The Role of ATP9A (c.1091G>C; p.(Arg364Thr)) Variant in Cognitive Impairment: Diagnostic Insight from Whole Exome Sequencing

The Role of ATP9A (c.1091G>C; p.(Arg364Thr)) Variant in Cognitive Impairment: Diagnostic Insight from Whole Exome Sequencing | 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 The Role of ATP9A (c.1091G>C; p.(Arg364Thr)) Variant in Cognitive Impairment: Diagnostic Insight from Whole Exome Sequencing Cuneyd Yavas, Asmaa Abuaisha, Emir Nekay, Alper Gezdirici, Halil Ibrahim Yilmaz, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8263851/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jan, 2026 Read the published version in Molecular Biology Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Background: The ATP9A gene encodes a P4-type ATPase involved in phospholipid translocation, essential for vesicular trafficking and neuronal development. Pathogenic ATP9A variants cause autosomal recessive neurodevelopmental disorders characterized by intellectual disability and microcephaly, yet the impact of missense variants remains poorly understood. Methods: A 7-year-old female patient with cognitive impairment, microcephaly, and developmental delay was admitted to Başakşehir Çam and Sakura City Hospital. Whole exome sequencing (WES) using Illumina technology identified a novel homozygous ATP9A variant, confirmed by Sanger sequencing and segregation analysis. In silico tools (RosettaFold, DynaMut, mCSM, SDM, DUET, AggreScan3D) assessed its structural impact. Quantitative real-time PCR (qPCR) was conducted to evaluate the relative expression levels of ATP9A . Results: WES revealed a homozygous missense variant, ATP9A (NM_006045.3):c.1091G > C p.(Arg364Thr), classified as variant of uncertain significance (ACMG: PP2, PM2, PM3). Protein modeling demonstrated reduced stability (ΔΔG = − 1.51 to − 0.26 kcal/mol), increased flexibility, and a 2.4-fold decrease in solvent accessibility. The mutation altered polar and hydrophobic interactions within the P-type ATPase IV domain, enhancing aggregation propensity. Expression analysis showed elevated ATP9A mRNA levels, suggesting a compensatory response. Conclusion: This novel ATP9A variant broadens the mutational spectrum of ATP9A -related neurodevelopmental disorders. Structural destabilization of the p.(Arg364Thr) protein may contribute to the patient’s cognitive impairment and microcephaly, warranting further functional studies. ATP9A p.(Arg364Thr) Novel Variant Cognitive Impairment Whole Exome Sequencing Neurodevelopmental Disorders Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The ATPase Phospholipid Transporting 9A ( ATP9A ) gene is mapped to chromosome 20q13.2 (GRCh37/hg19). Its primary transcript (ENST00000338821.5, NM_006045.3) contains 28 exons and encodes a protein of 1047 amino acids, with predominant expression in the brain. However, the precise physiological role and subcellular localization of endogenous ATP9A remain largely unclear ( 1 ). Dysfunction or mutations in the ATP9A gene have been associated with a wide range of clinical manifestations, including neurological disorders, neurodevelopmental abnormalities such as cognitive impairment (CI), and liver diseases ( 2 ). CI is common and highly heterogeneous phenotypes of genetic origin, affecting 1–2% of the general population ( 3 ). It is one of the most common pediatric disorders and is identified in childhood, manifesting itself early in the phenotype. Neurodevelopmental delay problems are associated with other deformities or clinical features such as behavioral abnormalities, epilepsy and microcephaly ( 3 , 4 ). A relevant variant causing hereditary or genetic diseases can be found in approximately 40–60% of cases using Next Generation Sequencing. In particular, data from genetic studies have recently become important for the diagnosis of idiopathic or novel diseases. The presence of disease-causing variants in the relevant genes is now required for diagnosis ( 5 , 6 ). Currently, more than 1300 genes are classified as main CI-related genes, with around 25% of these genes connected with microcephaly ( 7 ). Proteins encoded by microcephaly-associated genes participate in a wide range of biological processes and molecular pathways, including transcriptional control, DNA repair, microtubule organization, and endosome regulation ( 8 ). Genes expressing proteins of the Golgi and endosomal trafficking machinery are particularly interesting because they are essential for brain development, and abnormalities in these genes are linked to postnatal microcephaly ( 9 ). In this study, we report a novel homozygous missense variant in ATP9A identified in a patient with cognitive impairment, microcephaly, and developmental delay. To date, only a few reports have described ATP9A variants associated with human neurodevelopmental disorders, and the molecular mechanisms underlying their pathogenic effects remain poorly understood. Our study integrates clinical, molecular, and bioinformatic analyses to explore the potential pathogenicity of the identified variant and its impact on protein structure, stability, and gene expression, thereby contributing to the expanding genotype-phenotype spectrum of ATP9A -related disorders. Material and methods Study Design This study was approved by the Ethics Committee of Başakşehir Çam and Sakura City Hospital (Approval No: KAEK/26.08.2023.355). Informed consent was obtained from all participants prior to genetic testing. Whole exome sequencing (WES) results, together with the clinical data of a patient diagnosed with CI, were investigated. Sanger sequencing and family segregation analyses were subsequently performed for variant confirmation. All procedures were performed under strict ethical principles, and written informed consent was obtained from participants after clear explanation of the study design, methodology, and potential implications. WES Analysis Genomic DNA was isolated from peripheral blood samples. WES was performed by the Illumina platform. Initially, the isolated DNA was fragmented, and barcoded adapters were attached to allow multiplexed sequencing. The prepared libraries were then amplified by PCR and evaluated for fragment size and quality. For cluster generation, the DNA fragments were immobilized on a flow cell and underwent bridge amplification to form dense clusters. Sequencing was performed through the stepwise incorporation of fluorescently labeled nucleotides, with real-time detection of emitted signals used for base calling and sequence reconstruction. Bioinformatic analyses were conducted using the Genome Analysis Toolkit (GATK) along with other bioinformatics tools to align the reads, identify sequence variants, and highlight candidate pathogenic alterations. Data Analysis and Interpretation Genetic data were analyzed in relation to clinical presentation using a systematic evaluation strategy. Variants were classified in accordance with the American College of Medical Genetics and Genomics (ACMG) recommendations into five categories: benign, likely benign, variant of uncertain significance (VUS), likely pathogenic, and pathogenic ( 10 ). Each variant was interpreted in the context of patient symptoms and family background, and functional evidence, when available, was incorporated to strengthen interpretation. Multidisciplinary discussions including geneticists, neurologists, and bioinformatic experts were held to validate the findings and ensure accurate assessment. Protein Modeling and Stability Analysis As no experimentally resolved NMR or X-ray crystal structure was available for ATP9A (NM_006045.3), protein models were modeled using RosettaFold modeling algorithm with ab-initio modeling approach ( 11 ). RosettaFold employs a deep learning-based three-way neural network algorithms, providing comparative models of both ab initio and protein domains. Using the obtained wild-type (wt) structure as a template, the identified mutation (mt) in ATP9A gene was modeled with the same tool. The quality evaluations of the created protein models were made with QMEANDisco ( 12 ). Topological differences between wt and mutant (mt) protein structures were analyzed by Tm-Score ( 13 ). The changes in protein structure and stability after mutation were analyzed with DynaMut, mCSM, SDM, and DUET tools ( 14 – 17 ). Protein aggregation assessments were made with AggreScan3D. Protein aggregation analysis provides important data in the evaluation of proteins associated with diseases ( 18 ). Distance of aggregation was selected 5Å. PyMOL and Arpeggio were used to visualize atomic interactions ( 19 ). Quantitative Real-time PCR for ATP9A Gene Expression Total RNA was isolated from peripheral blood using the GeneAll® Hybrid-R™ Blood RNA kit (GeneAll, Korea). RNA purity and concentration were measured with a NanoDrop ND-2000c spectrophotometer (Thermo Fisher Scientific Inc., USA). Complementary DNA (cDNA) was synthesized from the extracted RNA using the OneScript Plus cDNA Synthesis Kit (Applied Biological Materials Inc., Richmond, Canada) on a T100 Thermal Cycler (Bio-Rad, Singapore), following the manufacturer’s instructions. Quantitative real-time PCR (qPCR) amplification was then carried out with BlasTaq 2X qPCR MasterMix (Applied Biological Materials Inc., Richmond, Canada). All reactions were performed in duplicate on CFX96 Real-Time System (Bio-Rad, Singapore). GAPDH was used as the reference gene, and relative expression levels were calculated using the 2 −∆∆Ct method. The primer sequences used for the studied genes are as follows: For the ATP9A gene, the forward primer was 5′-GTGTACAGCTGGGTGATTCG-3′ and the reverse primer was 5′-GGGTAAGAGTGCCTGTCTTGT-3′. For the GAPDH gene, the forward primer was 5′-CCACCCATGGCAAATTCC-3′ and the reverse primer was 5′-TGGGATTTCCATTGATGACAAG-3′. Statistical Analysis All statistical analyses were performed using GraphPad Prism software (version 10.4.1). Data are presented as mean ± standard deviation (SD). One way ANOVA test was applied to assess overall statistical significance. A p-value of less than 0.05 was considered statistically significant (*p < 0.05, ns: not significant). Results The patient was born via cesarean section at the 36th gestational week with a birth weight of 2300 grams. There were no additional notable features in the pregnancy history. A consanguinity history exists between the parents at the level of second cousins There is a history of consanguinity between parents at the second-degree cousin. Postnatal follow-up revealed microcephaly, prominent forehead and blue sclera. Neurodevelopmental evaluation showed significant developmental delay; the patient is unable to walk in terms of motor functions, and no language development has been observed. Chromosome analysis and chromosomal microarray studies were reported as normal. In addition, no pathological findings were detected in the metabolic evaluation. WES identified a homozygous variant in the ATP9A (NM_006045.3):c.1091G > C p.(Arg364Thr). The identified variant, located in exon 12, was absent from known mutation databases, suggesting it is a novel variant, and leads to the amino acid substitution p.(Arg364Thr) (arginine to threonine) at codon 364. This missense mutation is predicted to affect the P-type_ATPase_IV domain located between codons 53-1045. The pathogenicity assessment of the detected mutation is summarized in Table 1. The ATP9A protein models were created with the deep learning-based RosettaFold algorithm. The model confidence scores for wt and mt ATP9A were 0.83 and 0.82, respectively. The QMEAN quality scores for wt and mt ATP9A were 0.67 ± 0.05 and 0.66 ± 0.05, respectively. The wt and mt ATP9A proteins revealed high topological similarity (Fig. 1 ). Tm-score and root mean square deviation (rmsd) were 0.97 and 0.66 angstrom (Å), respectively. Protein stability analyzes revealed that the mutation caused a decrease in protein stability and an increase in molecular flexibility. The ΔΔG for mCSM stability, SDM, DUET, and DynaMut were − 0.073, -1.51, -0.365, and − 0.260 kcal.mol -1 , respectively. The vibrational entropy energy (ΔΔS vib ) between wt and mt-ATP9A was 0.285 kcal.mol -1 .K -1 . Before the mutation, there were seven polar interactions between Arg364 and Ser366 (one), Ile362 (one), Val361 (one), and Trp360 (four). The Arg364Thr mutation changed the interaction pattern of the 364th position. After the mutation, the interaction occurred between Thr364 and Ser366 (one polar), 361 (two polar), Ile362 (one polar and one van der-walls), and Trp360 (one polar and one hydrogen) (Fig. 1 ). The surface area for solvent accessible decreased 2.4-fold after mutation. The solvent accessible surface area for the 364th residue decreased from 175.019 Å 2 to 73.080 Å 2 . Aggregation analysis showed that, despite a 6% (the aggregation score was − 0.4224 and − 0.399 for wt and mt ATP9A, respectively) reduction in collective protein stability, the decrease in stability at residue 364 was approximately 2.8-fold (the aggregation score was − 2.5852 and − 0.9379 for wt and mt ATP9A, respectively). The p.(Arg364Thr) mutation increased the aggregation propensity (Fig. 2 ). Discussion ATP9A gene encodes a member of the group of P4-ATPases that regulate asymmetric membrane lipid distribution via ATP-dependent translocation of phospholipids from the exofascial to cytosolic leaflet ( 20 ). P4-ATPases are transmembrane lipid flippases, that function in vesicles formation and trafficking. They regulate the asymmetric distribution of phospholipids in membranes of eukaryotic cells ( 21 ). There is substantial evidence to suggest that ATP9A, located both in the intracellular compartments and the plasma membrane, is localized in early and recycling endosomes, which play an essential role in vesicle formation and trafficking ( 1 ). In addition to the endocytic vesicle formation process, ATP9A is known to have regulatory roles in the release of extracellular vesicles ( 22 ). Numerous biological processes are regulated by intercellular communication mediated by extracellular vesicles released from cells. A considerable increase in the release of extracellular vesicles, particularly the exosome, has been linked to ATP9A downregulation. Extracellular vesicle release allows for the transmission of a variety of signaling molecules, including proteins and RNA, without the need for direct interaction between cells. It is involved in a wide range of biological processes, such as blood coagulation and immune response ( 23 ). Extracellular vesicles have been reported to have different physiological roles in the central nervous system, including neurite outgrowth and neuronal survival ( 24 – 26 ). It was recently reported that ATP9A mutations are associated with neurodevelopmental disorder and microcephaly ( 20 , 23 , 26 ). We report a novel p.(Arg364Thr) mutation for ATP9A , which we suggest is associated with neurodevelopmental delay. Since ATP9A does not have an existing NMR or X-ray structure, the wt and mt-ATP9A protein models were modeled using deep learning algorithms. The possible functional effects of the mutation were analyzed by considering the tertiary and quaternary structure data of P4-ATPase flippase members whose NMR and X-ray structures are known ( 27 ). The p.(Arg364Thr) mutation reduced stability of ATP9A protein. The mutation decreased solvent access and increased aggregation tendency can trigger changes in protein stability and functional properties. The p.(Arg364Thr) mutation caused an extreme increase in vibration at 364th position and adjacent residues, and a partial increase in vibration at the membrane spanning helices and nucleotide binding site (Fig. 3 ). The p.(Arg364Thr) mutation, located at the C-terminal downstream end of the M4 helix, is located in the phospholipid binding site (Fig. 4 ). The mutation changed the interaction pattern of the 364th residue with other residues in the formation of the protein tertiary structure. While there was one polar interaction in wtATP9A with Val361 located in the phospholipid binding pocket, this relationship was maintained by two polar interactions in mtATP9A (Fig. 1 ). The formational integrity of the helices in the transmembrane region is provided by the interactions established between each helix. The distance between atoms is decisive in the establishment of these interactions. The formational integrity of the helices in the transmembrane region is provided by the interactions established between each helix. The distance between atoms is decisive in the establishment of these interactions. After the mutation, the distance between wtATP9A or mtATP9A and the helix in the membrane-spanning helices appear to have diverged by approximately 4 Å. In addition, end region measurements of wtATP9A and mtATP9A indicate a mean difference of 5 Å (Fig. 4 ). It is possible that the RMSD value between wtATP9A and mtATP9A is 0.66 Å, and the distance variations up to an average of 5 Å have effects on protein-ligand and protein substrate functional interactions achieved at very short distances. Conclusive evidence is needed on how the variation in interaction distances between helixes will affect the heterodimerization process of ATP9A and CDC50 required for functional activity. Segregation analysis and Sanger sequencing confirmed the presence of the ATP9A c.1091G > C p.(Arg364Thr) variant in the proband and family members. The pedigree revealed an autosomal recessive inheritance pattern, with the proband carrying the variant in a homozygous state, resulting in the substitution of arginine with threonine at codon 364. Both parents were identified as heterozygous carriers of the same missense variant. Sanger sequencing chromatograms clearly showed the guanine-to-cytosine substitution at nucleotide position 1091, consistent with the segregation of this variant within the family and supporting its pathogenic role in the observed phenotype (Fig. 5 ). In our study, quantitative expression analysis from peripheral blood samples of the proband carrying the homozygous ATP9A c.1091G > C p.(Arg364Thr) variant revealed a clear upregulation of ATP9A mRNA (Fig. 6 ). This observation parallels the findings of Cordovado et al. (2025), who demonstrated ATP9A overexpression in HeLa and HEK293T cell models following transfection with both wild-type and mutant constructs, indicating that ATP9A transcription may increase as a compensatory response to disrupted protein stability or endosomal dysfunction ( 28 ). Although their experiments were performed in immortalized cell lines rather than human tissues, the consistency between our in vivo blood-derived expression and their in vitro data suggests a shared cellular mechanism that upregulates ATP9A to counterbalance partial loss of function caused by missense variants. Conversely, Vogt et al. (2022) observed a pronounced downregulation of ATP9A expression in skin fibroblasts derived from patients with biallelic truncating or splice-site mutations, with transcript levels reduced to only 4–14% of control values due to nonsense-mediated mRNA decay (NMD) ( 20 ). The opposing regulatory patterns underscore the influence of both mutation type and tissue context on ATP9A expression: truncating mutations trigger transcript degradation and complete loss of function, whereas missense variants such as p.(Arg364Thr) appear to elicit a compensatory transcriptional upregulation in peripheral tissues. Collectively, these findings support the notion that elevated ATP9A expression in our case likely reflects a feedback response to reduced protein activity rather than true functional enhancement. Apart from the mutation we reported in this study, many ATP9A mutations were shown to be associated with neurodevelopmental disorders (Table 2 ). The fact that P4-ATPase mutations other than ATP9A are also associated with neurodevelopmental disorders increases the reliability of the data obtained for ATP9A ( 29 – 31 ). Meng et al. ( 2 ) revealed that mutation or deficiency of ATP9A leads to inactivation of RAB5 and RAB11, resulting in abnormal endosomal recycling in neural cells. They reported that this condition leads to synaptic dysfunction in the primary motor cortex and hippocampus of the brain, resulting in hypotonia, intellectual disability, hyperactivity disorders, and neurological disorders. Tanaka et al. ( 1 ) revealed that ATP9A is indispensable for endocytic recycling of transferrin receptor and glucose transporter 1 (GLUT1) to the plasma membrane. Depletion of ATP9A reduces plasma membrane expression of GLUT1 and alters its recycling, increasing its level in the endosome. GLUT1 deficiency has been associated with a neurological disorder with a variable phenotype, including epilepsy, movement disorders, mild or severe intellectual disability, and in some cases, acquired microcephaly. Table 1 Patogenicity of detected variant Gene ATP9A Transkript id NM_006045.3 Dbsnp Novel Variant c.1091G > C p.(Arg364Thr) Variant locus chr20-50287743 Exon 12 Variant type Missense Mutation taster Disease causing DANN Deleterious (0.95) PrimateAI Deleterious (Supporting) (0.8) Seq.genomize Not found Clinvar - Affected Domains P-type_ATPase_IV Conservation Conserved ACMG classification Variant of uncertain significance ACMG pathogenicity criteria PP2, PM2, PM3 ACMG: The American College Of Medical Genetics And Genomics, DANN: Prediction score based on a deep neural network. Gnomad: Genome Aggregation Database, PrimateAI: Predicts the pathogenicity of missense variants using deep learning trained on humans and non-human primate species, Seq.Genomize: Web Based Anutation Programme-Turkish Populastion Variant Database Table 2 ATP9A gene pathogenic variants reported in the literature and clinical findings of patients. Reference Nationality Gender Age (year) Variant Z Brain MRI anomalies DD SD MC S ADHD St. FA Proband Turkish F 7 c.1091G > C p.(Arg364Thr). HM Lateral ventricular asymmetry + + + - NA + + Mattioli et al. 2021 ( 23 ) P 1: Pakistani F 28 c.799 + 1 G > T HM ND + + + - + + + P 2: Pakistani F 21 c.799 + 1 G > T HM ND + + - - + + + P 3: Iranian M 18 c.327 + 1 G > T HM - + + - + ND - + Vogt et al. 2021 ( 20 ) P 1: Syrian M 12 c.868C > T p.(Arg290*) HM - + + + + P 2: Syrian M 4 c.868C > T p.(Arg290*) HM ND + + + - P 3: Turkish M 9 c.642 + 1G > A p. (Ser184Profs*16) HM + + + + - Meng et al. 2023 ( 2 ) P 1: Chinese M 16 c.658C > T/ c.433C > T p.(Arg220*p. Arg145*) Comp HT Mild abnormality + + - - + ND - P 2: Chinese F 11 c.658C > T/c.433C > T p. (Arg220*/p. Arg145*) Comp HT Mild abnormality + + - - + ND - P 3: Chinese F 11 c.983G > A/c.983G > A p. (Trp328*/p. Trp328*) HM Mild abnormality + + + - + ND + Cordovado et al. 2025 ( 28 ) P 1: Caucasian M 14 c.1178C > G p.(Thr393Arg) HT Cortical atrophy + + + + + + + P 2: Caucasian F 44 c.1198G > C p.(Glu400Gln) HT Sequela of callosotomy mild asymmetry of hippocampi + + NA + + - - P 3: North Africa F 10 c.1655G > C p.(Gly552Ala) HT Partial agenesis of corpus callosum, global cortical atrophy, myelination delay, epiphyseal cyst, pars intermedia cyst + + - + + + + P 4: Caucasian M 2.5 c.2137C > G p.(His713Asp) HT Mild nonspecific bilateral flair hypersignal of the posterior parietal periventricular white matter + + - - - - + P 5: Caucasian F 10 c.433C > T & c.2701G > T p.(Arg145*) and p. (Glu901*) HT Normal + + NA + + - - P 6: Caucasian NA NA c.1381A > G p.(Lys461Glu) HT NA + NA + NA NA NA + Comp HT: Compound heterozygous, DD: Developmental delay, FA: Facial Anomaly, FD: Feeding difficulities, GR: Growth retardation, HM: Homozygous, HT: Heterozygous, MC: Microcephaly, PA: Prenatal anomalies, SD: Speech delay, S: Seizure, Z: Zygosity, ND: not determined, St: Strabismus, ADHD: attention deficit hyperactivity disorder, NA: Not avaiable Variants in the ATP9A gene reported to date are predominantly biallelic truncating or splice-site mutations that result in a loss-of-function (LoF) mechanism ( 20 , 23 ). These alterations introduce premature stop codons or disrupt canonical splicing, thereby preventing the synthesis of a functional protein. Meng et al. (2023) further identified compound heterozygous truncating variants p.(Arg145*/p.Arg220*) leading to a similar functional outcome ( 2 ). More recently, Cordovado et al. (2025) described heterozygous missense variants p.(Thr393Arg) and p.(Glu400Gln) associated with dominantly inherited non-syndromic intellectual disability, demonstrating impaired dendritic spine maturation and altered postsynaptic morphology ( 28 ). In this context, the homozygous p.(Arg364Thr) variant identified in our proband represents a polar amino acid substitution predicted to alter the structural integrity of the ATP9A protein, suggesting a LoF-like effect despite its missense nature (Table 2 ). Clinically, ATP9A -related patients reported in the literature exhibit similar phenotypes. Mattioli et al. (2021) and Vogt et al. (2022) reported developmental delay (DD), speech delay (SD), and microcephaly (MC) as the cardinal manifestations ( 20 , 23 ). Additional features, such as strabismus, feeding difficulties, seizures, and facial dysmorphism, were occasionally observed. Meng et al. (2023) described patients with compound heterozygous truncating variants presenting with attention-deficit/hyperactivity disorder (ADHD)-like symptoms and mild structural brain abnormalities ( 2 ). Similarly, Cordovado et al. (2025) found that heterozygous missense variants may cause cortical atrophy, hippocampal asymmetry, and corpus callosum abnormalities ( 28 ). Collectively, these findings suggest that pathogenic variants of ATP9A disrupts endosomal recycling and phospholipid asymmetry, thereby impairing synaptic transmission and neuronal maturation. Our proband exhibited microcephaly, developmental and speech delay, lateral ventricular asymmetry, strabismus, and mild facial dysmorphism, similar to other reported patients who have biallelic pathogenic variants. Despite being a missense substitution, p.(Arg364Thr) appears to exert a deleterious impact comparable to that of truncating mutations, indicating that this residue may lie within a functionally critical domain (Table 2 ). Conclusion This study suggests that the ATP9A (c.1091G > C; p.(Arg364Thr)) variant may be associated with a neurodevelopmental disorder, particularly cognitive impairment. The mutation likely affects ATP9A’s structural stability and function, potentially leading to protein aggregation and subsequent cellular dysfunction. These findings highlight the possible contribution of ATP9A to neuronal pathology and emphasize the need for further molecular and functional studies to elucidate its pathogenic role. Declarations Funding: None. The authors state no conflict of interest. Data Availability Statement: The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Details of Ethical Approval and Informed Consents: This study adhered to the ethical principles outlined in the Helsinki Declaration for the investigation of human subjects. Ethical approval for this study was obtained from the Non-Interventional Ethics Committee of Istanbul Başakşehir Çam and Sakura City Hospital (KAEK/26.08.2023.355). Additionally, informed consent was obtained from all participants prior to the study. Acknowledgments: Not Applicable Authors Contribution: Conceptualization: C.Y., A.A., E.N., A.G.; Formal Analysis: C.Y., A.A., E.N., E.A.; Investigation: C.Y., A.A., A.G., H.I.Y., E.N., E.A., P.A.; Data Curation: C.Y., A.A., A.G., P.A. Methodology: A.A., C.Y., A.G., E.N., H.I.Y.; Writing-original draft: C.Y., A.A.; Review and Editing: C.Y., A.A., A.G., E.N., H.I.Y., E.A., P.A. Supervision: C.Y., A.A, A.G. All the authors have read and approved the final version of the manuscript and agreed to be held accountable for all aspects of the work. References Tanaka Y, Ono N, Shima T, Tanaka G, Katoh Y, Nakayama K et al (2016) The phospholipid flippase ATP9A is required for the recycling pathway from the endosomes to the plasma membrane. 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PLoS Genet 8(8):e1002853 Fazia T, Marzanati D, Carotenuto AL, Beecham A, Hadjixenofontos A, McCauley JL et al (2021) Homozygosity Haplotype and Whole-Exome Sequencing Analysis to Identify Potentially Functional Rare Variants Involved in Multiple Sclerosis among Sardinian Families. Curr Issues Mol Biol 43(3):1778–1793 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 28 Jan, 2026 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 28 Dec, 2025 Reviews received at journal 27 Dec, 2025 Reviews received at journal 24 Dec, 2025 Reviewers agreed at journal 10 Dec, 2025 Reviewers agreed at journal 10 Dec, 2025 Reviewers invited by journal 05 Dec, 2025 Editor assigned by journal 03 Dec, 2025 Submission checks completed at journal 03 Dec, 2025 First submitted to journal 02 Dec, 2025 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. 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12:44:03","extension":"png","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13028,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8263851/v1/99c30cdfceae9bf24507cf89.png"},{"id":97703886,"identity":"65a57859-0bbc-4012-8286-f3e4c3ae2d09","added_by":"auto","created_at":"2025-12-08 12:44:04","extension":"xml","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":117488,"visible":true,"origin":"","legend":"","description":"","filename":"069f730b0105475da8b945fce64d76c31structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8263851/v1/454dc7d3fbb1b0920bc5c290.xml"},{"id":97703888,"identity":"476fe35e-bdfc-480a-bce1-9b31d2a714a9","added_by":"auto","created_at":"2025-12-08 12:44:04","extension":"html","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126837,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8263851/v1/b0396d17c68fc00625a32684.html"},{"id":97703850,"identity":"2af17c66-7f10-4890-b8ac-205633647221","added_by":"auto","created_at":"2025-12-08 12:44:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":13416863,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentation of the tertiary structure of ATP9A. a) Superimpose cartoon representation, b) \u0026nbsp;Superimpose mesh representation of topological differences (blue and red color indicate wtATP9A and mtATP9A, respectively), c) Representation of atomic interaction of position 364 in wtATP9A pArg364Thr mutation, d) Representation of atomic interaction of position 364 in mtATP9A pArg364Thr mutation (Orange, red and cyan dashes indicate polar, hydrogen bound and van der walls interactions, respectively).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8263851/v1/31fbd3af22e20ba8b7e7193d.png"},{"id":97894695,"identity":"af7fea3f-3b7a-4228-936c-3bf261daed71","added_by":"auto","created_at":"2025-12-10 15:32:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12908834,"visible":true,"origin":"","legend":"\u003cp\u003eAggregation plot of wt- and mtATP9A.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8263851/v1/d741698f5bbb451b249671a5.png"},{"id":97703847,"identity":"22c37ab6-9ad6-4757-8ce2-e01668be7bb2","added_by":"auto","created_at":"2025-12-08 12:44:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":379250,"visible":true,"origin":"","legend":"\u003cp\u003eCartoon illustration of vibrational entropy change of ATP9A after p.(Arg464Thr) mutation.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8263851/v1/da92c01b8bdb4a46fe09add9.png"},{"id":97703855,"identity":"b88b2407-f7ed-4c16-9218-61dd9b431148","added_by":"auto","created_at":"2025-12-08 12:44:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":277161,"visible":true,"origin":"","legend":"\u003cp\u003eCartoon representation of functional domains of ATP9A. a) wt- and mt-ATP9A atomic interaction -end distance measurements at position 364. Colors indicates, red-mutant residue, cyan-actuator domain, purple-phosphorylation domain, blue-nucleotide binding domain, yellow-M1 membrane spanning helix, pink-M2 membrane spanning helix, orange-M3 membrane spanning helix, wheat-M4 membrane spanning helix, green-between M5 and M10 membrane spanning helices.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8263851/v1/3684f79b58f593f93852f158.png"},{"id":97703848,"identity":"45084ef6-c0d6-481a-98df-b30bd163c241","added_by":"auto","created_at":"2025-12-08 12:44:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":42589,"visible":true,"origin":"","legend":"\u003cp\u003eSegregation analysis and Sanger sequencing chromatograms of the \u003cem\u003eATP9A\u003c/em\u003ec.1091G\u0026gt;C p.(Arg364Thr) variant. Pedigree shows an autosomal recessive inheritance pattern. The proband carries a homozygous missense variant\u003cstrong\u003e \u003c/strong\u003ec.1091G\u0026gt;C, resulting in the amino acid substitution p.(Arg364Thr). Both parents are heterozygous carriers for the same variant. Sanger sequencing chromatograms demonstrate the guanine-to-cytosine substitution at position 1091 (highlighted), confirming the segregation of the variant within the family.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8263851/v1/9a088d3cae44216238736110.png"},{"id":97892847,"identity":"2d3599df-438b-4c7d-aa66-eb5de6dfaf2a","added_by":"auto","created_at":"2025-12-10 15:23:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":100792,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of \u003cem\u003eATP9A\u003c/em\u003e gene in the proband and carrier parents compared to control. Quantitative real-time\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003ePCR analysis revealed increased \u003cem\u003eATP9A\u003c/em\u003e expression in peripheral blood samples from the proband carrying the homozygous c.1091G\u0026gt;C (p.(Arg364Thr)) variant and from both heterozygous carrier parents compared to control. Data are presented as mean ± SD, normalized to \u003cem\u003eGAPDH\u003c/em\u003e expression.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8263851/v1/b3dae20b02af101f78c77004.png"},{"id":101690714,"identity":"2885702d-7202-49c5-a245-6fed79023da5","added_by":"auto","created_at":"2026-02-02 16:08:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":22588846,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8263851/v1/34956f37-c4ae-4831-88bb-ce7bee1506d5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Role of ATP9A (c.1091G\u003eC; p.(Arg364Thr)) Variant in Cognitive Impairment: Diagnostic Insight from Whole Exome Sequencing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe ATPase Phospholipid Transporting 9A (\u003cem\u003eATP9A\u003c/em\u003e) gene is mapped to chromosome 20q13.2 (GRCh37/hg19). Its primary transcript (ENST00000338821.5, NM_006045.3) contains 28 exons and encodes a protein of 1047 amino acids, with predominant expression in the brain. However, the precise physiological role and subcellular localization of endogenous ATP9A remain largely unclear (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Dysfunction or mutations in the \u003cem\u003eATP9A\u003c/em\u003e gene have been associated with a wide range of clinical manifestations, including neurological disorders, neurodevelopmental abnormalities such as cognitive impairment (CI), and liver diseases (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCI is common and highly heterogeneous phenotypes of genetic origin, affecting 1\u0026ndash;2% of the general population (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). It is one of the most common pediatric disorders and is identified in childhood, manifesting itself early in the phenotype. Neurodevelopmental delay problems are associated with other deformities or clinical features such as behavioral abnormalities, epilepsy and microcephaly (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). A relevant variant causing hereditary or genetic diseases can be found in approximately 40\u0026ndash;60% of cases using Next Generation Sequencing. In particular, data from genetic studies have recently become important for the diagnosis of idiopathic or novel diseases. The presence of disease-causing variants in the relevant genes is now required for diagnosis (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCurrently, more than 1300 genes are classified as main CI-related genes, with around 25% of these genes connected with microcephaly (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Proteins encoded by microcephaly-associated genes participate in a wide range of biological processes and molecular pathways, including transcriptional control, DNA repair, microtubule organization, and endosome regulation (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Genes expressing proteins of the Golgi and endosomal trafficking machinery are particularly interesting because they are essential for brain development, and abnormalities in these genes are linked to postnatal microcephaly (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we report a novel homozygous missense variant in \u003cem\u003eATP9A\u003c/em\u003e identified in a patient with cognitive impairment, microcephaly, and developmental delay. To date, only a few reports have described \u003cem\u003eATP9A\u003c/em\u003e variants associated with human neurodevelopmental disorders, and the molecular mechanisms underlying their pathogenic effects remain poorly understood. Our study integrates clinical, molecular, and bioinformatic analyses to explore the potential pathogenicity of the identified variant and its impact on protein structure, stability, and gene expression, thereby contributing to the expanding genotype-phenotype spectrum of \u003cem\u003eATP9A\u003c/em\u003e-related disorders.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cb\u003eStudy Design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study was approved by the Ethics Committee of Başakşehir \u0026Ccedil;am and Sakura City Hospital (Approval No: KAEK/26.08.2023.355). Informed consent was obtained from all participants prior to genetic testing. Whole exome sequencing (WES) results, together with the clinical data of a patient diagnosed with CI, were investigated. Sanger sequencing and family segregation analyses were subsequently performed for variant confirmation. All procedures were performed under strict ethical principles, and written informed consent was obtained from participants after clear explanation of the study design, methodology, and potential implications.\u003c/p\u003e\n\u003ch3\u003eWES Analysis\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was isolated from peripheral blood samples. WES was performed by the Illumina platform. Initially, the isolated DNA was fragmented, and barcoded adapters were attached to allow multiplexed sequencing. The prepared libraries were then amplified by PCR and evaluated for fragment size and quality. For cluster generation, the DNA fragments were immobilized on a flow cell and underwent bridge amplification to form dense clusters. Sequencing was performed through the stepwise incorporation of fluorescently labeled nucleotides, with real-time detection of emitted signals used for base calling and sequence reconstruction. Bioinformatic analyses were conducted using the Genome Analysis Toolkit (GATK) along with other bioinformatics tools to align the reads, identify sequence variants, and highlight candidate pathogenic alterations.\u003c/p\u003e\n\u003ch3\u003eData Analysis and Interpretation\u003c/h3\u003e\n\u003cp\u003eGenetic data were analyzed in relation to clinical presentation using a systematic evaluation strategy. Variants were classified in accordance with the American College of Medical Genetics and Genomics (ACMG) recommendations into five categories: benign, likely benign, variant of uncertain significance (VUS), likely pathogenic, and pathogenic (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Each variant was interpreted in the context of patient symptoms and family background, and functional evidence, when available, was incorporated to strengthen interpretation. Multidisciplinary discussions including geneticists, neurologists, and bioinformatic experts were held to validate the findings and ensure accurate assessment.\u003c/p\u003e\n\u003ch3\u003eProtein Modeling and Stability Analysis\u003c/h3\u003e\n\u003cp\u003eAs no experimentally resolved NMR or X-ray crystal structure was available for ATP9A (NM_006045.3), protein models were modeled using RosettaFold modeling algorithm with ab-initio modeling approach (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). RosettaFold employs a deep learning-based three-way neural network algorithms, providing comparative models of both ab initio and protein domains. Using the obtained wild-type (wt) structure as a template, the identified mutation (mt) in \u003cem\u003eATP9A\u003c/em\u003e gene was modeled with the same tool. The quality evaluations of the created protein models were made with QMEANDisco (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Topological differences between wt and mutant (mt) protein structures were analyzed by Tm-Score (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The changes in protein structure and stability after mutation were analyzed with DynaMut, mCSM, SDM, and DUET tools (\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Protein aggregation assessments were made with AggreScan3D. Protein aggregation analysis provides important data in the evaluation of proteins associated with diseases (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Distance of aggregation was selected 5\u0026Aring;. PyMOL and Arpeggio were used to visualize atomic interactions (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantitative Real-time PCR for\u003c/b\u003e \u003cb\u003eATP9A\u003c/b\u003e \u003cb\u003eGene Expression\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was isolated from peripheral blood using the GeneAll\u0026reg; Hybrid-R\u0026trade; Blood RNA kit (GeneAll, Korea). RNA purity and concentration were measured with a NanoDrop ND-2000c spectrophotometer (Thermo Fisher Scientific Inc., USA). Complementary DNA (cDNA) was synthesized from the extracted RNA using the OneScript Plus cDNA Synthesis Kit (Applied Biological Materials Inc., Richmond, Canada) on a T100 Thermal Cycler (Bio-Rad, Singapore), following the manufacturer\u0026rsquo;s instructions. Quantitative real-time PCR (qPCR) amplification was then carried out with BlasTaq 2X qPCR MasterMix (Applied Biological Materials Inc., Richmond, Canada). All reactions were performed in duplicate on CFX96 Real-Time System (Bio-Rad, Singapore). \u003cem\u003eGAPDH\u003c/em\u003e was used as the reference gene, and relative expression levels were calculated using the 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e method. The primer sequences used for the studied genes are as follows:\u003c/p\u003e\u003cp\u003eFor the \u003cem\u003eATP9A\u003c/em\u003e gene, the forward primer was 5\u0026prime;-GTGTACAGCTGGGTGATTCG-3\u0026prime; and the reverse primer was 5\u0026prime;-GGGTAAGAGTGCCTGTCTTGT-3\u0026prime;.\u003c/p\u003e\u003cp\u003eFor the \u003cem\u003eGAPDH\u003c/em\u003e gene, the forward primer was 5\u0026prime;-CCACCCATGGCAAATTCC-3\u0026prime; and the reverse primer was 5\u0026prime;-TGGGATTTCCATTGATGACAAG-3\u0026prime;.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using GraphPad Prism software (version 10.4.1). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). One way ANOVA test was applied to assess overall statistical significance. A p-value of less than 0.05 was considered statistically significant (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ns: not significant).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe patient was born via cesarean section at the 36th gestational week with a birth weight of 2300 grams. There were no additional notable features in the pregnancy history. A consanguinity history exists between the parents at the level of second cousins There is a history of consanguinity between parents at the second-degree cousin. Postnatal follow-up revealed microcephaly, prominent forehead and blue sclera. Neurodevelopmental evaluation showed significant developmental delay; the patient is unable to walk in terms of motor functions, and no language development has been observed. Chromosome analysis and chromosomal microarray studies were reported as normal. In addition, no pathological findings were detected in the metabolic evaluation.\u003c/p\u003e\u003cp\u003eWES identified a homozygous variant in the \u003cem\u003eATP9A\u003c/em\u003e (NM_006045.3):c.1091G\u0026thinsp;\u0026gt;\u0026thinsp;C p.(Arg364Thr). The identified variant, located in exon 12, was absent from known mutation databases, suggesting it is a novel variant, and leads to the amino acid substitution p.(Arg364Thr) (arginine to threonine) at codon 364. This missense mutation is predicted to affect the P-type_ATPase_IV domain located between codons 53-1045. The pathogenicity assessment of the detected mutation is summarized in Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003eThe ATP9A protein models were created with the deep learning-based RosettaFold algorithm. The model confidence scores for wt and mt ATP9A were 0.83 and 0.82, respectively. The QMEAN quality scores for wt and mt ATP9A were 0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 and 0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, respectively. The wt and mt ATP9A proteins revealed high topological similarity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Tm-score and root mean square deviation (rmsd) were 0.97 and 0.66 angstrom (\u0026Aring;), respectively. Protein stability analyzes revealed that the mutation caused a decrease in protein stability and an increase in molecular flexibility. The ΔΔG for mCSM stability, SDM, DUET, and DynaMut were \u0026minus;\u0026thinsp;0.073, -1.51, -0.365, and \u0026minus;\u0026thinsp;0.260 kcal.mol\u003csup\u003e-1\u003c/sup\u003e, respectively. The vibrational entropy energy (ΔΔS\u003csub\u003evib\u003c/sub\u003e) between wt and mt-ATP9A was 0.285 kcal.mol\u003csup\u003e-1\u003c/sup\u003e.K\u003csup\u003e-1\u003c/sup\u003e. Before the mutation, there were seven polar interactions between Arg364 and Ser366 (one), Ile362 (one), Val361 (one), and Trp360 (four). The Arg364Thr mutation changed the interaction pattern of the 364th position. After the mutation, the interaction occurred between Thr364 and Ser366 (one polar), 361 (two polar), Ile362 (one polar and one van der-walls), and Trp360 (one polar and one hydrogen) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The surface area for solvent accessible decreased 2.4-fold after mutation. The solvent accessible surface area for the 364th residue decreased from 175.019 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e to 73.080 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e. Aggregation analysis showed that, despite a 6% (the aggregation score was \u0026minus;\u0026thinsp;0.4224 and \u0026minus;\u0026thinsp;0.399 for wt and mt ATP9A, respectively) reduction in collective protein stability, the decrease in stability at residue 364 was approximately 2.8-fold (the aggregation score was \u0026minus;\u0026thinsp;2.5852 and \u0026minus;\u0026thinsp;0.9379 for wt and mt ATP9A, respectively). The p.(Arg364Thr) mutation increased the aggregation propensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eATP9A\u003c/em\u003e gene encodes a member of the group of P4-ATPases that regulate asymmetric membrane lipid distribution via ATP-dependent translocation of phospholipids from the exofascial to cytosolic leaflet (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). P4-ATPases are transmembrane lipid flippases, that function in vesicles formation and trafficking. They regulate the asymmetric distribution of phospholipids in membranes of eukaryotic cells (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). There is substantial evidence to suggest that ATP9A, located both in the intracellular compartments and the plasma membrane, is localized in early and recycling endosomes, which play an essential role in vesicle formation and trafficking (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In addition to the endocytic vesicle formation process, ATP9A is known to have regulatory roles in the release of extracellular vesicles (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Numerous biological processes are regulated by intercellular communication mediated by extracellular vesicles released from cells. A considerable increase in the release of extracellular vesicles, particularly the exosome, has been linked to ATP9A downregulation. Extracellular vesicle release allows for the transmission of a variety of signaling molecules, including proteins and RNA, without the need for direct interaction between cells. It is involved in a wide range of biological processes, such as blood coagulation and immune response (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Extracellular vesicles have been reported to have different physiological roles in the central nervous system, including neurite outgrowth and neuronal survival (\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). It was recently reported that \u003cem\u003eATP9A\u003c/em\u003e mutations are associated with neurodevelopmental disorder and microcephaly (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe report a novel p.(Arg364Thr) mutation for \u003cem\u003eATP9A\u003c/em\u003e, which we suggest is associated with neurodevelopmental delay. Since ATP9A does not have an existing NMR or X-ray structure, the wt and mt-ATP9A protein models were modeled using deep learning algorithms. The possible functional effects of the mutation were analyzed by considering the tertiary and quaternary structure data of P4-ATPase flippase members whose NMR and X-ray structures are known (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The p.(Arg364Thr) mutation reduced stability of ATP9A protein. The mutation decreased solvent access and increased aggregation tendency can trigger changes in protein stability and functional properties. The p.(Arg364Thr) mutation caused an extreme increase in vibration at 364th position and adjacent residues, and a partial increase in vibration at the membrane spanning helices and nucleotide binding site (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The p.(Arg364Thr) mutation, located at the C-terminal downstream end of the M4 helix, is located in the phospholipid binding site (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The mutation changed the interaction pattern of the 364th residue with other residues in the formation of the protein tertiary structure. While there was one polar interaction in wtATP9A with Val361 located in the phospholipid binding pocket, this relationship was maintained by two polar interactions in mtATP9A (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The formational integrity of the helices in the transmembrane region is provided by the interactions established between each helix. The distance between atoms is decisive in the establishment of these interactions. The formational integrity of the helices in the transmembrane region is provided by the interactions established between each helix. The distance between atoms is decisive in the establishment of these interactions. After the mutation, the distance between wtATP9A or mtATP9A and the helix in the membrane-spanning helices appear to have diverged by approximately 4 \u0026Aring;. In addition, end region measurements of wtATP9A and mtATP9A indicate a mean difference of 5 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It is possible that the RMSD value between wtATP9A and mtATP9A is 0.66 \u0026Aring;, and the distance variations up to an average of 5 \u0026Aring; have effects on protein-ligand and protein substrate functional interactions achieved at very short distances. Conclusive evidence is needed on how the variation in interaction distances between helixes will affect the heterodimerization process of ATP9A and CDC50 required for functional activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSegregation analysis and Sanger sequencing confirmed the presence of the \u003cem\u003eATP9A\u003c/em\u003e c.1091G\u0026thinsp;\u0026gt;\u0026thinsp;C p.(Arg364Thr) variant in the proband and family members. The pedigree revealed an autosomal recessive inheritance pattern, with the proband carrying the variant in a homozygous state, resulting in the substitution of arginine with threonine at codon 364. Both parents were identified as heterozygous carriers of the same missense variant. Sanger sequencing chromatograms clearly showed the guanine-to-cytosine substitution at nucleotide position 1091, consistent with the segregation of this variant within the family and supporting its pathogenic role in the observed phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn our study, quantitative expression analysis from peripheral blood samples of the proband carrying the homozygous \u003cem\u003eATP9A\u003c/em\u003e c.1091G\u0026thinsp;\u0026gt;\u0026thinsp;C p.(Arg364Thr) variant revealed a clear upregulation of ATP9A mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This observation parallels the findings of Cordovado et al. (2025), who demonstrated \u003cem\u003eATP9A\u003c/em\u003e overexpression in HeLa and HEK293T cell models following transfection with both wild-type and mutant constructs, indicating that \u003cem\u003eATP9A\u003c/em\u003e transcription may increase as a compensatory response to disrupted protein stability or endosomal dysfunction (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Although their experiments were performed in immortalized cell lines rather than human tissues, the consistency between our \u003cem\u003ein vivo\u003c/em\u003e blood-derived expression and their \u003cem\u003ein vitro\u003c/em\u003e data suggests a shared cellular mechanism that upregulates \u003cem\u003eATP9A\u003c/em\u003e to counterbalance partial loss of function caused by missense variants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConversely, Vogt et al. (2022) observed a pronounced downregulation of \u003cem\u003eATP9A\u003c/em\u003e expression in skin fibroblasts derived from patients with biallelic truncating or splice-site mutations, with transcript levels reduced to only 4\u0026ndash;14% of control values due to nonsense-mediated mRNA decay (NMD) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The opposing regulatory patterns underscore the influence of both mutation type and tissue context on \u003cem\u003eATP9A\u003c/em\u003e expression: truncating mutations trigger transcript degradation and complete loss of function, whereas missense variants such as p.(Arg364Thr) appear to elicit a compensatory transcriptional upregulation in peripheral tissues. Collectively, these findings support the notion that elevated \u003cem\u003eATP9A\u003c/em\u003e expression in our case likely reflects a feedback response to reduced protein activity rather than true functional enhancement.\u003c/p\u003e\u003cp\u003eApart from the mutation we reported in this study, many \u003cem\u003eATP9A\u003c/em\u003e mutations were shown to be associated with neurodevelopmental disorders (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The fact that P4-ATPase mutations other than \u003cem\u003eATP9A\u003c/em\u003e are also associated with neurodevelopmental disorders increases the reliability of the data obtained for \u003cem\u003eATP9A\u003c/em\u003e (\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Meng et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) revealed that mutation or deficiency of ATP9A leads to inactivation of RAB5 and RAB11, resulting in abnormal endosomal recycling in neural cells. They reported that this condition leads to synaptic dysfunction in the primary motor cortex and hippocampus of the brain, resulting in hypotonia, intellectual disability, hyperactivity disorders, and neurological disorders. Tanaka et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) revealed that \u003cem\u003eATP9A\u003c/em\u003e is indispensable for endocytic recycling of transferrin receptor and glucose transporter 1 (GLUT1) to the plasma membrane. Depletion of \u003cem\u003eATP9A\u003c/em\u003e reduces plasma membrane expression of GLUT1 and alters its recycling, increasing its level in the endosome. GLUT1 deficiency has been associated with a neurological disorder with a variable phenotype, including epilepsy, movement disorders, mild or severe intellectual disability, and in some cases, acquired microcephaly.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePatogenicity of detected variant\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eATP9A\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTranskript id\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNM_006045.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDbsnp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNovel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariant\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1091G\u0026thinsp;\u0026gt;\u0026thinsp;C p.(Arg364Thr)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariant locus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003echr20-50287743 Exon 12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariant type\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMissense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMutation taster\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDisease causing\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDANN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDeleterious (0.95)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimateAI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDeleterious (Supporting) (0.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSeq.genomize\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNot found\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinvar\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAffected Domains\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP-type_ATPase_IV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConservation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConserved\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eACMG classification\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVariant of uncertain significance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eACMG pathogenicity criteria\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePP2, PM2, PM3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eACMG: The American College Of Medical Genetics And Genomics, DANN: Prediction score based on a deep neural network. Gnomad: Genome Aggregation Database, PrimateAI: Predicts the pathogenicity of missense variants using deep learning trained on humans and non-human primate species, Seq.Genomize: Web Based Anutation Programme-Turkish Populastion Variant Database\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003eATP9A\u003c/em\u003e gene pathogenic variants reported in the literature and clinical findings of patients.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"14\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNationality\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAge (year)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eVariant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eZ\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eBrain MRI anomalies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eDD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eMC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003eADHD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003eSt.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c14\"\u003e\u003cp\u003eFA\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProband\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTurkish\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.1091G\u0026thinsp;\u0026gt;\u0026thinsp;C p.(Arg364Thr).\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLateral ventricular asymmetry\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eMattioli et al. 2021 (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 1: Pakistani\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.799\u0026thinsp;+\u0026thinsp;1\u0026thinsp;G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 2: Pakistani\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.799\u0026thinsp;+\u0026thinsp;1\u0026thinsp;G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 3: Iranian\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.327\u0026thinsp;+\u0026thinsp;1\u0026thinsp;G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eVogt et al. 2021 (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 1: Syrian\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.868C\u0026thinsp;\u0026gt;\u0026thinsp;T p.(Arg290*)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 2: Syrian\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.868C\u0026thinsp;\u0026gt;\u0026thinsp;T p.(Arg290*)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 3: Turkish\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.642\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A p. (Ser184Profs*16)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eMeng et al. 2023 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 1: Chinese\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.658C\u0026thinsp;\u0026gt;\u0026thinsp;T/ c.433C\u0026thinsp;\u0026gt;\u0026thinsp;T p.(Arg220*p. Arg145*)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eComp HT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMild abnormality\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 2: Chinese\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.658C\u0026thinsp;\u0026gt;\u0026thinsp;T/c.433C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003cp\u003ep. (Arg220*/p. Arg145*)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eComp HT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMild abnormality\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 3: Chinese\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.983G\u0026thinsp;\u0026gt;\u0026thinsp;A/c.983G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003ep. (Trp328*/p. Trp328*)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMild abnormality\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003eCordovado\u0026nbsp;et al. 2025 (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 1: Caucasian\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.1178C\u0026thinsp;\u0026gt;\u0026thinsp;G p.(Thr393Arg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCortical atrophy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 2: Caucasian\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.1198G\u0026thinsp;\u0026gt;\u0026thinsp;C p.(Glu400Gln)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSequela of callosotomy mild asymmetry of hippocampi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 3: North Africa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.1655G\u0026thinsp;\u0026gt;\u0026thinsp;C p.(Gly552Ala)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePartial agenesis of corpus callosum, global cortical atrophy, myelination delay, epiphyseal cyst, pars intermedia cyst\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 4: Caucasian\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.2137C\u0026thinsp;\u0026gt;\u0026thinsp;G p.(His713Asp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMild nonspecific bilateral flair hypersignal of the posterior parietal periventricular white matter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 5: Caucasian\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.433C\u0026thinsp;\u0026gt;\u0026thinsp;T \u0026amp; c.2701G\u0026thinsp;\u0026gt;\u0026thinsp;T p.(Arg145*) and p. (Glu901*)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNormal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP 6: Caucasian\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec.1381A\u0026thinsp;\u0026gt;\u0026thinsp;G p.(Lys461Glu)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"14\"\u003eComp HT: Compound heterozygous, DD: Developmental delay, FA: Facial Anomaly, FD: Feeding difficulities, GR: Growth retardation, HM: Homozygous, HT: Heterozygous, MC: Microcephaly, PA: Prenatal anomalies, SD: Speech delay, S: Seizure, Z: Zygosity, ND: not determined, St: Strabismus, ADHD: attention deficit hyperactivity disorder, NA: Not avaiable\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eVariants in the \u003cem\u003eATP9A\u003c/em\u003e gene reported to date are predominantly biallelic truncating or splice-site mutations that result in a loss-of-function (LoF) mechanism (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). These alterations introduce premature stop codons or disrupt canonical splicing, thereby preventing the synthesis of a functional protein. Meng et al. (2023) further identified compound heterozygous truncating variants p.(Arg145*/p.Arg220*) leading to a similar functional outcome (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). More recently, Cordovado et al. (2025) described heterozygous missense variants p.(Thr393Arg) and p.(Glu400Gln) associated with dominantly inherited non-syndromic intellectual disability, demonstrating impaired dendritic spine maturation and altered postsynaptic morphology (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In this context, the homozygous p.(Arg364Thr) variant identified in our proband represents a polar amino acid substitution predicted to alter the structural integrity of the ATP9A protein, suggesting a LoF-like effect despite its missense nature (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eClinically, \u003cem\u003eATP9A\u003c/em\u003e-related patients reported in the literature exhibit similar phenotypes. Mattioli et al. (2021) and Vogt et al. (2022) reported developmental delay (DD), speech delay (SD), and microcephaly (MC) as the cardinal manifestations (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Additional features, such as strabismus, feeding difficulties, seizures, and facial dysmorphism, were occasionally observed. Meng et al. (2023) described patients with compound heterozygous truncating variants presenting with attention-deficit/hyperactivity disorder (ADHD)-like symptoms and mild structural brain abnormalities (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Similarly, Cordovado et al. (2025) found that heterozygous missense variants may cause cortical atrophy, hippocampal asymmetry, and corpus callosum abnormalities (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Collectively, these findings suggest that pathogenic variants of ATP9A disrupts endosomal recycling and phospholipid asymmetry, thereby impairing synaptic transmission and neuronal maturation. Our proband exhibited microcephaly, developmental and speech delay, lateral ventricular asymmetry, strabismus, and mild facial dysmorphism, similar to other reported patients who have biallelic pathogenic variants. Despite being a missense substitution, p.(Arg364Thr) appears to exert a deleterious impact comparable to that of truncating mutations, indicating that this residue may lie within a functionally critical domain (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study suggests that the \u003cem\u003eATP9A\u003c/em\u003e (c.1091G\u0026thinsp;\u0026gt;\u0026thinsp;C; p.(Arg364Thr)) variant may be associated with a neurodevelopmental disorder, particularly cognitive impairment. The mutation likely affects ATP9A\u0026rsquo;s structural stability and function, potentially leading to protein aggregation and subsequent cellular dysfunction. These findings highlight the possible contribution of \u003cem\u003eATP9A\u003c/em\u003e to neuronal pathology and emphasize the need for further molecular and functional studies to elucidate its pathogenic role.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eNone.\u003c/p\u003e\n\u003cp\u003eThe authors state no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetails of Ethical Approval and Informed Consents:\u003c/strong\u003e This study adhered to the ethical principles outlined in the Helsinki Declaration for the investigation of human subjects. Ethical approval for this study was obtained from the Non-Interventional Ethics Committee of Istanbul Başakşehir \u0026Ccedil;am and Sakura City Hospital (KAEK/26.08.2023.355). Additionally, informed consent was obtained from all participants prior to the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution:\u003c/strong\u003e Conceptualization: C.Y., A.A., E.N., A.G.; Formal Analysis: C.Y., A.A., E.N., E.A.; Investigation: C.Y., A.A., A.G., H.I.Y., E.N., E.A., P.A.; Data Curation: C.Y., A.A., A.G., P.A. Methodology: A.A., C.Y., A.G., E.N., H.I.Y.; Writing-original draft: \u0026nbsp;C.Y., A.A.; Review and Editing: C.Y., A.A., A.G., E.N., H.I.Y., E.A., P.A. Supervision: C.Y., A.A, A.G. All the authors have read and approved the final version of the manuscript and agreed to be held accountable for all aspects of the work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTanaka Y, Ono N, Shima T, Tanaka G, Katoh Y, Nakayama K et al (2016) The phospholipid flippase ATP9A is required for the recycling pathway from the endosomes to the plasma membrane. Mol Biol Cell 27(24):3883\u0026ndash;3893\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeng T, Chen X, He Z, Huang H, Lin S, Liu K et al (2023) ATP9A deficiency causes ADHD and aberrant endosomal recycling via modulating RAB5 and RAB11 activity. Mol Psychiatry 28(3):1219\u0026ndash;1231\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIqbal Z, van Bokhoven H (2014) Identifying genes responsible for intellectual disability in consanguineous families. 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Rev Assoc Med Bras 2022;68(9):1282-7\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKochinke K, Zweier C, Nijhof B, Fenckova M, Cizek P, Honti F et al (2016) Systematic Phenomics Analysis Deconvolutes Genes Mutated in Intellectual Disability into Biologically Coherent Modules. Am J Hum Genet 98(1):149\u0026ndash;164\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDawidziuk M, Gambin T, Bukowska-Olech E, Antczak-Marach D, Badura-Stronka M, Buda P et al (2021) Exome Sequencing Reveals Novel Variants and Expands the Genetic Landscape for Congenital Microcephaly. Genes (Basel). ;12(12)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl Ghouzzi V, Boncompain G (2022) Golgipathies reveal the critical role of the sorting machinery in brain and skeletal development. 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Curr Issues Mol Biol 43(3):1778\u0026ndash;1793\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ATP9A, p.(Arg364Thr), Novel Variant, Cognitive Impairment, Whole Exome Sequencing, Neurodevelopmental Disorders","lastPublishedDoi":"10.21203/rs.3.rs-8263851/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8263851/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eATP9A\u003c/em\u003e gene encodes a P4-type ATPase involved in phospholipid translocation, essential for vesicular trafficking and neuronal development. Pathogenic \u003cem\u003eATP9A\u003c/em\u003e variants cause autosomal recessive neurodevelopmental disorders characterized by intellectual disability and microcephaly, yet the impact of missense variants remains poorly understood.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e\u003cp\u003eA 7-year-old female patient with cognitive impairment, microcephaly, and developmental delay was admitted to Başakşehir \u0026Ccedil;am and Sakura City Hospital. Whole exome sequencing (WES) using Illumina technology identified a novel homozygous \u003cem\u003eATP9A\u003c/em\u003e variant, confirmed by Sanger sequencing and segregation analysis. \u003cem\u003eIn silico\u003c/em\u003e tools (RosettaFold, DynaMut, mCSM, SDM, DUET, AggreScan3D) assessed its structural impact. Quantitative real-time PCR (qPCR) was conducted to evaluate the relative expression levels of \u003cem\u003eATP9A\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e\u003cp\u003eWES revealed a homozygous missense variant, \u003cem\u003eATP9A\u003c/em\u003e (NM_006045.3):c.1091G\u0026thinsp;\u0026gt;\u0026thinsp;C p.(Arg364Thr), classified as variant of uncertain significance (ACMG: PP2, PM2, PM3). Protein modeling demonstrated reduced stability (ΔΔG = \u0026minus;\u0026thinsp;1.51 to \u0026minus;\u0026thinsp;0.26 kcal/mol), increased flexibility, and a 2.4-fold decrease in solvent accessibility. The mutation altered polar and hydrophobic interactions within the P-type ATPase IV domain, enhancing aggregation propensity. Expression analysis showed elevated \u003cem\u003eATP9A\u003c/em\u003e mRNA levels, suggesting a compensatory response.\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e\u003cp\u003eThis novel \u003cem\u003eATP9A\u003c/em\u003e variant broadens the mutational spectrum of \u003cem\u003eATP9A\u003c/em\u003e-related neurodevelopmental disorders. Structural destabilization of the p.(Arg364Thr) protein may contribute to the patient\u0026rsquo;s cognitive impairment and microcephaly, warranting further functional studies.\u003c/p\u003e","manuscriptTitle":"The Role of ATP9A (c.1091G\u0026gt;C; p.(Arg364Thr)) Variant in Cognitive Impairment: Diagnostic Insight from Whole Exome Sequencing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 12:43:58","doi":"10.21203/rs.3.rs-8263851/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-28T17:08:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-27T18:31:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-24T19:23:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"208916141425565540854749556867026788758","date":"2025-12-10T17:31:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180243294609285381570072190743224009894","date":"2025-12-10T15:21:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-05T11:56:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-04T04:46:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-04T04:43:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2025-12-02T19:50:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"041d8f33-60ab-46bb-9e5d-be0150d4cfc3","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:04:02+00:00","versionOfRecord":{"articleIdentity":"rs-8263851","link":"https://doi.org/10.1007/s11033-026-11496-5","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2026-01-28 15:59:21","publishedOnDateReadable":"January 28th, 2026"},"versionCreatedAt":"2025-12-08 12:43:58","video":"","vorDoi":"10.1007/s11033-026-11496-5","vorDoiUrl":"https://doi.org/10.1007/s11033-026-11496-5","workflowStages":[]},"version":"v1","identity":"rs-8263851","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8263851","identity":"rs-8263851","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}