De novo mutations in MED13, a component of the Mediator complex, are associated with a novel neurodevelopmental disorder

Abstract

Many genetic causes of developmental delay and/or intellectual disability (DD/ID) are extremely rare, and robust discovery of these requires both large-scale DNA sequencing and data sharing. Here we describe a GeneMatcher collaboration which led to a cohort of 13 affected individuals harboring protein-altering variants, 11 of which are de novo, in MED13; the only inherited variant was transmitted to an affected child from an affected mother. All patients had intellectual disability and/or developmental delays, including speech delays or disorders. Other features that were reported in two or more patients include autism spectrum disorder, attention deficit hyperactivity disorder, optic nerve abnormalities, Duane anomaly, hypotonia, mild congenital heart abnormalities, and dysmorphisms. Six affected individuals had mutations that are predicted to truncate the MED13 protein, six had missense mutations, and one had an in-frame-deletion of one amino acid. Out of the seven non-truncating mutations, six clustered in two specific locations of the MED13 protein: an N-terminal and C-terminal region. The four N-terminal clustering mutations affect two adjacent amino acids that are known to be involved in MED13 ubiquitination and degradation, p.Thr326 and p.Pro327. MED13 is a component of the CDK8-kinase module that can reversibly bind Mediator, a multi-protein complex that is required for Polymerase II transcription initiation. Mutations in several other genes encoding subunits of Mediator have been previously shown to associate with DD/ID, including MED13L, a paralog of MED13. Thus, our findings add MED13 to the group of CDK8-kinase module-associated disease genes.

Fig. 1

Facial phenotypes of seven individuals with a MED13 variant. Overlapping facial characteristics include peri-orbital fullness, narrow palpebral fissures, a broad and high nasal bridge, full nasal tip, synophrys, flat philtrum, wide mouth and a thin upper lip

Fig. 2

Analysis of mutations: location, conservation and codon usage of variant sites. a Identified mutations are shown within a linear representation of the MED13 protein, consisting of 2174 amino acids. Missense mutations and the in-frame deletion are shown in blue, and nonsense and frameshift mutations in green. Six of the seven non-truncating mutations in our MED13 cohort cluster in two small regions within the N-terminal and C-terminal domains of the MED13 protein. Affected amino acids p.Thr326 and p.Pro327 and are part of a conserved phosphodegron (CPD), which is shown in orange. Two LxxLL nuclear receptor-binding motifs are also noted. b Analysis of conservation throughout the protein was performed using amino acid selection scores as previously published (Prokop et al. 2017), using a 21 codon sliding window for both MED13 and MED13L aligned such that the most selected motifs of a protein are identified as peaks. The center of each highly conserved linear motif is labeled and those containing variants described in this paper are boxed. c Codon usage throughout evolution for the locations of all missense mutations and the in-frame deletion. All five sites are under high selection with multiple synonymous (Syn, gray) amino acids in 352 open reading frames (ORFs) of MED13 and MED13L with only a single nonsynonymous (Nonsyn, red) change. Numbers indicate instances where ORFs in other species deviate from the conserved codon usage. Of note, for three locations (326, 327 and 540) the codon used differs between MED13 and MED13L with the amino acid conserved. In these cases, numbers indicate where ORFs in other species deviate from conserved codon usage in their respective ortholog

Fig. 3

Location of missense mutations and in-frame deletion in three-dimensional structure of MED13 and conservation of affected amino acids. A full model of MED13 protein created with I-TASSER modeling was combined with 152 species sequences for MED13 using ConSurf mapping. Amino acid coloring is as followed: gray = not conserved, yellow = conserved hydrophobic, green = conserved hydrophilic, red = conserved polar acidic, blue = conserved polar basic, magenta = conserved human variants of interest. A zoomed in view of the three different affected regions are shown, along with amino acid alignments from MED13 and MED13L. An asterisk (*) indicates 100% conservation in all sequences and a colon (:) indicates functional conservation. Linear motifs mapped with the Eukaryotic Linear Motif tool are shown below sites for 326–327 and 540

Fig. 4

Analysis of transcript and protein levels in patient with nonsense mutation. a Level of MED13 transcript was measured by qPCR and normalized to GAPDH and proband (patient J). No differences were detectable between groups (One-way ANOVA p = 0.5913). An additional loading control (AGPAT) produced very similar results (data not shown). b Representative Sanger traces from cDNA amplicons demonstrating the presence of the variant in the proband, and absence in the father and mother. c Quantification of the chromatograms of all Sanger sequences reveals less signal from the base on the mutant allele (p < 0.0001 by paired t-test compared to the wildtype base signal by trace). The father and mother do not have any signal at the mutant base above the level of noise. d Western blot for MED13 (and HSP90 and HDAC2 as loading controls) from nuclear extracts of patient peripheral blood mononuclear cells or a neural precursor cell line (present to demonstrate antibody specificity with a knockdown (KD) control). If the nonsense mutation resulted in a stable protein, a product at approximately 150 kDa would be expected, which is not present. No protein was recoverable from the blood sample from the mother