Novel truncating mutations in CTNND1 cause a dominant craniofacial and cardiac syndrome

Abstract CTNND1 encodes the p120-catenin (p120) protein, which has a wide range of functions, including the maintenance of cell–cell junctions, regulation of the epithelial-mesenchymal transition and transcriptional signalling. Due to advances in next-generation sequencing, CTNND1 has been implicated in human diseases including cleft palate and blepharocheilodontic (BCD) syndrome albeit only recently. In this study, we identify eight novel protein-truncating variants, six de novo, in 13 participants from nine families presenting with craniofacial dysmorphisms including cleft palate and hypodontia, as well as congenital cardiac anomalies, limb dysmorphologies and neurodevelopmental disorders. Using conditional deletions in mice as well as CRISPR/Cas9 approaches to target CTNND1 in Xenopus, we identified a subset of phenotypes that can be linked to p120-catenin in epithelial integrity and turnover, and additional phenotypes that suggest mesenchymal roles of CTNND1. We propose that CTNND1 variants have a wider developmental role than previously described and that variations in this gene underlie not only cleft palate and BCD but may be expanded to a broader velocardiofacial-like syndrome.


56
Genetic variation in CTNND1, which encodes for the armadillo-repeat protein p120-catenin 57 (p120), is associated with human birth defects, most notably non-syndromic cleft palate and 58 blepharocheilodontic (BCD) syndrome, which involves eyelid, lip and tooth anomalies [MIM: 59 617681] 1-3 . In contrast, CTNND1 has broader developmental roles in animal models. For example, 60 conditional deletions in mice demonstrate the importance of CTNND1 for development not only for 61 skin and teeth, but also for kidneys and other structures [4][5][6][7][8][9][10] , and complete deletion of CTNND1 leads 62 to prenatal lethality 5,9 . Similarly, loss-of-function experiments in Xenopus implicate CTNND1 in 63 craniofacial development 11,12 . Here, we describe a series of patients with CTNND1 variants, all of 64 whom present with multisystem involvement that demonstrates a broad spectrum craniofacial and 65 cardiac syndrome. 66 p120-catenin is a member of the catenin superfamily of proteins studied in catenin-cadherin 67 interactions; notably, it binds to and stabilizes E-cadherin (CDH1) at junctional complexes in 68 epithelia [13][14][15][16][17] . This binding is via the p120-catenin armadillo repeat domain, and displacement of 69 p120-catenin from E-cadherin is a key regulatory event at the adherens junction, that results in 70 endocytosis of E-cadherin and loss of the junction. The protein has a second function as a scaffolding 71 protein for the GTPase RhoA and associated Rho regulatory proteins 18,19 . In addition, it can also 72 directly interact with the zinc finger transcriptional repressor Kaiso (ZBTB33), facilitating Wnt signal 73 transduction 20,21 . Thus, p120-catenin appears to be a multi-functional protein, promoting epithelial 74 stability when in complex with E-cadherin, and regulating RhoA and transcriptional activities. p120-75 catenin is also able to associate with mesenchymal cadherins such as N-cadherin and cadherin-76 11 17,22 . In mesenchymal cells, p120-catenin associates with non-epithelial cadherins, regulating 77 motility and invasion via cytoskeletal events and transcription. Given its functions in both epithelia 78 and mesenchyme, it is unsurprising that both loss and gain of p120-catenin have been associated 79 with oncogenesis 23-25 . 80 In humans, the CTNND1 gene is located at 11q11 and consists of 21 exons, of which exons 81 11, 18 and 20 are alternatively spliced. Inclusion of exon 11, which is predominantly neural, disrupts 82 a nuclear localization signal (NLS), while exon 20 contains a nuclear export signal (NES) 26 . In addition, 83 there are four additional isoforms of the protein, which vary in their transcriptional start sites. Of the 84 four major isoforms, isoform 1 is abundant in mesenchymal cells, while isoform 3 appears 85 preferentially expressed in epithelial cells [27][28][29][30] . The other two isoforms are less well characterized. 86 The p120 superfamily includes p120-catenin itself, δ-catenin (CTNND2) and ARVCF 87 (armadillo repeat gene deleted in velocardiofacial syndrome) all of which can compete for E-88 cadherin binding. Although it is unclear whether they substitute for one another in other cellular 89 functions 31,32 , evidence from animal studies suggests some compensatory roles. For instance, δ-90 catenin (CTNND2) knockdown phenotypes can be rescued with p120-catenin, and the combined 91 depletion of δ-catenin and p120 generates more pronounced effects. However, levels of p120 are 92 not altered by reducing δ-catenin protein levels 33 . In humans, CTNND2 variants have been associated 93 with autism spectrum disorders and other neurodevelopmental conditions [34][35][36][37][38][39] . Interestingly, the 94 other p120 family member, ARVCF, lies in 22q11. While loss of TBX1 in 22q11 is thought to cause the 95 key malformations associated with velocardiofacial (VCF) syndrome [MIM: 192430], evidence from 96 animal models suggests that ARVCF may also play a role in craniofacial development 40-43 . 97 Although both p120-catenin and its binding partner E-cadherin have been proposed as 98 causative genes in non-syndromic palatal clefting and BCD syndrome 1-3 , the patients that we 99 describe here present with a multisystem condition broader than the previously described p120-

117
Medical and dental histories were taken, as well as detailed phenotyping by clinical 118 geneticists with expertise in dysmorphology. Saliva for DNA extraction was collected from family 119 trios using the Oragene® DNA (OG-500) kit. All patients also underwent high-resolution analysis for 120 copy number abnormalities using array-based comparative genomic hybridization. Informed consent 121 from all participants was obtained for publication of data and photographs in the medical literature.      (Table 2), and further details can be found in (Table   269   S1). Photographs from participants show a number of shared craniofacial and oral features (   Table 2 and Table S1).  (Table S1). In addition, one participant had velopharyngeal insufficiency (VPI) and a bifid uvula. Of interest, three individuals presented with 285 vocalization defects causing stridor and hoarseness or nasal speech.

286
Upon dental examination, all subjects were found to have intra-oral anomalies ( Figure 3). In 287 particular, congenital tooth agenesis (hypodontia) was frequently seen, with eight subjects missing 288 between three and twelve adult teeth (Figure 3G-L; Table S2). Other anomalies included retained 289 primary teeth and delayed eruption of the permanent teeth (6/13) (Table S1) Table S1).

292
Beyond the craniofacial structures, the majority of the participants had limb and heart 293 anomalies. Mild limb phenotypes (9/13) were present, including shorter fifth fingers, single 294 transverse palmar crease, mild syndactyly between the 2,3 toes, sandal gaps and camptodactyly of 295 the toes ( Figure S1C). Congenital cardiac defects, which have not previously been associated with 296 CTNND1 variants, consistently occurred in our cohort. Six subjects had cardiovascular anomalies 297 including tetralogy of Fallot, hypoplastic aortic arch, coarctation of the aorta, ventricular septal 298 defect, atrial septal defect, mitral valve stenosis, patent ductus arteriosus and patent foramen ovale 299 ( Table 2 and Table S1). Finally, in addition to the craniofacial and cardiac anomalies, individuals 300 presented with other phenotypes that added to the complexity of their conditions. Developmental 301 delay and other neurodevelopmental problems were also observed (8/13). These often appeared 302 from early toddler and school years and included mild learning difficulties, autism spectrum disorder, 303 speech and language delay, and behavioral problems (Table S1). One individual was diagnosed with 304 ovarian dysgerminoma stage III in the left ovary at the age of 12 years, which was treated with left 305 oophorectomy followed by chemotherapy. Other infrequent anomalies included urogenital 306 problems, scoliosis and partial agenesis of the corpus callosum (Table S1).  (Table S1 and Table 2), a phenotype described in 357 patients with velocardiofacial syndrome [56][57][58] . Antibody staining confirmed presence of p120-catenin 358 protein during development of the laryngeal and pharyngeal tissues in the mouse ( Figure S3A). We 359 then examined the laryngeal structures of mutant mice compared to their littermate controls at 360 E16.5, P1 and P2.5 ( Figure 6). To do this, we crossed a mouse carrying the ubiquitous β-actin::cre driver with Ctnnd1 fl/fl mice in order to generate heterozygous mutants 59,60 ( Figure 6C, 6H, 6M, 6R).

362
Because we previously showed that the vocal ligaments originated from the neural crest 61 , we also 363 generated tissue-specific Ctnnd1 heterozygotes using the neural crest specific driver, Wnt1::cre 62 364 ( Figure 6E, 6J, 6O). We found identical laryngeal anomalies in the heterozygous mutants in both 365 mouse crosses, confirming the neural crest-specificity of these phenotypes.  Figure 6M). Finally, in mutants, the muscles were ectopically fused to 386 the levator veli palatini muscles, which were then fused to the cranial base ( Figure 6M). This, in turn, gave the impression of a high-arched epiglottal area; a defect also found in the Wnt1::cre 388 heterozygous mutants ( Figure 6O).

389
We also explored other craniofacial phenotypes in our heterozygous mouse model.

390
Compared to their littermate controls ( Figure S3B

396
While genetic mutation of p120-catenin in mouse models revealed a role for the neural crest 397 in oropharyngeal development, analysis of multi-system involvement of p120-catenin was difficult 398 due to embryonic lethality of the homozygous null mice 5,9 . We therefore turned to the frog Xenopus, 399 where in vivo function of p120-catenin has been well studied 11,12,63 . Previous analyses of p120-400 catenin requirements were mainly performed with antisense morpholino oligonucleotide (MO) 401 knockdowns, which transiently prevent protein translation 11 . Instead, to create genetic mutants, we 402 used CRISPR/Cas9 approaches, allowing us to specifically delete different p120-catenin isoforms 64 .

403
As noted in the introduction, isoform 1 (full length at 968 amino acids (aa)) is most abundant in 404 mesenchymal cells, while isoform 3 (start at aa 102) is preferentially expressed in epithelial cells 27-30 . 405 Isoforms 2 and 4, which start at 55 aa and 324 aa, respectively, are less well characterized.

406
Embryos were injected at the one cell stage with single guide RNAs (sgRNAs) targeting either 407 of two coding exons, exon 3 or exon 7 (sgRNA1 and sgRNA2 respectively, Figure 7A). Disruptions in 408 exon 3 are predicted to only affect isoform 1, while sgRNA2 targeting exon 7 disrupt all four isoforms.

409
When embryos were scored at gastrula stages following sgRNA1 injections, disrupted or 410 delayed blastopore closure was evident (n=30/42 vs. 2/30 in the controls) ( Figure 7B). Furthermore, 411 we noted severe early lethality ( Figure 7D), especially using sgRNA2 which blocked all isoforms ( Figure 7D). Notably, by neurula stages the majority of these mutants died due to a loss of integrity 413 in the epithelium (data not shown).

414
Since the most well-established epithelial role for p120-catenin is in complex with E-415 cadherin at cell-cell junctions, we first examined E-cadherin localization in the neurectoderm at 416 stage 11, as gastrulation was concluding. Indeed, in uninjected controls, high levels of p120-catenin 417 and E-cadherin were found co-localized at the cell interface ( Figure 7C, a-d). E-cadherin is expressed 418 throughout the cell membrane ( Figure 7C, b), whereas p120-catenin, though localized to the cell 419 membrane, appears distributed in puncta ( Figure 7C

442
Finally, since the participants (6/13) had a high frequency of congenital heart defects and 443 because p120 is strongly expressed in the heart of human, mouse and frog embryos, we examined 444 the hearts in the CRISPR-knockout tadpoles. Notably, the strong expression of p120 seen in the 445 different heart chambers in the control tadpoles was lost when p120 was knocked down ( Figure 8B [65][66][67][68] . All of these syndromes could be 462 collectively considered to be neurocristopathies. Notably, the neural crest specific disruption of 463 CTNND1 in our animal models supports this role for CTNND1 as a candidate neurocristopathy gene and we suggest that these newly identified variants likely highlight both epithelial and mesenchymal 465 roles for p120-catenin.

466
Prior to our study, the majority of the participants did not have a recognizable or a 467 diagnosed condition when they were recruited. Here, we demonstrate that they collectively share 468 consistent characteristic phenotypic features that suggest that mutations in CTNND1 may lead to a 469 much broader phenotypic spectrum than previously described 1,2 . For instance, low set ears were 470 reported in one case of BCD by Kievit and colleagues 1 ; we find multiple participants with auricular 471 anomalies particularly the low-set ears and over-folded helices ( Figure S1B, Table S1). Similarly, 472 syndactyly was reported in one of the CTNND1 patients described in Ghoumid et al. 2 , and 473 clinodactyly (one patient) and camptodactyly (two patients) were reported by Kievit et al 1 . Again, we 474 find limb anomalies consistently associated with CTNND1 variation ( Figure S1C, Table S1). The 475 cardinal features of BCD include ectropion of the lower eyelids, euryblepharon and 476 lagopthalmos 69,70 ; while five of our patients showed these eye manifestations ( Figure 2; Table 2), we 477 also found short up-slanting palpebral fissures, hooded eyelids, high arched eyebrows and 478 telecanthus ( Figure S1A, Table 2 and Table S1). As BCD is associated with both CTNND1 and CDH1 (E-  Table S2).

486
Beyond the known phenotypes associated with CTNND1 and CDH1, we note the novel 487 phenotypes seen in our patients, which include the heart anomalies and behavioral disorders. These  3 . As these C-terminal truncations would all be predicted to retain E-cadherin 505 binding, but lose crucial RhoGAP interactions 24 , one might hypothesize that a mutation in this region 506 prevents p120 clearing from the epithelial complex, which is necessary for seam dissolution during 507 palate closure. Therefore, future analyses should focus on whether these C-terminal truncations are 508 acting in a dominant-negative manner, and preventing clearance of E-cadherin from the seam.

509
With regards to non-epithelial functions of p120, some of the phenotypes that this study, 510 and others, have reported, could be explained by the known interactions of p120 in the Wnt 511 signaling pathway 20 . Epithelial-specific knockouts of p120 (using a keratin-14 promoter) did not show 512 tooth agenesis 10 , suggesting that the tooth anomalies in our patients do not arise from the epithelial 513 functions of p120. In support of this, two key genes implicated in tooth agenesis are the Wnt ligand,

514
Wnt10A and a Wnt target gene Axin2 74-78;78-84 . The Wnt signaling pathway may also explain the 515 laryngeal findings (Figure 6), as knockout of the Wnt transducer β-catenin is also known to lead to similar vocal fold anomalies 85 as those seen in our neural crest specific p120-catenin heterozygotes 517 ( Figure 6). Furthermore, knockout of the mesenchymal form of p120 (isoform 1) in Xenopus ( Figure 7 518 and Figure 8), confirm prior studies on p120-catenin in the neural crest, where the p120-catenin 519 association with Wnt signaling is well-established 32,86,87 . Thus, we hypothesize that a subset of p120 520 phenotypes can also be attributed to Wnt perturbation in the neural crest ( Figure 9). The heart 521 defects seen in our patients could also be attributed to a failure in neural crest development, which 522 is known to be crucial for development of the septum and valves [88][89][90][91][92] .

523
In addition to the phenotypes shared commonly across our cohort, some participants in this 524 study had scoliosis, and one family reported two deceased children, who had bifid uvula, congenital 525 cardiac disease (VSD, PDS), eye anomalies, developmental delay and chronic bowel immotility and 526 gastroesophageal reflux disease; however, no genetic testing had been carried out. One patient 527 presented at a young age with an ovarian dysgerminoma. To our knowledge, this is the first patient 528 with a CTNND1 variant associated with an early onset cancer, though p120 has been associated with 529 cancer and tumorigenesis 23