A novel de novo frameshift deletion in EHMT1 in a patient with Kleefstra Syndrome results in decreased H3K9 dimethylation

Abstract

Background: Kleefstra Syndrome (KS) (MIM# 610253) is an autosomal dominant disorder caused by haploinsufficiency of euchromatic histone methyltransferase-1 (EHMT1, GLP). EHMT1 (MIM# 607001) encodes a histone methyltransferase that heterodimerizes with EHMT2 (also known as G9a, MIM# 604599), which together are responsible for mono- and dimethylation of H3 lysine 9 (H3K9me1 and -me2), resulting in transcriptional repression of target genes.

Methods: This report describes an 18-year-old woman with intellectual disability, severely limited speech, hypotonia, microcephaly, and facial dysmorphisms, who was found to have a novel de novo single-base frameshift deletion in EHMT1.

Results: Functional studies using patient fibroblasts showed decreased H3K9me2 compared to wild-type control cells, thus providing a rapid confirmatory test that complements molecular studies.

Conclusion: Whole exome sequencing revealed a novel frameshift deletion in EHMT1 after a lengthy diagnostic odyssey in this patient. Functional testing using this patient's fibroblasts provides proof-of-concept for the analysis of variants of uncertain significance that are predicted to impact EHMT1 enzymatic activity.

Keywords: EHMT1; GLP; Kleefstra Syndrome; functional validation; p.Arg310Aspfs*4; whole exome sequencing.

Figure 1

Patient photographs at 14 years of age showing features typical of Kleefstra syndrome. The patient has microcephaly, eyes widely spaced with epicanthal folds, ears that are overfolded and posteriorly rotated, with a deep notch on the left ear lobe (not shown), a short nose that is wide at the base, synophrys, and relative prognathism.

Figure 2

Novel patient EHMT1 variant results in reduced H3K9 dimethylation. (A–F) Immunofluorescence microscopy analysis of H3K9me2 levels in wild‐type and EHMT1^+/−‐mutant human fibroblasts illustrates a marked reduction in this histone mark in the mutant cells. (A, D) DAPI nuclear stain, (B, E) H3K9me2, (C, F) overlap of DAPI and H3K9me2. (H) Western blot analysis of total Histone H3 and H3K9me2 protein levels in the same cells confirms the reduction of H3K9me2 in the mutant cells relative to the wild‐type control. (I) Quantification of n = 3 western blots using ImageJ.