Functional null allele

Additional file 2 : Figure S11 summarizes the various intergenic and intragenic deletions identified in this study. Phe50Leu , c. Leu77Val , c. ArgTrp , c. GlyVal , c. All of the altered residues are evolutionarily conserved in species of the animal kingdom Fig. Notably, the p. Leu77Val variant family 3 affects the same amino acid as a previously reported variant p. Leu77Arg [ 13 ]. In both cases, the variant was inherited in trans to a deletion or stopgain variant, and resulted in a severe clinical presentation including congenital cataract or corneal clouding, brain abnormalities, and seizures.

This further supports the pathogenicity of the p. Leu77Val variant. The p. On the contrary, Arg is predicted to be surface exposed. Hence, the p. ArgTrp variant may increase surface hydrophobicity, reducing solubility and resulting in a less stable protein. GlyVal variant may affect the interaction between this potential transmembrane helix and the mitochondrial inner or outer membrane or between neighboring transmembrane helices of the ATAD3A hexamer due to the longer side chain.

Arg is located next to the ATP binding pocket. The side chain of Arg forms a salt bridge with the carboxylate of the Glu side chain and also makes a hydrogen bond interaction with the main chain carbonyl of Met Fig. This interaction would be abolished by the p.

ArgPro variant, resulting in structural changes that could impact ATP binding. K is a part of the long helix near the C-terminus which is solvent-exposed toward the C-terminus of the helix.

K is followed by 4 hydrophobic residues of which 3 are predicted to be part of the helix. In addition to losing one charged residue, deletion of K may also cause structural disruption of the helix and the following loop and expose those hydrophobic residues to the solvent, resulting in low protein stability.

These flies produce an N-terminal portion of the dATAD3A protein as well as the Gal4 protein whose expression is under control of endogenous cis -elements of dAtad3a. Gal4 is a transcriptional activator that drives the expression of transgenes by binding upstream activating sequence UAS Fig. The T2A-Gal4 cassette consists of a splice acceptor SA, light gray followed by a ribosomal skipping T2A peptide sequence pink , a Gal4 coding sequence green , and a polyadenylation signal light blue.

These data indicate that dAtad3a-T2A-Gal4 is a loss-of-function mutant. Five replicates were quantified. Error bars indicate SEM. To test whether the dAtad3a-T2A-Gal4 allele is a loss-of-function mutation, we performed complementation studies. Hence, the results indicate that dAtad3a-T2A-Gal4 is a severe loss of function allele.

All transgenes carry a C-terminal V5 tag. Western blot analysis for adult heads revealed that no protein was detected from dAtad3a GV expression, and the protein levels of dAtad3a RP and dAtad3a Kdel were lower than those in wild-type control dAtad3a WT Fig. Hence, these results indicate that the GV variant is a protein null allele and that RP and Kdel moderately affect protein levels. We found that expression of dAtad3a F56L , dAtad3a GV , dAtad3a RP , or dAtad3a Kdel completely failed to rescue the lethality caused by loss of dAtad3a , indicating that these four variants are severe loss of function alleles Fig.

Failure of lethality rescue by GV is consistent with the Western results showing complete loss of the protein with this mutation Fig. These results indicate that the flies carrying L83V and RW variants did not exhibit developmental defects. To investigate the phenotypic strength of F56L, GV, and RP, we decided to characterize phenotypes during embryogenesis from dAtad3a null mutants as well as each mutant because most animals expressing these variants die before the 1st instar larvae stage.

No reports for phenotypes caused by dAtad3a loss during embryogenesis have been documented so far. Biallelic deletion of ATAD3A and adjacent ATAD3 paralogs in humans causes severe neuro-developmental defects including congenital pontocerebellar hypoplasia and neonatal death [ 7 , 12 ] Thus, we sought to determine whether dAtad3a loss causes defects in neurodevelopment in Drosophila embryos using anti-Elav a neuronal marker and anti-HRP a marker for neuronal membranes antibodies.

We examined stage 15 embryos in which the central nervous system CNS including the brains and ventral nerve cord VNC , and the peripheral neurons and their neuronal projections are well established Fig. The dAtad3a null mutants exhibit a wide range of neurogenesis defects. Finally, the null mutants and three loss-of-function LOF alleles exhibited an abnormal increase in mitochondrial content and size, as compared to the null mutant embryos complemented with wild-type dAtad3a and as compared to wild-type control embryos Additional file 2 : Figure S14, S Hence, we discovered that dAtad3a loss leads to severe neurodevelopmental defects in Drosophila embryos.

Elav green -stained neurons and anti-HRP red -stained neuronal membranes. Br indicates brain, and VNC indicates ventral nerve cord. Arrowheads indicate shrunken and twisted VNC. Arrows indicate misguided and loss of neurons in the PNS. To investigate the phenotypes of L83V and RW, we sought to characterize post-developmental phenotypes such as behavioral and age-associated phenotypes because the expression of these variants did not exhibit developmental defects Fig.

To test this, we performed a climbing assay. We found that both variants exhibited age-dependent locomotion defects, and RW showed more severe locomotion defects compared to L83V flies Fig. We also performed a flight assay that is more sensitive than the climbing assay. Flies expressing RW exhibited a flight defect at a young age day 5 and failed to fly at all in old age day35 , whereas flies expressing L83V showed normal flight at young age, but mildly defective flight in old ages Fig.

Collectively, these results indicate that both L83V and RW variants are partial loss-of-function alleles and that RW has more defective gene function compared to L83V. L83V and RW variants cause behavioral defects in adult flies. We previously showed that human fibroblasts carrying the de novo variant p.

ArgTrp exhibited an increase in the mitophagic vesicles and expression of dAtad3a carrying p. ArgTrp, the homologous mutation of human p. ArgTrp, leads to small mitochondria with aberrant cristae as well as an increase in autophagic vesicles in larvae muscles [ 12 ]. These findings suggest that aberrant autophagy or mitophagy may underlie the behavioral defects in dAtad3a mutant flies expressing L83V or RW Fig.

To test this, we sought to examine mitochondria morphology and autophagy in adult thorax muscles. First, we assessed mitochondrial morphology using an antibody for ATP5A in both young 5-day-old and old day-old adult flies.

We found that RW leads to smaller mitochondria with a rounded shape compared to those in wild-type controls Additional file 2 : Figure S On the contrary, the animals expressing L83V exhibited irregular size of mitochondria with slightly longer mitochondria on average Additional file 2 : Figure S These results indicate that both RW and L83V variants affect mitochondrial dynamics, which in turn may lead to increased autophagy or mitophagy.

To test whether dAtad3a carrying RW or L83V variants cause an increase in autophagy, we measured the levels of ref 2 P, the Drosophila orthologue of p62, an autophagy marker, in adult muscles. While flies expressing RW or L83V exhibited comparable levels of ref 2 P as compared to wild-type controls as young animals 7-day-old , the older 8-week-old mutants expressing RW or L83V, exhibited significantly higher levels of ref 2 P than those in wild-type controls Fig.

To further characterize this, we performed transmission electron microscopy TEM. TEM in day-old animals revealed that RW led to small mitochondria and increased autophagic intermediates, whereas wild-type rescue animals exhibited normal mitochondria with lower numbers of autophagic intermediates Fig. Interestingly, the muscles expressing L83V showed many normal mitochondria Additional file 2 : Figure S17 , but parts of the muscles were filled with autophagic intermediates Fig. Collectively, the data indicate that both RW and L83V variants lead to increased autophagy and mitochondria loss and aberrant cristae and that the detrimental effect of dAtad3a RW is more severe than dAtad3a L83V.

L83V and RW variants cause increased p62 levels in the thorax in aged flies. ATP5A green labels mitochondria. Ref 2 P is the Drosophila homolog of p62 red. Three biological replicates were quantified. L83V and RW variants cause aberrant mitochondrial morphology, increased autophagic and mitophagic vesicles.

Arrows show autophagosomes i, ii, and v , autolysosomes v and viii , lysosomes vi and ix , and mitophagosomes iv and vii. Functional studies for the five missense variants in Drosophila were consistent with bioinformatic predictions, where the human variants p.

Leu77Val and p. ArgTrp had more benign prediction scores as compared to the other missense variants Additional file 2 : Table S3. However, caution must be exercised with bioinformatics prediction scores, as the p. Indeed, the family with the most mild phenotype, family 5, exhibited the p.

ArgTrp allele the mild variant in the Drosophila functional studies in trans to a frameshift variant c. On the contrary, homozygosity for the p. ArgPro allele a severe variant in the Drosophila functional studies led to a severe, infantile lethal phenotype family 8. Genotype-phenotype correlations must take into account both alleles—the p. Phe50Leu allele was associated with a severe phenotype when inherited in trans to an intragenic deletion of two exons family 4 , yet with a mild phenotype when observed with a non-frameshift single amino acid deletion family 7.

Overall, Drosophila studies provided support of variant pathogenicity and correlated reasonably well with the severity of the clinical phenotype in humans. The spectrum of ATAD3A variants has thus far focused on monoallelic gain-of-function variants and biallelic loss-of-function variants. We report eight families with biallelic variants, ranging from biallelic CNVs to biallelic SNVs and combinations thereof.

The relatively wide phenotypic and genotypic spectrum of ATAD3A- associated variation calls for caution in the interpretation of the clinical significance of missense variants.

To address this, we systematically analyzed the functional effect of six SNVs identified in affected individuals using Drosophila models.

We showed that F56L, GV, RP, and Kdel are severe loss of function alleles which fail to rescue the lethality of dAtad3a null mutants, whereas L83V and RW are mild hypomorph variants which rescued developmental lethality but exhibited behavioral defects in adult flies. The results from Drosophila studies correlated with the clinical severity of affected individuals—orthologs of the three more severe loss-of-function alleles F56L, GV, and RP in Drosophila , or F50L, GV, RP in humans led to severe phenotypes including hypotonia, global developmental delay, cataracts, cardiomyopathy, and structural brain abnormalities families 4, 6, and 8.

Although the clinical phenotype in family 7 is overtly consistent with ATAD3A- associated disorders, a major limitation of this study was that we could not phase the variants one inherited, the other de novo in the proband due to technical issues. This relatively weak variant Additional file 2 : Table S3 may have been overlooked or discarded in variant filtering of the exome; nonetheless, functional modeling combined with a clinical phenotype compatible with the mild range of the ATAD3A disease spectrum implicates the variant as probably disease-causing.

Members of this family form oligomers and have positive cooperativity in ATP binding and hydrolysis, whereby alteration of subunits i. Previous data from studies in Drosophila and in patient-derived fibroblasts with heterozygous variants i.

ArgTrp; RW and p. GlyAsp; GD suggested that both alleles act in a dominant-negative fashion [ 12 , 15 ], consistent with the location of the mutated residues in the key motifs required for the ATPase activity Gly, the Walker A motif [ 15 ]; R, the Sensor 2 motif, personal communication with Sukyeong Lee.

Here, we show that six SNVs five missense variants and one non-frameshift indel act as hypomorphic or loss-of-function alleles rather than dominant-negative. However, human carriers for ArgPro parents of family 8 are unaffected, suggesting that this allele does not function in a dominant negative manner in humans. On the contrary, the other five alleles moderately or do not affect protein levels Fig. The first 50 amino acids were reported to be important to form contact sites between the mitochondria and the ER membrane [ 1 ], implicating that the F50L variant may cause defects in mitochondria-ER communication.

Further molecular studies of the pathogenic variants and identification of ATAD3A-interacting proteins will provide insight as to how genetic variants cause the etiology at the molecular and cellular levels. Previous studies in Drosophila and in patient-derived fibroblasts with heterozygous variants i. ArgTrpand p. GlyAsp revealed a defect in mitochondrial dynamics, possibly triggering mitophagy and resulting in a significant reduction of mitochondria [ 12 ]. Here, we also demonstrated that aged muscles expressing RW and L83V variants exhibited defective mitochondrial membrane dynamics and increased mitophagic vesicles Fig.

Thus, these findings suggest that proper ATAD3A function is required for homeostasis of mitochondrial dynamics and mitophagy. Interestingly, in Drosophila embryonic stages, we found that the null and severe LOF alleles F56L, GV, and RP exhibited increased mitochondrial content Additional file 2 : Figure SS15 , which may result from the compensatory mechanisms for abnormal mitochondria.

One mechanism for mitophagy was documented in mouse hematopoietic stem cells, in which increased mitophagy in ATAD3A- deficient cells has been attributed to perturbation of Pink1-mediated mitophagy [ 34 ].

Abnormal regulation of nutrition and metabolism-sensing machineries such as the mechanistic target of rapamycin mTOR could be implicated in the etiology caused by loss of ATAD3A as mTOR is a major regulator for autophagy and mitophagy.

Indeed, Cooper et al. In mice, Atad3a and mTOR have central functions in the biogenesis of mitochondria during development [ 2 , 3 , 35 ]. These include impaired mtDNA and segregation, and aberrant cholesterol channeling and steroidogenesis [ 37 — 39 ]. Moreover, fibroblasts demonstrated multiple indicators of altered cholesterol metabolism [ 7 ].

The associated disease pathology was proposed to result either from compromised rigidity of the inner mitochondrial membrane with impaired mtDNA segregation subsequent to inadequate cholesterol metabolism or from a shortage of cholesterol products in Purkinje cells.

Affected individuals whose fibroblasts exhibited impaired cholesterol metabolism often presented with elevated urine levels of 3-methyglutaconic acid 3-MGA [ 7 , 12 , 18 ]. Interestingly, SERAC1 deficiency presents with impaired cholesterol metabolism together with elevated 3-MGA levels [ 40 ], suggesting that defective cholesterol metabolism and mitochondrial lipid metabolism may be implicated in increased levels of 3-MGA.

Whether manipulating cholesterol metabolism and mitochondrial lipid metabolism may ameliorate ATAD3A pathologies remains to be investigated. Drosophila has been well established as a powerful genetic model organism [ 41 ]. We utilized this model organism to assess functional impacts of various SNVs in ATAD3A and showed that the allele severity in Drosophila correlates with the phenotypic severity in humans. This study further reiterates the clinical findings associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy.

We contribute to the growing disease-causing allelic spectrum at the ATAD3A locus, which includes biallelic NAHR-mediated deletions and a reciprocal monoalleleic duplication; monoallelic dominant-negative variants, biallelic SNVs, and splice-site variants; and now SNVs in trans to intergenic or intragenic deletion alleles. The authors wish to thank the families for their participation in this study. We appreciate Dr. Abstract Purpose: To characterize the spectrum of RPE65 mutations present in patients with retinal dystrophy with an interest in understanding the range of functional deficits attributable to sequence variants in this gene.

Publication types Research Support, Non-U. Gov't Research Support, U. Gov't, P. Most classical HLA-class I genes are translated into one isoform. Therefore, a mutation in the gene yields a null allele, which results in the disappearance of this HLA molecule. In this respect, it would be important to determine the function of each HLA-G isoform and whether a specific role may be attributed to each of them in pregnancy as well as in tumors and transplants.

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