Molecular and functional analysis of a novel MEK2 mutation in cardio-facio-cutaneous syndrome: transmission through four generations

Abstract

Cardio-facio-cutaneous (CFC) syndrome is one of the RASopathies and is caused by alteration of activity through the Ras/mitogen-activated protein kinase (MAPK) pathway due to heterozygous de novo mutations in protein kinases BRAF, MEK1, or MEK2. CFC is a rare multiple congenital anomaly disorder in which individuals have characteristic dysmorphic features, cardiac defects, ectodermal anomalies and developmental delay.We report a 7(1/2)-month-old boy with a clinical diagnosis of CFC. Bidirectional sequence analysis of MEK2 revealed a novel c.383C-->A transversion in exon 3 resulting in a nonsynonymous missense substitution, p.P128Q. Other family members, including the proband's mother and half-sibling, displayed phenotypic features of CFC and were also screened for the MEK2 mutation identified in the proband. Sorting Intolerant From Tolerant (SIFT) analysis determined the novel MEK2 p.P128Q to be deleterious. To corroborate the functional alteration of the novel mutant protein, transient transfection of HEK 293T cells with subsequent Western analysis was used to demonstrate increased kinase activity, as measured by ERK phosphorylation. This first reported case of a vertically transmitted functional CFC MEK mutation further expands our understanding of germline mutations within the Ras/MAPK pathway.

Figure 1

Clinical images of the family. A) Current clinical images of mutation positive family members. All family members who tested positive for the mutation have similar ectodermal findings as well as have learning disabilities. B) A photograph of the proband’s maternal grandmothers (III-6) family. Photo taken circa late-1960s.

Figure 2

Pedigree of the family. The proband (indicated by an arrow and black shading) has phenotypic features consistent with a clinical diagnosis of CFC syndrome and has a p.P128Q MEK2 missense mutation. Family members who participated in the study and who have phenotypic features consistent with a clinical diagnosis of CFC syndrome also harbored the same MEK2 mutation (indicated by a blackened symbol). Participating family members that did not have features of CFC tested negative for the mutation (white) and members with gray shading are presumed mutation carriers. I-2, who is shaded in gray, was reported to be born with no arms and had reported features similar to affected individuals in the family. III-3, also shaded in gray, did not want to have his blood drawn but has phenotypic features which are similar to family members who tested positive for the MEK2 mutation. III-5 is deceased but presumed to have been a mutation carrier since he has two affected offspring.

Figure 3

Electropherograms of MEK2 exon 3 sequencing. Electropherograms of the novel MEK2 mutation c.383C>A transition in exon 3 results in a nonsynonymous missense substitution p.P128Q. The proband, his half-sibling brother and mother all have the heterozygous germline mutation. The proband’s father has the wild-type MEK2 sequence.

Figure 4

Functional characterization of the MEK2 p.P128Q mutant. Human embryonic kidney 293T cells were transiently transfected with the MEK2 p.P128Q mutant plasmid and appropriate controls. ERK phosphorylation was assayed by Western blotting using phosphospecific antibodies. The p.P128Q MEK2 mutant protein had increased ERK phosphorylation compared to the level induced by empty vector, wild-type MEK2 and the kinase dead control. However, the p.P128Q MEK2 mutant protein was less active less than the CFC MEK2 p.F57C mutant which is known to have increased activity over wild-type. This indicates that p.P128Q MEK2 is a weak hypermorph. Myc-tagged MEK2 served as a marker for transfection efficiency and total ERK served as a loading control.