Whole-exome sequencing identifies ECPAS as a novel potentially pathogenic gene in multiple hereditary families with nonsyndromic orofacial cleft

Graphical abstract

Dear Editor,

Orofacial cleft (OFC), which includes cleft lip and/or palate (CL/P) and cleft palate (CP), is the most common congenital craniofacial structural disorder, with a prevalence of 1.416‰ among live infants worldwide (Massenburg et al., 2021). Nonsyndromic OFC (NSOFC), which does not contain other malformations as syndromic OFC (SOFC), accounts for 70% of cases and is believed to have complex etiologies. Notably, it has been established that genetic factors play a crucial role in the occurrence of NSOFC (Dixon et al., 2011).

Prior to the comprehensive analysis of the entire human genome, linkage analysis, and candidate gene association studies had been the predominant approaches for exploring the genetic basis of NSOFC. Along with advances in genetics, genome-wide association studies (GWAS) are capable of identifying risk loci associated with NSOFC across the entire genome, independently of predetermined candidate genes. Up to now, GWAS have unveiled more than 45 genetic risk loci, usually with minor allele frequency (MAF) higher than 5%, including 1q32 (IRF6), 3q27 (TP63), 9q (FOXE1), etc., that collectively account for 10%–30% of the heritability of NSOFC (Leslie, 2022). However, there is still a significant proportion of genetic contribution to NSOFC that remains unexplained, limiting the diagnostic utility of clinical genetic testing, as is the case with other congenital deformities (Lord et al., 2019).

The implementation of next-generation sequencing strategies and functional studies has markedly advanced the identification of genetic variants associated with NSOFCs. Previous studies have indicated the substantial role of de novo variants (DNVs) or rare variants within clinically relevant OFC genes such as PTCH1, GRHL3, and CTNND1, which significantly contributed to the genetic etiology of NSOFC (He et al., 2022; Huang et al., 2023; Leslie, 2022). While DNVs are more important in sporadic cases, rare variants appear to play a greater role in hereditary pedigrees (Bishop et al., 2020; Zuk et al., 2014). Therefore, a systematic study of rare variants in patients from hereditary families is likely to unveil causal genes for NSOFC.

The development of the lip and palate is a conserved process across vertebrates and is controlled by several key signaling pathways such as Hedgehog (HH), WNT, TGF-β, and FGF. Although previous studies have indicated that structural clefts can result from disturbances in these pathways (Reynolds et al., 2020), a comprehensive understanding of how dysregulation in these pathways contributes to NSOFC remains elusive.

We recruited 30 families with NSOFC over the past seven years, in which at least two members in each family were affected (Fig. S1). Among these families, 23 exhibited autosomal dominant (AD) inheritance and seven exhibited autosomal recessive (AR) inheritance (Fig. S1). To uncover rare variants that may confer the risk of NSOFC, we conducted whole-exome sequencing (WES) on these families. After applying filtering criteria, we identified a total of 394 candidate variants related to OFC/craniofacial development in these 30 hereditary families (Fig. 1A; Table S1), and delineated the genetic architecture (Fig. S2). Next, we turned our attention to those pathogenic/likely pathogenic variants known to have roles in the OFC-related morphogenic processes or pathways. Following the American College of Medical Genetics and Genomics (ACMG) criteria, we identified nine pathogenic/likely pathogenic variants from seven out of the 30 families (23.33%) (Fig. 1B and 1C; Table S2). These variants included PTCH1 (two families, 6.67%), GLI2 (one family, 3.33%), IRF6 (one family, 3.33%), PLEKHA5 (one family, 3.33%), CREBBP (one family, 3.33%), and FZD6 (one family, 3.33%) (Fig. 1B). These variants fell into four morphogenic pathways crucial for lip and palate development: HH (three families, 10.00%), epithelial-related (two families, 6.67%), TGF-β (one family, 3.33%), and WNT (one family, 3.33%) signaling pathway (Fig. 1C), confirming the critical roles of these pathways for the pathogenesis of OFC and validating the reliability of our data. Apart from these known causal variants, we also detected many novel variants, which, without known functions for either lip/palate development or pathogenesis of OFC, represent a rich resource for future research.

Noticing that no known pathogenic variants were detected in the majority of hereditary families exhibiting AR inheritance, we, therefore, focused on these families. In Family 30, a rare homozygous missense variant in the ECPAS gene (c.C1931G, p.T644S) came to our attention (Figs. 1D and S3). The proband (D1), a boy with left cleft lip (CL) and median CP, and one of his second cousins (D2), a girl with left CL, are the two individuals diagnosed with NSOFC in this four-generation family (Fig. 1D). Using PCR-Sanger sequencing, we confirmed an AR inheritance model in this family (Fig. 1E). Then, we conducted in silico analysis to evaluate the impact of T644S on the function of ECPAS, which suggested that T644S variant is possibly pathogenic (Fig. S4).

ECPAS, also known as ECM29, is a proteasome-associated protein that plays an important role in cell proliferation and migration (Gorbea et al., 2004; Miettinen et al., 2018). Considering the coordinated proliferation and migration of epithelial and mesenchymal cells are essential for craniofacial morphogenesis, we next assessed whether ECPAS might affect cell proliferation and migration and whether the T664S variant might alter this functional role. As expected, the knockdown of ECPAS significantly inhibited cell proliferation in both HEK-293T (epithelial) and HEPM (mesenchymal) cells (Fig. 1F–I). Besides, the knockdown of ECPAS also reduced the migratory capacity of HEPM cells (Fig. 1J–L). Conversely, overexpression of wild-type ECPAS significantly promoted cell proliferation in HEK-293T cells (Fig. 1M–O). In contrast, although overexpression of ECPAS T664S appeared to slightly inhibit cell proliferation compared to the GFP control, this difference was not statistically significant, indicating that overexpressed ECPAS T644S mutant behaved like the GFP control (Fig. 1M–O). These results corroborate the role of ECPAS in boosting cell proliferation and migration as previously reported (Miettinen et al., 2018), and the bi-allelic T644S variant is a loss-of-function mutation that might be pathogenic for NSOFC.

We notice that the Ecpas-null mice exhibited impaired disassembly of the 26S proteasome under oxidative stress conditions and no craniofacial defect has been observed (Haratake et al., 2016). However, since the penetrance of clefts in knockout mice models is typically low and can sometimes be neglected (Peyrard-Janvid et al., 2014), a potential connection between ECPAS and craniofacial development cannot be entirely ruled out. We examined the spatiotemporal expression pattern of ECPAS in mice from E11.5 to E16.5. Using qPCR and immunohistochemistry (IHC), we found that ECPAS expression increased steadily during lip development (Fig. S5), suggesting a possible involvement of ECPAS in mouse lip development.

To determine whether ECPAS plays a role in craniofacial development, we turned to the zebrafish model. We first employed in situ hybridization (ISH) to investigate the expression pattern of ecpas in zebrafish embryos, and our findings demonstrated that ecpas is expressed maternally at one and two-cell stages and later in the craniofacial region from 36 h postfertilization (hpf) to 3 days postfertilization (dpf) (Fig. S6). Next, we designed translation-blocking and splicing-blocking antisense morpholino oligonucleotides (MOs) targeting the zebrafish ecpas gene, which effectively and specifically inhibited the expression of Ecpas protein (Figs. S7 and S8A). Compared to the negative control MO (CMO), microinjection of ecpas ATG MO or ecpas splicing MO caused craniofacial dysplasia, characterized by a less protruding mouth, at 4 dpf (Figs. 2A and S8B). Notably, a similar phenotype was observed when ecpas MO was co-injected with p53 MO, indicating that this phenotype was not due to nonspecific cytotoxicity mediated by the p53 pathway (Robu et al., 2007). We then performed Alcian blue staining to visualize the craniofacial cartilage. In comparison to embryos injected with control MO, ecpas morphants had significantly smaller ethmoid plates and palatoquadrates. Moreover, the depletion of ecpas also led to a reduction in the size of Meckel’s cartilages, as well as developmental abnormalities in the rest of the pharyngeal region, including shortened arches and enlarged arch angles (Figs. 2B, S8C and S9).

As craniofacial cartilage originates from cranial neural crest cells (CNCCs) (Cordero et al., 2011) and ECPAS promotes cell proliferation and migration, we sought to determine the effect of ecpas loss on these cells. In Tg(sox10:EGFP) transgenic zebrafish embryos, which enabled us to visualize CNCCs with GFP fluorescence, we observed that knockdown of ecpas led to a reduction in the number of CNCCs in the ethmoid plate, Meckel’s and palatoquadrate cartilages at 3 dpf (Fig. 2C). We next conducted a BrdU assay to assess the proliferative status of CNCCs. Immunofluorescence analysis revealed a significant decrease in the ratio of BrdU-positive cells in the pharyngeal region of ecpas morphants at 40 hpf (Fig. 2D and 2E), indicating impaired proliferation of CNCCs upon ecpas loss. However, except for the maternal expression of ecpas (Fig. S6), we did not observe any effect of ecpas disruption on the early stage of CNCCs migration at 24 hpf (Fig. S10A), or the later stage of CNCCs migration into the pharyngeal region at 36 hpf (Fig. S10B).

To examine whether the T644S variant of ECPAS has a loss-of-function effect on the proliferation of CNCCs in zebrafish embryos, akin to its role observed in cellular experiments, we performed a rescue experiment in zebrafish embryos. Tg(fli1:EGFP) transgenic zebrafish embryos, where CNCCs and blood vessels are labeled with GFP, were used in this experiment. We co-injected a sox10 promoter-driven recombinant Tol2 vector containing either the wild-type or T644S variant of human ECPAS tagged with mCherry at its C-terminal and transposase mRNA to ensure CNCCs-specific overexpression of these two constructs. We observed that overexpression of wild-type ECPAS could rescue the impaired proliferation of CNCCs induced by ecpas MO. However, the T644S variant, despite being overexpressed at levels comparable to the wild-type, failed to rescue the ecpas MO-induced weakened proliferation of CNCCs in zebrafish embryos (Fig. 2F). These results suggest that disruption of ecpas impairs craniofacial development in zebrafish by inhibiting the proliferation of CNCCs and the T644S variant is indeed a loss-of-function mutation that impairs the proliferation-promoting capacity of ECPAS in vivo.

In this study, we made three main findings. First, we delineate the genetic architecture in patients with NSOFC and are able to uncover many novel candidate variants by WES in multiple Chinese hereditary families. Second, we identified a bi-allelic loss-of-function variant in the ECPAS gene, T644S, in a hereditary NSOFC family showing an AR inheritance. Third, ECPAS promotes cell proliferation in mammalian cells and loss of ecpas impairs craniofacial development in zebrafish by inhibiting the proliferation of CNCCs.

Several limitations of this study should be mentioned. First, the use of MO in zebrafish presents challenges, such as incomplete suppression of gene expression, which limits the ability to observe phenotypes that require complete gene knockout or extended suppression. In addition, off-target effects can obscure the interpretation of MO-induced phenotypes, even with proper controls. In future studies, an effective CRISPR/Cas9 system could be utilized to create loss-of-function mutants, providing clearer links between the genotype and phenotype. Moreover, it’s important to recognize that when it comes to mimicking missense variants, knock in mouse models are more accurate compared to zebrafish models. Therefore, our future research will focus on establishing a knock in mouse model, to corroborate our conclusions and enhance the understanding of the underlying mechanisms.

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The Author(s) 2024. Published by Oxford University Press on behalf of Higher Education Press.