Using VAAST to identify an X-linked disorder resulting in lethality in male infants due to N-terminal acetyltransferase deficiency

Abstract

We have identified two families with a previously undescribed lethal X-linked disorder of infancy; the disorder comprises a distinct combination of an aged appearance, craniofacial anomalies, hypotonia, global developmental delays, cryptorchidism, and cardiac arrhythmias. Using X chromosome exon sequencing and a recently developed probabilistic algorithm aimed at discovering disease-causing variants, we identified in one family a c.109T>C (p.Ser37Pro) variant in NAA10, a gene encoding the catalytic subunit of the major human N-terminal acetyltransferase (NAT). A parallel effort on a second unrelated family converged on the same variant. The absence of this variant in controls, the amino acid conservation of this region of the protein, the predicted disruptive change, and the co-occurrence in two unrelated families with the same rare disorder suggest that this is the pathogenic mutation. We confirmed this by demonstrating a significantly impaired biochemical activity of the mutant hNaa10p, and from this we conclude that a reduction in acetylation by hNaa10p causes this disease. Here we provide evidence of a human genetic disorder resulting from direct impairment of N-terminal acetylation, one of the most common protein modifications in humans.

Figure 1

Triptych of Individual III-4 from Family 1 These pictures demonstrate the prominence of eyes, down-slanted palpebral fissures, thickened lids, large ears, flared nares, hypoplastic alae, short columella, protruding upper lip, and microretrognathia.

Figure 2

Pedigree Drawing and Pictures of Families 1 and 2 (A) Pedigree drawing for family 1. The most recent deceased individual, III-4, is the most well-studied subject in the family and is indicated by an arrow. Genotypes are marked for those in which DNA was available and tested. The following abbreviations are used: SB, stillborn; +, normal variant; mt, rare mutant variant. (B) Pictures of four affected and deceased boys in this family, showing the aged appearance. (C) Sanger sequencing results of NAA10 in individual III-4 from family 1. (D) Pedigree for family 2. Individual III-2 is the most well-studied subject in the family and is indicated by an arrow. (E) Picture of individuals II-I and III-2 in family 2 at ∼1 year of age.

Figure 3

IBD Analysis of the Two Families in the Genomic Region Surrounding c.109T>C Informative SNVs consist of two classes: rare SNVs present in the proband from family 1 in genomic regions with high coverage in the second family, and common SNVs present in the first proband and at least one member of the second family. Variant c.109T>C is indicated by the triangle. We imputed the genotype of the proband from family 2 from the genotypes of the mother and unaffected sibling of the second family (see Table 7). SNVs inconsistent with IBD, in which the imputed genotype of the second proband does not match the first proband, are indicated with an X. After allowing for multiple sequencing errors, the largest genomic segment consistent with IBD is around 700 kb in length.

Figure 4

NAT Activity of Recombinant hNaa10p WT or p.Ser37Pro toward Synthetic N-Terminal Peptides (A and B) Purified MBP-hNaa10p WT or p.Ser37Pro were mixed with the indicated oligopeptide substrates (200 μM for SESSS and 250 μM for DDDIA) and saturated levels of acetyl-CoA (400 μM). Aliquots were collected at indicated time points and the acetylation reactions were quantified with reverse phase HPLC peptide separation. Error bars indicate the standard deviation based on three independent experiments. The five first amino acids in the peptides are indicated, for further details see Subjects and Methods. Time-dependent acetylation reactions were performed to determine initial velocity conditions when comparing the WT and Ser37Pro NAT activities toward different oligopeptides. (C) Purified MBP-hNaa10p WT or p.Ser37Pro were mixed with the indicated oligopeptide substrates (200 μM for SESSS and AVFAD and 250 μM for DDDIA and EEEIA) and saturated levels of acetyl-CoA (400 μM) and incubated for 15 min (DDDIA and EEEIA) or 20 min (SESSS and AVFAD) at 37°C in acetylation buffer. The acetylation activity was determined as above. Error bars indicate the standard deviation based on three independent experiments. Black bars indicate the acetylation capacity of the MBP-hNaa10p WT, whereas white bars indicate the acetylation capacity of the MBP-hNaa10p mutant p.Ser37Pro. The five first amino acids in the peptides are indicated.