Clinical Characteristics and Induced Pluripotent Stem Cells (iPSCs) Disease Model of Harel-Yoon Syndrome Caused by Compound Heterozygous ATAD3A Variants

Clinical Characteristics and Induced Pluripotent Stem Cells (iPSCs) Disease Model of Harel-Yoon Syndrome Caused by Compound Heterozygous ATAD3A Variants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clinical Characteristics and Induced Pluripotent Stem Cells (iPSCs) Disease Model of Harel-Yoon Syndrome Caused by Compound Heterozygous ATAD3A Variants Ziyi Jiang, Hongyu Chen, Xianghong Zhang, Xiaoling Jiang, Zhengqing Tong, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5370879/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Apr, 2025 Read the published version in Human Cell → Version 1 posted 5 You are reading this latest preprint version Abstract ATPase family AAA-domain-containing protein 3A (ATAD3A) is enriched on the mitochondrial membrane and is essential to the maintenance of mitochondrial structure and function. Variants of the ATAD3A gene can lead to Harel-Yoon syndrome (HAYOS), a developmental defect in neurological, cardiovascular, and other systems. This study aims to develop induced pluripotent stem cells (iPSCs) from the somatic cells of a patient (ZJUCHYLi001-A) and a negative control (ZJUCHYLi002-A) as effective tools for further investigations into the etiology of ATAD3A variant-related disease. We described and analyzed the clinical manifestations of the proband and her family members. Somatic cells from the proband and a negative control were collected and reprogrammed into iPSCs. Furthermore, we measured the ATAD3A expression levels in the iPSCs to confirm the validity of these cell lines. The proband and her elder sister were both critically ill and harbored compound heterozygous ATAD3A variants (F459S/T498Nfs*13). Their parents were carriers of these variants without any clinical manifestations. Both variants are located on the ATPase domain of the ATAD3A protein. Cell lines ZJUCHYLi001-A and ZJUCHYLi002-A presented typical features of pluripotent stem cells. The ATAD3A expression levels of ZJUCHYLi001-A were significantly reduced compared with ZJUCHYLi002-A. This study generated iPSCs from a patient with compound heterozygous variants of ATAD3A and a negative control as valuable tools for clarifying the molecular mechanisms underlying ATAD3A variant-related diseases. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction ATPase family AAA-domain-containing protein 3 (ATAD3) is a member of the AAA + superfamily, which is characterized by NTPases with a conserved AAA + module. In human and non-human primates, ATAD3 has three paralogs: ATAD3A, ATAD3B, and ATAD3C.[ 1 ] Human ATAD3A is located in the 1p36.33. The predominant ATAD3A isoform has 586 amino acids with two coiled-coil domains, two transmembrane domains, a Walker A motif, a Walker B motif, and an ATPase domain.[ 2 ] ATAD3A is anchored in the inner mitochondrial membrane by its transmembrane domains, with its ATPase catalytic core facing the mitochondrial matrix.[ 3 ] It interacts with the displacement loop of mitochondrial DNA, assisting in the formation or segregation of mitochondrial nucleoids.[ 4 ] ATAD3A plays a critical role in the maintenance of normal mitochondrial cristae structure. Organ-specific knockout of ATAD3 in mice significantly disrupted mitochondrial cristae morphology in the corresponding organ.[ 5 , 6 ] ATAD3A also exerts various functions through interactions with other proteins in the mitochondrial or endoplasmic reticulum (ER) membrane. It interacts with mitochondrial fission protein DRP1, and fusion proteins MFN1 and MFN2, regulating the mitochondrial dynamics.[ 7 – 9 ] ATAD3A also plays a role in the PINK1-dependent mitophagy, exerting different effects in different cells.[ 10 – 12 ] During ER stress, ATAD3A can protect active protein translation in mitochondria by interacting with PERK.[ 13 ] In addition, in vitro studies have revealed that ATAD3A is essential for the transport of cholesterol at the mitochondrial membrane[ 3 , 14 ] and its metabolism[ 12 , 15 ]. Previous studies have reported various developmental defects and clinical conditions that are related to ATAD3A gene alterations. Harel-Yoon syndrome (HAYOS) is a neurodevelopmental disorder associated with ATAD3A variants, featuring psychomotor delay, truncal hypotonia, appendicular spasticity, and peripheral neuropathy.[ 16 , 17 ] Typical HAYOS can have neonatal- or childhood-onset, with disease severity varying across cases.[ 16 , 18 , 19 ] In addition to HAYOS, defects owing to ATAD3A gene alterations can also involve the cardiovascular system and metabolism. Yap et al.[ 20 ] reported 13 individuals from 8 unrelated families with biallelic ATAD3A variants. Common clinical features of these patients include developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Similarly, Skopkova et al.[ 21 ] described four individuals from two families with compound heterozygous variants of ATAD3A and a clinical diagnosis of pontocerebellar hypoplasia. They found a consistent clinical picture and severity in patients sharing the same variant combinations. Cooper et al.[ 22 ] identified a dominantly inherited heterozygous variant that produced ATPase-deficient ATAD3A protein in two patients with childhood-onset hereditary spastic paraplegia. In more severe cases, patients with duplications in the ATAD3 gene locus can present with lethal cardiomyopathy, metabolic disorders, corneal opacities, and encephalopathy.[ 17 , 23 ] Large biallelic deletions affecting the ATAD3A gene can lead to fatal congenital pontocerebellar hypoplasia[ 24 , 25 ] or lethal spinal cord hypoplasia[ 26 ], whereas deletions not involving ATAD3A cause later-onset encephalopathy, cerebellar atrophy, ataxia, and dystonia.[ 24 ] Many case reports of patients with ATAD3A variants have primarily focused on the clinical and genetic data of the patients.[ 16 , 18 , 21 , 26 – 29 ] There is a notable lack of in vitro and in vivo disease models. Primary fibroblasts generated from the patient skin samples are the most commonly used in vitro tool to investigate the phenotype and pathogenicity of ATAD3A variants.[ 17 , 22 – 25 , 30 – 32 ] While harboring the patients’ genetic background, the skin fibroblasts may not accurately reflect the pathophysiology of the affected tissue or organs. Some researchers have generated transgenic Drosophila models expressing the cDNA of Drosophila dAtad3a with homologous variants of the patients and carried out immunostaining of the muscle and axon of the larvae.[ 17 , 20 ] This model provides a deeper insight into the disease mechanism associated with ATAD3A variants in muscular and neural tissue. However, significant anatomical and physiological differences between Drosophila and humans limit the model’s effectiveness for studying genetic variants. Induced pluripotent stem cells (iPSCs), which are embryonic-like stem cells reprogrammed from somatic cells,[ 33 , 34 ] have the potential to generate any tissues in the body while keeping the patient-specific genetic background. This technique enables researchers to develop disease-specific cell lines and differentiate them into functional somatic cells making it a promising platform for in vitro patient-specific disease modeling, drug testing, toxicity screening, and cell replacement therapy.[ 35 , 36 ] However, up till now, patient-specific iPSCs have not been applied in the studies of the ATAD3A variants and the disease mechanism of Harel-Yoon Syndrome. In this study, we identified two novel compound heterozygous variants of ATAD3A (F459S/T498Nfs*13) in a term infant who was diagnosed with Harel-Yoon Syndrome.[ 37 ] The child presented with coughing, tachycardia, and tachypnea, ultimately dying from heart failure. We generated iPSCs from this patient (ZJUCHYLi001-A) and a matched control child without ATAD3A variants (ZJUCHYLi002-A). The iPSCs presented typical pluripotency characteristics. Notably, we found that the ATAD3A protein level was lower in ZJUCHYLi001-A. Our study highlights the clinical manifestations of the affected patient and establishes patient-specific iPSCs as a valuable model for further investigation into the underlying molecular mechanisms of ATAD3A-related disorders. Materials and methods Proband, pedigree, and clinical assessments The patient was admitted to the Children’s Hospital of Zhejiang University School of Medicine on the 11th day after birth due to neonatal pneumonia. The negative control was a neonatal girl from the Department of Neonatology. The study was ethically approved by the Human Subjects Committees of Children's Hospital, Zhejiang University School of Medicine (2023-IRB-0177-IR-01). Written informed consent was obtained from the parents of each baby. Pathogenic variants analysis Genomic DNA was isolated from peripheral blood for each individual. A total of 200 ng of genomic DNA was fragmented into 150–200 bp using a focused ultrasonicator (Covaris, USA). The DNA libraries were then constructed through end-repair, poly(A) addition, adapters ligation, and PCR amplification using a VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, China). The targeted whole exome regions were enriched using a human total exon sequence capture kit KAPA HyperExome (Roche, USA) and sequenced on the DNBSEQ-T7 gene sequencer with 150 base paired-end reads. The original FASTQ files were processed by filtering out low-quality reads using Fastp.[ 38 ] Clean reads were then mapped to the reference genome GRCh37 using Burrows-Wheeler Aligner Maximal Exact Match (BWA-MEM)[ 39 ] and variants were called following the Genome Analysis Toolkit (GATK 4.1.7.0) best practice pipeline[ 40 ] and annotated with minor allele frequency (MAF) and functional predictions by Annovar.[ 41 ] Variants were filtered and prioritized based on an MAF of less than 0.8% in East Asian gnomAD population datasets, with functional annotations focusing on missense, in-frame, frameshift indels, canonical splice site, or nonsense variants. Pathogenic (P), likely pathogenic (LP) variants, and variants of uncertain significance (VUS) were prioritized according to the guidelines of the American College of Medical Genetics and Genomics (ACMG).[ 42 ] Protein structure analysis Protein sequence conservation analysis was performed using the constraint-based multiple alignment tool.[ 43 ] Protein sequencing conservation among different species was analyzed. The three-dimensional structure of the ATP-binding domain of ATAD3A based on the UniProt Q9NVI7 model was predicted by AlphaFold.[ 44 ] The structure was presented using PyMOL[ 45 ]. Isolation of fibroblast or Peripheral blood mononuclear cells The minced patient skin biopsy was digested with collagenase Ⅱ (Solarbio) for 6–8 hours and filtered through a 40 µm cell strainer. The isolated fibroblasts were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37°C in a 5% CO 2 atmosphere. The peripheral blood mononuclear cells (PBMCs) were isolated following the method described by Agu et al.[ 46 ] In brief, peripheral blood was diluted with phosphate-buffered saline (PBS) and gently layered over FICOLL PAQUE PLUS (GE Life). The mixture was then centrifuged at 500g for 30 minutes. The mononuclear cell layer was carefully collected and washed with PBS. The isolated PBMCs were cultured in RPMI1640 medium supplemented with 10% FBS and 1% antibiotics at 37°C in a 5% CO 2 incubator. Generation of iPSC lines Reprogramming of the fibroblast was conducted using the Reproeasy hiPSC Reprogramming Kit (Cellapy, Cat. No. CA5002003). On day − 1, fibroblasts were seeded in Matrigel(Corning, Cat. No. 354277)-coated 96-well plates at densities of 0.5, 1.0, or 1.5 × 10 4 per well. On day 0, wells containing 1 ~ 2 × 10 5 cells were selected and transduced with Reprogramming Media B. On day 3, cells were digested with 0.25% trypsin and replated into Matrigel-coated six-well plates at appropriate gradients using Reprogramming Media C. The following day, the media was replaced with DMEM/F12. Gradually changed the media from DMEM/F12 to Reproeasy Somatic Cell Reprogramming Media and Pluripotency Growth Master 1(PGM1) as the colonies formed and enlarged. By around day 21, large colonies were picked using pipette tips and transferred into a new Matrigel-coated plate in PGM1 media supplied with 10 µM Y-27632 (StemCell, Cat. No. 72304). Reprogramming of PBMCs was conducted in a similar procedure as described above. PBMCs were seeded into a 24-well plate with PBMC Activating Media (Cellapy, Cat. No. CA3408025). Fresh media were changed daily for 4 to 6 days. On day 0, 5 ~ 10 × 10 5 cells were collected and seeded into a 96-well plate with Reprogramming Media B. The following day, cells were collected and replated into 2 to 4 Matrigel-coated six-well plates with Reprogramming Media C. A half-media change with PBMC Activating Media was performed on day 3 and every other day thereafter. When small colonies appeared, the media was half changed with Reproeasy Somatic Cell Reprogramming Media every other day. Around day 11 and every other day thereafter, half of the media was replaced with PGM1. Once large colonies developed, the media was changed with PGM1 daily. Colony picking was conducted as described above. iPSCs maintenance and culture iPSCs were cultured in PGM1 media on Matrigel-coated six-well plates. For passaging, when the iPSCs reached approximately 80% confluent, they were digested with PSCeasy Human Pluripotent Stem Cell Dissociation Reagent (Cellapy, Cat. No. CA3001100) for 3 to 4 minutes in a 37℃ incubator. Cells were dissociated into small clumps and seeded into new Matrigel-coated six-well plates at a ratio of 1:6 in PGM1 media supplemented with 10 µM Y-27632. The media was changed to PGM1 media the following day and replaced daily until iPSCs reached 80% confluence. Immunofluorescence staining Cells cultured on Matrigel-coated 14mm coverslips were fixed in 4% paraformaldehyde for 15 minutes and permeabilized with 1 ~ 3‰ Triton-X-100 for 10 minutes at room temperature. Blocking buffer was prepared by dissolving 0.3 ~ 0.5g of bovine serum albumin in 10 ml PBST buffer. Coverslips were blocked for one hour at room temperature, followed by overnight incubation at 4℃ with primary antibodies (anti-Oct4 antibody, Huaan, SD0750; anti-SOX2 antibody, Huaan, PO00-28; SSEA4 mouse mAb, CST, #4755). The next day, coverslips were incubated with the appropriate secondary antibodies and mounted with DAPI-containing mounting media. The slides were observed under an inverted fluorescence microscope (Zeiss Axio Observer Z1). Reverse transcription-quantitative PCR Total RNA was extracted using FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, Cat. No. RC101-01) according to the manufacturer’s instructions. The purity and concentration of the extracted RNA were assessed using the BioDrop µLite + Microvolume Spectrophotometer. Reverse transcription of RNA was carried out with HiScript III RT SuperMix (Vazyme, Cat. No. R323-01) on Mastercycler® nexus GSX1 (Eppendorf). Quantitative PCR (qPCR) was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Cat. No. Q711-02) on CFX Opus 96 Real-Time PCR System (Bio-Rad). All primers used in this study are listed in Supplementary Table 1. Gene expression was analyzed using the 2 −ΔΔCt method. Trilineage differentiation assay Trilineage differentiation of iPSCs was conducted using STEMdiff Trilineage Differentiation Kit (StemCell, Cat.No. #05230) following the manufacturer’s instruction. After differentiation, biomarker expression for each lineage was assessed using quantitative PCR (qPCR) method as described above. Genetic variants validation Genomic DNA was extracted using the FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme, Cat. No. DC102-01) according to the manufacturer’s instructions. ATAD3A amplification was carried out using Phanta Max Master Mix (Vazyme, Cat. No. P515-01) with primers listed in Supplementary Table 1. The polymerase chain reaction (PCR) product was sent for Sanger Sequencing to detect ATAD3A variants. Karyotype analysis Eighty percent confluent iPSCs were incubated with 50ng/ml demecolcine at 37℃ for 6 to 8 hours. The cells were then harvested and incubated in a hypotonic potassium chloride solution for 20 to 40 minutes at 37℃. Fixation was carried out using a 3:1 mixture of methanol and acetic acid. Slides were prepared using gradual drips, followed by Giemsa staining. Sendai virus detection Total RNA extracted and transcribed into cDNA as described above. Detection of the Sendai virus was conducted using Phanta Max Master Mix (Vazyme, Cat. No. P515-01) with the primer pair listed in Supplementary Table 1. The PCR products were separated by agarose gel electrophoresis and visualized under a chemiluminescence imaging system (Bio-rad, ChemiDoc MP). Mycoplasma detection Mycoplasma detection was conducted using Myco-Lumi™ Luminescent Mycoplasma Detection Kit (Beyotime, Cat. No. C0298S) according to the manufacturer’s instructions. The cell culture supernatant was collected and incubated with Mycoplasma Detection Reagent A for five minutes, followed by Reagent B for ten minutes at room temperature. Luminescence was measured using a microplate reader (TECAN-SPARK). Western blot Cultured cells were lysed using radioimmunoprecipitation assay buffer (RIPA buffer). Protein concentrations were determined using the BCA Protein Quantification Kit (Vazyme, Cat. No. E112-01). Proteins were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were incubated overnight at 4 ℃ with ATAD3A primary antibodies (Novus, Cat. No. H00055210-D01P) or GAPDH primary antibodies (Abcam, Cat. No. ab313650), followed by incubation with secondary antibodies for 2 hours at room temperature. Chemiluminescence detection was performed using the G:Box chemi XRQ gel documentation system (Syngene). Gray values of the protein bands were quantified using ImageJ software. Results Clinical characteristics The proband (Fig. 1 A, II-2) was a term infant with an Apgar score of 10 and a birth weight of 3,170g. The patient exhibited poor responsiveness to sound and visual stimuli, tachycardia, weak heart sounds, and tachypnea. Hypermyotonia was observed in all four limbs, predominantly in the upper limbs. Cardiac ultrasonography revealed hypertrophic cardiomyopathy with reduced contractility, atrial septal defect, patent foramen ovale, and an ejection fraction ranging from 32–69% (Fig. 1 B). Cardiac magnetic resonance imaging (MRI) showed heterogeneous thickening of the left ventricular myocardium with abnormal signals and fibrosis (Fig. 1 C). Electrocardiograms showed T-wave changes and high lateral wall ST-segment changes, as well as sinus tachycardia and intraventricular block (Fig. 1 D). Brain MRI revealed high T2 signals in the white matter of the cerebrum and pons, along with a slightly widened subarachnoid space in the frontotemporal region (Fig. 1 E). Additionally, laboratory tests revealed elevated levels of creatine kinase isoenzyme and plasma lactate. Blood tandem mass spectrometry acylcarnitine assay indicated elevated levels of octanoylcarnitine and decanoylcarnitine. At 3 months, she died from heart failure. Notably, the proband’s elder sister (Fig. 1 A, II-1), who was born two and a half years earlier and died at the age of 18 days, displayed similar clinical features. Genetic findings WES was performed initially on the patient and her parents ( Supplementary Fig. 1 ). A VUS missense variant c.1376T > C (NM_0011 70535.3 p.F459S) compound with a likely pathogenic frameshift variant c.1492dup (NM_0011 70535.3 p.T498N fs*13) in SRD5A2 gene were identified. Segregation analysis showed that the c.1376T > C variant was inherited from the mother, while the c.1492dup variant was inherited from the father. The c.1376T > C variant was absent from population databases, and most predictive algorithms classified it as damaging. Based on the ACMG guidelines, this variant was categorized as a VUS. The c.1492dup has no frequency as well in population databases and was predicted to produce a presumed loss of function change. According to ACMG guidelines, it was classified as a likely pathogenic variant. The proband’s elder sister’s DNA was retrieved from the hospital’s Birth Cohort Biobank. Genetic testing revealed that she carried the same compound heterozygous variants inherited from their parents, respectively (Fig. 1 A, II-1; Supplementary Fig. 1). ATAD3A protein structure Human ATAD3A is located in the 1p36.33. Figure 2 A illustrates the structure of predominant ATAD3A isoform. Conservation analysis revealed sequence conservation of both variants among humans, mice, chickens, rats, and caeel (Fig. 2 A). The comparison of the initial monomeric 3D structures of the wild type and variant ATAD3A proteins is shown in Fig. 2 B-E. Both the F459S and T498Nfs*13 variants might interfere with the correct folding of ATAD3A. Establishment of ATAD3A F459S/T498Nfs*13 and control iPSC cell lines We established iPSC cell line ZJUCHYLi001-A from the fibroblasts from the skin tissue of the proband (Fig. 1 A, II-2) and ZJUCHYLi002-A from the PBMCs from a negative control. Both cell lines exhibited typical iPSC morphology. Immunofluorescence confirmed the expression of key pluripotency markers of OCT4, SOX2, and Nanog (Fig. 3 A). Directed differentiation through trilineage differentiation assay successfully generated all three germ layers (Fig. 3 B). Both cell lines exhibited normal karyotypes (46, XX) (Fig. 3 C). Sanger sequencing confirmed the presence of the expected variants in ATAD3A in ZJUCHYLi001-A and the absence of variants in ZJUCHYLi002-A. (Fig. 3 D). No residual Sendai virus was detected after passage 11 (Fig. 3 E). Mycoplasma detection assay confirmed the absence of mycoplasma contamination (Fig. 3 F). Compound heterozygosity results in the reduction of ATAD3A protein level After the establishment of iPSCs from both the patient and control, we investigated whether the expression level of ATAD3A was impaired in the patient-derived cells. We found no significant difference in the mRNA level of ATAD3A between the ZJUCHYLi001-A and ZJUCHYLi002-A (Fig. 4 A). However, the expression of ATAD3A protein was significantly down-regulated in the ZJUCHYLi001-A than ZJUCHYLi002-A (0.18 ± 0.06 vs. 0.48 ± 0.12, respectively. p = 0.009. Figure 4 B and 4 C). Discussion ATAD3A is essential to the structure and function of mitochondria. Alterations in the ATAD3A gene can lead to a wide range of diseases and symptoms, including HAYOS, cerebellar dysfunction, pontocerebellar hypoplasia, hereditary spastic paraplegia, and cardiomyopathy.[ 2 ] In this study, we identified compound heterozygous variants of ATAD3A in a critically ill infant diagnosed with HAYOS. Clinical manifestations of the patient included coughing, feeding difficulties, heart failure, abnormal electrocardiograms, and abnormal brain MRI. The patient’s elder sister, who carried the same ATAD3A variants, exhibited a similar clinical course during her neonatal period. Structure analysis showed that both variants are located within the ATPase domain of the protein, indicating that the pathogenic mechanism may be related to disruption in this specific domain. Previous studies have reported other variants in the ATPase domain of ATAD3A,[ 3 , 17 ] which impair normal oligomer functions of the protein and may lead to mitochondria fragmentation, small mitochondria, or increased mitophagy.[ 47 ] Patients with defects in this domain can present with developmental delay, hypotonia, spasticity, myopia, and enlargement of subarachnoid spaces.[ 17 ] Given the importance of mitochondrial function in human development and physiology, our study paves the way for further investigations into the function of ATAD3A. In our pedigree, the patient’s mother carries a heterozygous missense variant (p.F459S) of ATAD3A, and her father carries a heterozygous frameshift variant (p.T498Nfs*13). Both parents had non-significant clinical findings, supporting the recessive inheritance pattern of both variants. The iPSCs are considered a useful platform for studying disease mechanisms and developing therapeutic strategies.[ 48 ] iPSCs maintain the genetic background of the donor while exhibiting self-renewal and trilineage differentiation potentials.[ 49 , 50 ] In this study, we generated iPSCs from both the patient and a negative control. The patient-derived iPSCs (ZJUCHYLi001-A) demonstrated significantly downregulated protein expression of ATAD3A, indicating that these cells mimic the in vivo expression patterns observed in the patient. Currently, treatment options for patients with ATAD3A variants are mostly supportive,[ 28 , 51 ] with limited therapeutic approaches available. Our iPSC models provide a useful tool for investigating the underlying mechanism of ATAD3A-related diseases and exploring potential targeted therapies. Future studies will dive into the molecular mechanisms by which ATAD3A mutations affect mitochondrial function. In conclusion, we reported a HAYOS patient with compound heterozygous variants of ATAD3A and developed iPSC models from the patient and negative control. These models offer valuable insights into the molecular mechanisms behind ATAD3A-related diseases and may serve as platforms for developing targeted therapies. Declarations Conflicts of interest The authors declare no competing financial interests. Funding This work was supported by the Natural Science Foundation of Zhejiang Province (Y24H020013). Author contributions Lan Yu and Qiang Shu conceived the experiment. Xianghong Zhang, Jingjing Ye, Shanshan Shi and Xucong Shi collected and analyzed the clinical data and treated the patients. Ziyi Jiang, Hongyu Chen, Xiaoling Jiang and Weiqin Shao performed the experiments and analyzed the data. Zhengqing Tong and Fengxia Li performed the bioinformatics analysis. Ziyi Jiang and Hongyu Chen wrote the manuscript. Lan Yu, Hongyu Chen and Ziyi Jiang revised the manuscript. All authors read and approved the final manuscript. Acknowledgement We would like to acknowledge the patients and their family for their support of this study. Data availability The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request. References Teng Y, Lang L, Shay C. ATAD3A on the Path to Cancer. In: Guest PC, editor. Reviews on Biomarker Studies of Metabolic and Metabolism-Related Disorders [Internet]. Cham: Springer International Publishing; 2019 [cited 2023 Aug 18]. pp. 259–69. http://link.springer.com/ 10.1007/978-3-030-12668-1_14 Chen L, Li Y, Zambidis A, Papadopoulos V. ATAD3A: A Key Regulator of Mitochondria-Associated Diseases. Int J Mol Sci. 2023;24:12511. Gilquin B, Taillebourg E, Cherradi N, Hubstenberger A, Gay O, Merle N, et al. The AAA + ATPase ATAD3A controls mitochondrial dynamics at the interface of the inner and outer membranes. Mol Cell Biol. 2010;30:1984–96. He J, Mao C-C, Reyes A, Sembongi H, Di Re M, Granycome C, et al. The AAA + protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization. J Cell Biol. 2007;176:141–6. Arguello T, Peralta S, Antonicka H, Gaidosh G, Diaz F, Tu Y-T, et al. ATAD3A has a scaffolding role regulating mitochondria inner membrane structure and protein assembly. Cell Rep. 2021;37:110139. Peralta S, Goffart S, Williams SL, Diaz F, Garcia S, Nissanka N et al. ATAD3 controls mitochondrial cristae structure in mouse muscle, influencing mtDNA replication and cholesterol levels. J Cell Sci. 2018;131. Zhao Y, Hu D, Wang R, Sun X, Ropelewski P, Hubler Z, et al. ATAD3A oligomerization promotes neuropathology and cognitive deficits in Alzheimer’s disease models. Nat Commun. 2022;13:1121. Zhao Y, Sun X, Hu D, Prosdocimo DA, Hoppel C, Jain MK, et al. ATAD3A oligomerization causes neurodegeneration by coupling mitochondrial fragmentation and bioenergetics defects. Nat Commun. 2019;10:1371. Goller T, Seibold UK, Kremmer E, Voos W, Kolanus W. Atad3 function is essential for early post-implantation development in the mouse. PLoS ONE. 2013;8:e54799. Lang L, Loveless R, Dou J, Lam T, Chen A, Wang F, et al. ATAD3A mediates activation of RAS-independent mitochondrial ERK1/2 signaling, favoring head and neck cancer development. J Exp Clin Cancer Res. 2022;41:43. Jin G, Xu C, Zhang X, Long J, Rezaeian AH, Liu C, et al. Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells. Nat Immunol. 2018;19:29–40. Chen L, Li Y, Sottas C, Lazaris A, Petrillo SK, Metrakos P, et al. Loss of mitochondrial ATPase ATAD3A contributes to nonalcoholic fatty liver disease through accumulation of lipids and damaged mitochondria. J Biol Chem. 2022;298:102008. Brar KK, Hughes DT, Morris JL, Subramanian K, Krishna S, Gao F et al. PERK-ATAD3A interaction provides a subcellular safe haven for protein synthesis during ER stress. Science. 2024;eadp7114. Issop L, Fan J, Lee S, Rone MB, Basu K, Mui J, et al. Mitochondria-associated membrane formation in hormone-stimulated Leydig cell steroidogenesis: role of ATAD3. Endocrinology. 2015;156:334–45. Peres de Oliveira A, Basei FL, Slepicka PF, de Castro Ferezin C, Melo-Hanchuk TD, de Souza EE, et al. NEK10 interactome and depletion reveal new roles in mitochondria. Proteome Sci. 2020;18:4. Tawfik CA, Zaitoun R, Farag AA. Harel Yoon syndrome: a novel mutation in ATAD3A gene and expansion of the clinical spectrum. Ophthalmic Genet. 2023;44:226–33. Harel T, Yoon WH, Garone C, Gu S, Coban-Akdemir Z, Eldomery MK, et al. Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes. Am J Hum Genet. 2016;99:831–45. Zhang S, Lin L, Li Y, Peng C, Lin Y, Liu Y, et al. Harel-Yoon syndrome caused by a novel variant in ATAD3A: A case report. Heliyon. 2024;10:e23669. Qi R, Li R, Yang W, Sang Y. Harel-Yoon syndrome caused by the c.368G > A variant in the ATAD3A gene: A case report. Asian J Surg. 2022;45:914–6. Yap ZY, Park YH, Wortmann SB, Gunning AC, Ezer S, Lee S, et al. Functional interpretation of ATAD3A variants in neuro-mitochondrial phenotypes. Genome Med. 2021;13:55. Skopkova M, Stufkova H, Rambani V, Stranecky V, Brennerova K, Kolnikova M, et al. ATAD3A-related pontocerebellar hypoplasia: new patients and insights into phenotypic variability. Orphanet J Rare Dis. 2023;18:92. Cooper HM, Yang Y, Ylikallio E, Khairullin R, Woldegebriel R, Lin K-L, et al. ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia. Hum Mol Genet. 2017;26:1432–43. Frazier AE, Compton AG, Kishita Y, Hock DH, Welch AE, Amarasekera SSC, et al. Fatal perinatal mitochondrial cardiac failure caused by recurrent de novo duplications in the ATAD3 locus. Med (N Y). 2021;2:49–73. Desai R, Frazier AE, Durigon R, Patel H, Jones AW, Dalla Rosa I, et al. ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism. Brain. 2017;140:1595–610. Peralta S, González-Quintana A, Ybarra M, Delmiro A, Pérez-Pérez R, Docampo J, et al. Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities. Mol Genet Metab. 2019;128:452–62. Ebihara T, Nagatomo T, Sugiyama Y, Tsuruoka T, Osone Y, Shimura M, et al. Severe spinal cord hypoplasia due to a novel ATAD3A compound heterozygous deletion. Mol Genet Metab Rep. 2022;33:100912. Hanes I, McMillan HJ, Ito Y, Kernohan KD, Lazier J, Lines MA, et al. A splice variant in ATAD3A expands the clinical and genetic spectrum of Harel-Yoon syndrome. Neurol Genet. 2020;6:e452. Chen Y, Rong S, Luo H, Huang B, Hu F, Chen M, et al. Ketogenic Diet Attenuates Refractory Epilepsy of Harel-Yoon Syndrome With ATAD3A Variants: A Case Report and Review of Literature. Pediatr Neurol. 2023;143:79–83. Abdul-Raheem J, Nikkola E, Chen Z, Rohena L. Expanding the phenotype of Harel‐Yoon syndrome: A case report suggesting a genotype/phenotype correlation. Am J Med Genet Pt A. 2024;e63647. Lepelley A, Della Mina E, Van Nieuwenhove E, Waumans L, Fraitag S, Rice GI et al. Enhanced cGAS-STING-dependent interferon signaling associated with mutations in ATAD3A. J Exp Med. 2021;218. Gunning AC, Strucinska K, Muñoz Oreja M, Parrish A, Caswell R, Stals KL, et al. Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism. Am J Hum Genet. 2020;106:272–9. Dorison N, Gaignard P, Bayot A, Gelot A, Becker PH, Fourati S, et al. Mitochondrial dysfunction caused by novel ATAD3A mutations. Mol Genet Metab. 2020;131:107–13. Lowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, Sridharan R, et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci USA. 2008;105:2883–8. Saha K, Jaenisch R. Technical Challenges in Using Human Induced Pluripotent Stem Cells to Model Disease. Cell Stem Cell. 2009;5:584–95. Grskovic M, Javaherian A, Strulovici B, Daley GQ. Induced pluripotent stem cells — opportunities for disease modelling and drug discovery. Nat Rev Drug Discov. 2011;10:915–29. Paik DT, Chandy M, Wu JC. Patient and Disease–Specific Induced Pluripotent Stem Cells for Discovery of Personalized Cardiovascular Drugs and Therapeutics. Ohlstein EH, editor. Pharmacol Rev. 2020;72:320–42. Zheng Y, Yu X, Zhang T, Hu L, Zhou D, Huang X. ATAD3A gene variations in a family with Harel-Yoon syndrome. J Zhejiang Univ (Med Sci). 2023;52:738–43. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884–90. Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25:1754–60. Van Der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, Levy-Moonshine A et al. From FastQ Data to High‐Confidence Variant Calls: The Genome Analysis Toolkit Best Practices Pipeline. CP in Bioinformatics [Internet]. 2013 [cited 2024 Sep 20];43. https://currentprotocols.onlinelibrary.wiley.com/doi/ 10.1002/0471250953.bi1110s43 Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010;38:e164–164. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Sci. 2015;17:405–24. Papadopoulos JS, Agarwala R. COBALT: constraint-based alignment tool for multiple protein sequences. Bioinformatics. 2007;23:1073–9. The UniProt Consortium. ATPase family AAA domain-containing protein 3A [Internet]. https://www.uniprot.org/uniprotkb/Q9NVI7/entry Schrödinger LLC. The PyMOL Molecular Graphics System, Version 1.8. 2015. Agu CA, Soares FAC, Alderton A, Patel M, Ansari R, Patel S, et al. Successful Generation of Human Induced Pluripotent Stem Cell Lines from Blood Samples Held at Room Temperature for up to 48 hr. Stem Cell Rep. 2015;5:660–71. Bagli E, Zikou AK, Agnantis N, Kitsos G. Mitochondrial Membrane Dynamics and Inherited Optic Neuropathies. Vivo. 2017;31:511–25. Cerneckis J, Cai H, Shi Y. Induced pluripotent stem cells (iPSCs): molecular mechanisms of induction and applications. Sig Transduct Target Ther. 2024;9:112. Nagy A, Turksen K, editors. Induced pluripotent stem (iPS) cells: methods and protocols. Second edition. New York, NY: Humana Press; 2022. Lyra-Leite DM, Gutiérrez-Gutiérrez Ó, Wang M, Zhou Y, Cyganek L, Burridge PW. A review of protocols for human iPSC culture, cardiac differentiation, subtype-specification, maturation, and direct reprogramming. STAR Protocols. 2022;3:101560. Lang L, Loveless R, Teng Y. Emerging Links between Control of Mitochondrial Protein ATAD3A and Cancer. Int J Mol Sci. 2020;21. Supplementary Files supplementary.pdf Cite Share Download PDF Status: Published Journal Publication published 17 Apr, 2025 Read the published version in Human Cell → Version 1 posted Editorial decision: Major Revisions Needed 18 Nov, 2024 Reviewers agreed at journal 04 Nov, 2024 Reviewers invited by journal 03 Nov, 2024 Editor assigned by journal 03 Nov, 2024 First submitted to journal 31 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5370879","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":373539005,"identity":"11f5107d-617f-4897-83b2-623623c6f136","order_by":0,"name":"Ziyi Jiang","email":"","orcid":"","institution":"Zhejiang University School of Medicine Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ziyi","middleName":"","lastName":"Jiang","suffix":""},{"id":373539006,"identity":"dca75dbf-6244-4ecd-95df-7635a22a4c21","order_by":1,"name":"Hongyu Chen","email":"","orcid":"","institution":"Zhejiang University School of Medicine Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongyu","middleName":"","lastName":"Chen","suffix":""},{"id":373539007,"identity":"7ba836f7-9170-4c22-8f43-24dfda711cd0","order_by":2,"name":"Xianghong Zhang","email":"","orcid":"","institution":"Zhejiang University School of Medicine Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xianghong","middleName":"","lastName":"Zhang","suffix":""},{"id":373539008,"identity":"8022bce3-5ab1-4f7b-a0e2-507d2a9d7fa6","order_by":3,"name":"Xiaoling Jiang","email":"","orcid":"","institution":"Zhejiang University School of Medicine Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Jiang","suffix":""},{"id":373539009,"identity":"bdf2a33d-8cd4-4342-b200-ada70246b3f7","order_by":4,"name":"Zhengqing Tong","email":"","orcid":"","institution":"Snow Lake Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhengqing","middleName":"","lastName":"Tong","suffix":""},{"id":373539010,"identity":"c11fa2c6-7349-4c25-970a-41321d1e0f85","order_by":5,"name":"Jingjing Ye","email":"","orcid":"","institution":"Zhejiang University School of Medicine Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Ye","suffix":""},{"id":373539011,"identity":"2c7ba1e6-304d-400a-af3b-06d6bdbbfac7","order_by":6,"name":"Shanshan Shi","email":"","orcid":"","institution":"Zhejiang University School of Medicine Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shanshan","middleName":"","lastName":"Shi","suffix":""},{"id":373539012,"identity":"0009a945-edc7-4d9f-8235-52d649e87ab7","order_by":7,"name":"Xucong Shi","email":"","orcid":"","institution":"Zhejiang University School of Medicine Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xucong","middleName":"","lastName":"Shi","suffix":""},{"id":373539013,"identity":"9d393d37-e790-4a3b-82f8-cb40724cff75","order_by":8,"name":"Fengxia Li","email":"","orcid":"","institution":"Zhejiang University School of Medicine Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Fengxia","middleName":"","lastName":"Li","suffix":""},{"id":373539014,"identity":"79b7a4f9-37b7-43ce-b105-58fa60ff8b55","order_by":9,"name":"Weiqin Shao","email":"","orcid":"","institution":"Zhejiang University School of Medicine Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Weiqin","middleName":"","lastName":"Shao","suffix":""},{"id":373539015,"identity":"548343b3-08f5-446b-898e-da7f0682a180","order_by":10,"name":"Qiang Shu","email":"","orcid":"","institution":"Zhejiang University School of Medicine Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Shu","suffix":""},{"id":373539016,"identity":"affb3206-d7d3-40b0-9c88-f3b57494f4c5","order_by":11,"name":"Lan Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYFAC5gYGhgoJOQaGA0AOG1FaGIFaztgYk6iFsS0tsQHMIUaLwY3Exs8FbIfT5zeeMWD4UHaYgX92A0EtzdIzeA7nNjacMWCcce4wg8SdAwS1NEjzSBzObWY4Y8DM23aYwUAigbAtv3kMDqezgbT8JVJLmzRPQloCD0gLIzFaJM88bLPmOWBjOIPhWMHBnnPpPBI3CGjhO558+DbvPwl5+RmHNz74UWYtxz+DgBaFAzCWxAFwZPLgVw8E8g0wFn8DblWjYBSMglEwsgEAGqhG0Usa7O4AAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang University School of Medicine Children's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Lan","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-11-01 06:05:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5370879/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5370879/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13577-025-01214-x","type":"published","date":"2025-04-17T15:57:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70379248,"identity":"412bc679-4b5a-47b8-9bc2-dba1f513ddbd","added_by":"auto","created_at":"2024-12-02 15:45:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":263809,"visible":true,"origin":"","legend":"\u003cp\u003eClinical examinations of the proband. (A) The pedigree of the patients indicated that the F459S variant was inherited from the mother and the T498Nfs*13 variant was inherited from the father of the patient. The arrow indicates the proband. (B) Echocardiography reveals reduced ventricle contractility and ejection fraction. (C) Cardiac MRI shows heterogeneous thickening of the left ventricular myocardium. (D) The electrocardiogram shows T-wave and ST-segment changes. (E) Brain MRI reveals T2 signal changes in the white matter and widened subarachnoid space.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5370879/v1/c6229ce317767c518b41f771.png"},{"id":70379246,"identity":"44bc1336-0c97-47b7-a0b6-8b7310d477fa","added_by":"auto","created_at":"2024-12-02 15:45:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":329454,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the protein sequence and structure of ATAD3A variants. (A) ATAD3A isoform and protein sequence conservation analysis. Both variants are conserved among vertebrates and invertebrates. (B-E) 3D protein structure modeling shows conformational differences of wild type and variant ATAD3A proteins.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5370879/v1/6ac593c5605e2facd39dd9a4.png"},{"id":70379244,"identity":"07a933fa-307f-46ee-85d3-0f6fccaed22a","added_by":"auto","created_at":"2024-12-02 15:45:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":455606,"visible":true,"origin":"","legend":"\u003cp\u003eEstablishment and verification of iPSC cell lines ZJUCHYLi001-A and ZJUCHYLi002-A. (A) Immunofluorescence confirmed the expression of pluripotency markers (OCT-4, SOX-2, and SSEA-4) in two iPSC cell lines. (B) qPCR analysis of the biomarkers of endoderm (CXCR4 and FOXA2), mesoderm (CXCR4 and Brachyury), and ectoderm (PAX6 and Nestin) demonstrate successful trilineage differentiation of both iPSC cell lines. (C) Karyotype analysis confirmed normal 46, XX karyotype in both patient and control iPSC cell lines. (D) Sanger sequencing confirmed the presence of ATAD3A c.1376T\u0026gt;C and c.1492dup variants in the patient’s fibroblasts and ZJUCHYLi001-A and fibroblast and the absence of both variants in the control’s PBMC and ZJUCHYLi002-A. (E) Electrophoresis demonstrates no residual Sendai virus in both iPSCs at passage 11. N: negative control without DNA. P: positive control with cDNA of ZJUCHYLi002-A at passage 3. (F) Mycoplasma detection assay verifies no contamination of mycoplasma. N: negative control with freshly prepared Pluripotency Growth Master 1(PGM1) media. P: positive control provided in the Myco-Lumi™ Luminescent Mycoplasma Detection Kit.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5370879/v1/c191f18240ab678f09a0f8fb.png"},{"id":70379245,"identity":"df1a94cb-3d92-4282-9386-ad4bf962c7ef","added_by":"auto","created_at":"2024-12-02 15:45:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":69025,"visible":true,"origin":"","legend":"\u003cp\u003eExpression level of ATAD3A in iPSC cell lines ZJUCHYLi001-A and ZJUCHYLi002-A. (A) A comparison of relative mRNA levels of ATAD3A suggested no significant difference between ZJUCHYLi001-A and ZJUCHYLi002-A. Data are presented as mean ± SD from three independent experiments. (B) Western blot showed downregulated expression of ATAD3A protein in ZJUCHYLi001-A compared with ZJUCHYLi002-A. (C) Comparison of relative protein levels of ATAD3A in ZJUCHYLi001-A and ZJUCHYLi002-A. Data are presented as mean ± SD from four independent experiments.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5370879/v1/e37666bda7ed873fefa3239f.png"},{"id":81050995,"identity":"264cb673-a9d8-43ba-a6bc-52beda44cfa1","added_by":"auto","created_at":"2025-04-21 16:09:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1912799,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5370879/v1/751f4d8a-9b11-4f0c-9718-fef8760f482f.pdf"},{"id":70379247,"identity":"9f9bef39-9ee5-426b-b189-b54ca51d5369","added_by":"auto","created_at":"2024-12-02 15:45:29","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":171587,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5370879/v1/bd78ad9416029ae61551a115.pdf"}],"financialInterests":"","formattedTitle":"Clinical Characteristics and Induced Pluripotent Stem Cells (iPSCs) Disease Model of Harel-Yoon Syndrome Caused by Compound Heterozygous ATAD3A Variants","fulltext":[{"header":"Introduction","content":"\u003cp\u003eATPase family AAA-domain-containing protein 3 (ATAD3) is a member of the AAA\u0026thinsp;+\u0026thinsp;superfamily, which is characterized by NTPases with a conserved AAA\u0026thinsp;+\u0026thinsp;module. In human and non-human primates, ATAD3 has three paralogs: ATAD3A, ATAD3B, and ATAD3C.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Human \u003cem\u003eATAD3A\u003c/em\u003e is located in the 1p36.33. The predominant ATAD3A isoform has 586 amino acids with two coiled-coil domains, two transmembrane domains, a Walker A motif, a Walker B motif, and an ATPase domain.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] ATAD3A is anchored in the inner mitochondrial membrane by its transmembrane domains, with its ATPase catalytic core facing the mitochondrial matrix.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] It interacts with the displacement loop of mitochondrial DNA, assisting in the formation or segregation of mitochondrial nucleoids.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] ATAD3A plays a critical role in the maintenance of normal mitochondrial cristae structure. Organ-specific knockout of \u003cem\u003eATAD3\u003c/em\u003e in mice significantly disrupted mitochondrial cristae morphology in the corresponding organ.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] ATAD3A also exerts various functions through interactions with other proteins in the mitochondrial or endoplasmic reticulum (ER) membrane. It interacts with mitochondrial fission protein DRP1, and fusion proteins MFN1 and MFN2, regulating the mitochondrial dynamics.[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] ATAD3A also plays a role in the PINK1-dependent mitophagy, exerting different effects in different cells.[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] During ER stress, ATAD3A can protect active protein translation in mitochondria by interacting with PERK.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] In addition, in vitro studies have revealed that ATAD3A is essential for the transport of cholesterol at the mitochondrial membrane[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and its metabolism[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies have reported various developmental defects and clinical conditions that are related to \u003cem\u003eATAD3A\u003c/em\u003e gene alterations. Harel-Yoon syndrome (HAYOS) is a neurodevelopmental disorder associated with \u003cem\u003eATAD3A\u003c/em\u003e variants, featuring psychomotor delay, truncal hypotonia, appendicular spasticity, and peripheral neuropathy.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] Typical HAYOS can have neonatal- or childhood-onset, with disease severity varying across cases.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] In addition to HAYOS, defects owing to \u003cem\u003eATAD3A\u003c/em\u003e gene alterations can also involve the cardiovascular system and metabolism. Yap et al.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] reported 13 individuals from 8 unrelated families with biallelic \u003cem\u003eATAD3A\u003c/em\u003e variants. Common clinical features of these patients include developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Similarly, Skopkova et al.[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] described four individuals from two families with compound heterozygous variants of \u003cem\u003eATAD3A\u003c/em\u003e and a clinical diagnosis of pontocerebellar hypoplasia. They found a consistent clinical picture and severity in patients sharing the same variant combinations. Cooper et al.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] identified a dominantly inherited heterozygous variant that produced ATPase-deficient ATAD3A protein in two patients with childhood-onset hereditary spastic paraplegia. In more severe cases, patients with duplications in the \u003cem\u003eATAD3\u003c/em\u003e gene locus can present with lethal cardiomyopathy, metabolic disorders, corneal opacities, and encephalopathy.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Large biallelic deletions affecting the \u003cem\u003eATAD3A\u003c/em\u003e gene can lead to fatal congenital pontocerebellar hypoplasia[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] or lethal spinal cord hypoplasia[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], whereas deletions not involving \u003cem\u003eATAD3A\u003c/em\u003e cause later-onset encephalopathy, cerebellar atrophy, ataxia, and dystonia.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eMany case reports of patients with \u003cem\u003eATAD3A\u003c/em\u003e variants have primarily focused on the clinical and genetic data of the patients.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] There is a notable lack of in vitro and in vivo disease models. Primary fibroblasts generated from the patient skin samples are the most commonly used in vitro tool to investigate the phenotype and pathogenicity of \u003cem\u003eATAD3A\u003c/em\u003e variants.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] While harboring the patients\u0026rsquo; genetic background, the skin fibroblasts may not accurately reflect the pathophysiology of the affected tissue or organs. Some researchers have generated transgenic Drosophila models expressing the cDNA of \u003cem\u003eDrosophila dAtad3a\u003c/em\u003e with homologous variants of the patients and carried out immunostaining of the muscle and axon of the larvae.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] This model provides a deeper insight into the disease mechanism associated with \u003cem\u003eATAD3A\u003c/em\u003e variants in muscular and neural tissue. However, significant anatomical and physiological differences between Drosophila and humans limit the model\u0026rsquo;s effectiveness for studying genetic variants. Induced pluripotent stem cells (iPSCs), which are embryonic-like stem cells reprogrammed from somatic cells,[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] have the potential to generate any tissues in the body while keeping the patient-specific genetic background. This technique enables researchers to develop disease-specific cell lines and differentiate them into functional somatic cells making it a promising platform for in vitro patient-specific disease modeling, drug testing, toxicity screening, and cell replacement therapy.[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] However, up till now, patient-specific iPSCs have not been applied in the studies of the \u003cem\u003eATAD3A\u003c/em\u003e variants and the disease mechanism of Harel-Yoon Syndrome. In this study, we identified two novel compound heterozygous variants of \u003cem\u003eATAD3A\u003c/em\u003e (F459S/T498Nfs*13) in a term infant who was diagnosed with Harel-Yoon Syndrome.[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] The child presented with coughing, tachycardia, and tachypnea, ultimately dying from heart failure. We generated iPSCs from this patient (ZJUCHYLi001-A) and a matched control child without \u003cem\u003eATAD3A\u003c/em\u003e variants (ZJUCHYLi002-A). The iPSCs presented typical pluripotency characteristics. Notably, we found that the ATAD3A protein level was lower in ZJUCHYLi001-A. Our study highlights the clinical manifestations of the affected patient and establishes patient-specific iPSCs as a valuable model for further investigation into the underlying molecular mechanisms of ATAD3A-related disorders.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProband, pedigree, and clinical assessments\u003c/h2\u003e \u003cp\u003eThe patient was admitted to the Children\u0026rsquo;s Hospital of Zhejiang University School of Medicine on the 11th day after birth due to neonatal pneumonia. The negative control was a neonatal girl from the Department of Neonatology. The study was ethically approved by the Human Subjects Committees of Children's Hospital, Zhejiang University School of Medicine (2023-IRB-0177-IR-01). Written informed consent was obtained from the parents of each baby.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePathogenic variants analysis\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was isolated from peripheral blood for each individual. A total of 200 ng of genomic DNA was fragmented into 150\u0026ndash;200 bp using a focused ultrasonicator (Covaris, USA). The DNA libraries were then constructed through end-repair, poly(A) addition, adapters ligation, and PCR amplification using a VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, China). The targeted whole exome regions were enriched using a human total exon sequence capture kit KAPA HyperExome (Roche, USA) and sequenced on the DNBSEQ-T7 gene sequencer with 150 base paired-end reads.\u003c/p\u003e \u003cp\u003eThe original FASTQ files were processed by filtering out low-quality reads using Fastp.[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] Clean reads were then mapped to the reference genome GRCh37 using Burrows-Wheeler Aligner Maximal Exact Match (BWA-MEM)[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and variants were called following the Genome Analysis Toolkit (GATK 4.1.7.0) best practice pipeline[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and annotated with minor allele frequency (MAF) and functional predictions by Annovar.[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] Variants were filtered and prioritized based on an MAF of less than 0.8% in East Asian gnomAD population datasets, with functional annotations focusing on missense, in-frame, frameshift indels, canonical splice site, or nonsense variants. Pathogenic (P), likely pathogenic (LP) variants, and variants of uncertain significance (VUS) were prioritized according to the guidelines of the American College of Medical Genetics and Genomics (ACMG).[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/p\u003e\n\u003ch3\u003eProtein structure analysis\u003c/h3\u003e\n\u003cp\u003eProtein sequence conservation analysis was performed using the constraint-based multiple alignment tool.[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] Protein sequencing conservation among different species was analyzed. The three-dimensional structure of the ATP-binding domain of ATAD3A based on the UniProt Q9NVI7 model was predicted by AlphaFold.[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] The structure was presented using PyMOL[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eIsolation of fibroblast or Peripheral blood mononuclear cells\u003c/h3\u003e\n\u003cp\u003eThe minced patient skin biopsy was digested with collagenase Ⅱ (Solarbio) for 6\u0026ndash;8 hours and filtered through a 40 \u0026micro;m cell strainer. The isolated fibroblasts were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u003c/p\u003e \u003cp\u003eThe peripheral blood mononuclear cells (PBMCs) were isolated following the method described by Agu et al.[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] In brief, peripheral blood was diluted with phosphate-buffered saline (PBS) and gently layered over FICOLL PAQUE PLUS (GE Life). The mixture was then centrifuged at 500g for 30 minutes. The mononuclear cell layer was carefully collected and washed with PBS. The isolated PBMCs were cultured in RPMI1640 medium supplemented with 10% FBS and 1% antibiotics at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\n\u003ch3\u003eGeneration of iPSC lines\u003c/h3\u003e\n\u003cp\u003eReprogramming of the fibroblast was conducted using the Reproeasy hiPSC Reprogramming Kit (Cellapy, Cat. No. CA5002003). On day \u0026minus;\u0026thinsp;1, fibroblasts were seeded in Matrigel(Corning, Cat. No. 354277)-coated 96-well plates at densities of 0.5, 1.0, or 1.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e per well. On day 0, wells containing 1\u0026thinsp;~\u0026thinsp;2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were selected and transduced with Reprogramming Media B. On day 3, cells were digested with 0.25% trypsin and replated into Matrigel-coated six-well plates at appropriate gradients using Reprogramming Media C. The following day, the media was replaced with DMEM/F12. Gradually changed the media from DMEM/F12 to Reproeasy Somatic Cell Reprogramming Media and Pluripotency Growth Master 1(PGM1) as the colonies formed and enlarged. By around day 21, large colonies were picked using pipette tips and transferred into a new Matrigel-coated plate in PGM1 media supplied with 10 \u0026micro;M Y-27632 (StemCell, Cat. No. 72304).\u003c/p\u003e \u003cp\u003eReprogramming of PBMCs was conducted in a similar procedure as described above. PBMCs were seeded into a 24-well plate with PBMC Activating Media (Cellapy, Cat. No. CA3408025). Fresh media were changed daily for 4 to 6 days. On day 0, 5\u0026thinsp;~\u0026thinsp;10 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were collected and seeded into a 96-well plate with Reprogramming Media B. The following day, cells were collected and replated into 2 to 4 Matrigel-coated six-well plates with Reprogramming Media C. A half-media change with PBMC Activating Media was performed on day 3 and every other day thereafter. When small colonies appeared, the media was half changed with Reproeasy Somatic Cell Reprogramming Media every other day. Around day 11 and every other day thereafter, half of the media was replaced with PGM1. Once large colonies developed, the media was changed with PGM1 daily. Colony picking was conducted as described above.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eiPSCs maintenance and culture\u003c/h2\u003e \u003cp\u003eiPSCs were cultured in PGM1 media on Matrigel-coated six-well plates. For passaging, when the iPSCs reached approximately 80% confluent, they were digested with PSCeasy Human Pluripotent Stem Cell Dissociation Reagent (Cellapy, Cat. No. CA3001100) for 3 to 4 minutes in a 37℃ incubator. Cells were dissociated into small clumps and seeded into new Matrigel-coated six-well plates at a ratio of 1:6 in PGM1 media supplemented with 10 \u0026micro;M Y-27632. The media was changed to PGM1 media the following day and replaced daily until iPSCs reached 80% confluence.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eCells cultured on Matrigel-coated 14mm coverslips were fixed in 4% paraformaldehyde for 15 minutes and permeabilized with 1\u0026thinsp;~\u0026thinsp;3\u0026permil; Triton-X-100 for 10 minutes at room temperature. Blocking buffer was prepared by dissolving 0.3\u0026thinsp;~\u0026thinsp;0.5g of bovine serum albumin in 10 ml PBST buffer. Coverslips were blocked for one hour at room temperature, followed by overnight incubation at 4℃ with primary antibodies (anti-Oct4 antibody, Huaan, SD0750; anti-SOX2 antibody, Huaan, PO00-28; SSEA4 mouse mAb, CST, #4755). The next day, coverslips were incubated with the appropriate secondary antibodies and mounted with DAPI-containing mounting media. The slides were observed under an inverted fluorescence microscope (Zeiss Axio Observer Z1).\u003c/p\u003e\n\u003ch3\u003eReverse transcription-quantitative PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, Cat. No. RC101-01) according to the manufacturer\u0026rsquo;s instructions. The purity and concentration of the extracted RNA were assessed using the BioDrop \u0026micro;Lite\u0026thinsp;+\u0026thinsp;Microvolume Spectrophotometer. Reverse transcription of RNA was carried out with HiScript III RT SuperMix (Vazyme, Cat. No. R323-01) on Mastercycler\u0026reg; nexus GSX1 (Eppendorf). Quantitative PCR (qPCR) was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Cat. No. Q711-02) on CFX Opus 96 Real-Time PCR System (Bio-Rad). All primers used in this study are listed in Supplementary Table\u0026nbsp;1. Gene expression was analyzed using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTrilineage differentiation assay\u003c/h2\u003e \u003cp\u003eTrilineage differentiation of iPSCs was conducted using STEMdiff Trilineage Differentiation Kit (StemCell, Cat.No. #05230) following the manufacturer\u0026rsquo;s instruction. After differentiation, biomarker expression for each lineage was assessed using quantitative PCR (qPCR) method as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGenetic variants validation\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted using the FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme, Cat. No. DC102-01) according to the manufacturer\u0026rsquo;s instructions. \u003cem\u003eATAD3A\u003c/em\u003e amplification was carried out using Phanta Max Master Mix (Vazyme, Cat. No. P515-01) with primers listed in Supplementary Table\u0026nbsp;1. The polymerase chain reaction (PCR) product was sent for Sanger Sequencing to detect \u003cem\u003eATAD3A\u003c/em\u003e variants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eKaryotype analysis\u003c/h2\u003e \u003cp\u003eEighty percent confluent iPSCs were incubated with 50ng/ml demecolcine at 37℃ for 6 to 8 hours. The cells were then harvested and incubated in a hypotonic potassium chloride solution for 20 to 40 minutes at 37℃. Fixation was carried out using a 3:1 mixture of methanol and acetic acid. Slides were prepared using gradual drips, followed by Giemsa staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSendai virus detection\u003c/h2\u003e \u003cp\u003eTotal RNA extracted and transcribed into cDNA as described above. Detection of the Sendai virus was conducted using Phanta Max Master Mix (Vazyme, Cat. No. P515-01) with the primer pair listed in Supplementary Table\u0026nbsp;1. The PCR products were separated by agarose gel electrophoresis and visualized under a chemiluminescence imaging system (Bio-rad, ChemiDoc MP).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMycoplasma detection\u003c/h2\u003e \u003cp\u003eMycoplasma detection was conducted using Myco-Lumi\u0026trade; Luminescent Mycoplasma Detection Kit (Beyotime, Cat. No. C0298S) according to the manufacturer\u0026rsquo;s instructions. The cell culture supernatant was collected and incubated with Mycoplasma Detection Reagent A for five minutes, followed by Reagent B for ten minutes at room temperature. Luminescence was measured using a microplate reader (TECAN-SPARK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eCultured cells were lysed using radioimmunoprecipitation assay buffer (RIPA buffer). Protein concentrations were determined using the BCA Protein Quantification Kit (Vazyme, Cat. No. E112-01). Proteins were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were incubated overnight at 4 ℃ with ATAD3A primary antibodies (Novus, Cat. No. H00055210-D01P) or GAPDH primary antibodies (Abcam, Cat. No. ab313650), followed by incubation with secondary antibodies for 2 hours at room temperature. Chemiluminescence detection was performed using the G:Box chemi XRQ gel documentation system (Syngene). Gray values of the protein bands were quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eClinical characteristics\u003c/h2\u003e \u003cp\u003eThe proband (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, II-2) was a term infant with an Apgar score of 10 and a birth weight of 3,170g. The patient exhibited poor responsiveness to sound and visual stimuli, tachycardia, weak heart sounds, and tachypnea. Hypermyotonia was observed in all four limbs, predominantly in the upper limbs. Cardiac ultrasonography revealed hypertrophic cardiomyopathy with reduced contractility, atrial septal defect, patent foramen ovale, and an ejection fraction ranging from 32\u0026ndash;69% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Cardiac magnetic resonance imaging (MRI) showed heterogeneous thickening of the left ventricular myocardium with abnormal signals and fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Electrocardiograms showed T-wave changes and high lateral wall ST-segment changes, as well as sinus tachycardia and intraventricular block (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Brain MRI revealed high T2 signals in the white matter of the cerebrum and pons, along with a slightly widened subarachnoid space in the frontotemporal region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Additionally, laboratory tests revealed elevated levels of creatine kinase isoenzyme and plasma lactate. Blood tandem mass spectrometry acylcarnitine assay indicated elevated levels of octanoylcarnitine and decanoylcarnitine. At 3 months, she died from heart failure. Notably, the proband\u0026rsquo;s elder sister (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, II-1), who was born two and a half years earlier and died at the age of 18 days, displayed similar clinical features.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGenetic findings\u003c/h2\u003e \u003cp\u003eWES was performed initially on the patient and her parents (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). A VUS missense variant c.1376T\u0026thinsp;\u0026gt;\u0026thinsp;C (NM_0011 70535.3 p.F459S) compound with a likely pathogenic frameshift variant c.1492dup (NM_0011 70535.3 p.T498N fs*13) in \u003cem\u003eSRD5A2\u003c/em\u003e gene were identified. Segregation analysis showed that the c.1376T\u0026thinsp;\u0026gt;\u0026thinsp;C variant was inherited from the mother, while the c.1492dup variant was inherited from the father. The c.1376T\u0026thinsp;\u0026gt;\u0026thinsp;C variant was absent from population databases, and most predictive algorithms classified it as damaging. Based on the ACMG guidelines, this variant was categorized as a VUS. The c.1492dup has no frequency as well in population databases and was predicted to produce a presumed loss of function change. According to ACMG guidelines, it was classified as a likely pathogenic variant.\u003c/p\u003e \u003cp\u003eThe proband\u0026rsquo;s elder sister\u0026rsquo;s DNA was retrieved from the hospital\u0026rsquo;s Birth Cohort Biobank. Genetic testing revealed that she carried the same compound heterozygous variants inherited from their parents, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, II-1; Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eATAD3A protein structure\u003c/h2\u003e \u003cp\u003eHuman \u003cem\u003eATAD3A\u003c/em\u003e is located in the 1p36.33. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA illustrates the structure of predominant ATAD3A isoform. Conservation analysis revealed sequence conservation of both variants among humans, mice, chickens, rats, and caeel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The comparison of the initial monomeric 3D structures of the wild type and variant ATAD3A proteins is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-E. Both the F459S and T498Nfs*13 variants might interfere with the correct folding of ATAD3A.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of ATAD3A\u003csup\u003eF459S/T498Nfs*13\u003c/sup\u003e and control iPSC cell lines\u003c/h2\u003e \u003cp\u003eWe established iPSC cell line ZJUCHYLi001-A from the fibroblasts from the skin tissue of the proband (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, II-2) and ZJUCHYLi002-A from the PBMCs from a negative control. Both cell lines exhibited typical iPSC morphology. Immunofluorescence confirmed the expression of key pluripotency markers of OCT4, SOX2, and Nanog (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Directed differentiation through trilineage differentiation assay successfully generated all three germ layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Both cell lines exhibited normal karyotypes (46, XX) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Sanger sequencing confirmed the presence of the expected variants in \u003cem\u003eATAD3A\u003c/em\u003e in ZJUCHYLi001-A and the absence of variants in ZJUCHYLi002-A. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). No residual Sendai virus was detected after passage 11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Mycoplasma detection assay confirmed the absence of mycoplasma contamination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCompound heterozygosity results in the reduction of ATAD3A protein level\u003c/h2\u003e \u003cp\u003eAfter the establishment of iPSCs from both the patient and control, we investigated whether the expression level of ATAD3A was impaired in the patient-derived cells. We found no significant difference in the mRNA level of ATAD3A between the ZJUCHYLi001-A and ZJUCHYLi002-A (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, the expression of ATAD3A protein was significantly down-regulated in the ZJUCHYLi001-A than ZJUCHYLi002-A (0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 vs. 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, respectively. p\u0026thinsp;=\u0026thinsp;0.009. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eATAD3A is essential to the structure and function of mitochondria. Alterations in the \u003cem\u003eATAD3A\u003c/em\u003e gene can lead to a wide range of diseases and symptoms, including HAYOS, cerebellar dysfunction, pontocerebellar hypoplasia, hereditary spastic paraplegia, and cardiomyopathy.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] In this study, we identified compound heterozygous variants of \u003cem\u003eATAD3A\u003c/em\u003e in a critically ill infant diagnosed with HAYOS. Clinical manifestations of the patient included coughing, feeding difficulties, heart failure, abnormal electrocardiograms, and abnormal brain MRI. The patient\u0026rsquo;s elder sister, who carried the same \u003cem\u003eATAD3A\u003c/em\u003e variants, exhibited a similar clinical course during her neonatal period.\u003c/p\u003e \u003cp\u003eStructure analysis showed that both variants are located within the ATPase domain of the protein, indicating that the pathogenic mechanism may be related to disruption in this specific domain. Previous studies have reported other variants in the ATPase domain of ATAD3A,[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] which impair normal oligomer functions of the protein and may lead to mitochondria fragmentation, small mitochondria, or increased mitophagy.[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] Patients with defects in this domain can present with developmental delay, hypotonia, spasticity, myopia, and enlargement of subarachnoid spaces.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] Given the importance of mitochondrial function in human development and physiology, our study paves the way for further investigations into the function of ATAD3A. In our pedigree, the patient\u0026rsquo;s mother carries a heterozygous missense variant (p.F459S) of ATAD3A, and her father carries a heterozygous frameshift variant (p.T498Nfs*13). Both parents had non-significant clinical findings, supporting the recessive inheritance pattern of both variants.\u003c/p\u003e \u003cp\u003eThe iPSCs are considered a useful platform for studying disease mechanisms and developing therapeutic strategies.[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] iPSCs maintain the genetic background of the donor while exhibiting self-renewal and trilineage differentiation potentials.[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] In this study, we generated iPSCs from both the patient and a negative control. The patient-derived iPSCs (ZJUCHYLi001-A) demonstrated significantly downregulated protein expression of ATAD3A, indicating that these cells mimic the in vivo expression patterns observed in the patient. Currently, treatment options for patients with \u003cem\u003eATAD3A\u003c/em\u003e variants are mostly supportive,[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] with limited therapeutic approaches available. Our iPSC models provide a useful tool for investigating the underlying mechanism of ATAD3A-related diseases and exploring potential targeted therapies. Future studies will dive into the molecular mechanisms by which \u003cem\u003eATAD3A\u003c/em\u003e mutations affect mitochondrial function.\u003c/p\u003e \u003cp\u003eIn conclusion, we reported a HAYOS patient with compound heterozygous variants of \u003cem\u003eATAD3A\u003c/em\u003e and developed iPSC models from the patient and negative control. These models offer valuable insights into the molecular mechanisms behind ATAD3A-related diseases and may serve as platforms for developing targeted therapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Natural Science Foundation of Zhejiang Province (Y24H020013).\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eLan Yu and Qiang Shu conceived the experiment. Xianghong Zhang, Jingjing Ye, Shanshan Shi and Xucong Shi collected and analyzed the clinical data and treated the patients. Ziyi Jiang, Hongyu Chen, Xiaoling Jiang and Weiqin Shao performed the experiments and analyzed the data. Zhengqing Tong and Fengxia Li performed the bioinformatics analysis. Ziyi Jiang and Hongyu Chen wrote the manuscript. Lan Yu, Hongyu Chen and Ziyi Jiang revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eWe would like to acknowledge the patients and their family for their support of this study.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTeng Y, Lang L, Shay C. ATAD3A on the Path to Cancer. In: Guest PC, editor. Reviews on Biomarker Studies of Metabolic and Metabolism-Related Disorders [Internet]. Cham: Springer International Publishing; 2019 [cited 2023 Aug 18]. pp. 259\u0026ndash;69. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://link.springer.com/\u003c/span\u003e\u003cspan address=\"http://link.springer.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-030-12668-1_14\u003c/span\u003e\u003cspan address=\"10.1007/978-3-030-12668-1_14\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Li Y, Zambidis A, Papadopoulos V. ATAD3A: A Key Regulator of Mitochondria-Associated Diseases. Int J Mol Sci. 2023;24:12511.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGilquin B, Taillebourg E, Cherradi N, Hubstenberger A, Gay O, Merle N, et al. The AAA\u0026thinsp;+\u0026thinsp;ATPase ATAD3A controls mitochondrial dynamics at the interface of the inner and outer membranes. Mol Cell Biol. 2010;30:1984\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe J, Mao C-C, Reyes A, Sembongi H, Di Re M, Granycome C, et al. The AAA\u0026thinsp;+\u0026thinsp;protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization. J Cell Biol. 2007;176:141\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArguello T, Peralta S, Antonicka H, Gaidosh G, Diaz F, Tu Y-T, et al. ATAD3A has a scaffolding role regulating mitochondria inner membrane structure and protein assembly. Cell Rep. 2021;37:110139.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeralta S, Goffart S, Williams SL, Diaz F, Garcia S, Nissanka N et al. ATAD3 controls mitochondrial cristae structure in mouse muscle, influencing mtDNA replication and cholesterol levels. J Cell Sci. 2018;131.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Y, Hu D, Wang R, Sun X, Ropelewski P, Hubler Z, et al. ATAD3A oligomerization promotes neuropathology and cognitive deficits in Alzheimer\u0026rsquo;s disease models. Nat Commun. 2022;13:1121.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Y, Sun X, Hu D, Prosdocimo DA, Hoppel C, Jain MK, et al. ATAD3A oligomerization causes neurodegeneration by coupling mitochondrial fragmentation and bioenergetics defects. Nat Commun. 2019;10:1371.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoller T, Seibold UK, Kremmer E, Voos W, Kolanus W. Atad3 function is essential for early post-implantation development in the mouse. PLoS ONE. 2013;8:e54799.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLang L, Loveless R, Dou J, Lam T, Chen A, Wang F, et al. ATAD3A mediates activation of RAS-independent mitochondrial ERK1/2 signaling, favoring head and neck cancer development. J Exp Clin Cancer Res. 2022;41:43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin G, Xu C, Zhang X, Long J, Rezaeian AH, Liu C, et al. Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells. Nat Immunol. 2018;19:29\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Li Y, Sottas C, Lazaris A, Petrillo SK, Metrakos P, et al. Loss of mitochondrial ATPase ATAD3A contributes to nonalcoholic fatty liver disease through accumulation of lipids and damaged mitochondria. J Biol Chem. 2022;298:102008.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrar KK, Hughes DT, Morris JL, Subramanian K, Krishna S, Gao F et al. PERK-ATAD3A interaction provides a subcellular safe haven for protein synthesis during ER stress. Science. 2024;eadp7114.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIssop L, Fan J, Lee S, Rone MB, Basu K, Mui J, et al. Mitochondria-associated membrane formation in hormone-stimulated Leydig cell steroidogenesis: role of ATAD3. Endocrinology. 2015;156:334\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeres de Oliveira A, Basei FL, Slepicka PF, de Castro Ferezin C, Melo-Hanchuk TD, de Souza EE, et al. NEK10 interactome and depletion reveal new roles in mitochondria. Proteome Sci. 2020;18:4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTawfik CA, Zaitoun R, Farag AA. Harel Yoon syndrome: a novel mutation in ATAD3A gene and expansion of the clinical spectrum. Ophthalmic Genet. 2023;44:226\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarel T, Yoon WH, Garone C, Gu S, Coban-Akdemir Z, Eldomery MK, et al. Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes. Am J Hum Genet. 2016;99:831\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Lin L, Li Y, Peng C, Lin Y, Liu Y, et al. Harel-Yoon syndrome caused by a novel variant in ATAD3A: A case report. Heliyon. 2024;10:e23669.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQi R, Li R, Yang W, Sang Y. Harel-Yoon syndrome caused by the c.368G\u0026thinsp;\u0026gt;\u0026thinsp;A variant in the ATAD3A gene: A case report. Asian J Surg. 2022;45:914\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYap ZY, Park YH, Wortmann SB, Gunning AC, Ezer S, Lee S, et al. Functional interpretation of ATAD3A variants in neuro-mitochondrial phenotypes. Genome Med. 2021;13:55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkopkova M, Stufkova H, Rambani V, Stranecky V, Brennerova K, Kolnikova M, et al. ATAD3A-related pontocerebellar hypoplasia: new patients and insights into phenotypic variability. Orphanet J Rare Dis. 2023;18:92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCooper HM, Yang Y, Ylikallio E, Khairullin R, Woldegebriel R, Lin K-L, et al. ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia. Hum Mol Genet. 2017;26:1432\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrazier AE, Compton AG, Kishita Y, Hock DH, Welch AE, Amarasekera SSC, et al. Fatal perinatal mitochondrial cardiac failure caused by recurrent de novo duplications in the ATAD3 locus. Med (N Y). 2021;2:49\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDesai R, Frazier AE, Durigon R, Patel H, Jones AW, Dalla Rosa I, et al. ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism. Brain. 2017;140:1595\u0026ndash;610.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeralta S, Gonz\u0026aacute;lez-Quintana A, Ybarra M, Delmiro A, P\u0026eacute;rez-P\u0026eacute;rez R, Docampo J, et al. Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities. Mol Genet Metab. 2019;128:452\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEbihara T, Nagatomo T, Sugiyama Y, Tsuruoka T, Osone Y, Shimura M, et al. Severe spinal cord hypoplasia due to a novel ATAD3A compound heterozygous deletion. Mol Genet Metab Rep. 2022;33:100912.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanes I, McMillan HJ, Ito Y, Kernohan KD, Lazier J, Lines MA, et al. A splice variant in ATAD3A expands the clinical and genetic spectrum of Harel-Yoon syndrome. Neurol Genet. 2020;6:e452.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Rong S, Luo H, Huang B, Hu F, Chen M, et al. Ketogenic Diet Attenuates Refractory Epilepsy of Harel-Yoon Syndrome With ATAD3A Variants: A Case Report and Review of Literature. Pediatr Neurol. 2023;143:79\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdul-Raheem J, Nikkola E, Chen Z, Rohena L. Expanding the phenotype of Harel‐Yoon syndrome: A case report suggesting a genotype/phenotype correlation. Am J Med Genet Pt A. 2024;e63647.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLepelley A, Della Mina E, Van Nieuwenhove E, Waumans L, Fraitag S, Rice GI et al. Enhanced cGAS-STING-dependent interferon signaling associated with mutations in ATAD3A. J Exp Med. 2021;218.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGunning AC, Strucinska K, Mu\u0026ntilde;oz Oreja M, Parrish A, Caswell R, Stals KL, et al. Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism. Am J Hum Genet. 2020;106:272\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDorison N, Gaignard P, Bayot A, Gelot A, Becker PH, Fourati S, et al. Mitochondrial dysfunction caused by novel ATAD3A mutations. Mol Genet Metab. 2020;131:107\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, Sridharan R, et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci USA. 2008;105:2883\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaha K, Jaenisch R. Technical Challenges in Using Human Induced Pluripotent Stem Cells to Model Disease. Cell Stem Cell. 2009;5:584\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrskovic M, Javaherian A, Strulovici B, Daley GQ. Induced pluripotent stem cells \u0026mdash; opportunities for disease modelling and drug discovery. Nat Rev Drug Discov. 2011;10:915\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaik DT, Chandy M, Wu JC. Patient and Disease\u0026ndash;Specific Induced Pluripotent Stem Cells for Discovery of Personalized Cardiovascular Drugs and Therapeutics. Ohlstein EH, editor. Pharmacol Rev. 2020;72:320\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng Y, Yu X, Zhang T, Hu L, Zhou D, Huang X. ATAD3A gene variations in a family with Harel-Yoon syndrome. J Zhejiang Univ (Med Sci). 2023;52:738\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Durbin R. Fast and accurate short read alignment with Burrows\u0026ndash;Wheeler transform. Bioinformatics. 2009;25:1754\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, Levy-Moonshine A et al. From FastQ Data to High‐Confidence Variant Calls: The Genome Analysis Toolkit Best Practices Pipeline. CP in Bioinformatics [Internet]. 2013 [cited 2024 Sep 20];43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://currentprotocols.onlinelibrary.wiley.com/doi/\u003c/span\u003e\u003cspan address=\"https://currentprotocols.onlinelibrary.wiley.com/doi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/0471250953.bi1110s43\u003c/span\u003e\u003cspan address=\"10.1002/0471250953.bi1110s43\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010;38:e164\u0026ndash;164.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Sci. 2015;17:405\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapadopoulos JS, Agarwala R. COBALT: constraint-based alignment tool for multiple protein sequences. Bioinformatics. 2007;23:1073\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThe UniProt Consortium. ATPase family AAA domain-containing protein 3A [Internet]. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/uniprotkb/Q9NVI7/entry\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/uniprotkb/Q9NVI7/entry\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchr\u0026ouml;dinger LLC. The PyMOL Molecular Graphics System, Version 1.8. 2015.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgu CA, Soares FAC, Alderton A, Patel M, Ansari R, Patel S, et al. Successful Generation of Human Induced Pluripotent Stem Cell Lines from Blood Samples Held at Room Temperature for up to 48 hr. Stem Cell Rep. 2015;5:660\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBagli E, Zikou AK, Agnantis N, Kitsos G. Mitochondrial Membrane Dynamics and Inherited Optic Neuropathies. Vivo. 2017;31:511\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCerneckis J, Cai H, Shi Y. Induced pluripotent stem cells (iPSCs): molecular mechanisms of induction and applications. Sig Transduct Target Ther. 2024;9:112.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagy A, Turksen K, editors. Induced pluripotent stem (iPS) cells: methods and protocols. Second edition. New York, NY: Humana Press; 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLyra-Leite DM, Guti\u0026eacute;rrez-Guti\u0026eacute;rrez \u0026Oacute;, Wang M, Zhou Y, Cyganek L, Burridge PW. A review of protocols for human iPSC culture, cardiac differentiation, subtype-specification, maturation, and direct reprogramming. STAR Protocols. 2022;3:101560.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLang L, Loveless R, Teng Y. Emerging Links between Control of Mitochondrial Protein ATAD3A and Cancer. Int J Mol Sci. 2020;21.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5370879/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5370879/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eATPase family AAA-domain-containing protein 3A (ATAD3A) is enriched on the mitochondrial membrane and is essential to the maintenance of mitochondrial structure and function. Variants of the ATAD3A gene can lead to Harel-Yoon syndrome (HAYOS), a developmental defect in neurological, cardiovascular, and other systems. This study aims to develop induced pluripotent stem cells (iPSCs) from the somatic cells of a patient (ZJUCHYLi001-A) and a negative control (ZJUCHYLi002-A) as effective tools for further investigations into the etiology of ATAD3A variant-related disease. We described and analyzed the clinical manifestations of the proband and her family members. Somatic cells from the proband and a negative control were collected and reprogrammed into iPSCs. Furthermore, we measured the ATAD3A expression levels in the iPSCs to confirm the validity of these cell lines. The proband and her elder sister were both critically ill and harbored compound heterozygous ATAD3A variants (F459S/T498Nfs*13). Their parents were carriers of these variants without any clinical manifestations. Both variants are located on the ATPase domain of the ATAD3A protein. Cell lines ZJUCHYLi001-A and ZJUCHYLi002-A presented typical features of pluripotent stem cells. The ATAD3A expression levels of ZJUCHYLi001-A were significantly reduced compared with ZJUCHYLi002-A. This study generated iPSCs from a patient with compound heterozygous variants of ATAD3A and a negative control as valuable tools for clarifying the molecular mechanisms underlying ATAD3A variant-related diseases.\u003c/p\u003e","manuscriptTitle":"Clinical Characteristics and Induced Pluripotent Stem Cells (iPSCs) Disease Model of Harel-Yoon Syndrome Caused by Compound Heterozygous ATAD3A Variants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-02 15:45:21","doi":"10.21203/rs.3.rs-5370879/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2024-11-18T10:13:31+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-11-05T01:29:43+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-03T23:35:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-03T15:01:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Cell","date":"2024-11-01T02:05:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3134e8c5-34bc-4475-90c2-94b1c5a44bdc","owner":[],"postedDate":"December 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-21T16:04:02+00:00","versionOfRecord":{"articleIdentity":"rs-5370879","link":"https://doi.org/10.1007/s13577-025-01214-x","journal":{"identity":"human-cell","isVorOnly":false,"title":"Human Cell"},"publishedOn":"2025-04-17 15:57:44","publishedOnDateReadable":"April 17th, 2025"},"versionCreatedAt":"2024-12-02 15:45:21","video":"","vorDoi":"10.1007/s13577-025-01214-x","vorDoiUrl":"https://doi.org/10.1007/s13577-025-01214-x","workflowStages":[]},"version":"v1","identity":"rs-5370879","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5370879","identity":"rs-5370879","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}