Novel large deletion involving EVC and EVC2 in Ellis–van Creveld syndrome

Ellis–van Creveld syndrome is an autosomal recessive skeletal dysplasia that is characterized by thoracic hypoplasia, polydactyly, oral abnormalities, and congenital heart disease. It is caused by pathogenic variants in the EVC or EVC2 genes. We report a case of a newborn with a compound heterozygous variant comprising NM_147127.5: c.1991dup:[p.Lys665Glufs*10] in the EVC2 gene and a novel large deletion involving exon 1 in EVC and exons 1–7 in EVC2.

limbs. The birth weight was 3230 g (+0.4 SD), birth length was 50.0 cm (+0.3 SD), and head circumference was 35.0 cm (+1.2 SD) (SDs based on Japanese neonatal standards) 6 . Chest X-ray revealed bell-shaped thoracic hypoplasia, left clavicle fracture, and pulmonary air leakage in the right lung (Fig. 1B). Cardiac ultrasonography showed a complete atrioventricular septal defect (Rastelli type A) and bidirectional shunting (right-to-left dominant) at the ductus arteriosus and massive tricuspid valve regurgitation, suggesting persistent pulmonary hypertension of newborn syndrome. Because of his lung hypoplasia and multiple congenital anomalies, the patient was transferred from the secondary neonatal intensive care unit to the tertiary neonatal intensive care unit at Tokushima University Hospital.
On admission, mechanical ventilation with high-frequency oscillation and inhaled nitric oxide for persistent pulmonary hypertension of the newborn were initiated. At 3 days of age, inhaled nitric oxide and sedative agents were discontinued. At 4 days of age, he was extubated and switched to a high-flow nasal cannula. At 25 days of age, he began receiving nasal oxygen. At 43 days, he was discharged with home oxygen therapy (1 L/min). Due to inadequate oral intake, he required tube feeding since he was 2 months old. At 12 months, he showed severe growth delay (body weight, -3.2 SD; height, -2.9 SD) and mild developmental delay (creeping along the floor) and still required home oxygen therapy and tube feeding. His chromosomal karyotype was 46, XY with inv(9)(p11q13) (normal benign variant).
In our patient, we identified the compound heterozygous variant c.1991dup (exon 13) in EVC2 and a large deletion involving exon 1 in EVC and exons 1-7 in EVC2. The Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php) includes 8 gross deletions in EVC and 6 gross deletions in EVC2. Three pedigrees of EVC with large deletions in EVC and EVC2 as well as in the contiguous genes C4orf6 and STK32B have been reported 9,10 . However, these patients showed borderline intelligence, which is not typical for EVC and probably depends on C4orf6 or STK32B. To our knowledge, the present deletion is a novel contiguous deletion that is limited to EVC and EVC2. EVC and EVC2 form a head-to-head configuration on 4p16.2. They are separated by 2.6 kb of genomic sequence and share a common transcriptional promoter 11 . Therefore, in the present case, the large deletion encompassing exon 1 of EVC and exons 1-7 of EVC2 included the shared promoter of EVC and EVC2. Both alleles of our patient's EVC2 gene and one allele of his EVC gene are nonfunctional due to the promoter deletion and frameshift variant of the gene.
Ohashi et al. 8 reported a lethal thoracic hypoplasia case of EVC that had the compound heterozygous variants c.1991dup and c.871-3C > G (intron 7) on EVC2. However, in the present case, thoracic hypoplasia was mild, and the patient was able to be discharged with home oxygen therapy. Thus, the phenotypes of EVC do not always match the genotypes of EVC and/or EVC2.
EVC is a type of ciliopathy. In wild-type mice, Evc, Evc2, and Smo form a complex, which is located in the EvC zone of primary cilia in the presence of Sonic Hedgehog (Shh) ligands 12 . This complex promotes Hedgehog (Hh) signaling, thereby promoting transcription. In the case of EVC, the Hh signal is not transmitted even in the presence of Shh ligands 13 . Shh -/mice are nonviable, and Shh -/mouse embryos display several heart defects, including atrioventricular septal defects 14,15 . Hh-responsive cells in the growth plate comprise osteogenic progenitors that can directly differentiate into osteoblasts 16 . Shh signaling regulates the formation of various tooth components, including enamel, dentin, cementum, and various soft tissues 17 .
Other skeletal ciliopathies with overlapping features include short rib polydactyly syndrome 18 , Jeune syndrome 19 , and cranioectodermal dysplasia 2,20 , all of which show thoracic hypoplasia and short ribs. McKusick-Kaufman syndrome is characterized by polydactyly and congenital heart disease 18 , and Weyers acrofacial dysostosis is characterized by dental  abnormalities, nail dystrophy, and polydactyly 21 . Identifying gene alterations is important because these diseases are difficult to differentiate based on their clinical phenotypes 2 .
Concerning our patient's family, the recurrence risk for subsequent pregnancies is 25%, and we have provided this genetic information to the patient's parents. Although the patient had not been diagnosed in the fetal period, the fetus of any consequent pregnancy can be diagnosed prenatally according to the existence of polydactyly, thoracic hypoplasia, and cardiac malformations, such as atrioventricular septal defects. This report has several limitations. First, we did not perform any confirmatory array comparative genomic hybridization to identify the deletion point. Second, the follow-up period was short. Our patient had mild developmental delay, which should be continued to be monitored.
In conclusion, we describe a novel large deletion involving both EVC and EVC2. The phenotype of this variant seems to be typical for EVC.

HGV DATABASE
The relevant data from this Data Report are hosted at the Human Genome Variation Database at https://doi.org/10.6084/m9.figshare.hgv.3157.