Dissecting KMT2D missense mutations in Kabuki syndrome patients

Abstract

Kabuki syndrome is a rare autosomal dominant condition characterized by facial features, various organs malformations, postnatal growth deficiency and intellectual disability. The discovery of frequent germline mutations in the histone methyltransferase KMT2D and the demethylase KDM6A revealed a causative role for histone modifiers in this disease. However, the role of missense mutations has remained unexplored. Here, we expanded the mutation spectrum of KMT2D and KDM6A in KS by identifying 37 new KMT2D sequence variants. Moreover, we functionally dissected 14 KMT2D missense variants, by investigating their impact on the protein enzymatic activity and the binding to members of the WRAD complex. We demonstrate impaired H3K4 methyltransferase activity in 9 of the 14 mutant alleles and show that this reduced activity is due in part to disruption of protein complex formation. These findings have relevant implications for diagnostic and counseling purposes in this disease.

Figure 1.

Missense variants distribution across the entire length or FUSION–KMT2D gene. (A) Schematic representation of the KMT2D protein (PHD, plant homeodomain; HMG, high-mobility group; Coiled Coil domain; LXXLL domain, motifs with the consensus sequence L–X–X–L–L motif; Poli Q region, Poli Q reach region identified in this study; ZF domain, Zinc Finger Domain; FYRN, FY-rich, N-terminal; FYRC, FY-rich, C-terminal; WIN, WDR5 Interaction domain, SET, Su(var)3-9, Enhancer-of-zeste, Trithorax). In black, missense variants found in our cohort of KS patients. De novo variants are indicated in bold, pathogenic variants with P, likely pathogenic with LP, likely benign with LP, and VOUS with V. (B) Distribution of the functionally analyzed KMT2D missense variants across the FUSION–KMT2D construct composed of PHD4–5–6 (amino acids 1358–1572) and ZF-PHD7–FYRN–FYRC–WIN–SET–post-SET domains (amino acids 4507–5537). De novo variants are indicated in bold, pathogenic variants are indicated with P, likely pathogenic with LP, likely benign with LB and VOUS with V.

Figure 2.

KMT2D missense variants are associated with defective methyltransferase activity and diminished H3K4 methylation. (A) H3K4 Lysine methyltransferase activity of mutated FLAG-KMT2D proteins on HeLa nucleosomes. (B) Relative H3K4 methyltransferase activity of semi-purified FLAG-KMT2D proteins on HeLa nucleosomes (mean±SD; n=two independent biological replicates). Data are expressed as fold differences compared with the activity of the wild-type control, arbitrarily set as 1. Pathogenic variants are indicated with P, likely pathogenic with LP, likely benign with LB, and VOUS with V. Student’s T-test is indicated as *P<0.05; **P<0.01; ***P<0.001. (C) Schematic representation of the epigenetic reporter allele encoding the two halves of GFP separated by a flexible linker region with a histone tail (H3) and a histone reader (TAF3 PHD) at the N and C termini, respectively (23). Modification of the histone tail by (KMT2D-mediated) trimethylation leads to reconstitution of the GFP structure and function, which can be measured by fluorescence. (D) The H3K4me3 indicator demonstrated a decreased H3K4me3 activity for the 6 tested missense variants compared with the WT. Pathogenic variants are indicated with P, likely pathogenic with LP, likely benign with LB, and VOUS with V. Student’s t-test is indicated as *P<0.001.

Figure 3.

Interaction of KMT2D missense mutants with ASH2L, RbBP5 and WDR5. (A) On the left, immunoblot analysis of KMT2D, ASH2L and RbBP5 in HEK293T cell line transfected with wild-type KMT2D or KMT2D missense mutants followed by immunoprecipitation with anti-Flag antibody. On the right, relative interaction of the indicated KMT2D missense mutants with ASH2L (top) and RbBP5 (bottom) (mean±SD, n=2 independent replicates). Pathogenic variants are indicated with P, likely pathogenic with LP, likely benign with LB, and VOUS with V. Student’s T-test is indicated as *P<0.05, **P<0.01 and ***P<0.001. (B) On the left, the predicted amino acid tolerance for KMT2D p.5340: the presence of Methionine (M) is very slightly tolerated. On the right, immunoblot analysis of KMT2D and WDR5 in HEK293 cell line transfected with wild-type WDR5, wild-type C-Ter KMT2D or the p.R5340Q KMT2D missense mutant before (input) and after immunoprecipitation with the anti-Flag antibody. EV, empty vector.