MED13L pathogenic missense variants impair protein stability and interaction, underlying diverse clinical outcomes

Abstract

Heterozygous pathogenic variants in the Mediator complex subunit 13-like gene located in the locus 12q21.21 (MED13L) are associated with intellectual disability, developmental delay, and distinctive facial features. While nonsense and frameshift variants typically cause haploinsufficiency, resulting in a well-characterized clinical presentation, missense variants have been associated with a broader range of phenotypes, including epilepsy and severe motor delay. In this study, we investigated five pathogenic missense variants in MED13L-c.2597C>T p.Pro866Leu, c.2605C>T p.Pro869Ser, c.3392G>A p.Cys1131Tyr, c.5695G>A p.Gly1899Arg, and c.6485C>T p.Thr2162Met-associated with different clinical severities. We identified significant reductions in protein stability across these variants, with some exhibiting aberrant cytoplasmic localization, suggesting disruptions in structural integrity and function. In particular, exon 15 variants (p.Pro866Leu and p.Pro869Ser) correlated with severe phenotypes, including epilepsy and severe motor impairment, whereas p.Gly1899Arg and p.Thr2162Met were associated with milder manifestations. 3D protein modeling suggested that these missense variants may disrupt MED13L's interaction with the CDK8 kinase module, leading to functional deficits. Our findings highlight different pathogenic mechanisms, ranging from protein instability to altered molecular interactions, that contribute to the clinical variability observed in MED13L-related disorders.

Keywords: MED13L; functional approach; intellectual disability; mediator complex; missense variants.

Figure 1

Localization, stability, and integration of pathogenic missense variants (A) Western blot showing protein levels in cells expressing wild-type or mutant MED13L. (B) Relative quantification of total MED13L protein levels under different conditions. A significant decrease in MED13L levels was observed in all mutant conditions, with the greatest instability associated with p.Pro866Leu, p.Cys1131Tyr, and p.Gly1899Arg. Statistical comparisons between wild-type and mutant MED13L were performed using a t test (∗∗∗p < 0.005). (C and D) Immunofluorescence studies revealed a nuclear localization for the wild-type condition, while all protein alterations except p.Pro869Ser showed an altered localization. (E and F) Co-immunoprecipitation assays showed a reduced interaction between the p.Gly1899Arg variant of MED13L and MED12, CDK8, and cyclin—C. The 3xFLAG blot of input samples confirms equal expression of wild-type and mutant MED13L.

Figure 2

MED13L conservation and structural predictions (A) Schematic representation of human MED13L highlighting conserved N- and C-terminal domains, a potential intrinsically disordered region (IDR), and the predicted PIWI-like domain. (B) Multiple sequence alignment showing the conservation of Pro866, Pro869, Cys1131, Gly1899, and Thr2162 residues across species. (C and D) Wild-type and predicted mutant conformations and interactions for the p.Cys1131Tyr substitution. The predicted hydrogen bond between Cys1131 and Phe1128 stabilizes an α helix formed by residues Arg1126-Asn1127-Phe1128-Asp1129-Ser1130, which participates in interactions with both the N- and C-terminal regions of the protein. Substitution with Tyr1131 is predicted to create a new interaction with residue Cys1136. (E and F) Wild-type and predicted mutant conformations and interactions for the p.Gly1899Arg substitution. The Gly1899 residue is part of a β strand and contributes to an 8-stranded antiparallel β sheet structure. Substitution of the buried Gly1899 with Arg1899 is predicted to cause a steric clash with surrounding residues within the β sheet structure. (G and H) Wild-type and predicted mutant conformations and interactions for the p.Thr2162Met substitution. The side chain of Thr2162 forms hydrogen bonds with residues Leu2166 and Leu2095, the latter being lost upon the introduction of Met2162. Reference and alternative residues are underlined in (C)–(H).