MED12-Related (Neuro)Developmental Disorders: A Question of Causality

Abstract

MED12 is a member of the Mediator complex that is involved in the regulation of transcription. Missense variants in MED12 cause FG syndrome, Lujan-Fryns syndrome, and Ohdo syndrome, as well as non-syndromic intellectual disability (ID) in hemizygous males. Recently, female patients with de novo missense variants and de novo protein truncating variants in MED12 were described, resulting in a clinical spectrum centered around ID and Hardikar syndrome without ID. The missense variants are found throughout MED12, whether they are inherited in hemizygous males or de novo in females. They can result in syndromic or nonsyndromic ID. The de novo nonsense variants resulting in Hardikar syndrome that is characterized by facial clefting, pigmentary retinopathy, biliary anomalies, and intestinal malrotation, are found more N-terminally, whereas the more C-terminally positioned variants are de novo protein truncating variants that cause a severe, syndromic phenotype consisting of ID, facial dysmorphism, short stature, skeletal abnormalities, feeding difficulties, and variable other abnormalities. This broad range of distinct phenotypes calls for a method to distinguish between pathogenic and non-pathogenic variants in MED12. We propose an isogenic iNeuron model to establish the unique gene expression patterns that are associated with the specific MED12 variants. The discovery of these patterns would help in future diagnostics and determine the causality of the MED12 variants.

Keywords: Hardikar syndome; MED12 variants; expression profile; iNeuronal model; intellectual disability; transcription regulation.

Figure 1

Schematic overview of the structure of the Mediator kinase subunit and the functions of individual proteins. The kinase subunit (light blue) consists of MED13, MED12, Cyclin C (CCNC), and cyclin-dependent kinase 8 (CDK8). Paralogs have been discovered for all subunit components expect CCNC. These paralogs are MED13L, MED12L, and CDK19. Adjusted from Aranda-Orgilles et al. [2].

Figure 2

Schematic overview of variants distributed across the predicted functional domains in the MED12 protein (not to scale). Previously described ID associated hemizygous variants found in males are presented below the protein scheme. Patient numbers in red describe nonsense mutations. Above the protein scheme all known variants in females are described. Patient numbers in red describe patients with Hardikar syndrome. The MED12 domain that is part of the kinase section of Mediator, the LCEWAV domain with no known function, and the catenin-binding PQL domain are indicated. Only domains predicted by the PFAM database (https://pfam.xfam.org/ (accessed on 1 April 2021) were included in this figure. Figure was adapted from [16] and supplemented with data from [17]. Descriptions of individual patients can be found in Table S1.

Figure 3

Model of how MED12 activates CDK8. Step 1: Cyclin C binds to CDK8 and pushes the αC-helix of CDK8 into the “pushed-in” conformation. This binding event is crucial for the formation of the active site of CDK8 and results in basal kinase activity. Step 2: MED12 binding to CDK8/CCNC stabilizes and activates the MED12-CCNC-CDK8 complex. In particular, an activation helix in MED12 contacts and stabilizes the T-loop of CDK8, thereby activating the kinase. Moreover, MED12 binding favors remodeling of the active site of CDK8 to enhance its activity. Adapted from Klatt et al. [13].

Figure 4

Model for the pathogenic effect of MED12 variants. MED12 recruitment to its target gene is regulated via interaction with ncRNAs, eRNAs, and cofactors such as REST. We propose that specific mutations in MED12 disrupt its interactions with either ncRNAs, eRNAs, or cofactors, depending on where the mutation is present in the protein. This leads to reduced recruitment of MED12 to the target genes, which will in turn result in lower MED12-mediated recruitment of additional factors, such as G9a, as well as other Mediator subunits. This will lead to loss of regulation over developmental pathways, loss of regulation over chromatin state (leading to loss of active enhancers), as well as loss of long-range transcriptional interactions.

Figure 5

General approach to determine pathogenicity of MED12 variants. Firstly, an isogenic male iPSC is generated which also serves as a control. Then by use of CRISPR-Cas, the novel variants are introduced in the cells (e.g., p.Arg621Gln, c.1249-1G>C), pTyr1973*). These cell lines are subsequently sequenced and the differentially expressed genes as compared to a control are selected through an AI algorithm.