Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes

Abstract

Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries—a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5′ untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5′ UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females.

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

“…Seven of these patients have been described in prior clinical studies, and the mutations of 13 patients have been published before. 6,32,33 Design and procedure Complete series of standardized photographs of all patients were collected, combined with a review of the patient's medical file and physical examinations. If available, radiological images were also consulted.…”

“…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.…”

Expanding the mutation spectrum in 182 Spanish probands with craniosynostosis: identification and characterization of novel TCF12 variants

“…5 The syndrome associated with mutations in EFNB1 is distinctive as females and male mosaics are more affected than males. 6,7 Mutations in TWIST1 (MIM 601622) result in haploinsufficiency of the transcription factor TWIST1, and are associated with Saethre-Chotzen syndrome (SCS). In the early development of the coronal suture, TWIST1 is expressed in the sutural mesenchyme between the proliferating osteoblasts of the frontal and parietal bone edges, and overlapping with these two populations, consistent with roles in separating the two bone-forming tissues and with initiating and maintaining transcription of FGFR2.…”

A Family with Craniofrontonasal Syndrome and a Mutation (p.G151S) in the EFNB1 Gene: Expanding the Phenotype

“…Cleft lip/palate was absent in our family and in the 3 previous sporadic cases [Orr et al, 1997;Twigg et al, 2004]; nevertheless, it was present in 2 families with CFNS [Twigg et al, 2004;Özylmaz et al, 2015], denoting that the p.G151S mutation has clinical heterogeneity. Familial mutations in the EFNB1 gene have been observed in about 25%, cleft lip/palate is present in 30% and cutaneous syndactyly of the hands and/or feet occurs in about 25% of the cases [Twigg et al, 2004[Twigg et al, , 2013. Dental anomalies, such as crowding of the teeth, are observed in 23% of CFNS cases.…”

“…The X-linked dominant inheritance of CFNS is clinically unexpected because women are more severely affected than men; apparently, this stems from the random X inactivation in heterozygous females. Males with severe phenotypes similar to those of heterozygous women may have postzygous mutations of the EFNB1 gene brought about through the mechanism of metabolic or cell interference [Wieland et al, 2005;Twigg et al, 2013]. The inter-and intrafamilial clinical variability could correlate with the patchy or mosaic distribution of EFNB1 expression observed in the limbs of heterozygous mice with random X chromosome inactivation.…”