- Research article
- Open Access
- Open Peer Review
Molecular and clinical analysis of Ellis-van Creveld syndrome in the United Arab Emirates
© Ali et al; licensee BioMed Central Ltd. 2010
- Received: 8 April 2009
- Accepted: 25 February 2010
- Published: 25 February 2010
Ellis-van Creveld (EvC) syndrome is an autosomal recessive chondrodysplastic condition with clinical manifestations that include short-limbs and ribs, postaxial polydactyly and dysplastic nails and teeth. In about two thirds of patients, mutations in either EVC or EVC2 genes have been found to be the underlying cause.
In this paper, we describe the molecular (DNA sequencing) and clinical analysis of six children diagnosed with EvC from four different families from the United Arab Emirates (UAE).
All the children had the common clinical and radiological features of this syndrome. However, DNA sequence analysis of the genes shown to be involved (EVC and EVC2) revealed a novel splice site mutation (c.2047-1G>T) in intron 13 of EVC2 gene in one family. In addition, we confirm previous mutational analyses that showed a truncating mutation in exon 13 of EVC gene (c.1813C>T; p.Q605X) in the second family and a single nucleotide deletion (c.981delG; p.K327fs) in exon 8 of EVC2 gene in the third family. No mutations in the exons, splice sites or the promoter regions of either gene have been found in the index case of the fourth family who exhibited "EvC-like" features.
Given the small population size of UAE, our data illustrates further the molecular heterogeneity observed in EvC patients and excludes the possibility of a common founder effect for this condition in the UAE reflecting the current ethnic diversity of the country.
- Atrial Septal Defect
- United Arab Emirate
- Splice Site Mutation
- Oral Manifestation
Ellis-van Creveld syndrome (EvC, MIM 225500) is a chrondro-ectodermal dysplasia characterized by Richard Ellis and Simon van Creveld . This condition is inherited in an autosomal recessive manner and typically involves malformations of certain cartilages especially those derived from "neural crest" cells, polydactyly and congenital heart defects in about 50 to 60% of the affected individuals . EvC is a relatively rare disorder with only around 300 cases reported worldwide and is most prevalent in the Amish population of USA and some Arab populations [3–5]. Consanguinity has been reported in about 30% of the cases . The birth prevalence in non-Amish population is estimated to be 0.7/100,000 of live birth . However, in the UAE, Al-Gazali et al.  calculated the birth rate of this syndrome to be 5.2/100,000 of live births, representing a useful baseline for this group of birth defects in the local society.
The EvC underlying gene was mapped to chromosome 4p16  and mutations have been found in the EVC gene by Ruiz-Perez et al. . Mutations in a second gene EVC2, which is arranged in a head to head orientation with the EVC gene on chromosome 4, have also been identified as causative of the disease in a number of patients . More recently, more mutations have been reported in both EVC and EVC2 in two thirds of EvC patients suggesting molecular heterogeneity of this condition and the possibility of the involvement of other gene(s) . While EVC gene has 21 coding exons that produce a 992 amino acid protein, EVC2 gene has 22 coding exons and encodes a 1,308 amino acid protein without any homology to each other or to any other protein family . In most cases, affected individuals with mutations in EVC and EVC2 have the typical spectrum of features and are phenotypically indistinguishable . However, recent findings by Ulucan et al.  indicated that there is a considerable phenotypic variation in EvC highlighted by the analysis of a large family that exhibited complex septal cardiac defects, rhizomelic limb shortening and polydactyly without the typical lip, dental or nail abnormalities. The underlying genotype for this milder phenotype was reported to be a missense mutation (p.L623P) in EVC gene . In this study we report the molecular analysis and clinical phenotyping of six individuals from the UAE with EvC. Our main findings are the identification of a novel splice site mutation in EVC2 gene and the illustration of the molecular heterogeneity of this condition among UAE patients.
Genomic DNA Isolation
Genomic DNA was extracted from whole blood using the Flexigene DNA Kit (Qiagen, GmbH) according to the manufacturer's instructions or other standard technique.
Polymerase Chain Reaction (PCR)
The primers shown in additional file 1 were designed using Primer3 http://frodo.wi.mit.edu/primer3/ and used to screen genomic DNA for mutations in the promoter regions and all coding exons and at least 100 bp of the flanking intron regions including the splice sites of EVC and EVC2 genes http://genome.ucsc.edu/. The promoter regions of EVC and EVC2 genes were predicted using the free online Proscan Version 1.7 software http://www-bimas.cit.nih.gov/molbio/proscan/. PCR reactions consisted of 1.5 μM MgCl2, 5 μM each of forward and reverse primers, 100 ng DNA template, 0.2 mM of each dNTP, 0.5 Units Taq DNA Polymerase (Qiagen, GmbH) and 1× PCR buffer (Qiagen, GmbH), in a total volume of 20 μl. Reactions were started by 10 min DNA denaturation at 95°C, followed by 40 cycles of the following PCR programme: 30 sec at 95°C, 45 sec at 58°C, and 45 sec at 72°C; the PCR was completed by a single cycle of 7 min at 72°C.
After PCR, products were cleaned by treatment with ExoSAP-IT according to the manufacturer's protocol (USB, USA). Both strands of each amplicon were sequenced using the corresponding forward and reverse PCR primers (additional file 1). 10 μl of cycle sequencing reactions consisted of: 4 μl BigDye Mix (ABI, Foster City, CA); 1 μl primer (3.2 μM); and 1-3 μl PCR product were cycled 25 times10 sec at 96°C, 5 sec at 50°C, and 4 min at 60°C. Purified reactions were then analyzed on an ABI 3130xl Genetic Analyzer (ABI, Foster City, CA) based at the Faculty of Medicine and Health Sciences, UAE University.
Clinical features of cases from 4 families with EvC syndrome.
Common atrium with dilated coronary sinus
AV canal defect
Short/thin upper lip
Irregular alveolar ridge
Short broad nose
Hands and feet, bilateral
Short long bones
Short ribs with narrow chest
Small iliac bones with downward spike
The parents are first cousin once removed (Figure 1) originally from Sudan and they had 5 children, 2 of them were affected. There was a history of one miscarriage. The first affected had the typical features of EvC and died on the second day of life. No DNA was available from this child. The second affected was diagnosed prenatally by ultrasound. She died half an hour after birth. No birth measurements were available. There was distal shortening of the limbs, postaxial polydactyly of both hands with hypoplastic nails, narrow chest, and distended abdomen with enlarged liver. She had the facial and oral features of EvC syndrome (Table 1). Skeletal survey showed short long bones with short ribs with narrow and long thorax. The iliac bones appeared small with a downward spike (Table 1).
This patient was the third child of unrelated parents of Egyptian origin (Figure 1). The first 2 children were healthy. There was a history of postaxial polydactyly in the paternal grandfather. The pregnancy and delivery were normal. The patient birth weight was 3600 gm (>25%), length 45 cm (<5%), and head circumference 34 cm (50%). At birth she was noted to have a narrow chest with shortening of the distal segments of all limbs with bilateral postaxial polydactyly of the hands and hypoplastic fingernails and toenails. Echocardiography showed complete AV canal defect. This was corrected at 6 months of age. Ultrasound of the brain and kidneys were normal. Skeletal survey confirmed the diagnosis of EVC syndrome (Table 1).
This is a highly inbred UAE family originally from Afghanistan (Figure 1). There were 2 affected children in 2 branches (the proband and her uncle from the father's side). The proband was a female and the product of normal pregnancy and delivery. Her birth weight was 2500 gm (>5%) and length was 42 cm (<5%). She was noted to have dysmorphic features with short limbs with bilateral postaxial polydactyly of the upper and lower limbs, very narrow chest with hypoplastic lungs (Table 1). The nails were noted to be normal. Echocardiography revealed atrial septal defect (ASD). Skeletal survey showed short ribs with long and narrow thorax, short long bones and typical changes in the pelvis. She had repeated chest infections requiring intensive care unit (ICU) care on many occasions. At 2 years of age she was oxygen dependant and had normal intelligence. She died at the age of 3 years due to respiratory complications. The uncle of the proband who was the product of first cousin marriage was also affected. He was the product of normal pregnancy and delivery. At birth he was noted to have short limbs with bilateral post axial polydactyly of upper and lower limbs, hypoplastic nails, narrow chest. There were short frenula and natal teeth. There was a murmur at the left sternal edge suggestive of ASD. The baby died in the first few weeks of life due to respiratory complications. Due to lack of an obvious pathogenic mutation in either EVC or EVC2 genes we consider the diagnosis of EvC in this family tentative and therefore we refer to it as "EvC-Like" syndrome. Unfortunately, X-rays are not available for the affected members of this family.
SNPs identified in EVC and EVC2 genes in the index case of family 1 with the novel mutation
Minor Allele Frequencya
0.351 +/- 0.229
0.480 +/- 0.098
0.500 +/- 0.014
0.461 +/- 0.135
0.441 +/- 0.162
0.499 +/- 0.020
0.496 +/- 0.045
0.482 +/- 0.093
Summary of mutations found in EvC patients from UAE.
Tompson et al., 2007
Tompson et al., 2007
Molecular study on the 4 families identified a novel splice site mutation (c.2047-1G>T) in intron 13 of EVC2 gene in one of these families and confirmed two mutations in two other families; a nonsense mutation (c.1813C>T; p.Q605X) in exon 13 of EVC gene and a single nucleotide deletion (c.981delG; p.K327fs) in exon 8 of EVC2 gene . RNA from the effected members of the family with the novel splice site mutation was not available for to demonstrate its altered RNA splicing effects. The lack of mutation in EVC and EVC2 genes in the 4th family could indicate incorrect diagnosis or molecular heterogeneity of the condition. However, both affected children in this family had postaxial polydactyly, oral manifestations of EvC syndrome, limb shortening, congenital heart defect (ASD) typically seen in EvC syndrome and one of them had dysplastic nails making the diagnosis of EvC syndrome most likely and hence we refer to it to "EvC-like" syndrome. However, it is possible that lack of mutation in EVC and EVC2 genes in this family is due to genetic heterogeneity of this disorder as was suggested by Tompson et al.  who sequenced EVC and EVC2 genes in 65 individuals affected by EvC. They reported mutations in either EVC or EVC2 in only two thirds of their patients with the remaining of unknown molecular cause. From these studies, we anticipate the existence of another EvC causative gene(s). Further studies are needed to clarify the underlying cause of EvC in this group of patients.
We conclude that EvC in the UAE population is heterogeneous at the molecular level which reflects the ethnic diversity of the current population in the country.
We thank the Dubai Harvard Foundation for Medical Research for the financial support of this project. We also thank the affected families for their cooperation on this project.
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