Dissecting the molecular mechanisms in craniofrontonasal syndrome: differential mRNA expression of mutant EFNB1 and the cellular mosaic.
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.
Wieland
Ilse
I
Institut für Humangenetik, Otto-von-Guericke-Universität, Magdeburg, Germany. ilse.wieland@medizin.uni-magdeburg.de
Makarov
Roman
R
Reardon
William
W
Tinschert
Sigrid
S
Goldenberg
Alice
A
Thierry
Patrick
P
Wieacker
Peter
P
D000328:Adult; D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D002648:Child; D018389:Codon, Nonsense / Q000235:genetics; D019465:Craniofacial Abnormalities / Q000235:genetics* / Q000473:pathology; D036387:Ephrin-B1 / Q000096:biosynthesis / Q000235:genetics*; D005260:Female; D016368:Frameshift Mutation / Q000502:physiology; D006579:Heterozygote; D006801:Humans; D007223:Infant; D008297:Male; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000502:physiology; D022821:RNA Splice Sites / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D011897:Random Allocation; D013577:Syndrome
D018389:Codon, Nonsense; C484288:EFNB1 protein, human; D036387:Ephrin-B1; D022821:RNA Splice Sites; D012333:RNA, Messenger