Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Aug 8;111(8):1605-1625.
doi: 10.1016/j.ajhg.2024.06.008. Epub 2024 Jul 15.

Comprehensive EHMT1 variants analysis broadens genotype-phenotype associations and molecular mechanisms in Kleefstra syndrome

Dmitrijs Rots  1 , Arianne Bouman  2 , Ayumi Yamada  3 , Michael Levy  4 , Alexander J M Dingemans  5 , Bert B A de Vries  5 , Martina Ruiterkamp-Versteeg  5 , Nicole de Leeuw  5 , Charlotte W Ockeloen  5 , Rolph Pfundt  5 , Elke de Boer  5 , Joost Kummeling  5 , Bregje van Bon  5 , Hans van Bokhoven  5 , Nael Nadif Kasri  5 , Hanka Venselaar  6 , Marielle Alders  7 , Jennifer Kerkhof  4 , Haley McConkey  8 , Alma Kuechler  9 , Bart Elffers  10 , Rixje van Beeck Calkoen  11 , Susanna Hofman  12 , Audrey Smith  13 , Maria Irene Valenzuela  14 , Siddharth Srivastava  15 , Zoe Frazier  16 , Isabelle Maystadt  17 , Carmelo Piscopo  18 , Giuseppe Merla  19 , Meena Balasubramanian  20 , Gijs W E Santen  21 , Kay Metcalfe  13 , Soo-Mi Park  22 , Laurent Pasquier  23 , Siddharth Banka  24 , Dian Donnai  13 , Daniel Weisberg  13 , Gertrud Strobl-Wildemann  25 , Annemieke Wagemans  26 , Maaike Vreeburg  27 , Diana Baralle  28 , Nicola Foulds  29 , Ingrid Scurr  30 , Nicola Brunetti-Pierri  31 , Johanna M van Hagen  32 , Emilia K Bijlsma  33 , Anna H Hakonen  34 , Carolina Courage  34 , David Genevieve  35 , Lucile Pinson  36 , Francesca Forzano  37 , Charu Deshpande  13 , Maria L Kluskens  38 , Lindsey Welling  38 , Astrid S Plomp  7 , Els K Vanhoutte  27 , Louisa Kalsner  39 , Janna A Hol  40 , Audrey Putoux  41 , Johanna Lazier  42 , Pradeep Vasudevan  43 , Elizabeth Ames  44 , Jessica O'Shea  44 , Damien Lederer  45 , Julie Fleischer  46 , Mary O'Connor  46 , Melissa Pauly  47 , Georgia Vasileiou  48 , André Reis  48 , Catherine Kiraly-Borri  49 , Arjan Bouman  50 , Chris Barnett  51 , Marjan Nezarati  52 , Lauren Borch  53 , Gea Beunders  54 , Kübra Özcan  55 , Stéphanie Miot  56 , Catharina M L Volker-Touw  57 , Koen L I van Gassen  57 , Gerarda Cappuccio  58 , Katrien Janssens  59 , Nofar Mor  60 , Inna Shomer  60 , Dan Dominissini  61 , Matthew L Tedder  62 , Alison M Muir  63 , Bekim Sadikovic  4 , Han G Brunner  64 , Lisenka E L M Vissers  65 , Yoichi Shinkai  66 , Tjitske Kleefstra  67
Affiliations

Comprehensive EHMT1 variants analysis broadens genotype-phenotype associations and molecular mechanisms in Kleefstra syndrome

Dmitrijs Rots et al. Am J Hum Genet. .

Abstract

The shift to a genotype-first approach in genetic diagnostics has revolutionized our understanding of neurodevelopmental disorders, expanding both their molecular and phenotypic spectra. Kleefstra syndrome (KLEFS1) is caused by EHMT1 haploinsufficiency and exhibits broad clinical manifestations. EHMT1 encodes euchromatic histone methyltransferase-1-a pivotal component of the epigenetic machinery. We have recruited 209 individuals with a rare EHMT1 variant and performed comprehensive molecular in silico and in vitro testing alongside DNA methylation (DNAm) signature analysis for the identified variants. We (re)classified the variants as likely pathogenic/pathogenic (molecularly confirming Kleefstra syndrome) in 191 individuals. We provide an updated and broader clinical and molecular spectrum of Kleefstra syndrome, including individuals with normal intelligence and familial occurrence. Analysis of the EHMT1 variants reveals a broad range of molecular effects and their associated phenotypes, including distinct genotype-phenotype associations. Notably, we showed that disruption of the "reader" function of the ankyrin repeat domain by a protein altering variant (PAV) results in a KLEFS1-specific DNAm signature and milder phenotype, while disruption of only "writer" methyltransferase activity of the SET domain does not result in KLEFS1 DNAm signature or typical KLEFS1 phenotype. Similarly, N-terminal truncating variants result in a mild phenotype without the DNAm signature. We demonstrate how comprehensive variant analysis can provide insights into pathogenesis of the disorder and DNAm signature. In summary, this study presents a comprehensive overview of KLEFS1 and EHMT1, revealing its broader spectrum and deepening our understanding of its molecular mechanisms, thereby informing accurate variant interpretation, counseling, and clinical management.

Keywords: DNA methylation; EHMT1; H3K9; Kleefstra syndrome; NDD; neurodevelopmental disorders.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests A.M.M. is an employee of GeneDx, LLC. B.S. is a shareholder in EpiSign Inc., a biotechnology company involved in commercialization of EpiSign technology.

Figures

Figure 1
Figure 1
EHMT1 LP/P variants and VUSs identified in this study (A) Intragenic location of the EHMT1 point and structural non-deletion variants that were classified as LP/P or VUS. (B) Genomic location of the identified 9q34.3 deletions affecting EHMT1 (region highlighted in blue).
Figure 2
Figure 2
EHMT1 PAV position on the protein 3D structure (A) Ankyrin repeat domain (cyan) with H3 tail (orange) (PDB: 6BY9 and 3B95). Amino acids affected by variants predicted to disrupt the domain’s structure or binding to the H3 tail are shown in magenta and neutral variants are shown in green. (B) SET domain (with pre-SET and post-SET domains) (green) with H3 tail and SAH (in dark gray) and Zn atoms (gray) (PDB: 2RFI). Amino acids affected by variants predicted to disrupt the domain’s structure are shown in magenta, variants that are predicted to disrupt the enzymatic activity only are shown in purple, and neutral variants are shown in cyan. (C) RING-like domain (yellow) with Zn atoms (gray). The region removed by skipping the inframe exon due to c.1791G>A is shown in cyan, while the region of insertion c.1647_1648ins643_1170 is shown in dark gray.
Figure 3
Figure 3
In vitro assay results for the EHMT1 PAVs (A) In vitro methylation assay was performed using recombinant MBP-EHMT1 (635–1298 aa)-His proteins. Methylated histone H3 was examined by western blotting. Graph showed relative amount of H3K9me2 (n = 3 independent experiments; means ± SD; ∗p < 0.05, ∗∗p < 0.01; one-way ANOVA analysis; Dunnett’s multiple comparison test). (B) FLAG-tagged EHMT1 proteins were expressed together with GFP-tagged EHMT2 in 293T cells. EHMT2 bound to EHMT1 was precipitated using FLAG M2 beads from cell lysates. Graph showed relative amount of co-precipitated EHMT2 with EHMT1 (n = 3 independent experiments, means ± SD, ∗∗∗p < 0.001, one-way ANOVA analysis, Dunnett’s multiple comparison test). (C) FLAG-tagged EHMT1 wild type and mutants were stably expressed in EHMT1 knockout HeLa cells. Histone H3 dimethylation level was analyzed by western blotting. Graph showed relative level of H3K9me2 (means ± SD, ∗∗p < 0.01, ∗∗∗p < 0.001).
Figure 4
Figure 4
Flowchart of the individuals included and excluded in this study
See this image and copyright information in PMC

References

    1. Jansen S., Vissers L.E.L.M., de Vries B.B.A. The Genetics of Intellectual Disability. Brain Sci. 2023;13 doi: 10.3390/brainsci13020231. - DOI - PMC - PubMed
    1. Wilczewski C.M., Obasohan J., Paschall J.E., Zhang S., Singh S., Maxwell G.L., Similuk M., Wolfsberg T.G., Turner C., Biesecker L.G., Katz A.E. Genotype first: Clinical genomics research through a reverse phenotyping approach. Am. J. Hum. Genet. 2023;110:3–12. doi: 10.1016/j.ajhg.2022.12.004. - DOI - PMC - PubMed
    1. Rots D., Chater-Diehl E., Dingemans A.J.M., Goodman S.J., Siu M.T., Cytrynbaum C., Choufani S., Hoang N., Walker S., Awamleh Z., et al. Truncating SRCAP variants outside the Floating-Harbor syndrome locus cause a distinct neurodevelopmental disorder with a specific DNA methylation signature. Am. J. Hum. Genet. 2021;108:1053–1068. doi: 10.1016/j.ajhg.2021.04.008. - DOI - PMC - PubMed
    1. Cuvertino S., Hartill V., Colyer A., Garner T., Nair N., Al-Gazali L., Canham N., Faundes V., Flinter F., Hertecant J., et al. A restricted spectrum of missense KMT2D variants cause a multiple malformations disorder distinct from Kabuki syndrome. Genet. Med. 2020;22:867–877. doi: 10.1038/s41436-019-0743-3. - DOI - PMC - PubMed
    1. Menke L.A., van Belzen M.J., Alders M., Cristofoli F., DDD Study. Ehmke N., Fergelot P., Foster A., Gerkes E.H., Hoffer M.J.V., et al. CREBBP mutations in individuals without Rubinstein-Taybi syndrome phenotype. Am. J. Med. Genet. 2016;170:2681–2693. doi: 10.1002/ajmg.a.37800. - DOI - PubMed

Substances

Supplementary concepts

LinkOut - more resources