FUNCTIONAL STUDY OF THE EHMT2 GENE (CANDIDATE FOR A NOVEL KLEEFSTRA-LIKE SYNDROME)

• Grant number: 25/00171-7
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: May 29, 2025
• End date: May 28, 2026
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Ana Cristina Victorino Krepischi
• Grantee: Laura Machado Lara Carvalho
• Supervisor: Maria Jose Barrero Nuñez
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: Institut de Salud Carlos III, Majadahonda, Spain
• Associated to the scholarship: 22/03980-5 - GENOMIC AND FUNCTIONAL STUDY OF SYNDROMIC FORMS OF INTELLECTUAL DISABILITY, BP.PD

Abstract

In my postdoctoral project, one of the objectives is to investigate the splice site variant c.328+2T>G in the EHMT2 gene, identified in homozygosity in a patient with syndromic intellectual disability. We found that the patient presents an epigenetic signature compatible with that validated for patients with pathogenic variants in EHMT1 (Kleefstra syndrome 1). Using RNAseq, we identified the use of two cryptic splice donor sites located in exon 3, with no detection of wild-type transcripts. The presence of an in-frame isoform may explain the viability of this homozygous mutation. We believe that the mutation does not cause complete loss of EHMT2 function, suggesting the existence of alternative isoforms. Together, the clinical, genetic, and epigenetic data indicate a LoF mechanism for the variant, positioning the EHMT2 gene as a new candidate for a Kleefstra-like syndrome with a recessive inheritance pattern. These results were reported in a paper recently published in Molecular Neurobiology, with Dr. Barrero as a coauthor.Currently, Dr. Barrero is working on functional studies proposing that heterozygous missense mutations in the EHMT2 gene may also lead to a Kleefstra-like syndrome, but under a dominant inheritance model, when mapped to the SET domain, which has methyltransferase activity. In a preprint article, Dr. Barrero and collaborators present the hypothesis and functional evidence based on a case with the heterozygous missense variant p.Ala1077Ser. This variant impaired the affinity for the H3 tail, leading to a three- to fivefold reduction in EHMT2 enzymatic activity. Furthermore, the EpiSign assay revealed a profile similar to that observed in Kleefstra syndrome 1. Histone methylation analysis by mass spectrometry and RNAseq showed considerable overlap between fibroblast cultures with the p.Ala1077Ser variant of EHMT2 and a pathogenic variant of EHMT1 (p.His620ThrfsTer12). Thus, a dominant inheritance pattern was proposed due to the missense mutation in EHMT2 with a haploinsufficiency mechanism caused by reduced enzymatic activity, although a dominant negative effect cannot be excluded. In addition to the patient reported in the preprint article, Dr. Barrero identified, through collaborations, six other promising heterozygous missense variants in the SET domain in patients with clinical features similar to Kleefstra syndrome. Her team is developing cellular models using iPSCs from these patients and working on their characterization.I plan to spend one year in Dr. Barrero's group, aiming to investigate cellular models derived from seven patients with clinical overlap with Kleefstra syndrome and heterozygous variants in the SET domain of EHMT2. Additionally, I plan to add our cellular model based on iPSCs from the patient with the homozygous c.328+2T>G mutation to this ongoing study in Dr. Barrero's laboratory.During the internship abroad, I will have the opportunity to learn and work with the following methodologies:i) Development and characterization of brain organoids with EHMT2 mutations;ii) In vitro evaluation of EHMT2 enzymatic activity;iii) Evaluation of histone marks by ChIP-Seq;iv) Correction of variants by CRISPR-Cas9 to assess potential rescue of the wild-type phenotype.