Introduction

Weyers acrofacial dysostosis (MIM 193530) or Curry-Hall syndrome, is an autosomal dominant disorder clinically characterized by mild short stature, hypotelorism, cleft of mandibular symphysis (infancy), postaxial polydactyly type A or type B, onychodystrophy. The oral manifestation consists of irregular, small, peg-shaped teeth, a single central incisor or hypodontia (Weyers 1952; Curry and Hall 1979; Roubicek and Spranger 1984; Shapiro et al. 1984). Ellis–van Creveld syndrome (chondroectodermal dysplasia; EvC; MIM 225500) is recognized as an autosomal recessive skeletal dysplasia with associated multisystemic involvement. It is highly prevalent in the Old Order Amish community of Lancaster County, Pennsylvania. Prominent features of this syndrome are short-limbed, disproportionate dwarfism, short ribs, thoracic dysplasia, postaxial polydactyly and nail dystrophy (Ellis and van Creveld 1940; McKusick et al. 1964; Biggerstaff and Mazaheri 1968; Taylor 1984). Other features include oral (multiple oral frenula, neonatal teeth, delayed teeth eruption, hypodontia) and genitourinary anomalies (epispadias or hypospadias). Distinct cardiac defects, frequently atrioventricular septation or a single atrium, occur in approximately 60% of the affected individuals and are the major cause of shortened life expectancy (Digilio et al. 1999; Katsouras et al. 2003). Despite strong similarities mentioned above, the distinguishing characteristics between these two syndromes are their mode of inheritance and degree of phenotypic severity (Gorlin et al. 1990; Howard et al. 1997).

The EvC gene was localized on chromosome 4p16.1 among geographically diverse families (Francomano et al. 1995; Polymeropoulos et al. 1996; Ide et al. 1996). In a large family with features of Weyers acrofacial dysostosis, the disease locus was mapped to a region which overlapped the EvC locus (Howard et al. 1997). Mutations in the EVC (MIM 604831) were found to be responsible for both Weyers acrofacial dysostosis and EvC syndrome (Ruiz-Perez et al. 2000; McKusick 2000). However, in a considerable number of EvC cases, it was not possible to detect the underlying mutations for even one allele (Ruiz-Perez et al. 2003; Takamine et al. 2004). Recently, homozygous mutations were found in seven EvC kindreds in a novel gene, EVC2 (MIM 607261), which is nonhomologous and is adjacent, but oriented in the opposite direction to the EVC (Galdzicka et al. 2002; Ruiz-Perez et al. 2003).

In this study, we present a novel heterozygous deletion in the EVC2 that is associated with autosomal dominant Weyers acrofacial dysostosis in a Chinese family. This is the first report of Weyers acrofacial dysostosis caused by this gene. The L1265fsX1266 (c.3793delC), which leads to a premature stop codon, provides conclusive evidence that Weyers acrofacial dysostosis and EvC are allelic conditions.

Materials and methods

Enrollment of subjects

The proband was recruited during a routine dental examination and family members were interviewed and examined. Patients were examined by at least two experienced dentists. Cardinal signs of Weyers acrofacial dysostosis were present in the familial cases, and other diagnoses such as EvC, asphyxiating thoracic dystrophy (ATD), the short-rib polydactyly syndromes (SRPS) and McKusick–Kaufman syndrome (MKKS) were excluded (Krakow et al. 2000; Elcioglu et al. 2002; Stone et al. 2000). All procedures were performed in accordance with an Institutional Review Board protocol on human studies approved by the School of Stomatology, Wuhan University. Informed consent was obtained from all participants or their guardians. Family histories, radiographs were taken and venous blood samples were drawn from four affected (III:1, IV:1, IV:3, V:3) and three unaffected relatives (III:2, IV:4, IV:5). In addition, 150 unrelated local healthy individuals of Han nationality were selected as controls.

Polymerase chain reaction and DNA sequencing

Genomic DNA was isolated by using the standard SDS-proteinase K salt chloroform techniques. Based on a candidate gene approach, all exons and the flanking intron–exon boundaries of both EVC and EVC2 were amplified in the proband by polymerase chain reactions (primer sequences and annealing temperature conditions are available on request). The genomic sequences containing the EVC2 were identified and analyzed by performing a Blast search with the EVC2 mRNA sequence (accession no. AY185210) at NCBI Blast server (http://www.ncbi.nlm.nih.gov). PCR products were purified using a Sequencer (Applied Biosystem, Foster City, CA, USA). To further confirm the mutant allele with EVC2 deletion, the amplified PCR products were subsequently cloned directly into the pGEM-T easy vector (Promega, Madison, WI, USA), and the sequence was confirmed by automated DNA sequencing.

Restriction enzyme analysis

To confirm that the deletion was a causal mutation but not a rare polymorphism, we used restriction enzyme digestion as an assay and performed segregation analysis. Exon 22 of the EVC2 was amplified in 25 μl reaction volume with primers E22F: 5′-AAATGGCACTGGGTTGGGG-3′ and E22R: 5′-GCAGAGGATGGGGTGTGG-3′. Ten microliters of purified PCR products were digested with 5 U of MboI restriction endonuclease (TaKaRa, Dalian, China) at 37°C for 2 h in 20 μl volume, according to the recommendations of the manufacturer. Digestion products were analyzed by 2% agarose gel electrophoresis in 0.5×TBE buffer at 5 V/cm for 2 h. DNA samples from 150 unrelated normal controls were also included in the restriction analysis with MboI enzyme.

Results

Clinical and radiographic investigation

The family was a large nonconsanguineous kindred of Han nationality. Autosomal dominant inheritance mode was suggested by the presence of affected individuals in each of the five generations, and male-to-male transmission. The proband, an 8-year-old boy, together with his two relatives (IV:6, V:4) were the most severely affected individuals. The boy, with a height of 110 cm, was referred to the pediatric dental clinic for a comprehensive treatment of delayed teeth eruption and hypodontia. General examinations revealed scars on his four extremities (postaxial polydactyly type A were removed after birth), partial syndactyly of the second and third toes and apparent dysplastic nails. His oral manifestations demonstrated multiple oral frenula, small, conical incisors and absence of bilateral incisors with wide spaces. (Fig. 1a–g). The proband’s mother (IV:3) was 155 cm tall, with postaxial polydactyly type B in her hands but type A in her feet, syndactyly of the toes and nail dystrophy. She had a single central incisor and several molars agenesis from her young age. The remaining permanent teeth were of abnormal size and shape. However, due to several extractions to accommodate prostheses, we could not ascertain the exact number or types of missing teeth. The grandmother (III:1) was 62 years old and 160 cm tall. Symptoms in the proband’s grandmother and uncle (IV:1) were milder than other affected individuals. They share a number of similar manifestations, including postaxial polydactyly on the feet, syndactyly in the toes, irregular size or shape of teeth and dysplastic nails, but clinical and X-ray examinations showed no postaxial polydactyly on their hands. Although the affected uncle (36 years old, 170 cm tall) was shorter than his unaffected brother (IV:5, 32 years old, 180 cm tall), we could not make a definitive conclusion of mild short stature in this family, due to the average height of adults in the Chinese population (The height distribution of Chinese males between 18 and 44 years old is 169.8±6.5 cm) (Yang et al. 2005). All patients underwent thorough cardiovascular and radiographic examinations. The electrocardiogram (ECG), chest X-ray and transthoracic echocardiogram results showed no features of atrioventricular canal defects or thoracic anomalies (data not shown). Intelligence was normal in all patients. No dyspnea upon exertion and fatigue were identified.

Fig. 1
figure 1

Clinical photographs and radiographs of the proband (a–g). a, c Bilateral postaxial polydactyly type A were removed at birth by surgical operations on both hands and feet, only scars (black arrows) were seen after amputation. b, d Apparent dysplastic fingernails and toenails. d, e Partial syndactyly at the second and third toes on both feet (black arrows). X-ray show that the phalanges were not affected. f Oral examination of the upper lip reveals multiple oral frenula, diastemas, teeth which were abnormal in size and shape, delayed teeth eruption. g Panoramic radiographs showed the absence of permanent incisors (white arrows)

Full size image

Mutation analysis of EVC and EVC2

We selected EVC as the first candidate gene in this family. However, no pathogenic mutation was detected. Since the EVC2 has previously been shown to be associated with the EvC syndrome, we then sequenced all the coding exons and intron–exon boundaries of this entire gene from both directions and the affected allele was also cloned for sequencing (Fig. 2a–e). A novel heterozygous deletion, L1265fsX1266 (c.3793delC) in exon 22 of EVC2 was identified in the affected but not in unaffected family members. This mutation is predicted to result in an immediate premature stop codon. By restriction enzyme analysis, it was confirmed that this sequence change was not present in at least 300 normal chromosomes (data not shown).

Fig. 2
figure 2

a Pedigree structure of the five-generation family. b The L1265fsX1266 (c.3793delC) mutation was confirmed by MboI enzyme digestion. The heterozygous deletion abolishes an MboI site (GATC→GATT), resulting in four bands (357, 207, 141 and 115 bp) in the heterozygous patients. MboI digested products derived from all seven available family members are indicated by the corresponding numbers. Lane 1 DNA Marker (M), lanes 2–5 the affected individuals; lanes 6–8 the unaffected family members, lane 9 control undigested amplicon of EVC2 exon 22 (C). c, d DNA sequence analysis of exon 22 in the EVC2 showed a heterozygous cytosine deletion at position 3793 in the proband (right panel). The deletion (pointed by the red arrow) resulted in an immediate premature stop codon. His unaffected father was normal (left panel). e Sequencing result of the cloned mutant allele from the proband

Full size image

Discussion

Because of phenotypic and radiographic similarities, there has been longstanding speculation that Weyers acrofacial dysostosis and EvC syndrome are allelic. The affected individuals described here have an autosomal dominant condition as well as features consistent with Weyers acrofacial dysostosis, and a heterozygous deletion L1265fsX1266 (c.3793delC) was identified in EVC2. Thus, mutations in both EVC and EVC2 can cause a wide variety of manifestations, ranging from mild Weyers acrofacial dysostosis to severe EvC syndrome, and patients carrying either gene mutations are clinically indistinguishable. The remarkable instance of combined phenotypic and genetic heterogeneity may suggest direct interactions or indirect effects between EVC and EVC2. However, other than predicted transmembrane domains, no obvious homologies or motifs give clues regarding their functions (Galdzicka et al. 2002). Limited knowledge and lack of a common structure for these two genes makes it rather hard to elucidate the functional relationship between them.

The coupled EVC and EVC2 lie in a syntenic region with conserved gene order and are arranged with transcription start sites separated by only 1.6 kb (Galdzicka et al. 2002). In the human genome, gene pairs located in such proximity are common (Shimada et al. 1989; Platzer et al. 1997; Adachi and Lieber 2002). These tightly linked head-to-head gene pairs are often functionally related and regulated by a common promoter (PÖschl et al. 1988). For example, ABCG5 and ABCG8 are two homologous genes neighboring in a head-to-head orientation that, when mutated, cause autosomal recessive sitosterolemia (Berge et al. 2000; Lu et al. 2001). In contrast, HADHA and HADHB, which are nonhomologous genes, cause long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (Brackett et al. 1995). These two genes exist side by side in a head-to-head arrangement with a common bidirectional promoter region (Orii et al. 1999). Another example is EVER1 and EVER2, which are also nonhomologous genes in a head-to-head orientation that cause autosomal recessive epidermodysplasia verruciformis (Ramoz et al. 2002). The EVC and EVC2 likewise, do not have significant homologous sequences, and either gene defect can cause both dominant and recessive conditions with variable presentations. So far, unique functions of the bidirectional sequences between the EVC and EVC2 have not been identified definitively. One possibility is that it might permit the two genes to be coregulated by a common promoter (Ruiz-Perez et al. 2003). Though the two genes do not have an obvious homologous relationship, they may be coordinately modulated for certain cellular responses or function in a similar signal pathway. Furthermore, we speculated that in positional cloning strategies, whether those tightly linked head-to-head genes are homologous or not, both should be considered as candidates in that both of them might be involved in the same disease rather than only one.

Previous reports concerning the EVC and EVC2 mutations are summarized in Table 1. The majority of identical homozygous or compound heterozygous mutations in two genes in EvC syndrome often lead to truncated protein or absence of the protein, which correlates with a more severe phenotype. On the contrary, only one heterozygous missense mutation was found in the EVC associated with Weyers acrofacial dysostosis: a man with Weyers acrodental dysostosis inherited S307P to his EvC daughter (Ruiz-Perez et al. 2000). The mutation identified in the EVC2 in our patients, however, is unusual in location and type compared to the previous cases. The L1265fsX1266 (c.3793delC) is the most 3′ frameshift mutation found so far in both Weyers acrofacial dysostosis and EvC syndrome. Additionally, the obligate heterozygous carriers with mutations in either the EVC or EVC2 did not manifest features of EvC syndrome (Ruiz-Perez et al. 2000, 2003). EVC and EVC2 proteins produced by only one normal allele is sufficient for development and maintenance of limbs, skeleton and teeth, yet in homozygotes, null alleles may result in the EvC syndrome (Spranger and Tariverdian 1995). Heterozygosity for at least some mutations in the EVC and EVC2 cause symptoms as well (like the mutation described in this context), even in the presence of a normal second allele. The potential mechanism that a heterozygous mutation causes an abnormal phenotype remains obscure. The specific heterozygous mutations in Weyers acrofacial dysostosis may have a more severe effect than those found associated with EvC. The abnormal protein may interfere with the function of the normal allele (act through a dominant-negative effect) (Torres et al. 2004). It may also be possible that the effect of the abnormal protein may manifest only when a specific cis- or trans-modifying gene(s) or genetic background is present (Thompson et al. 2005).

Table 1 Identified EVC and EVC2 mutations in Ellis–van Creveld syndrome and Weyers acrofacial dysostosis
Full size table

In summary, this study presents the first description of the EVC2 underlying Weyers acrofacial dysostosis. Thus, the spectrum of malformation syndromes due to EVC2 abnormalities is further extended. Our data further highlight the importance of the EVC2 in the limb, skeleton and teeth development. Further studies are needed to elucidate the functions and pathways involving EVC and EVC2 pairs. These may clarify the pathogenesis of diseases due to EVC and EVC2 mutations.