Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism

Abstract

Recent studies have identified both recessive and dominant forms of mitochondrial disease that result from ATAD3A variants. The recessive form includes subjects with biallelic deletions mediated by non-allelic homologous recombination. We report five unrelated neonates with a lethal metabolic disorder characterized by cardiomyopathy, corneal opacities, encephalopathy, hypotonia, and seizures in whom a monoallelic reciprocal duplication at the ATAD3 locus was identified. Analysis of the breakpoint junction fragment indicated that these 67 kb heterozygous duplications were likely mediated by non-allelic homologous recombination at regions of high sequence identity in ATAD3A exon 11 and ATAD3C exon 7. At the recombinant junction, the duplication allele produces a fusion gene derived from ATAD3A and ATAD3C, the protein product of which lacks key functional residues. Analysis of fibroblasts derived from two affected individuals shows that the fusion gene product is expressed and stable. These cells display perturbed cholesterol and mitochondrial DNA organization similar to that observed for individuals with severe ATAD3A deficiency. We hypothesize that the fusion protein acts through a dominant-negative mechanism to cause this fatal mitochondrial disorder. Our data delineate a molecular diagnosis for this disorder, extend the clinical spectrum associated with structural variation at the ATAD3 locus, and identify a third mutational mechanism for ATAD3 gene cluster variants. These results further affirm structural variant mutagenesis mechanisms in sporadic disease traits, emphasize the importance of copy number analysis in molecular genomic diagnosis, and highlight some of the challenges of detecting and interpreting clinically relevant rare gene rearrangements from next-generation sequencing data.

Keywords: ATAD3; ATAD3 gene cluster; Harel-Yoon; NAHR; cardiomyopathy; cholesterol; metabolic disorder; mitochondrial DNA; non-allelic homologous recombination.

Figure 1

NAHR between ATAD3C Exon 8 and ATAD3A Exon 11 Produces a Fusion Gene, with Variants at Key Functional Residues within the ATPase Domain Gene intron-exon structures are shown in cartoon format; open boxes indicate UTRs while closed boxes indicate coding regions. Arrows following the gene name indicate reading direction, and the first exon is labeled. Genes are shown in their relative position on chromosome 1 in a 5′ to 3′ direction from left to right. (A) Nucleotide sequence identity between ATAD3A (chr1:1512151–1534687:1) and ATAD3C (chr1:1449689–1470158:1) in a sliding 500 bp window. ATAD3A and ATAD3C exon positions are represented below according to their relative position within the KAlign alignment; this includes alignment gaps. The 398 bp region of 100% sequence identity is marked in yellow. (B) Reference arrangement of the ATAD3 cluster showing the exon structures of ATAD3C (purple), ATAD3B (orange), and ATAD3A (green). The duplicated region is highlighted in red. (C) The reference arrangement of the ATAD3 cluster above the predicted configuration following duplication. (D) The exon structure of the ATAD3A-C fusion gene, with exons 1–11 derived from ATAD3A (green) and exons 12–16 derived from ATAD3C (purple). The ATPase domain is underlined (Asn347-Leu475; PFam PF00004), with the position of a key functional residue, Arg466, indicated by an arrow. (E) Amino acid sequence of the ATPase domain of ATAD3A (top) and the predicted amino acids sequence of the ATAD3A-C fusion protein (bottom). The green residues are derived from ATAD3A, while the purple residues are derived from ATAD3C. A vertical bar (|) indicates an identical amino acid, a colon (:) indicates a strongly conservative amino acid change (score > 0.5 in PAM250 matrix), and a period (.) indicates a weakly conservative amino acid change (score = < 0.5 in PAM250 matrix). The sequences differ at seven positions.

Figure 2

Elevated ATAD3 and Free Cholesterol Levels in Fibroblasts Harboring the ATAD3 Gene Cluster Duplication (A) Level of ATAD3 in fibroblast of subject 1 compared to control subjects (Fiji ImageJ densitometric analysis). The data are the mean of n = 6 independent experiments using three different control cell lines. Error bars show 1 standard deviation (^∗∗p < 0.01; Welch’s t test). (B) A representative ATAD3 immunoblot using a pan-specific antibody in fibroblasts. Levels of GAPDH were used as indicators of protein loading. The increased signal of the upper band [B] is consistent with the duplication of ATAD3B. ATAD3A isoform 2 and the predicted ATAD3A-C fusion protein are of identical size; hence, the increased signal of the lower band [A] is consistent with the fusion gene being expressed and stable. (C) Chart showing mean filipin signal of cells quantified by ImageJ. Subject 1: fibroblasts of an individual with the ATAD3 gene cluster duplication; Deletion: fibroblasts of an individual with a biallelic ATAD3 gene cluster deletion (see Desai et al. for details); U18: U18666A is an inhibitor of cholesterol trafficking; Filipin is a fluorescent marker, which binds specifically to unesterified cholesterol. Data are the results of 8 independent experiments for subject 1 and control subject(s) and n = 6 for the “deletion.” Error bars show 1 standard deviation (^∗∗∗p < 0.001; ^∗∗p ≤ 0.01; one-way ANOVA). (D) Representative images of filipin-stained cells. Scale bar 10 μm.

Figure 3

Abnormal Mitochondrial Morphology and mtDNA Organization in Cells with an ATAD3 Gene Cluster Duplication (A) Confocal images showing the mitochondria of control cell lines (C2) and fibroblasts from subject 1 (S1) labeled with an antibody to the outer mitochondrial membrane protein TOMM20 (red). Proportion of cells with clumped mitochondria for subject 1 versus 2 control subjects (n = 2 independent experiments, ≥50 cells per cell line, per experiment). (B) Fibroblast cells from control subject (C1) and subject 1 (S1) labeled with an antibody against TOMM20 (red), a DNA antibody (green), and DAPI (blue); arrows indicate mtDNA aggregation. Scale bars 10 μm. Error bars show 1 standard deviation.

Figure 4

Protein Modeling of ATAD3 Hexamer and 3D Alignment against SPAST ATPase Domain (A) Hexameric structure of ATAD3A ATPase domain (amino acids 348–474), modeled in SwissModel using PDB: 6f0x (H. sapiens, TRIP13) as a template. A single monomer is highlighted in violet. (B) Single ATAD3A monomer (violet) aligned to H. sapiens SPAST ATPase domain (blue). (C) The ATAD3A arginine finger, Arg466 (yellow) which is changed to a cysteine in the ATAD3A-C fusion gene, is overlaid with the SPAST arginine finger (Arg499; orange).