Mexican patient with Ellis-van Creveld syndrome and cleft palate: Importance of functional hemizygosity and phenotype expansion

Abstract

Background

Ellis-van Creveld syndrome (EvCS) is a chondroectodermal dysplasia caused by germline pathogenic variants in ciliary complex subunit 1 and 2 genes (EVC, EVC2) on chromosome 4p16.2. This disease has a broad phenotype, and there are few described phenotype–genotype correlations.

Methods

Ethical Compliance: Written informed consent was obtained from the parents. Here, we report a genetically confirmed Mexican patient with EvCS having two inherited pathogenic variants in trans in EVC2: c.[1195C>T];[2161delC].

Results

This patient allowed a genotypic–phenotypic comparison with another Mexican subject who presented a more attenuated phenotype; furthermore, our patient also presented cleft palate, a rarely reported feature.

Conclusion

Our case shows the importance of comparing functional hemizygosity between patient's phenotypes when they share a variant, and our case also supports the association of alterations in the palate as part of the EvCS phenotype.

INTRODUCTION

Ellis-van Creveld syndrome (EvCS, OMIM#225500) is an autosomal recessive chondroectodermal dysplasia caused by germline pathogenic variants in ciliary complex subunit 1 and 2 genes (EVC [OMIM#604831] and EVC2 [OMIM#607261]). EvCS has a prevalence of 7 in 1,000,000 and it is characterized by short limbs, polydactyly, nail dysplasia, short ribs, and dental anomalies, and up to 64.5% congenital heart disease (Da Silva et al., 2023). The EVC and EVC2 genes are located on chromosome 4p16.2 in divergent orientation and separated by about 2.6 Kb. EVC has 21 exons and encodes a protein of 992 amino acids, while EVC2 has 22 exons and encodes a protein of 1308 amino acids (Da Silva et al., 2023). EVC and EVC2 are membrane proteins anchored at the N-terminus that form a protein complex and require each other for their ciliary localization, acting through the activation of other proteins such as Smoothened (SMO) and Glioma-associated oncogene homolog (GLI), and mainly intervening in Hedgehog (Hh)-mediated signaling through intraflagellar transport (Da Silva et al., 2023; Louie et al., 2020; Thomas et al., 2022). Here, we report a Mexican patient carrying two inherited pathogenic variants in trans in EVC2 (NM_147127.4), c.[1195C>T];[2161delC]. This patient allowed a genotypic–phenotypic comparison with another Mexican patient who presented a more attenuated phenotype; and illustrated the importance of using bioinformatic tools to analyze variants when other molecular or functional studies are not available. Furthermore, our patient also presented cleft palate, a rarely reported feature.

CASE REPORT

Written informed consent was obtained from the parents and they consented to the submission of the case report to the journal, publishing their data and photographs. Patient was an 8-month-old boy born to Mexican parents. There is no known consanguinity in the family, including the parents. Prenatal ultrasound at 16 weeks of gestation showed a fetus with micromelic skeletal dysplasia, short rib syndrome, polydactyly, and probable atrioventricular canal. Gestational diabetes was diagnosed in the second trimester. A history of a previous pregnancy with the same partner ended in fetal death at 26 weeks of gestation. The fetus was reported with a similar phenotype. No invasive study was carried out by decision of the parents. Follow-up was carried out by structural ultrasound (Figure S1).

The patient was born at 37.6 weeks of gestation by cesarean section, with a birth weight of 2230 g (<5th centile), length of 43 cm (<5th centile), and occipital-frontal circumference of 33 cm (10th centile). He had cleft palate, presence of neonatal teeth (one upper and one lower incisor teeth), narrow and short thorax, upper extremities with rhizomelic shortening, right hand with postaxial polydactyly, syndactyly of the sixth and seventh fingers, left hand with postaxial polydactyly, and a seventh finger as a pedunculated appendage. Postnatal echocardiogram showed congenital heart disease of the common atrium type, atrioventricular septation defect, and persistent left vena cava. X-rays showed restrictive chest, bilateral shortening of the humerus, acetabular dysplasia with the presence of a trident-shaped acetabulum, and the presence of six metacarpal bones in both hands. Renal ultrasound showed no alterations.

At 8 months of age, he showed weight of 4400 g (<1st centile), length of 63 cm (<1st centile), occipital-frontal circumference of 41 cm (<1st centile), chest circumference of 33 cm (<3rd centile), internipple distance of 10 cm (25th–50th centile), and sternal length of 8.5 cm (25th–50th centile). In addition to the neonatal phenotype, ectodermal abnormalities were added: scant and very fine hair, sparse eyebrows, and nail hypoplasia on hands and feet (Figure 1).

FIGURE 1. Clinical features of patient at 8 months of age. (a) Right hand with postaxial polydactyly, and syndactyly of the sixth and seventh fingers (b) Left hand with postaxial polydactyly and a seventh finger as a pedunculated appendage (c) Front view of patient with scant and very fine hair, sparse eyebrows, and wide nasal bridge. (d) Right foot with nail hypoplasia.

Multigene panel based on next generation sequencing of the genomic DNA derived from the peripheral blood of the patient and his parents identified two trans variants in EVC2 gene (NM_147127.4): c.1195C>T p.[(Arg399*)] inherited from the father and c.2161delC p.[(Leu721Trpfs*19)] inherited from the mother. When the identified variant c.1195C>T p.[(Arg399*)] was evaluated using the American College of Medical Genetics and Genomics recommendations, PVS1, PS4, PP4_Strong, PM3, and PM2_Supporting were applied, indicating it was “pathogenic.” On the other hand, PVS1, PP4_Strong, PM3, and PM2_Supporting were applied in the c.2161delC p.[(Leu721Trpfs*19)] variant, indicating it was “pathogenic” also (Abou Tayoun et al., 2018; Biesecker et al., 2024; Richards et al., 2015; Sequence Variant Interpretation Working Group, 2019, 2020). At 9 months of age, the patient died due to complications secondary to COVID-19 pneumonia.

DISCUSSION

Here, we report a Mexican patient with EvCS in which Next-generation sequencing identified two inherited trans variants in EVC2. Da Silva et al. (2023) published a systematic review to establish a genotype–phenotype correlation in EvCS, finding that 88.7% of the variants in EVC and EVC2 are truncated (nonsense, frameshift, splicing, or copy number variants [CNVs]), and compound heterozygotes represent only 30.5%. They found an increase in the frequency of thoracic anomalies when the EVC2 gene was the cause; or when the variants were nonsense and CNVs, and less common in missense and splicing variants. Cardiac alterations were the phenotypic group that had the least variation between the different types of variants.

The phenotypic consequences of variants depend on the combined functional effects in the biallelic genotypic context, so the opportunity to study functional hemizygosity in autosomal recessive diseases caused by quantitative variants helps to determine the impact of a novel or rare variant when a loss of function variant is found in the other allele. In these cases, if the observed phenotype is “severe,” the variant in question would surely be loss of function, while if we observe an attenuated phenotype, we would be dealing with a variant with residual or hypomorphic function (Zschocke et al., 2023). One of the patients published by Negrete-Torres et al. (2023) allowed us to make a comparison of this type with our patient because they shared the frameshift variant in EVC2: c.2161delC p.[(Leu721Trpfs*19)] on one allele and differed in the variant of the second allele. In the patient described by Negrete, a splicing variant is observed in the other allele c.519_519+1delinsT, while our patient had a nonsense variant c.1195C>T p.[(Arg399*)].

The patient reported by Negrete-Torres et al. (2023) shows an attenuated phenotype and a delayed diagnosis compared to our patient (Table 1). Although the variants are found in the same EVC2 gene, we could observe the absence of thoracic anomalies, congenital heart disease, and prenatal data in the previously reported patient. Zaka et al. (2021) described a case of three brothers affected by a homozygous in-frame deletion variant in the EVC gene that also caused attenuated phenotypes due to its hypomorphic nature.

TABLE 1 Clinical and molecular features of Ellis-van Creveld syndrome patients in Mexican families.

	Negrete-Torres et al. (2023)	This report
Sex	Female	Male
Age at diagnosis	14 years old	8 months old
Sibling with congenital heart disease history	+	+
Consanguinity	−	−
EVC2 variant	+	+
Variant combination	Splicing, frameshift	Nonsense, frameshift
DNA sequence variants*	c.[519_519+1delinsT];[2161delC]	c.[1195C>T];[2161delC]
Protein variants	p.[?];[(Leu721Trpfs*19)]	p.[(Arg399*)];[(Leu721Trpfs*19)]
Diagnostic test	Exome sequencing	NGS panel
Prenatal features	−	+
Feature
Short stature	+	+
Upper-lip defect	−	−
Neonatal teeth	NA	+
Hypodontia/delayed eruption	+	−
Congenital heart defects	−	+
Narrow chest	−	+
Short, poorly developed ribs	−	+
Limb shortening	NA	+
Postaxial polydactyly (hands)	+	+
Postaxial polydactyly (feet)	−	−
Nail dysplasia/hypoplasia	+	+

Note: NA—not available or not assessed. (*):NM_147127.4.

In addition, Piceci-Sparascio et al. (2020) reported a familial case of an attenuated EvCS phenotype in which father and daughter shared a nonsense variant in the EVC gene and differed in the second allele: the father had a missense variant in the other allele while the daughter had a splicing variant. The functional studies carried out confirmed the nature of loss of function of the nonsense variant and demonstrated that the missense and splicing variants were hypomorphic, even finding the presence of these two hypomorphic variants in an unaffected member of the family. These reports support the conclusion of Da Silva et al. (2023) which indicate that this condition is gene dose dependent because the presence of one completely functional allele is sufficient for the disease to manifest significantly milder.

This comparison of functional hemizygosity in these two Mexican patients allowed us to assume that both variants in our patient were null variants due to the more severe phenotype observed. Moreover, the c.519_519+1delinsT variant could be a hypomorphic variant that would attenuate the phenotype observed in the previously reported patient. This novel variant was originally reported as likely pathogenic by Negrete-Torres et al. (2023) in accordance with PVS1 and PM2 criteria. The bioinformatic analysis they did of this variant allowed them to conclude that this variant alters the donor site of exon 4 and creates a premature stop codon, hypothetically generating a truncated protein of 173 amino acids. Rather than producing such truncated protein, this variant most likely undergoes nonsense-mediated mRNA decay (NMD). Both scenarios result in a null variant.

Our comparison of functional hemizygosity suggests a hypomorphic behavior, so when we reanalyzed this variant with SpliceAI, we observed that it is predicted to disrupt the donor site of exon 4 (score: 1), although it is also predicted to disrupt the acceptor site of exon 4 (score: 0.89). Exon skipping is inferred if both splice sites flanking an exon have a loss score above threshold. Given this scenario, our hypothesis is that in splicing, this variant causes a skip of exon 4, which has 69 nucleotides, so it does not alter the reading frame (Dawes et al., 2023; Jaganathan et al., 2019).

Using Mutalyzer tool, we observed at mRNA level NM_147127.4:r.(451_519del) and at protein NM_147127.4(NP_667338.3): p.(Tyr151_Leu173del), which would imply a loss of 23 amino acids from the wild-type protein without altering the reading frame (Figure S2) (Jumper et al., 2021; Lefter et al., 2021; Varadi et al., 2022). This deleted region does not correspond to any coiled-coil region or any intrinsically disordered domain and is topologically located in the extracellular domain (The UniProt Consortium, 2023; Zahn-Zabal et al., 2020).

EVC2 has alternative splicing, and four isoforms are annotated in Ensembl (Martin et al., 2023). We analyzed this region in SpliceVault which uses data from the Genotype-Tissue Expression dataset (GTEx) and the Sequence Read Archive (SRA), and we observed some transcripts that jump exon 4 (Dawes et al., 2023). This new bioinformatics and RNAseq data evidence allowed us to suggest that the c.519_519+1delinsT variant could be reclassified as likely pathogenic with the following ACMG criteria: PVS1_Moderate, PM3, PP4_Moderate, and PM2_Supporting (Abou Tayoun et al., 2018; Biesecker et al., 2024; Richards et al., 2015; Sequence Variant Interpretation Working Group, 2019, 2020; Walker et al., 2023). An RNAseq study would be necessary to confirm this hypothesis but at least this perspective offers a bioinformatic explanation for the likely hypomorphic nature of this variant. We did not rule out that variable expressivity may also be due to other factors such as modifying genes, epigenetic factors, and environmental influences.

Moreover, only 8 of the 190 patients reported by Da Silva showed alterations in the palate, mainly a high-arched palate or a narrow palate, but none of them had cleft palate like our patient. Likewise, alterations in the palate, since they are not part of the initially described clinical phenotype, may be biased at the time of reporting, and therefore, be found in a lower percentage (Da Silva et al., 2023).

The main role of the Hh signaling pathway is during embryonic development: Hh signaling molecules are differentially expressed in various tissues, resulting in tissue-specific regulatory functions (Dilower et al., 2023). It has been shown that defects in Hh pathway signaling in the craniofacial region during embryonic development can lead to alterations not only in the lips but also in the palate (Louie et al., 2020). Therefore, we propose paying attention to even minimal alterations of the palate, such as a submucosal cleft, to integrate them into the EvCS phenotype.

In conclusion, our case shows the importance of comparing functional hemizygosity between patient phenotypes when they share a variant, and how the use of bioinformatics tools allows us to create predictions about the behavior of these variants. However, we recognize the limitations of these predictions given the need to perform molecular and functional studies to corroborate them. Our case also supports the association of alterations in the palate as part of the EvCS phenotype.

AUTHOR CONTRIBUTIONS

León-Madero: Conceptualization, Methodology, Software, Formal analysis & Writing - Original Draft, Review & Editing. Fregoso-Ron: Validation, Resources & Visualization. De León-Carbajal: Data Curation & Supervision. Valdés-Miranda: Supervision & Project administration.

ACKNOWLEDGMENTS

To the patient’s family for contributing to expanding knowledge. To the Genetics department of the Hospital General de México for allowing us to provide high-quality clinical care. To the Universidad Nacional Autónoma de México for supporting the dissemination of science.

FUNDING INFORMATION

Universidad Nacional Autónoma de México.

CONFLICT OF INTEREST STATEMENT

None declared.

DATA AVAILABILITY STATEMENT

Luis Felipe León Madero and Juan Manuel Valdés Miranda had full access to all the clinical data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

ETHICS STATEMENT

Written informed consent was obtained from the parents and they consented to the submission of the case report to the journal, publishing their data and photographs.