Mutations in ACY1, the gene encoding aminoacylase 1, cause a novel inborn error of metabolism

Abstract

N-terminal acetylation of proteins is a widespread and highly conserved process. Aminoacylase 1 (ACY1; EC 3.5.14) is the most abundant of the aminoacylases, a class of enzymes involved in hydrolysis of N-acetylated proteins. Here, we present four children with genetic deficiency of ACY1. They were identified through organic acid analyses using gas chromatography-mass spectrometry, revealing increased urinary excretion of several N-acetylated amino acids, including the derivatives of methionine, glutamic acid, alanine, leucine, glycine, valine, and isoleucine. Nuclear magnetic resonance spectroscopy analysis of urine samples detected a distinct pattern of N-acetylated metabolites, consistent with ACY1 dysfunction. Functional analyses of patients' lymphoblasts demonstrated ACY1 deficiency. Mutation analysis uncovered recessive loss-of-function or missense ACY1 mutations in all four individuals affected. We conclude that ACY1 mutations in these children led to functional ACY1 deficiency and excretion of N-acetylated amino acids. Questions remain, however, as to the clinical significance of ACY1 deficiency. The ACY1-deficient individuals were ascertained through urine metabolic screening because of unspecific psychomotor delay (one subject), psychomotor delay with atrophy of the vermis and syringomyelia (one subject), marked muscular hypotonia (one subject), and follow-up for early treated biotinidase deficiency and normal clinical findings (one subject). Because ACY1 is evolutionarily conserved in fish, frog, mouse, and human and is expressed in the central nervous system (CNS) in human, a role in CNS function or development is conceivable but has yet to be demonstrated. Thus, at this point, we cannot state whether ACY1 deficiency has pathogenic significance with pleiotropic clinical expression or is simply a biochemical variant. Awareness of this new genetic entity may help both in delineating its clinical significance and in avoiding erroneous diagnoses.

Figure 1

A, Deacetylation of N-acetylamino acids that is catalyzed by ACY1. For R=(CH₂)₂SCH₃ and R^′=CH₃, the N-acetylamino acid is N-acetylmethionine, a preferred substrate of ACY1. B and C, Organic acids detected by GC-MS in the urine from (B) a patient (OS-104 II-1) with ACY1 deficiency and (C) a parent as a control. Abbreviations used for peak labels are as follows: ME = methyl ester; IS = internal standard (isopropylmalonic acid diME); 1 = N-acetylalanine ME; 2 = N-acetylvaline ME; 3 = N-acetylisoleucine ME; 4 = N-acetylglycine ME; 5 = N-acetylleucine ME; 6 = N-acetylmethionine ME, 7 = N-acetylglutamic acid diME. Consistent with lack of enzymatic deacetylation, the urinary concentrations of N-acetylamino acids are increased in the ACY1-deficient individual.

Figure 2

Assessment of ACY1 activity in lymphoblasts of 3 patients (OS 104 II-1, OS 124 II-1, and OS 127 II-1) and of 19 controls revealed much lower enzyme activity in the patients’ cells (P=.001 in Student’s t test).

Figure 3

Results of the mutational analysis of ACY1 in four patients with ACY1 deficiency. A, Sequence chromatograph from the affected individual OS-124 II-1 and his consanguineous parents (father, OS-124 I-1; mother, OS-124 I-2), showing a homozygous G→A transversion at position −1 of the obligatory exon 6 splice-acceptor site [IVS5−1G→A] in the patient and heterozygous mutations in both parents, consistent with homozygosity by descent. B, Sequence chromatograph from a control individual and the affected individual OS-104 II-1, depicting a homozygous 2-bp insertion in exon 15 resulting in a frame shift [1105^1106insAC] and a postmature stop codon [369PfsX46]. C, Two sequence chromatographs of the affected individual OS-120 II-1 depicting her compound heterozygous missense mutations 699A→C in exon 10 (E233D) and 1057C→T in exon 14 (R353C), which predicts the substitutions of evolutionary conserved amino acid residues E233D and R353C, respectively. D, Sequence chromatograph from the affected individual OS-127 II-1, showing the homozygous missense mutation 1057C→T in exon 14 (R353C).

Figure 4

Sequence alignment of human ACY1 and corresponding orthologs from various species. Highlighted letters and letters on a gray background represent identical and conserved amino acid residues, respectively. Most parts of the orthologous proteins are highly conserved between species and predict a large peptidase domain, which includes a central peptidase dimerization region. Human ACY1 shares amino acid sequence identity and similarity, respectively, with M. musculus (85%/91%), R. norvegicus (87%/93%), X. laevis (60%/76%), and D. rerio (61%/77%). The amino acids affected by the missense mutations (E233D and R353C) are marked with arrows. The obligatory acceptor splice-site mutation [IVS5−1G→A] is marked with an asterisk (*) and predicts the skipping of exon 6 (aa 121 to aa 146) and a premature stop codon [120QfsX1]. The 2-bp insertional mutation [1105^1106insAC] is marked with an arrowhead and predicts a frame shift leading to a severely altered C-terminus [369PfsX46]. Ortholog proteins were identified by BLAST search. The multiple-protein alignment was constructed using the MAP program. Functional domains were predicted with the Scansite program. The corresponding accession numbers of the protein sequences are: Homo_sapiens AAH14112, Mus_musculus AAH05631, Rattus_norvegicus AAH78930, Xenopus_laevis AAH77639, and Danio_rerio NP_957289.

Figure 5

Human adult tissue northern blot of the ACY1 gene. The ACY1 probe detects a single band of ∼1.6 kb, which corresponds to the predicted size of the human cDNA. Expression is highest in kidney, strong in brain, and weaker in placenta and spleen. ACY1 mRNA is also expressed in uterus and lung. RNA size markers are indicated on the left side.