1. ¹Institut für Humangenetik, Otto-von-Guericke-Universität, Magdeburg, Germany
2. ²National Centre for Medical Genetics, Our Lady's Hospital for Sick Children, Dublin, Ireland
3. ³Institut für Klinische Genetik, Medizinische Fakultät Carl Gustav Carus, Technische Universität (TU), Dresden, Germany
4. ⁴Institut für Medizinische Genetik, Charité – Universitätsmedizin, Berlin, Germany
5. ⁵Centre Hospitalier Universitaire de Rouen, Unité de Génétique Clinique, Hôpital Charles Nicolle, Rouen, France
6. ⁶Service de Pédiatrie, Centre Hospitalier Intercommunal de la Haute-Saône, Vesoul, France
7. ⁷Institut für Humangenetik, Westfälische Wilhelms-Universität, Münster, Germany

Correspondence: Dr I Wieland, Institut für Humangenetik, Otto-von-Guericke-Universität, Leipziger Strasse 44, Magdeburg D-39120, Germany. Tel: +49 391 67 15381; Fax: +49 391 67 15066; E-mail: ilse.wieland@medizin.uni-magdeburg.de

Received 28 August 2007; Revised 30 October 2007; Accepted 31 October 2007; Published online 28 November 2007.

Abstract

Craniofrontonasal syndrome (CFNS) is an X-linked malformation syndrome with variable phenotype that is caused by mutations in the ephrin-B1 gene (EFNB1). Over 50% of EFNB1 mutations result in premature termination codons that may elicit mRNA degradation by the nonsense-mediated decay pathway. To assess the effects of various mutations at the transcript level, expression of EFNB1 mRNA was studied by RT-PCR in fibroblast cultures established from CFNS female patients. Compared to the wild-type and two missense mutation alleles, severe depletion of transcripts was observed for mutant alleles harbouring either splice site mutation c.407-2A>T at the exon 2/3 boundary or frameshift mutation c.377_384delTCAAGAAG in exon 2. In contrast, escape from mRNA decay was observed for mutation c.614_615delCT, which generates a premature termination codon close to the 3'-end of the penultimate exon 4 disobeying the '50–55 bp' rule. These results suggest differential degradation of mutant EFNB1 transcripts by the nonsense-mediated mRNA decay pathway. Although the clinical phenotypes of the patients were not highly suggestive of a phenotype–genotype correlation, the two female patients were diagnosed with diaphragmatic hernia harbouring putative ephrin-B1 truncating mutations. Previously, disease manifestation in heterozygous females had been attributed mainly to cellular interference of divergent cell populations expressing wild-type or mutant EFNB1, depending on the pattern of X-inactivation. Upon clonal expansion of patient cells with either the wild-type or mutant EFNB1 on the active X-chromosome, we were able to separate mutant and wild-type EFNB1-expressing cells in vitro, further supporting the concept of cellular interference in CFNS.