De Novo Missense Variants in TRAF7 Cause Developmental Delay, Congenital Anomalies, and Dysmorphic Features

Abstract

TRAF7 is a multi-functional protein involved in diverse signaling pathways and cellular processes. The phenotypic consequence of germline TRAF7 variants remains unclear. Here we report missense variants in TRAF7 in seven unrelated individuals referred for clinical exome sequencing. The seven individuals share substantial phenotypic overlap, with developmental delay, congenital heart defects, limb and digital anomalies, and dysmorphic features emerging as key unifying features. The identified variants are de novo in six individuals and comprise four distinct missense changes, including a c.1964G>A (p.Arg655Gln) variant that is recurrent in four individuals. These variants affect evolutionarily conserved amino acids and are located in key functional domains. Gene-specific mutation rate analysis showed that the occurrence of the de novo variants in TRAF7 (p = 2.6 × 10^-3) and the recurrent de novo c.1964G>A (p.Arg655Gln) variant (p = 1.9 × 10^-8) in our exome cohort was unlikely to have occurred by chance. In vitro analyses of the observed TRAF7 mutations showed reduced ERK1/2 phosphorylation. Our findings suggest that missense mutations in TRAF7 are associated with a multisystem disorder and provide evidence of a role for TRAF7 in human development.

Keywords: ERK1/2; MAPKs; RASopathy; TRAF7; cancer; congenital heart defects; de novo missense variants; developmental delay; exome sequencing; limb and digit anomalies.

Figure 1

Facial Features and Limb Phenotypes for Subjects with TRAF7 Variants Shown are subject 7 (A), subject 4 (B), subject 3 (C), subject 5 (D), subject 6 (E), and subject 1 (F). Shared dysmorphic features included epicanthal folds, ptosis, abnormally set or dysplastic ears, a low hairline, excess nuchal skin, and multiple hair whorls. Overlapping toes are shown in (C) and (F).

Figure 2

In Silico Analysis of the Four TRAF7 Variants Identified in This Study (A) The R655, T601, R371, and K346 residues are conserved from human to zebrafish (prepared from UCSC genome browser Multiz Alignments of 100 Vertebrates track). (B) Schematic view of the TRAF7 exon-intron structure. Blue boxes represent exons and yellow fields represent introns. The identified cDNA changes are listed. (C) Schematic view of the TRAF7 protein and its domains based on data extracted from Zotti et al. Domains are represented by blue shapes. The domain names and the identified amino acid changes are listed.

Figure 3

The Effect of TRAF7 Mutants on ERK1/2 Signaling (A) Phosphorylation of ERK1/2 was analyzed by transiently expressing the FLAG-tagged wild-type and mutant constructs in HEK293T cells. Twenty-four hours after transfection, cells were stimulated with 10 ng ml⁻¹ TNFα for 30 min. Cells were then collected and lysed and immunoblotting was performed. Expression of the TRAF7 mutants resulted in decreased phosphorylation of ERK1/2 when compared with cells expressing wild-type protein, either with or without TNFα treatment. Asterisk and pound symbols indicate ERK1 and ERK2, respectively. (B–E) Quantification of the effect of TRAF7 mutants on ERK1/2 signaling. pERK1 (B) and pERK2 (C) signaling without TNFα treatment. pERK1 (D) and pERK2 (E) signaling with 10 ng ml⁻¹ TNFα stimulation for 30 min. All data were normalized to GAPDH protein levels, with the wild-type protein set at 1.0. The results are representative of three independent experiments. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001, one-way ANOVA with Dunnett’s multiple-comparisons test. Data were shown as mean ± standard error of the mean, n = 3.