Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities

Journal Pre-proof Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities

Susana Peralta, Adrián González-Quintana, Marta Ybarra, Aitor Delmiro, Rafael Pérez-Pérez, Jorge Docampo, Joaquín Arenas, Alberto Blázquez, Cristina Ugalde, Miguel A. Martín PII:

S1096-7192(19)30693-6

DOI:

https://doi.org/10.1016/j.ymgme.2019.10.012

Reference:

YMGME 6576

To appear in:

Molecular Genetics and Metabolism

Received date:

2 October 2019

Revised date:

29 October 2019

Accepted date:

30 October 2019

Please cite this article as: S. Peralta, A. González-Quintana, M. Ybarra, et al., Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities, Molecular Genetics and Metabolism (2019), https://doi.org/10.1016/j.ymgme.2019.10.012

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© 2019 Published by Elsevier.

Journal Pre-proof Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities

Running Title: Novel ATAD3A recessive variant

Susana Peralta1,*, Adrián González-Quintana1,2,*, Marta Ybarra3, Aitor Delmiro1, Rafael Pérez-

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Pérez1, Jorge Docampo1,2, Joaquín Arenas1,2, Alberto Blázquez1,2, Cristina Ugalde1,2,#, Miguel A.

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*These authors contributed equally to this work

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Martín1,2

Laboratorio de Enfermedades Raras, Mitocondriales y Neuromusculares, Instituto de

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Investigación Hospital 12 de Octubre (i+12), 28041 Madrid, Spain; 2Centro de Investigación

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Biomédica en Red de Enfermedades Raras (CIBERER), U723, Instituto de Salud Carlos III,

Corresponding author: Dr. Cristina Ugalde, Laboratorio de Enfermedades Raras,

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#

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28029 Madrid, Spain; 3Servicio de Neonatología, Hospital Infantil La Paz, 28046 Madrid, Spain.

Mitocondriales y Neuromusculares, Instituto de Investigación Hospital 12 de Octubre (i+12). Avda. de Córdoba s/n 28041 Madrid. Phone: +34 91 779 2784, FAX: +34 91 390 8544, e-mail: [email protected].

GRANT NUMBERS This work was funded by the ISCIII-ERDF grants PI14/00209, PI17/00048 and PI18/01374, and CAM-ERDF grants P2017/BMD-3721 and P2018/BAA-4403.

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Journal Pre-proof ABSTRACT Lethal neonatal encephalopathies are heterogeneous congenital disorders that can be caused by mitochondrial dysfunction. Biallelic large deletions in the contiguous ATAD3B and ATAD3A genes, encoding mitochondrial inner membrane ATPases of unknown function, as well as compound heterozygous nonsense and missense mutations in the ATAD3A gene have been recently associated with fatal neonatal cerebellar hypoplasia. In this work, whole exome

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sequencing (WES) identified the novel homozygous variant c.1217T>G in ATAD3A, predicting a

fatal

neonatal

cerebellar

hypoplasia,

seizures,

axial

hypotonia,

hypertrophic

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with

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missense p.(Leu406Arg) substitution, in four siblings from a consanguineous family presenting

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cardiomyopathy, hepatomegaly, congenital cataract, and dysmorphic facies. Biochemical phenotypes of the patients included hyperlactatemia and hypocholesterolemia. Healthy siblings

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and parents were heterozygous for this variant, which is predicted to introduce a polar chain

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within the catalytic domain of ATAD3A that shortens its beta-sheet structure, presumably affecting protein stability. Accordingly, patient’s fibroblasts with the homozygous variant

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displayed a specific reduction in ATAD3A protein levels associated with profound

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ultrastructural alterations of mitochondrial cristae and morphology. Our findings exclude the causative role of ATAD3B on this severe phenotype, expand the phenotypical spectrum of ATAD3A pathogenic variants and emphasize the vital role of ATAD3A in mitochondrial biogenesis.

KEYWORDS Mitochondrial disorder, ATAD3A, missense homozygous, fatal neonatal encephalopathy, cerebellar hypoplasia, mitochondrial ultrastructural alterations.

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ABREVIATIONS ATAD3: ATPase family AAA domain-containing protein 3 mtDNA: mitochondrial DNA OXPHOS: Oxidative Phosphorylation System

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CJs: cristae junctions

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Journal Pre-proof 1. INTRODUCTION Mitochondrial diseases are among the most common forms of inherited neurological disorders, with an incidence of 1:4300 adults [1] and of 1:5000 live births [2]. Their symptoms, heterogeneity and variable age of onset make these disorders challenging for diagnosis and therapy development. Pathogenic genetic variants in the mitochondrial ATAD3, ATPase family AAA domain-containing protein 3, gene family were recently associated with a diversity of

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neurological syndromes [MIM 612316, 612317] (summarized in Table 1). In hominids, the

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ancestral ATAD3 gene was duplicated twice in tandem, giving rise to a cluster of three paralog

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genes: ATAD3A, ATAD3B and ATAD3C (located on chromosome 1, map: 1p36.33). Biallelic

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large deletions in the contiguous ATAD3A and ATAD3B genes, as well as compound heterozygous nonsense and some missense variations in ATAD3A, were recently linked to fatal

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congenital cerebellar hypoplasia [3-5]. Exceptionally, an ATAD3A recessive missense variant in

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the amino terminal of the protein was associated with milder phenotypes [3], more commonly associated with dominant missense variants in ATAD3A [3, 6], or with deletions also involving

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ATAD3B and ATAD3C [4]. These phenotypes included milder cerebellar hypoplasia, global

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developmental delay, hypotonia, hereditary spastic paraplegia, axonal neuropathy, optic atrophy or hypertrophic cardiomyopathy [3, 4, 6]. The cellular pathophysiological consequences of the ATAD3 genetic variants and their relative contribution to mitochondrial disease remain unknown. Human ATAD3A is ubiquitous and particularly abundant in the central nervous system (CNS). ATAD3A is a mitochondrial inner membrane (IM) ATPase that simultaneously interacts with the outer membrane (OM) through its N-terminal domain [7], suggesting a role in mitochondrial intermembrane contacts [8, 9]. The N-terminal domain comprises two transmembrane domains (TM1 and TM2), two coiled-coil domains (Cc1 and Cc2) important for

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Journal Pre-proof protein-protein interactions and ATAD3A oligomerization, and a proline-rich domain (PR) of unknown function. Its C-terminal AAA ATPase domain, located in the mitochondrial matrix, contains two conserved Walker A and Walker B motifs for ATP binding and ATPase activity, respectively. ATAD3B is preferentially expressed in embryos, adult germinal zones of the brain, and astrocytoma cell lines [10-12], and differs from ATAD3A by a C-terminal extension of 62 amino acids (aa). ATAD3C is a truncated pseudogene that has lost the first 70 aa [13].

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Mounting evidence suggests overlapping roles of ATAD3A in the maintenance of

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mitochondrial cristae, lipid trafficking and mtDNA replication/segregation. This is supported by

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in vitro studies linking ATAD3A to mitochondrial DNA (mtDNA) maintenance [4, 14, 15], lipid

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biosynthesis/trafficking [4, 16], mitochondrial dynamics [3, 6, 8] and ER-mitochondria contacts preservation [16, 17]. In vivo studies also determined that ATAD3A is essential for mitochondrial

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function during development in C. elegans [18], D. melanogaster [8], and mice [19, 20]. Specific

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ablation of ATAD3 in mouse skeletal muscle caused muscle atrophy and OXPHOS deficiency associated with altered cholesterol trafficking, degeneration of mitochondrial cristae

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ultrastructure, and accumulation of mtDNA replication intermediates and mtDNA depletion at

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late stages [20]. Nevertheless, the primary function of ATAD3A remains to be proven. Here, we describe four siblings of a consanguineous family with lethal neonatal mitochondrial encephalopathy and multiorgan involvement associated with a novel recessive missense variant in the ATPase domain of the ATAD3A gene: NM_001170535.2: c.1217T>G, p.(Leu406Arg). Biochemical analyses in patient´s fibroblasts demonstrated a strong reduction in ATAD3A protein levels associated with decreased mitochondrial cristae content and reduced mitochondrial size, confirming the essential role of ATAD3A in mitochondrial biogenesis.

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Journal Pre-proof 2. SUBJECTS, MATERIALS AND METHODS 2.1. Editorial Policies and Ethical Considerations This study was approved by the Ethics Committee from ‘Hospital Universitario 12 de Octubre’, Madrid, Spain (approval numbers CEIC 11_347 and CEIC 18_487), and performed in accordance with the Declaration of Helsinki for Human Research. Written informed consent was obtained from the parents.

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2.2. Family clinical features

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The main clinical and genetic features of the affected individuals in this family are summarized

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in Table 1. The proband (subject II-4) was a newborn male of Moroccan origin resulting from a

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normal full-term pregnancy, whose parents were consanguineous first cousins. He exhibited dysmorphic facies with eyelashes absence, bilateral hydrocele, micropenis and left

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cryptorchidism. Neurological examination showed severe axial hypotonia and progressive

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encephalopathy. Brain magnetic resonance imaging (MRI) revealed cerebellar hypoplasia with megacisterna magna (Dandy-Walker variant), cortical dysplasia with pachygyria and lack of

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myelination. Cranial ultrasound showed ependymal cysts lesions and lenticulostriate

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vasculopathy. Electroencephalogram (EEG) displayed burst-suppression pattern. He had congenital cataracts and papillary depigmentation. Echocardiogram revealed hypertrophic cardiomyopathy. Hepatomegaly was also evident at age 5 days. Laboratory data revealed hyperlactatemia and decreased plasma cholesterol. At day 7 he died from cardiorespiratory arrest. The affected proband’s siblings (II-3, II-6 and II-8) displayed similar clinical features. In brief, the proband’s sister (II-3), who died at 3 days of age, showed dysmorphic features (short nose with anteverted nostrils and wide nasal root, microstomia and micrognathia, sunken eyes, mild blepharophimosis, eyelashes absence, sparse eyebrows, and thin scalp), neurological signs

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Journal Pre-proof (axial hypotonia, seizures with burst-suppression EEG pattern, and cerebellar hypoplasia, striated arteries vasculopathy and thin corpus callosum at cranial ultrasound), hypertrophic cardiomyopathy, hepatomegaly, hyperlactatemia, and hypocholesterolemia. The proband’s sister (II-6) presented with an acute encephalopathy, cerebellar hypoplasia and lenticulostriate vasculopathy, lactic acidosis and died at 19 days of age. The proband’s sister (II-8) presented hydrops fetalis, dysmorphic facies, axial hypotonia, clonic movements in limbs, congenital

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cataracts, cardiac hypertrophy (detected by ecography 3 days before birth) and ventricular

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dysfunction. Cranial ultrasound showed cerebellar hypoplasia, lenticulostriate vasculopathy and

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dysgenesis of corpus callosum, and lack of cerebral sulcation and subarachnoid hemorrhage in

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the interhemispheric fissure (Figure 1). The proband’s sister (II-1) is currently 19 and remains

2.3. Whole exome sequencing

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asymptomatic. Individuals II-2, 3, 5 and 7 could not be genetically studied (Figure 2).

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Whole exome sequencing (WES) was performed in the proband (subject II-4) using massive parallel sequencing core facilities at the “National Center for Genetic Analysis” (CNAG) through

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the Spanish CIBERER research program. Blood DNA was used to prepare a library using

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mechanic fragmentation (300 bp size). Exome capture was performed with the SeqCap EZ Exome Library (Roche Diagnostics) and sequenced in a HiSeq2000 platform (Illumina) (2x75bp reads, mean target coverage >75X). Sequencing data were mapped to human genome reference sequence hg19 using BWA aligner. GATK was used for calling single-nucleotide variants (SNV) and short indels. The variants called were annotated by Annovar [21]. To perform this analysis from sequence to annotated variants an in-house bioinformatic pipeline was used. Variants prioritization was performed assuming an autosomal recessive inheritance and homozygous status (parents were consanguineous) in a context of suspected mitochondrial dysfunction

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Journal Pre-proof (according to patients’ clinical and biochemical findings) following the next steps: i) analysis of variants in genes encoding for proteins with strong support of mitochondrial localization included in Mitocarta 2.0. database [22], ii) status and ranking of the variants in ClinVar database, and InterVar tool[23], iii) Minor Allele Frequency (MAF) < 0.05 in population databases such as 1000 genomes project, and Genome Aggregation Database (gnomAD), iv) variant pathogenicity predictors including SIFT, PolyPhen-2, LRT, MutationTaster, M-CAP,

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PROVEAN and CADD Phred; and v) assessment of phylogenetic conservation using GERP,

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2.4. PCR Amplification and Sanger sequencing

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PhyloP and PhastCons.

Exon 12 of the ATAD3A gene was amplified from genomic DNA by conventional PCR, with

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specific primers for ATAD3A. Primer F12-modified: (5’-ATGCTGATCGGCTTCTGTCGA-3’)

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(modified nucleotide underlined) and Primer R12: (5’-GAAACTGAACAGAGGGCTGCA-3’).

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PCR products were purified using Illustra GFX PCR DNA and gel purification kit (GEHealthcare) followed by Sanger sequencing in a 3130xl Genetic Analyzer (Applied Biosystems).

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2.6. 3D Modeling of protein structure

MODEL

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The AAA protein domains of wild-type and mutant ATAD3A were generated by SWISS(https://swissmodel.expasy.org/)

homology

modelling

using

6AZ0.1.F

pdb

(mitochondrial inner membrane i-AAA protease supercomplex subunit YME1) as a template. 2.7. Cell culture and mRNA analysis Primary skin fibroblasts from subject II-4 and neonatal controls were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37ºC in a 5% CO2 atmosphere. RNA was extracted using Trizol reagent by standard procedures. 2g RNA were retrotranscribed with SuperScript kit

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Journal Pre-proof (Invitrogen). The relative levels of ATAD3A and ATAD3B mRNAs were determined with SsoFast SYBR Green Supermix (Roche) following manufacturer’s PCR conditions in a CFX96 Real Time PCR system (Roche). Primer design and mRNA quantification analyses were done as described [4]. 2.8. Protein electrophoresis and Western-blot Whole cell lysates were extracted from cultured fibroblasts using standard methods. Protein

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concentration was quantified using BCA kit (Thermo Fisher Scientific). 30 µg of protein

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extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

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PAGE) in 7.5% polyacrylamide gels. Alternatively, mitochondrial solubilization and blue native

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analyses were performed as described before[24]. Proteins were transferred to nitrocellulose membranes and the following antibodies were used for immunodetection: ATAD3 (16610-1-AP,

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Proteintech); SDHA (ab14715), UQCRC2 (ab14745), COX1 (ab14705), ATP5A (ab14748), and

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VDAC1 (ab14734) from Abcam; beta Actin (A1978, Sigma). Secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technologies) were used, and the reactions were

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developed with ECLTM Prime (Amershan Biosciencies) in a ChemiDocTM MP Imager (BioRad).

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2.10. Transmission Electron Microscopy Human cultured fibroblasts were placed in a 50:50 media/Karnovski’s fixative solution (2% paraformaldehyde and 2.5% glutaraldehyde in 1% sodium cacodylate buffer) for 20 min, followed by 100% Karnovski’s fixative for 40 min. Cell pellets were collected, left in fresh fixative for 15 min and fixed with 1% aqueous Osmium Tetroxide. After dehydration in graded series of ethanol, cells were embedded in Epon 812 (Fluka) and polymerized at 60º C for 2 days. Ultrathin sections (70 nm) were stained with 1% uranyl acetate for 2 hours, followed by lead citrate for 5 min, and analyzed in a JEOL JEM1010 transmission electron microscope equipped

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Journal Pre-proof with a 4Kx4K TemCam-F416 digital camera (TVIPS, Gauting, Germany). The “Free hand selection” and “Analyze particles” tools from the ImageJ software (National Institutes of Health) were used to trace and quantify mitochondrial morphometric parameters after setting the pixel size value according to the scale bar of the .tiff images. Results were exported to Excel. For quantification of mitochondrial size, we averaged the mitochondrial area values. For quantification of cristae perimeter, we averaged the total cristae perimeters per mitochondrion,

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and then the ratios of controls’ and patient’s mitochondria. For Cristae Junction (CJ)

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quantification, we calculated the mitochondrial cristae surface that displays contacts with the

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outer membrane versus total cristae surface per mitochondrion. Mean values per mitochondrion

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(n=15 for controls and n=20 for patient) were expressed as percentages of control values. 2.11. Mitochondrial respiratory chain activities

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The mitochondrial respiratory chain (MRC) complexes and citrate synthase enzyme activities in

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skeletal muscle were determined spectrophotometrically (Table 2) using reported methods [25]. 2.12. Quantification of mtDNA

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The mitochondrial DNA content in skeletal muscle from proband II-4 and in proband’s sister II-6

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was calculated as the relative quantification of mtDNA versus nuclear DNA (nDNA) using TaqMan probes against the MTRNR1 and RNAseP genes, respectively, in a 7500 Real Time PCR System (Applied Biosystems, USA), as previously described [26]. 2.13. Statistical analysis For statistical analysis unpaired Student’s t-test (two-tailed) was used when comparing two groups, and One-way ANOVA followed by Bonferroni test when comparing three groups. Data were represented as mean ± s.e.m. and GraphPad Prism 6 software was used for presentation.

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Journal Pre-proof 3. RESULTS 3.1. Clinical and biochemical features associated with the biallelic ATAD3A variant c.1217T>G Four subjects, II-3 (female), II-4 (male), II-6 (female) and II-8 (female), were siblings born to healthy Moroccan consanguineous parents (Table 1). All pregnancies developed to term and clinical symptoms manifested either right after birth (3/4) or 3 days before delivery (1/4). The

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four patients presented respiratory distress at birth, requiring intubation. The phenotypic

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spectrum included: dysmorphic facies (4/4), severe cerebellar hypoplasia (4/4), seizures (4/4),

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axial hypotonia (4/4), congenital cataracts (3/4), elevated lactate in plasma (4/4), hypertrophic

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cardiomyopathy (4/4), and hepatomegaly (4/4). All patients died before 30 days of age. Cranial ultrasound images from patient II-8 revealed cerebellar hypoplasia (Figure 1). Mitochondrial

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dysfunction was suggested in patient II-3 by elevated plasma lactate, severe encephalopathy and

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multiorgan involvement. MRC enzyme activities in muscle from patient II-4 revealed normal values despite a significant mtDNA depletion (17% of control mtDNA/nDNA mean ratio).

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Subject II-6 showed a mild isolated decrease of complex I activity (Table 2) with 48% of control

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mtDNA/nDNA mean ratio in muscle, suggesting the severe mtDNA depletion is not a general feature. The main features of reported ATAD3A pathogenic variants are summarized in Table 1. 3.2. Whole exome sequencing and c.1217T>G variant assessment To identify the genetic cause of the neonatal lethal disorder, we performed WES on patient II-4. Out of 1572 variants identified in genes included in Mitocarta 2.0 [27], 141 showed MAF <0.05 in population databases, of which only 9 resulted homozygous. From those, 7 were classified as benign or likely benign in ClinVar or InterVar. The remaining 2 homozygous variants were localized either in the MRPS24 and ATAD3A genes. The MRPS24 gene variant was discarded,

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Journal Pre-proof since it was present in the GenomeAD Exomes African subpopulation database at 0.09 of allelic frequency and in 69 homozygote individuals. The ATAD3A gene homozygous variant, NC_000001.10: g.1460622T>G hg19 assembly, NM_001170535.2: c.1217T>G, was not found in the ExAC, GnomAD or 1000 genomes databases. The chromosome region encompassing the variant position presents a good coverage in GnomAD, with a reference allele number in the African subpopulation >8000. The variant was categorized as deleterious by in silico predictors

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such as SIFT, PolyPhen2, LRT, PROVEAN, MutationTaster, CADDPhred, GERP and PhyloP.

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The InterVar tool using ACMG-AMP guidelines classified this variant as likely pathogenic. The

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mutation is located in exon 12, and predicts a missense mutation leading to the amino acid

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change p.(Leu406Arg) in the ATPase domain of the ATAD3A protein (Figures 2A-2B). Sanger sequencing of exon 12 of ATAD3A (Figure S1) in blood DNA from the probands’ asymptomatic

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parents and a healthy sister (subjects I-1, I-2 and II-1, respectively) revealed the heterozygosity

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of the novel variant, whereas the affected siblings (II-4, II-6 and II-8) were homozygotes, supporting an autosomal recessive inheritance of this variant (Figures 2A and S2). A

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synonymous transversion c.1221G>C, p.(Leu407=) was also identified in those members of the

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family harboring the p.(Leu406Arg) mutated allele (Figure S2), which was filtered out through the variant-prioritization analysis as it was classified as likely benign by InterVar. Multiple sequence alignment of ATAD3A showed that the mutated Leu406 residue is conserved throughout evolution (Figures 2B and S3). Leu406 is located within the catalytic core of ATAD3A (AAA or ATPase domain), in the vicinity of the Walker B domain that hydrolyzes ATP. The arginine residue in mutated ATAD3A introduces a positively charged guanidine group in the side chain, in contrast to the hydrophobic side chain of leucine in an 8 hydrophobic amino acids stretch right before the Walker B domain. The predicted structure of the mutant ATAD3A-

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Journal Pre-proof Leu406Arg protein shows a shortening of the beta-sheet structure close to the Walker B domain that could potentially affect ATAD3A protein folding and stability (Figure 2C). 3.3. The biallelic c.1217T>G variant specifically decreases ATAD3A protein levels without affecting OXPHOS complexes To functionally characterize the novel variant, we analyzed ATAD3A transcript and protein levels in the proband´s (subject II-4) and two neonatal control fibroblasts. In parallel, ATAD3B

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transcripts and protein levels were analyzed as negative controls. The ATAD3A transcript

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variant-2 (NM_001170535.2) was largely expressed in the proband´s and control fibroblasts

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(Figure 3A), while the ATAD3A transcript variant-1 (NM_018188.4) was not detected (Figure

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S4), in agreement with previous studies reporting the unique expression of ATAD3A transcript variant-2 in human U373 and Hela cultured cells [28]. qRT-PCR analysis confirmed that

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ATAD3A transcript variant-2 expression levels were similar between proband´s and control

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fibroblasts (Figure 3B), indicating that the homozygous variant c.1217T>G does not alter ATAD3A transcription. The expression of the ATAD3B transcript in patient’s fibroblasts was also

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similar to controls (Figure 3B). Further immunoblotting experiments with an ATAD3 antibody

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showed two clear bands in control fibroblasts (Figure 3C), the lower intense band being compatible with the 66 kDa- ATAD3A protein, and the upper fainter band being compatible with the 72 kDa- ATAD3B protein. As reported before[4, 14], ATAD3A is more highly expressed than ATAD3B in human control fibroblasts. Interestingly, we detected a marked decrease of ATAD3A levels in the proband´s fibroblasts, while ATAD3B levels remained unaffected (Figure 3C). These results suggest that the homozygous variant c.1217T>G, p.(Leu406Arg) may induce the specific misfolding and degradation of ATAD3A without affecting ATAD3B expression.

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Journal Pre-proof Then we used BN-PAGE to analyze the structural state of ATAD3A-containing complexes using digitonized mitochondrial extracts from proband´s and control fibroblasts (Figure 3D). Similar to mice [19, 20], human control fibroblasts showed ATAD3 proteins in two major complexes of >900 kDa (arrows), as well as in less abundant complexes of ~600 kDa and <400 kDa (asterisks). A dramatic reduction in the levels of ATAD3-containing complexes was detected in the proband’s fibroblasts, as expected from the significant decrease in ATAD3A

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protein levels (Figure 3C). This reduction was not associated with structural alterations in the

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OXPHOS complexes (Figure 3D), in agreement with the normal respiratory chain enzyme

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activities measured in proband’s skeletal muscle (Table 2). Altogether, our results suggest that

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ATAD3A is not primarily implicated in the function of the OXPHOS complexes.

ATAD3A c.1217T>G variant

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3.4. Mitochondrial ultrastructural alterations in patient’s fibroblasts with the biallelic

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Next, we analyzed mitochondrial ultrastructure by transmission electron microscopy (TEM) on proband´s (subject II-4) and control fibroblasts (Figure 4). Ultrastructural abnormalities were

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observed in the mitochondrial cristae from patient´s fibroblasts, such as the loss of cristae

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junctions (contacts between the mitochondrial outer and inner membrane, CJs) (Figure 4A, white arrows) or concentric cristae (Figure 4A, arrowhead). Quantification analyses on TEM pictures (described in Methods) indicated that the relative amount of CJs was reduced to 69% in patient´s fibroblasts (Figure 4B), that the cristae perimeter per mitochondria was significantly decreased (Figure 4C), and that mitochondrial size was significantly smaller, to about one third of the mean control size (Figure 4D). These findings indicate that the homozygous c.1217T>G variant interferes with mitochondrial cristae shaping or maintenance.

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Journal Pre-proof 4. DISCUSSION In this study we report four siblings from a consanguineous family presenting with fatal neonatal cerebellar hypoplasia due to a novel recessive missense variant in ATAD3A, c.1217T>G. Additionally, all patients showed hypertrophic cardiomyopathy and hepatomegaly, expanding the phenotypical spectrum commonly associated with pathogenic ATAD3A variants. The c.1217T>G variant causes a p.(Leu406Arg) substitution in an evolutionarily conserved amino

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acid region predicted to disrupt the catalytic Walker B motif of ATAD3A. In agreement, the

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patient´s fibroblasts showed a severe ATAD3A depletion associated with mitochondrial cristae

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malformation without alterations in the OXPHOS complexes, cristae junction degeneration, and

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a significant reduction in mitochondrial size, supporting a role for ATAD3A in the maintenance of the mitochondrial ultrastructure [15, 20, 29]. These results contrast with previous work in a

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skeletal muscle-specific ATAD3A conditional knockout (KO) mouse model (Atad3-Mlc1f KO),

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where cristae morphology alterations were associated to a severe loss of complex V (CV) stability [20]. This apparent discrepancy may reflect species- or tissue-specific differences

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regarding the stabilization of CV upon cristae disruption, or it might be explained by the

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relatively higher expression levels of ATAD3A in the patient´s fibroblasts, which would allow a partial maintenance of cristae morphology with less detrimental effects for CV stability, which has been shown to rely on cristae morphology [30]. Although the precise molecular mechanism of ATAD3A function remains unknown, the alterations in cholesterol metabolism observed in patients ([4], and this work) suggest it could regulate the mitochondrial cholesterol transport relevant to maintain the mitochondrial membrane shape [15]. Defects in the ATAD3 genes were previously associated with fatal neonatal cerebellar hypoplasia [3-5] (Table 1). Several clinical features reported here replicate those associated with

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Journal Pre-proof large biallelic deletions/rearrangements in the ATAD3A/ATAD3B/ATAD3C gene cluster, causing fatal cerebellar dysfunction, where both ATAD3A and ATAD3B were barely expressed [3, 4, 31]. In our study, ATAD3A protein levels were strongly reduced in the patient’s fibroblasts, whereas those of ATAD3B remained unaffected, indicating the specificity of the p.(Leu406Arg) substitution for ATAD3A. These results associate ATAD3A loss with fatal neonatal cerebellar hypoplasia, putting aside a role of ATAD3B in the causation of this severe phenotype. This is

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further supported by milder clinical phenotypes and delayed disease onset and progression

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associated with the lack of ATAD3B and normal ATAD3A levels [4], or with ATAD3A genetic

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variants that did not alter ATAD3A protein levels [3, 6]. Although overall evidence suggests that

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genetic conditions that highly reduce ATAD3A expression are primarily associated with neonatal lethality, phenotype-genotype correlations in cases presenting missense variants are not

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straightforward [31]. Interestingly, mutations in the catalytic ATPase domain of ATAD3A seem

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to cause more severe disease phenotypes than those in the N-terminus [3, 6]. In agreement, over expression of mutated ATAD3A variants within the catalytic domain showed reduced activity of

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the wild type enzyme in a mixed solution [6], or led to premature death in Drosophila

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experimental models [3]. These mutations could potentially disrupt ATAD3A C-terminal domain- related functions, i.e., its mitochondrial targeting, functional oligomerization, membrane insertion, or positioning of the AAA-ATPase domain in the matrix compartment [8, 29]. In summary, we report a new family presenting with neonatal fatal cerebellar hypoplasia and multiorgan involvement associated with a novel biallelic ATAD3A genetic variant. Our study sheds light on the phenotypic impact of ATAD3A missense mutations, specifically associates ATAD3A (rather than ATAD3B) loss with infantile lethal cerebellar hypoplasia and emphasizes the vital role of ATAD3A in mitochondrial biogenesis.

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Journal Pre-proof ACKNOWLEDGEMENTS We thank Dr. Ann Frazier and Prof. David Thorburn for critically reading the manuscript. Part of this work was developed through a joint action on molecular-genetics diagnosis into the CIBERER-Mitochondrial Disorders Research Program (ISCIII). Research was supported by Instituto de Salud Carlos III-MINECO/European Regional Development Funds (ERDF) grants PI14-00209 and PI17-00048 (to CU) and PI18-00374 (to MAM), and by Comunidad Autónoma

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de Madrid/ERDF grant P2018/BAA-4403 (to CU) and P2017/BMD-3721 (to MAM). S.P. was a

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recipient of a grant from Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain;

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A.Q. from the CIBER of Rare Disorders (CIBERER), and J.D. from the Spanish Mitochondrial

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CONFLICT OF INTERESTS

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Patients Associations AEPMI /FACDM.

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The authors have no conflict of interest to declare.

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mitochondrial protein, re-expressed in cancer cells, that functions as dominant negative for the ubiquitous ATAD3A Mitochondrion 12 (2012) 441-448.

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oligomerization

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Qi,

Y. Zhao, X. Sun, D. Hu, D.A. Prosdocimo, C. Hoppel, M.K. Jain, R. Ramachandran, X. causes

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correlation in ATAD3A deletions: not just of scientific relevance Brain 140 (2017) e67.

22

Journal Pre-proof FIGURE LEGENDS

Figure 1. Cranial ultrasound from an affected individual with the variant c.1217T>G, p.(Leu406Arg) in ATAD3A. Cranial ultrasound images from patient II-8 harboring the homozygous c.1217T>G, p.(Leu406Arg) variant in the ATAD3A gene. Left: Coronal view showing mild ventriculomegaly (arrows) and widened interhemispheric fissure (arrowhead);

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Center: Sagittal view (Midline) revealing corpus callosum dysgenesis (arrowheads) and

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cerebellar hypoplasia (arrow); Right: Mastoid view displaying cerebellar hypoplasia (arrows).

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Figure 2. Family with a novel biallelic ATAD3A pathogenic variant c.1217T>G, p.(Leu406Arg). (A) The genetic pedigree and ATAD3A genotype were determined by whole-

lP

exome and Sanger sequencing for available family members. (B) Schematic representation of

na

ATAD3A protein (Uniprot Q9NVI7) indicating the proline rich domain (PR), coiled-coil domains (CC1-2), transmembrane domains (TM1-2), and Walker A and Walker B ATP-biding

ur

and ATPase domains, respectively. The localization of the mutated residue is indicated. The

Jo

p.(Leu406Arg) substitution alters a conserved residue within the ATPase domain of ATAD3A. (C) Modelling of the p.(Leu406Arg) amino acid substitution in the ATAD3A protein structure: panel a) superimposed wildtype (blue) and mutant (yellow) AAA domains shows the shortening in the beta-sheet structure near the walker B domain induced by the novel ATAD3A variant; panel b) wild type structure showing the Leu406 residue; panel c) mutant structure showing that Arg406, positively charged, alters the ATPase domain structure.

23

Journal Pre-proof Figure 3. The c.1217T>G, p.(Leu406Arg) homozygous variant induces a posttranscriptional decay of ATAD3A protein levels without affecting ATAD3B or OXPHOS complexes. (A) Full length ATAD3A cDNA (transcript variant-2, NM_001170535.2) was amplified using primers OT441 and OT443 on isolated mRNA from 2 controls’ and proband’s skin fibroblasts. (B) The relative expression levels of ATAD3A and ATAD3B transcripts were determined by quantitative RT-PCR on isolated mRNA from 2 controls’ and proband’s skin

of

fibroblasts. Results were normalized to the HPRT mRNA expression levels and presented as

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percentage of control 2; n=3, error=SEM. One-way ANOVA followed by Bonferroni test

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comparing the subject with each control showed no differences in ATAD3A or in ATAD3B mRNA expression levels. (C) ATAD3 proteins were detected by western blot using whole cell

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lysates and mitochondrial enriched fractions from controls’ and proband’s fibroblasts. Actin and

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VDAC were used as loading controls. ATAD3A (but not ATAD3B) protein levels were

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drastically reduced in the proband relative to the controls. (D) In-gel activity (IGA) of complex I and BN–PAGE analysis of digitonized mitochondria from controls’ and proband’s fibroblasts

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showing ATAD3-containing- and OXPHOS complexes I to V (CI-CV). Asterisks indicate

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ATAD3-containing complexes at ~900 and ~600 KDa that were dramatically reduced in the patient´s fibroblasts (arrow). OXPHOS complexes, supercomplexes (SC), and complex I-IGA were unaffected by the ATAD3A variant. Citrate synthase (CS) and complex II (CII) were used as loading controls. The positions in the gel of the ferritin monomer (440 kDa) and dimer (880 kDa) used as molecular mass standards are indicated on the left.

Figure 4. The ATAD3A c.1217T>G, p.(Leu406Arg) homozygous variant alters mitochondrial ultrastructure. (A) Representative mitochondrial electron micrographs from

24

Journal Pre-proof control and proband’s fibroblasts. Mitochondria from patient´s fibroblasts were smaller, showed less cristae content per mitochondria, less cristae junctions (CJs, white arrows), and showed disrupted cristae forming concentric circles (arrowhead). Scale bars are 200 nm for upper panels and 0.5 µm for lower panels. (B) Quantification of the cristae junctions (CJs), (C) of the cristae surface per mitochondria (expressed as cristae perimeter per mitochondria perimeter), and (D) of the mitochondrial area from >15 mitochondria from control and from 20 mitochondria form

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proband’s fibroblasts. Data are expressed as mean ± s.e.m. P-values were calculated by Student’s

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t-test. *P<0.05 and **P<0.01.

25

Journal Pre-proof TABLES Table 1. Clinical and biochemical findings in patients with mutations in the ATAD3 cluster gene. Cases

(Harel et al., 2016)

(Cooper et al., 2017)

F1,II2 NR

F2,II4 NR

F3,II1 Italia n

F4,II1 NR

F5,II1 NR

F6,II1 Italia n

F6,II2 Italia n

F7,II1 India n/Cau casia n

Sex Age at onset

F NR

F NR

M >3 mo.

F 12 days

M 37 w.g.

F >4 y.

M >3 y.

Age at death

Alive 9y

Alive 5y

Alive 3y

Alive 5y

Alive 26 y

-

-

-

-

Alive 23 mo +

-

-

+

+

+

+

+

+ + -

+ + -

+ NR

Origin

Consanguinity Foetal distress/ polyhydramnios/ early delivery Dysmorphic features Developmental delay Feeding difficulties Encephalopathy Cerebellar hypoplasia/atrophy Seizures Hypotonia Peripheral Spasticity Axonal neuropathy Eye defects Hypertrophic cardiomyopathy Hepatomegaly Brittle hair Elevated plasma lactate Decreased plasma cholesterol ATAD3Amutations * ATAD3A/ATAD3B deletion ATAD3A/ATAD3C deletion and ATAD3A/ATAD3B rearrangement Type of inheritance Type of allele

(Desai et al., 2017)

(Peeters-Scholte et al., 2017)

S1a

S1b

S2

S3

S4

S5

S1a

S1b

S2a

S2b

S3

S4

SII-3

SII-4

SII-6

SII-8

Chi nes e

Dutch

Dutch

Egy ptian

Moro ccan

Moro ccan

Moro ccan

Moro ccan

M 29 w.g . Da y1

M NR

M 30 w.g.

M or oc ca n F N R

M NR

F Birth

M Birth

F Birth

F 38 w.g.

Day 6

Day 3

Da y 7 +

Day 5

Day 3

Day 7

Day 19

Day 30

+ +

+ -

+ -

+ -

+ -

+

+

+

+

+

+

NR

+

+

+

Finni sh

Finni sh

Irani an

Irani an

Dutc h

India n

Jap ane se

Frenc h

Chine se

F 32 w.g.

F 1 y.

M 4 mo

F 38 w.g.

M 33 w.g.

F 33 w.g.

M 34 w.g.

F Ch.

F 30 w.g.

Alive 24 y

Day 13

Alive

Alive

Day 5

Day 1

Day 5

Day 2

M 37 w.g . 7 mo

-

-

+

-

-

+ +

+ +

+ +

+

+

+

+

+

+

+

+

+

NR

NR

NR

+ + -

+ + -

NR + -

+ +

+ +

+ +

+

+ + (m) + +

+ + (m) + +

NR + + (s) + +

+ + -

+ -

+ + +

+ + +

+ -

+ -

+ -

NR + +

-

+

+

+

NR

NR

+

+/R528 W -

+/R528 W -

+/R528 W -

+/R528 W -

+/R528 W -

+/+ T53I

+/+ T53I

-

-

-

-

-

-

-

-

-

-

AD

AD

AD

AD

AD

AR

Anti

Anti

Anti

Anti

Anti

Hypo

-

+ -

+ -

+ +

+ +

+ +

+ +

+ NR

+ +

+ + (s)

+ NR

NR

+

+ -

NR

NR

+

+

NR

+ + (s)

+

+ +

+

+

Alive 30 y

Day 1

-

+

+

+

+

+

+

+

+

+

+

+

+ + (s) -

+ + (m) -

+ NR -

+ + (s) -

+ + (s) +

+ + (s) +

+ + (s) + +

+ + (s) + +

+ + (s) + +

+ + (s) + -

+

NR

+

+

+

+

+

+ +

+ +

+ +

+ + +

+ + +

+ + +

+ + +

+

+

NR

NR

+/+ L406 R -

+/+ L406 R -

NR

NR

NR

+ + (s) + +

+ + (s)

+ + (m)

NR -

NR +

+

+

+/G355 D -

-

-

-

-

-

+

+/G355 D -

+

+

+

+

-

-

-

-

-

-

-

AR

AR

AD

AD

AR

AR

AR

Hypo

Null

Anti

Anti

Null

Null

Null

f o

o r p

e

r P

l a

n r u

Jo NR

-

Present study

NR

-

NR

NR

-

-

-

+

-

+

-

+

-

AR

AR

AD

Null

Nul l

Hypo

+

+

+

+

+/+ Q164* L77R -

-

-

NR

+

+/+ Q164* L77R -

+

+

-

+/+ L406 R -

-

-

-

-

-

-

-

-

-

AR

AR

AR

AR

AR

AR

AR

AR

AR

Null

Nul l

Null

Null

A R Nu ll

Null

Null

Null

Null

Null

26

Journal Pre-proof Others

Defic iency CI +CIII

Defic iency CII; CS; CII+ +CIII

47,X XY

Dela yed puber ty

Dela yed puber ty

mtD NA clust ering

mtDN A cluster ing

Pulmo nary hypop lasia. cornea l cloudi ng

Pul mo nar y hyp opl asia

Conge nital catarac t and corneal cloudin g

corneal cloudin g

co rn eal clo ud in g

corn eal clou ding

mtD NA deple tion, and cong enital catar act

CI defici ency and cong enital catar act

NR: not reported; F: female, M: male,; m: moderate, s: severe, ; w.g: weeks of gestation, mo: months, y: year/s, ch: childhood; AD: Autosomal Dominant, AR: Autosomal Recessive; +/+: Biallelic (homozygous or compound heterozygous), +/-: Heterozygous; Anti: Antimorph, Hypo:Hypomorph

f o

o r p

* ATAD3A RefSeq Transcript NM_001170535.2.

l a

r P

e

n r u

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27

Cong enital catar act and corne al cloud ing

Journal Pre-proof Table 2. Mitochondrial respiratory chain enzyme activities in skeletal muscle homogenates.

%2

II-6 (controls, n: 80)1

%2

II-4 (controls; n:110)1 14.0 (10-25)

140%

11.3 (15-37)

75%

CII (Succinate dehydrogenase)

5.0 (4.5-19.5)

111%

44 (26-65)

169%

CIII (DBH2-Cyt c oxidoreductase)

45 (31-127)

146%

244(40-89)

610%

CIV (Cytochrome c oxidase)

63 (30-125)

211%

249 (70-228)

356%

CS (Citrate synthase)3

138 (70-250)

-

161.2 (105-350)

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CI (NADH-DB oxidoreductase)

Complex activity (nmol min-1 mg-1 protein) expressed as percentage of CS specific activity. In parenthesis 2.5th - 97.5th percentile control range. Respiratory chain methods were slightly different for assessed MRC for Patient II-4 and II_6. 2 % of complex activity respect to the 2.5th percentile of controls 3 CS activity expressed as nmol min-1 mg-1 protein

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1

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Figure 1

Figure 2

Figure 3

Figure 4