Functional interpretation of ATAD3A variants in neuro-mitochondrial phenotypes

Background The ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane anchored protein involved in diverse processes including mitochondrial dynamics, mitochondrial DNA organization, and cholesterol metabolism. Biallelic deletions (null), recessive missense variants (hypomorph), and heterozygous missense variants or duplications (antimorph) in ATAD3A lead to neurological syndromes in humans. Objective To expand the mutational spectrum of ATAD3A variants and to provide functional interpretation of missense alleles in trans to deletion alleles. Methods Exome sequencing was used to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in ATAD3A in individuals with neurological and mitochondrial phenotypes. A Drosophila Atad3A Gal4 trap null allele was generated using CRISPR-Cas9 genome editing technology to aid interpretation of variants. Results We report 13 individuals from 8 unrelated families with biallelic ATAD3A variants. Four of the identified missense variants, p.(Leu77Val), p.(Phe50Leu), p.(Arg170Trp), p.(Gly236Val), were inherited in trans to loss-of-function alleles. A fifth missense variant, p.(Arg327Pro), was homozygous. Affected individuals exhibited findings previously associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Drosophila studies indicated that Phe50Leu, Gly236Val, and Arg327Pro are severe loss-of-function alleles leading to early developmental lethality and neurogenesis defects, whereas Leu77Val and Arg170Trp are partial loss of function alleles that cause progressive locomotion defects. Moreover, Leu77Val and Arg170Trp expression leads to an increase in autophagy and mitophagy in adult muscles. Conclusion Our findings expand the allelic spectrum of ATAD3A variants, and exemplify the use of a functional assay in Drosophila to aid variant interpretation.

pUASTattB-dAtad3a L83V -V5, pUASTattB-dAtad3a F56L -V5, pUASTattB-dAtad3a R176W -V5, 1 pUASTattB-dAtad3a G242V -V5, and pUASTattB-dAtad3a R333P -V5 were generated by site-directed 2 mutagenesis PCR using primers: (L83V)-F: 5'-  Western Blotting 20 Fly heads were homogenized in 1x Laemmli sample buffer containing 2.5% b-mercaptoethanol 21 (Sigma-Aldrich). After boiling for 10 min, samples were loaded into 4-20% Mini-PROTEAN® Drosophila Climbing Assay 1 Method was adapted from Madabattula et al. (2015). 25 25 flies were anesthetized using CO2 and 2 allowed to rest in fresh food vials 24 hours at 25°C prior to the assay. Male and female flies were 3 kept separately as gender difference on behavior might be significant. To prepare the climbing 4 apparatus, measure a distance of 8 cm from the bottom surface of an empty polystyrene vial and 5 mark the distance by drawing a line around the entire circumference of the vial. Flies were 6 transferred without using CO2 into different climbing apparatus for each genotype to prevent 7 cross contamination. The apparatus was closed off by vertically joining to another empty 8 polystyrene vial using tape and the flies were left to acclimatize to the surrounding for at least 10 9 min. Then, the apparatus was gently tapped five times to displace the flies to the bottom of the 10 apparatus and a video was recorded for 20 s to measure the number of flies able to cross the 11 height of 8 cm at each time point. After a 10 min rest, the assay was repeated. Three trials were 12 conducted. fixative. The pre-fixed thoraxes were then irradiated and fixed again, followed by 3x millipore 20 water rinses, post-fixed with 1% aqueous Osmium Tetroxide, and 1% Potassium Ferrocyanide 21 mixture in Millipore water. This was followed by 3X Millipore water rinses. Ethanol 22 concentrations from 30-100% were used for the initial dehydration series, followed with 100% 23 1 propylene oxide and Embed 812, finally going into 3 changes of pure resin under vacuum.

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Samples were allowed to infiltrate in pure resin overnight on a rotator. The samples were 3 embedded into flat silicone molds and cured in the oven at 62 o C for at least three days. The 4 polymerized samples were thin-sectioned at 48-50 nm and stained with 1% uranyl acetate for 5 thirteen minutes followed by 2.5% lead citrate for two and a half minutes the day before TEM 6 examination. Grids were viewed in a JEOL 1400 Plus transmission electron microscope at 7 80kV. Images were captured using an AMT XR-16 mid-mount 16 mega-pixel digital camera in 8 Sigma mode. Images were contrast adjusted in Image J.  Table 1 and Table S1 (Clinical information -Additional file 1). Briefly, individuals with 6 biallelic deletion CNVs showed a neonatal-lethal presentation with respiratory failure, 7 generalized hypotonia, seizures, congenital contractures, bilateral ophthalmologic findings, and 8 brain anomalies, consistent with the reported phenotype. 12 Individuals with a loss-of-function 9 allele (intergenic CNV, intragenic CNV, or frameshift SNV) inherited in trans to a missense 10 variant (Families 3-6) presented with varied severity of the phenotype ( Table 1, and Table S1 -     by one carbon. On the contrary, Arg170 is predicted to be surface exposed. Hence, the  To test expression patterns of dAtad3a, we generated flies having a dAtad3a-T2A-Gal4 allele with UAS-mCD8::GFP. We found that dATAD3A is expressed ubiquitously during 1 embryogenesis, and that the expression pattern includes neurons in the brain and ventral nerve 2 cord (VNC) ( Figure S1). dATAD3A remains highly expressed in brain in both larval and adult 3 stages. Moreover, dATAD3A is expressed in the adult thorax and in peripheral neurons in adult 4 wings ( Figure 2B).

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To test whether the dAtad3a-T2A-Gal4 allele is a loss of function mutation, we  Figure 2E). In addition, dAtad3a mutant larvae expressing dAtad3a L83V , or dAtad3a R176W 15 exhibited comparable mitochondrial content to those of flies expressing dAtad3a WT ( Figure S2). 16 These results indicate that the flies carrying L83V and R176W variants did not exhibit 17 developmental defects. Hence, the results indicate that F56L, G242V, and R333P are severe loss-18 of-function alleles, whereas L83V and R176W only mildly affect gene function.

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To investigate the phenotypic strength of F56L, G242V, and R333P, we decided to 20 characterize phenotypes during embryogenesis from dAtad3a null mutants as well as each  To investigate the phenotypes of L83V and R176W, we sought to characterize post-20 developmental phenotypes such as behavioral and age-associated phenotypes because expression 21 of these variants did not exhibit developmental defects ( Figure 2E, Figure S2). First, we 22 performed life-span assays and found that dAtad3a null flies expressing L83V, or R176W ( Figure S3). These results indicate that both R176W and L83V variants affect mitochondrial 1 dynamics, which in turn may lead to increased autophagy or mitophagy.

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To test whether dAtad3a carrying R176W or L83V variants cause an increase in in wild-type controls ( Figure 5B). We found that in muscles expressing R176W, most and L83V variants lead to increased autophagy and mitochondria loss, and aberrant cristae, and reading of this manuscript. We appreciate Dr. Sheng Zhang's kind gift of rabbit anti-Ref2(P)