Dissecting the molecular mechanisms in craniofrontonasal syndrome: differential mRNA expression of mutant EFNB1 and the cellular mosaic

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.407-2A4T 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 0 -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.

Phenotypes of craniofrontonasal syndrome in patients with a pathogenic mutation in EFNB1

“…In the other two families, the clinical features differed highly, suggesting that intrafamilial variability can be significant. A genotype-phenotype correlation seems to be unlikely, based on this intrafamilial variability and a previous study, 26 and was not found in this study.…”

“…24,28 A genotype-phenotype correlation has not been proven, and previous studies suggest that this is unlikely. 26 Taken together, around 20% of the patients screened for CFNS did not display a mutation in the EFNB1 gene. 21,23,25 Multiple explanations have been proposed, one of which is misdiagnosis of some of the included patients.…”

“…[16][17][18] The disease locus was finally claimed to be within Xq13.1 and loss of function mutations in EFNB1 were proven to cause CFNS. 10,[18][19][20][21][22][23][24][25][26][27][28][29][30] EFNB1 encodes ephrin-B1, which is a transmembrane ligand for Eph receptor tyrosine kinases. Because of random X-inactivation heterozygous females are uniquely mosaic and by consequence a cell either does or does not produce a functional protein.…”

“…10 An explanation for the few severely affected males reported in literature [6][7][8]32 could be a mosaicism in these patients, in which the wild type to mutant ratio should be similar to that in heterozygous CFNS females. 23,26,33 Additional mechanisms were recently added to the phenotypic manifestation. Not only cellular interference, but also an impaired signaling capacity of ephrin-B1 and improper regulation of gap junctional communication could be responsible for the pathogenic process in CFNS expression.…”

Regulation and misregulation of Eph/ephrin expression

“…Nonsense and frameshift mutations in the efnb1 gene were reported to undergo NMD. 81 In fact these mutations are considered to be contributors to the pathogenic mechanisms reported in the human X-linked malformation syndrome, craniofrontonasal syndrome (CFNS). Therefore, accumulating evidence implicates mRNA stability as a possible regulatory or deregulatory process in Eph/ephrin expression in cancer and other pathologies.…”