Identification of a novel EVC variant in a Han‐Chinese family with Ellis‐van Creveld syndrome

Abstract Background Ellis‐van Creveld syndrome (EVC), a very rare genetic skeletal dysplasia, is clinically characterized by a tetrad consisting of chondrodystrophy, polydactyly, ectodermal dysplasia, and cardiac anomalies. The aim of this study was to identify the genetic defect for EVC in a five‐generation consanguineous Han‐Chinese pedigree. Methods A five‐generation, 12‐member Han‐Chinese pedigree was enrolled in this study. Exome sequencing was applied in the proband to screen potential genetic variant(s), and then Sanger sequencing was used to identify the variant in family members and 200 unrelated ethnicity‐matched controls. Results A novel homozygous variant, c.2014C>T, p.(Q672*), in the EvC ciliary complex subunit 1 gene (EVC), was detected in the patient, which was cosegregated with the disease in the family and absent in the controls. Conclusion The identified novel homozygous EVC variant, c.2014C>T, p.(Q672*), was responsible for EVC in this Han‐Chinese pedigree. The findings in this study extend the EVC mutation spectrum and may provide new insights into EVC causation and diagnosis with implications for genetic counseling and clinical management.

This study sought to reveal the genetic factor giving rise to EVC in a five-generation consanguineous Han-Chinese family. A novel homozygous EVC c.2014C>T variant, which may result in a protein truncation (p.(Q672*)) and a loss of protein function, was identified. These may hinder normal hedgehog signaling pathway, and disrupt normal endochondral growth, and orofacial, limb, and skeletal development and maintenance (Chen, Chen, Chern, Su, & Wang, 2012;Ibarra-Ramirez et al., 2017;Ruiz-Perez & Goodship, 2009;Valencia et al., 2009).

| Ethical compliance
Written informed consent was obtained from participants or the legal guardian. This study had received approval from the Institutional Review Board of the Third Xiangya Hospital, Central South University, Changsha, China.

| Participators and clinical examination
A five-generation Han-Chinese family with EVC was recruited at the Third Xiangya Hospital, Central South University, Changsha, China ( Figure 1a). Six individuals, including the proband (IV:1), the unaffected first-cousin parents (III:1 and III:2), and three other unaffected family members (IV:2, IV:3, and V:1) were involved in this study, and blood samples were collected. Detailed clinical medical histories and physical examinations were collected from all participants. Blood specimens were also collected from 200 unrelated, ethnically matched controls (male/female: 100/100, age 33.4 ± 6.9 years) which lacked EVC diagnostic features. Each participant underwent thorough examination for clinical diagnosis or exclusion, including general examination such as polydactyly and dysplastic nails, oral check-up such as oral frenula, and teeth, limbs, and cardiovascular inspection.

| Exome capture
Genomic DNA (gDNA) was separated from peripheral blood lymphocytes via a phenol-chloroform extraction . Proband (IV:1) exome sequencing was performed for unveiling genetic causation of EVC in this pedigree, following the established protocol of BGI-Shenzhen (Fan et al., 2019;Yuan et al., 2016). A paired-end DNA library was constructed and whole-exome capture was performed utilizing BGI exome V4 kit. The qualified circular DNA library was sequenced on a BGISEQ-500 platform (Fan et al., 2019).

| Clinical findings
The EVC diagnosis was made using characteristic clinical manifestations. The proband (IV:1) was a 42-year-old female, born to an unaffected consanguineous couple. Physical examination revealed disproportionate dwarfism, bilateral ulnar polydactyly, distal phalanges hypoplasia, dysplastic nails, and genu valgus. Intraoral examination showed multiple oral frenula, absence of maxillary incisors, mandibular anodontia, and alveolar ridge defect (Figure 2). No congenital heart defect was found. All relatives were unaffected with no similar features.

| Genetic analysis
A total of 38,706.61 Mb mapped to reference genome were generated by exome sequencing on gDNA from the proband (IV:1), with a coverage of 99.93% in the target region. The mean sequencing depth of target region was 277.04-fold, and 99.17% of the target region was covered by at least 10-fold.  (Figure 1b). Sanger sequencing showed the variant heterozygosity in her unaffected first-cousin parents (Figure 1c). This variant was not present in three other unaffected family members (IV:2, IV:3, and V:1, Figure 1d), BGI in-house exome databases, NHLBI ESP6500, or 200 unrelated controls. No homozygote (0/117,630) was recorded in the ExAC database. The homozygous variant cosegregated with the disease in this family, suggesting it is likely to be the pathogenic variant. PROVEAN predicted it was deleterious, and MutationTaster revealed it was "disease causing" (classification due to nonsense-mediated mRNA decay, NMD), further supporting the identified EVC variant was deleterious for EVC. The variant is classified as "pathogenic" following the ACMG variant interpretation guidelines.

| DISCUSSION
This study identified, a novel homozygous variant, c.2014C>T, p.(Q672*), in the EVC present in a consanguineous Han-Chinese family with EVC. The patient manifested typical EVC clinical features, including disproportionate dwarfism, multiple oral frenula, absence of maxillary incisors, mandibular anodontia and alveolar ridge defect, bilateral ulnar polydactyly, distal phalanges hypoplasia, dysplastic nails, and genu valgus, but no congenital heart defect. Various symptoms were evidenced in early onset and a special attention was needed from birth. The homozygous variant cosegregated with the disorder in this family, and was absent in the 200 controls, public, and in-house exome databases, suggesting that it is pathogenic. Computer-based analysis further confirmed the predicted deleterious effect of the variant.
The EVC, located at chromosome 4p16.2, contains 21 exons and encodes a 992-amino acid protein (Ruiz-Perez et al., 2000;Shi et al., 2016). It orients head-to-head with its homolog gene EVC2, which also encodes a single-pass type I transmembrane protein (Vona et al., 2018). These two proteins interact directly to form a complex at the primary cilium membrane (EvC zone), which is essential for normal endochondral growth and intramembranous ossification (Shi et al., 2016;Umair et al., 2017).
At least 81 EVC mutations, in the homozygous or compound heterozygous state, have been reported as responsible for EVC in the HGMD and in published literature, including 25 missense/nonsense mutations, 19 splicing mutations, 14 small deletions, 9 small insertions, 1 small indel, 8 gross deletions, 2 gross insertions, and 3 complex rearrangements (D'Ambrosio et al., 2015;D'Asdia et al., 2013;Liu et al., 2018;Shi et al., 2016;Tompson et al., 2007). The common mutations usually produce premature termination codons, either directly or following a reading frame shift, and are thus predicted to lead to nonsensemediated decay of transcripts or nonfunctional truncated proteins (D'Asdia et al., 2013;Ibarra-Ramirez et al., 2017;Valencia et al., 2009). Weyers acrofacial dysostosis (WAD, OMIM 193530) and EVC are allelic disorders. WAD is a mild EVC phenotype caused by EVC or EVC2 heterozygous mutations, and the last exon of EVC2 may be a hot region for mutations (D'Asdia et al., 2013;Nguyen et al., 2016). WAD is distinguished from EVC by its inheritance mode and phenotypic severity, which does not have narrow chest or congenital cardiac anomalies (D' Asdia et al., 2013;Shi et al., 2016). The pathogenic mechanism of these two disorders is a cilia-mediated, diminished response to hedgehog ligands (Ruiz-Perez & Goodship, 2009). In EVC, biallelic nonsense and/or frameshift mutations in the EVC or EVC2 are likely to result in NMD or truncated proteins, and thus lead to EVC in a loss-of-function mechanism. In WAD, mutations in the last exon of EVC2 may escape NMD and the resulted, more stable EVC2 proteins may have a negative effect by gaining a function or affecting the normal protein (Ibarra-Ramirez et al., 2017;Shen, Han, Zhang, Zhao, & Feng, 2011;Shi et al., 2016;Valencia et al., 2009).
Evc −/− mice represent a precise EVC model, presenting short limbs, short ribs, and dental abnormalities, but no polydactyly . Abnormal epiphyseal and periosteal induction was observed in histology of cartilage plate growth, similar to the defects in Indian hedgehog signaling (Blair et al., 2011). Evc +/− mice have no obvious abnormalities . Genetic animal studies further establish a loss-of-function mechanism in EVC progress.
In summary, a novel homozygous nonsense alteration, c.2014C>T, p.(Q672*), is likely the responsible variant for the EVC present in this Han-Chinese family. The findings extend the EVC genotypic spectrum and have genetic counseling and clinical management implications. Due to the limited number of patients in this study and relative rarity of this disorder, more cases in additional families confirmed by genetic analysis, as well as functional evidence may strengthen the variant pathogenicity. Further and more extensive studies involving either, or both, in vitro and in vivo models with genetic deficiencies, as well as detection of disease-causing mutations, should help reveal pathogenic mechanisms and assist in developing targeted EVC gene therapeutic strategies.