Biochemical validation of EHMT1 missense mutations in Kleefstra syndrome

Abstract

Kleefstra syndrome (KS) (9q34 deletion syndrome) is a rare autosomal dominant disorder characterized by intellectual disability, frequently coupled with a spectrum of complex physical and clinical manifestations. As the euchromatic histone methyltransferase-1 gene (EHMT1, GLP, or KMT1D) within the 9q34 region is deleted or mutated in most of the individuals with KS, its absence or defect in one allele is speculated to cause the major symptoms of the syndrome. Most of the EHMT1 mutations are frameshift or nonsense mutations, but two individuals with KS were reported to possess EHMT1 missense mutations. These two mutations have been predicted to cause a defective enzymatic function, but precise biochemical validation was not conducted. Therefore, we validated these two mutations by performing in vitro histone methyltransferase (HMT) activity assay and found that C1073Y and R1197W mutations severely affected the HMT activity. Additionally, the same amino-acid substitutions in mouse GLP induced impairment of in vivo GLP function. Furthermore, these two EHMT1 mutants showed defective heterocomplex formation with G9a (partner HMT) which is essential for their in vivo HMT function. Conclusively, our biochemical characterization clearly demonstrates that the previously reported two missense mutations of EHMT1 deteriorate HMT activity and GLP function, which presumably cause KS.

Chiari 1 malformation and exome sequencing in 51 trios: the emerging role of rare missense variants in chromatin-remodeling genes

“…A posteriori, the malformative picture of our patient fits well with that of Kleefstra syndrome. The biochemical characterization of this variant, made on the previously reported patient, demonstrated that it deteriorates the histone methyltransferase-1 gene activity (Yamada 2018). It is noteworthy that in our patient the detection of the variant in a chromatin-remodeling gene, prompted us to investigate whether C1M, although never reported in Kleefstra syndrome, were present, as indeed it was the case (Fig.…”

EHMT1 pathogenic variants and 9q34.3 microdeletions share altered DNA methylation patterns in patients with Kleefstra syndrome

“…A considerable strength of previously reported DNAm signatures is their utility in predicting the pathogenicity of variants of uncertain significance (VUS) [10,11,15] . Pathogenic missense variants are rare but present in KS [49] , Thus, we sought to assess the power/utility of the KS signature in classifying missense variants. We included one patient in the discovery group used to derive the DNAm signature, who carried a pathogenic missense variant, P809R, and therefore expected the KS DNAm signature to have clinical utility in this regard.…”

Multiple Mongolian spots in an individual with Kleefstra syndrome caused by a novel nonsense euchromatin histone methyltransferase 1 variant

“…The EHMT1 gene encodes lysine methyltransferase, which can methylate histones. The lack of lysine methyltransferase impairs the proper control of the activity of certain genes in many body organs and tissues, leading to the developmental abnormalities and functional characteristics of KS ( Yamada et al , 2018 ). So far, most of the KS cases reported in the literature involve submicroscopic deletion of 9q34.3, but with the development of genetic testing technology, we can detect more intragenic pathogenic variant of EHMT1 gene responsible for KS, both result in haploinsufficiency of the EHMT1 gene.…”