relative expression levels of CTNND1 cDNA of the patient and three controls and f Sanger sequencing data of CTNND1 patient cDNA showing nonsense mediated decay of the mutated transcript. Variants identified in this study are reported as circles, variants identified by Ghoumid et al. [1] as triangles and variants identified by Nishi et al. [22] as squares

Fig. 3

Effect of human CDH1 variants on zebrafish early development. a Schematical representation of the experimental setup. Fertilized 1-cell zebrafish embryos were injected with mRNA corresponding to the human CDH1 and phenotyped at 1 day post fertilization (d.p.f.) b Phenotypical classification of 1 d.p.f. zebrafish embryos microinjected with hCDH1 variants. Arrowheads point at head hypoplasia in Class III and severe tail defect in Class IV, respectively. c Quantification of the phenotypical classes described in b for different hCDH1 variants. The p.(Pro373Leu) and p.Trp638* variants are found in hereditary diffuse gastric cancer patients. For all categories, number of embryos analyzed n: 112 < n < 276 d At 10 hpf (bud stage; wild type, upper panel), embryos expressing BCDS-related CDH1 variants often display severe developmental delay, denoted by absence of the tail bud (lower panel; arrowhead) e During somitogenesis (upper left panel; 13 somites) embryos expressing BCDS-related CDH1 variants may display severely delayed development with rough surface and detaching cells at the posterior end of the midline (right panels; lower panel is magnification of upper panel). Major midline defects (lower left panel) are also observed. f Numerous detaching cells (arrowheads) can be observed at the midline of affected embryos at 1 d.p.f. (Class II-V; embryo and magnifications shown are representative of the observed phenotype). Note abnormal development of the neural tube. g 5 d.p.f. zebrafish larvae microinjected with mRNA from BCDS-related CDH1 variants (p.(Asp254Asn) in the representrative picture) display severe jaw and craniofacial defects (arrowhead). Cartilage staining reveals major defective patterning and interrupted cartilage formation at the base of the palate (arrow)

Fig. 4

Dominant negative effects of the c.760 G > A; p.(Asp254Asn) BCDS-associated variant. Immunofluorescence images of MCF7 CDH1 KO cells co-expressing a GFP-CDH1 wild type (WT) and SNAP-CDH1 WT, b c.760 G > A; p.(Asp254Asn) or c p.Trp638*. Cells were labeled with SNAP surface 549 before fixation. In contrast to SNAP-CDH1 WT, which highly localizes with GFP-CDH1 at the plasma membrane, SNAP-CDH1 c.760 G > A; p.(Asp254Asn) inhibits the stable localization of GFP-CDH1 to the plasma membrane. SNAP-CDH1 p.Trp638* has no effect on GFP-CDH1 localization