Mechanisms of Congenital Heart Disease Caused by NAA15 Haploinsufficiency

Abstract

Rationale: NAA15 is a component of the N-terminal (Nt) acetyltransferase complex, NatA. The mechanism by which NAA15 haploinsufficiency causes congenital heart disease (CHD) remains unknown. To better understand molecular processes by which NAA15 haploinsufficiency perturbs cardiac development, we introduced NAA15 variants into human induced pluripotent stem cells (iPSCs) and assessed the consequences of these mutations on RNA and protein expression. Objective: We aim to understand the role of NAA15 haploinsufficiency in cardiac development by investigating proteomic effects on NatA complex activity, and identifying proteins dependent upon a full amount of NAA15. Methods and Results: We introduced heterozygous LoF, compound heterozygous and missense residues (R276W) in iPS cells using CRISPR/Cas9. Haploinsufficient NAA15 iPS cells differentiate into cardiomyocytes, unlike NAA15-null iPS cells, presumably due to altered composition of NatA. Mass spectrometry (MS) analyses reveal ~80% of identified iPS cell NatA targeted proteins displayed partial or complete Nt-acetylation. Between null and haploinsufficient NAA15 cells Nt-acetylation levels of 32 and 9 NatA-specific targeted proteins were reduced, respectively. Similar acetylation loss in few proteins occurred in NAA15 R276W iPSCs. In addition, steady-state protein levels of 562 proteins were altered in both null and haploinsufficient NAA15 cells; eighteen were ribosomal-associated proteins. At least four proteins were encoded by genes known to cause autosomal dominant CHD. Conclusions: These studies define a set of human proteins that requires a full NAA15 complement for normal synthesis and development. A 50% reduction in the amount of NAA15 alters levels of at least 562 proteins and Nt-acetylation of only 9 proteins. One or more modulated proteins are likely responsible for NAA15-haploinsufficiency mediated CHD. Additionally, genetically engineered iPS cells provide a platform for evaluating the consequences of amino acid sequence variants of unknown significance on NAA15 function.

Charting the N-Terminal Acetylome: A Comprehensive Map of Human NatA Substrates

“…In line with previous reports [ 21 , 27 , 37 ], and as evidenced from protein expression levels when probing for the NatA subunits by means of Western blotting, exclusive knockdown of NAA10 also results in a significant destabilization of NAA15 ( Figure S1 ).…”

“…By making use of CRISPR/Cas9 gene editing, NAA15 haplo-insufficient and deficient induced human pluripotent stem cells (iPSCs) could successfully be obtained. Interestingly, in these iPSCs, a mere partial effect on NatA-dependent NTA acetylation was observed [ 37 ]. In case of NAA15 deficiency and since a role for NAA16-NAA10 complexes has been proposed to act as a potential backup system for the NAA15-NAA10 NatA complex [ 38 ], akin NAA11 expression, expression of its homologue NAA16 in human cells may also (in part) be responsible for this observation.…”

Naa12 compensates for Naa10 in mice in the amino-terminal acetylation pathway

“…Although this was not a significant result statistically (p=0.09), it is worth noting that peptidyl-prolyl cis-trans isomerase A (PPIA), having a 10.3% decrease in amino-terminal acetylation, was previously identified with decreased amino-terminal acetylation in patient-derived B cells and fibroblasts in boys with the S37P mutation in NAA10 [25], along with being decreased in siNatA knockdown HeLa cells [1]. PPIA also had decreased amino-terminal acetylation in one sample from homozygous null NAA15 L314*/L314* induced pluripotent stem cells [47].…”

From Stem Cells to Populations—Using hiPSC, Next-Generation Sequencing, and GWAS to Explore the Genetic and Molecular Mechanisms of Congenital Heart Defects

“…A recent study demonstrated how the haploinsufficiency of NAA15 , a known CHD gene, perturbs cellular function in hiPS-CMs. Heterozygous loss of function, compound heterozygous, and missense NAA15 variants were introduced into hiPSCs using CRISPR/Cas9 gene editing, and the consequences of these mutations on hiPS-CM phenotype and RNA and protein expression were assessed [ 82 ]. The study identified four CHD-causing genes ( DHCR7 , MAP2K2 , NSD1 , and RPL5 ) that were differentially expressed in NAA15 haploinsufficiency, providing molecular-level mechanisms behind the pathogenicity.…”

“…The study identified four CHD-causing genes ( DHCR7 , MAP2K2 , NSD1 , and RPL5 ) that were differentially expressed in NAA15 haploinsufficiency, providing molecular-level mechanisms behind the pathogenicity. Importantly, the study provided a reference on how to estimate the pathogenicity of NAA15 variants of uncertain significance in patient-derived cells [ 82 ].…”