We collected detailed clinical information of 13 patients with rare, protein-altering MED13 variants. Eleven variants were confirmed to be de novo, and one patient (patient B) inherited the variant from her mother who is also affected (patient C). Phenotypic data summarizing the spectrum of features of this cohort of 13 patients are shown in Table 1.
All patients had developmental delays with varying severity and course. In the patients that underwent formal intelligence testing, total IQ levels varied from 85 (lower range of normal IQ) to an IQ between 35 and 50 (moderate ID). Five patients had an Autism Spectrum Disorder (ASD), and three patients were diagnosed with Attention Deficit Hyperactivity Disorder (ADHD). All patients had speech delays and/or disorders, with delayed milestones in speech and language development. While several patients had expressive and receptive language problems, in the majority of patients, speech production was significantly more impaired than language comprehension. Three patients (patient A, K and M) showed characteristics of speech apraxia, a developmental speech disorder in which affected individuals have difficulties accurately programming the motor sequences required to produce fluent speech. Patient A had a mild ID, but showed speech apraxia with a mixed receptive and expressive language disorder, and limited verbal expression at the age of 8 years. Patient M had a non-verbal IQ of 70 with a severe speech/language disorder. Her expressive speech was severely affected, with signs of speech apraxia. At the age of 8 years she only used single words and very short sentences. Patient K developed some speech capabilities, but showed regression at the age of 13 months and has since remained non-verbal.
Seven of 13 patients showed delays in motor development, most of which affected at least the gross motor skills (6 of 7), although one patient was reported to have only fine motor delays. Three patients had hypotonia (patient E, F and G). One patient (patient H) developed severe drug-resistant myoclonic-atonic epilepsy at 4 years of age with generalized clonic, myoclonic, atonic, tonic and atypical absence seizures. MRI screening of this patient showed a small abnormality in the left occipital lobe of his brain that did not correspond to the electrophysiological onset or the semiology of his seizures. In other patients, MRI scans were not performed or showed no clear abnormalities, except for mild frontal atrophy in patient M.
Eight patients (62%) presented with eye or vision abnormalities. Two patients (patients L and M) presented with Duane anomaly, a congenital type of strabismus that is characterized by non-progressive horizontal ophtalmoplegia and retraction of the globe with attempted adduction, together with narrowing of the palpebral fissure (Andrews et al. 1993). One patient (patient G) had strabismus, two patients had astigmatism (patient A and I), and one patient (patient E) had congenital nystagmus. While only one patient (patient D) had a visual impairment, three patients had optic nerve abnormalities: pale optic nerves in patient D, papilledema in patient G, and in patient E outer retinal atrophy temporal to both optic discs was reported with relatively small optic nerves on a MRI-scan.
Several other interesting phenotypes were observed in at least two patients in the cohort. Four patients presented with chronic obstipation (patients C, E, G and M). Two patients had conductive hearing loss (patients F and L). Two patients had congenital heart abnormalities: a mildly dilated aortic root and pulmonary artery (both improving over time) in patient F, and a subaortic stenosis in patient J. Two patients were reported to have chronic sleep issues (patient J and K).
Overlapping facial characteristics were reported, including widely spaced eyes with narrow palpebral fissures and peri-orbital fullness, a broad and high nasal bridge, full nasal tip, synophrys, a flat philtrum and a wide mouth with thin upper lip (Fig. 1).
Variants and predicted consequences
The MED13 transcript (NM_005121.2) encodes a large protein consisting of 2174 amino acids (NP_005112.2). The Pfam database characterizes two domains within the MED13 protein: an N-terminal domain (aa 11–383) and a C-terminal domain (aa 1640–2163), as shown in Fig. 2a. Analysis of conservation across the length of the protein indicates several highly conserved residues that lie between these two domains (Fig. 2b).
All 12 unique variants found in our patients are absent from the gnomAD database (Lek et al. 2016) and TOPMED Bravo database (https://bravo.sph.umich.edu/freeze3a/hg19/) and are predicted to be highly deleterious by CADD v1.3 (Kircher et al. 2014), with scores ranging from 20.5 to 41 (Table 1). Six patients had five unique variants that are predicted to be truncating: three nonsense mutations (p.Leu131* in Patients B and C, p.Leu582* in Patient I and p.Arg1400* in Patient J) and two frameshift variants leading to a premature stop codon (p.Pro42Leufs*6 in patient A and p.Thr1496Metfs*11 in Patient K). The remaining variants include six missense variants and a single amino acid deletion. These seven variants form two apparent clusters: one in the N-terminal conserved phosphodegron domain and the other in the C-terminal domain (Fig. 2a). These seven variants were all found to lie within motifs that are highly conserved between MED13 and MED13L (Fig. 2b) and affect sites under high codon selection (Fig. 2c). These missense variants and the in-frame deletion are each located on surface-exposed sites within a three-dimensional model of the MED13 protein (Fig. 3). The four mutations that cluster in the N-terminal domain affect two adjacent amino acids (p.Thr326 and p.Pro327) that are known to be part of a conserved phosphodegron that is required for binding with SCF-Fbw7 ubiquitin ligase for degradation (Davis et al. 2013). Using interaction data from Davis et al. and PDB structure 2OVQ, which has Fbw7 interacting with a similar motif as MED13, we modeled this interaction for MED13 followed by insertion of each variant and calculation of binding energy. All four variants (p.Thr326Ile, p.Thr326del, p.Pro327Ser, p.Pro327Gln) are predicted to alter the phosphorylation and Fbw7 interaction with drastic decreases in binding energy to Fbw7 (Supplementary Fig. 1). The two missense changes clustering in the C-terminal portion of the protein (p.Gln2060Lys and p.Ala2064Val; in patients L and M, respectively) were also studied in more detail. One of the changes (p.Ala2064Val) is predicted to be structure-altering through increasing hydrophobic collapse, secondary structure formation, and increasing aliphatic index of a surface exposed linear motif. This results in a decrease of the regions linear interacting peptide potential that is highly conserved and likely functional (Supplementary Fig. 2). The remaining missense variant (p.Pro540Thr in Patient H) lies within a highly conserved linear motif centered near amino acid 538 (Fig. 2b); it results in the formation of a high probability Casein Kinase 1 phosphorylation motif, which could lead to additional interaction with proteins containing forkhead-associated domains when analyzed through the ELM database (Dinkel et al. 2016) (Fig. 3).
Effects of truncating MED13 mutation on transcript and protein levels
As truncating mutations often lead to nonsense-mediated decay and haploinsufficiency, we aimed to examine the effects of a truncating MED13 mutation on levels of MED13 transcript and MED13 protein. We performed RT-PCR on cDNA transcribed from RNA of patient J, who was heterozygous for a nonsense mutation (c.4198C > T; p.Arg1400*). We compared the MED13 transcript level of the patient to her biological parents and two healthy controls (Fig. 4a). No differences in MED13 transcript levels were detectable between the affected patient and the unaffected parents or controls (One-way ANOVA p = 0.5913). Sanger sequencing of cDNA amplicons from the child demonstrated the presence of the aberrant transcript in the child (Fig. 4b), at ~ 70% levels relative to the normal transcript (Fig. 4c). To assess the effect of the nonsense mutation on protein levels, a western blot was performed on nuclear extracts from mononuclear blood cells of the patient and controls (Fig. 4d). While full-length MED13 protein was present in the patient (and in the controls), no truncated MED13 protein product could be detected. The MED13 protein level of the patient was not clearly different compared with the MED13 protein level of the father.
Enrichment of de novo MED13 variants in DD/ID cohorts
We quantified the extent of enrichment of de novo variants in MED13 within DD/ID-affected probands. We used only the two largest cohorts considered within this study, each of which yielded at least two de novo MED13 variants. Five patients described here (A, E, F, I, and K) come from a cohort of 11,149 affected individuals, and two patients, one of which is described here (patient L), were identified within the Deciphering Developmental Disorders (DDD) study of 4293 trios (Deciphering Developmental Disorders 2017). Both studies suggest a rate close to 1 de novo variant affecting MED13 per ~ 2200 DD/ID-affected individuals. When comparing the number of observed de novo mutations in MED13 to the expected number based on the gene specific mutation rate of MED13 for missense, splice-site, nonsense and frameshift mutations [6.237 × 10−5 per chromosome (Samocha et al. 2014)], we find evidence for a significant enrichment among DD/ID-affected individuals (7 variants in 30,884 alleles; p = 0.00371).