The patient is a girl, now 11 years old, born three weeks preterm by section due to transverse lie, birth weight 2720 g. There were no feeding difficulties in infancy or later. Motor and language development was delayed: she walked without support at age 2½ years, and at age 3 years she said her first words. At age 10 years she knew the alphabet and tried to put letters together. She used diapers until age 8–9 years. Her sleep pattern is mildly irregular with frequent awakenings. Congenital malformations or epilepsy have never been detected. She has normal stature with length along the 25th–50th centile and weight along the 25th centile, but her head circumference has been in the lower normal range (2.5th - 5th centile). Her facial features are also normal - she is not clearly dysmorphic. Behavior is normal without autistic traits, but bruxism is a problem. She prefers the company of younger children. Her intellectual level is judged to be comparable to mild-moderate ID, formal IQ testing has not yet been done. At age 9 years trio whole exome sequencing (WES), comparing child to parental DNA sequences, revealed a de novo NAA10 (NM_003491.3) c.332 T > G p.V111G variant which was further confirmed by Sanger sequencing. In line with other NAA10 missense variants, the V111G substitution affects a highly conserved amino acid. NAA10 is a 235 amino acid protein which adapts the characteristic GNAT fold common to NAT catalytic subunits. This fold consists of six or seven β strands and four α helices. β-strands 2-5 constitute the core of the protein. These four β strands, together with the α2 helix and the β6-β7 loop important for substrate binding, are highly conserved. V111 is located towards the end of the β5 strand, and a valine in this position is highly conserved in NAA10 homologues as well as in several other NAT catalytic subunits for which crystal structures have been solved, 4U9W (NAA40), 3FTY (NAA50), 5ICV (NAA60) and 4LX9 (ssNAT)) [–]. The side chain of V111 is forming a hydrophobic pocket together with Y145, M147, L119 and L109. It is also in close proximity (3.7 Å) to the sulphur group of Acetyl-CoA (AcCoA), which could indicate a role for V111 in positioning of AcCoA. A glycine in this position will not cause any steric clashes, but loss of the more bulky hydrophobic side chain of valine may possibly cause structural alterations affecting protein stability or AcCoA binding. In order to functionally assess NAA10-V111G, we first expressed His/MBP-NAA10-WT and His/MBP-NAA10-V111G in E.coli, purified enzymes and tested the in vitro NAT activity. Contrary to His/MBP-NAA10-WT, it was difficult to obtain good protein expression of His/MBP-NAA10-V111G, and a substantial fraction of the purified His/MBP-NAA10-V111G molecules eluted in the void volume of the size exclusion chromatography column. This indicate that parts of the protein aggregate in units larger than 200 kDa, most likely due to an alteration of the protein structure, or reduced protein stability. Enzymes that eluted at a column volume corresponding to monomeric His/MBP-NAA10-V111G were tested for catalytic activity and shown to have an approximately 85% reduction in catalytic activity compared to His/MBP-NAA10-WT. Due to the low expression levels and protein yield of NAA10-V111G from E.coli transfection and protein purification, we transfected HeLa cells with plasmids coding for either V5-tagged NAA10-WT or V5-tagged NAA10-V111G followed by immunoprecipitation (IP) of the overexpressed protein by an anti-V5 antibody. Thereafter, NAT activity in the precipitate was measured. NAA10 and NAA15 form a high-affinity protein complex, and as expected endogenous NAA15 co-immunoprecipitated with both overexpressed NAA10-WT-V5 and NAA10-V111G-V5. The amount of NAA10 and NAA15 present in each sample were determined by SDS-PAGE and Western blotting. Bands from the Western blot were quantified, and the measured catalytic activity was correlated with the amount of NAA10-V5 present in the sample (i.e. a mixture of monomeric NAA10 and NAA10 in complex with NAA15 – the NatA complex), and separately correlated with the amount of NAA15 present in the sample (i.e. the amount of the NatA complex only). Results from the IP-activity experiment corresponded well with our previous finding: the ability of NAA10-V111G to acetylate the acidic N-termini EEEI24 was greatly reduced. However, it also revealed a second interesting feature: As can be seen from the Western blot in Fig., more NAA15 co-immunoprecipitated with NAA10-V111G-V5 compared to NAA10-WT-V5. This was also reflected in our NAT-activity measurements where the immunoprecipitated NAA10-V111G-V5 sample showed higher NatA product formation (for the NatA substrate polypeptide SESS24) compared to the immunoprecipitated NAA10-WT-V5 sample when correlating for the amount of total NAA10-V5 present in the sample. However, when correlating for the amount of NAA15 present in each sample, the NatA product formation per NAA15 molecule (and thus NatA complex) was approximately equal. As monomeric NAA10 has a 1000-fold lower NAT-activity towards NatA substrates compared to the NatA complex [], the contribution of monomeric NAA10 on acetylation of SESS24 is minimal. Taken together, this suggest that NAA10-V111G has reduced catalytic activity in a monomeric form, but not in complex with NAA15. In order to assess protein turnover of NAA10-WT and NAA10-V111G, we expressed V5-tagged NAA10-WT and NAA10-V111G in HeLa cells and performed cycloheximide-chase experiments. As can be seen from Fig., NAA10-V111G-V5 has a higher turnover rate than NAA10-WT-V5. 2 h after cycloheximide treatment, the average amount of NAA10-V111G-V5 was reduced by approximately 80%, while NAA10-WT-V5 molecules was reduced by 20% relative to the amount of NAA10 molecules before cycloheximide treatment. Although the amount of NAA10-V111G-V5 is drastically decreased after 2 h, the level of NAA10-V111G-V5 seems to stabilize at around 20%. Most likely overexpressed NAA10-V111G-V5 is present both in an unstable monomeric form that is rapidly degraded and in complex with NAA15, which stabilizes the enzyme and protects it from degradation.