PurposeN-terminal acetyltransferases modify proteins by adding an acetylmoiety to the first amino acid and are vital for protein and cell function. TheNatB complex acetylates 20% of the human proteome and is composed of thecatalytic subunit NAA20 and the auxiliary subunit NAA25. In five individualswith overlapping phenotypes, we identified recessive homozygous missensevariants in NAA20. MethodsTwo different NAA20 variants wereidentified in affected individuals in two consanguineous families by exome andgenome sequencing. Biochemical studies were employed to assess the impact of theNAA20 variants on NatB complex formationand catalytic activity. ResultsTwo homozygous variants, NAA20p.Met54Val and p.Ala80Val (GenBank: NM_016100.4, c.160A>G andc.239C>T), segregated with affected individuals in two unrelatedfamilies presenting with developmental delay, intellectual disability, andmicrocephaly. Both NAA20-M54V and NAA20-A80V were impaired in their capacity toform a NatB complex with NAA25, and in vitro acetylation assays revealed reducedcatalytic activities toward different NatB substrates. Thus, both NAA20 variantsare impaired in their ability to perform cellular NatB-mediated N-terminalacetylation. ConclusionWe present here a report of pathogenic NAA20 variants causing human disease and data supporting anessential role for NatB-mediated N-terminal acetylation in human development andphysiology. Graphical Abstract [Display omitted]