De novo variants in NPTN cause a neurodevelopmental disorder with autism and neuroplastin-PMCA hypofunction

De novo variants in NPTN cause a neurodevelopmental disorder with autism and neuroplastin-PMCA hypofunction | 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 De novo variants in NPTN cause a neurodevelopmental disorder with autism and neuroplastin-PMCA hypofunction Yi Liang, Rodrigo Ormazabal-Toledo, Harini Srinivasan, Ayse Malci, and 19 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8079156/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract NPTN encodes human neuroplastin (hNp), a subunit of plasma membrane Ca 2+ -ATPases (PMCA). The critical importance of hNp and its associations with PMCA are unknown for the human brain. Here, we describe de novo NPTN variants in seven individuals with autism and mild-to-severe DD/ID and evaluate them using animal models and in silico , molecular and cellular approaches. We identified NPTN variants with dominant-negative (missense) or loss-of-function (nonsense/ frameshift) effect on hNp-PMCA expression and function. The missense variants caused structural and thermodynamic molecular abnormalities and lower expression of hNp in HEK cells. In neurons, hNp missense variants affected PMCA levels and cytosolic Ca²⁺ regulation. In Drosophila , a missense mutation with affected PMCA interaction failed to prevent a lethal phenotype caused by hNp ortholog elimination. In Nptn +/− mice, levels of Np and PMCA were reduced and insufficient for normal social behavior. Therefore, we show that de novo variants in NPTN cause a neurodevelopmental disorder with intellectual disability and autism, likely linked to PMCA dysfunction. Medical Genetics General Cell Biology & Physiology Neurology Cellular & Molecular Neuroscience autism epilepsy speech delay neuroplastin PMCA calcium homeostasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Developmental delay (DD), often linked to intellectual disability (ID) and autism spectrum disorder (ASD) 1 , has a prevalence of 1–3% which may vary from large international cohorts to local populations 1 – 3 . Due to its heterogeneity, the etiology of DD is not always diagnosed, as it remains uncertain for 60% of the affected children 1 , 3 . Exome and genome sequencing are being used to identify genetic variants as the plausible cause of DD, revealing that 40–60% of individuals with undiagnosed DD and 30–39% of the ASD cases may carry pathogenic de novo variants 3 – 5 . Discovering genetic variants associated with ID and ASD will facilitate early diagnosis and open avenues for their treatment. The gene NPTN encodes two isoforms of the type I transmembrane glycoprotein neuroplastin expressed in the brain, human neuroplastin55 (hNp55) and neuron-specific human neuroplastin65 (hNp65) 6 . High levels of neuroplastin mRNAs are detected at 19–24 post-coital weeks (PCW) in neurons across fetal brain regions 7 , with peak levels in the prefrontal cortex of 18-year-old individuals 6 . The isoforms hNp55 and hNp65 are enriched in human synapses 6 . NPTN is often deleted or duplicated in individuals with 15q24 microdeletion syndrome 8 – 10 . Also, a single nucleotide polymorphism in the NPTN promoter is associated with thinner frontal and temporal lobes in the left hemisphere of the brain, correlating with verbal and non-verbal abilities in adolescents 11 . Studies in Nptn −/− mutant mice provide direct evidence for the necessity of normal Np55/65 expression for multiple cognitive functions 6 , 12 . Neuroplastin55/65 are obligatory binding partners in protein complexes for more than 95% of the four plasma membrane Ca 2+ -ATPases (PMCA1-4) 13 . The abundance and importance of these associations remain unexplored for human brain. PMCA1-4 are ATP-fed Ca 2+ -H + co-transporters that pump Ca 2+ towards the extracellular space to reinstate resting cytosolic levels and regulate intracellular Ca 2+ signaling while setting the perisynaptic alkaline pH necessary for the activation of ionotropic glutamate receptors of the NMDA type (iGluNRs) 14 – 18 . Mutations in the PMCA1-4 genes ( ATP2B1-4 ) in individuals with DD, ASD, and other neurodevelopmental disorders decrease the expression and/or activity of these Ca 2+ pumps, leading to defective restoration of cytosolic Ca 2+ levels 19–30 . Importantly, the expression, stabilization, and activity of PMCA1-4 are strongly dependent on Np55/65 binding 6 , 12 , 13 , 31 – 33 and constitutive and inducible Nptn -deficient mice display massive PMCA1-4 loss associated with cognitive impairments and deficits in social and affective behaviours 6 , 12 . Here, we report mild to severe DD/ID and autism in seven individuals with de novo missense, nonsense or frameshift in NPTN . The missense variants cause structural abnormalities in hNp as evaluated in silico through protein modelling and molecular dynamics. We confirmed that, in a human cell line, cultured primary rodent neurons, and in vivo in Drosophila melanogaster , the missense variants are inefficiently expressed and exert dominant negative effects on PMCA levels resulting in failed cytosolic Ca 2+ regulation. Nptn +/− mice express reduced amounts of both Np and PMCA. In a social behavior test, Nptn +/− mice display loss of preference for a novel mouse representing an endophenotype analog to social deficits that characterize autism. We conclude that these de novo variants in NPTN cause a neurodevelopmental disorder likely through direct Np-PMCA hypofunction and Ca 2+ -deregulation in central neurons. Results Clinical and genetic analysis We describe a cohort of seven individuals with heterozygous variants in NPTN , six of which are of de novo origin. An overview of the clinical evaluation of all individuals is presented in table 1. Additional descriptions are provided as case reports in the supplementary data and in supplementary table 3 and 4. All seven individuals presented with developmental delay (DD) and/or intellectual disability (ID) ranging from mild to severe. Four individuals displayed severe, two moderate, and one mild DD/ID, respectively. All individuals were diagnosed with autism spectrum disorder. Other behavioral findings included poor social interactions, high pain threshold, automutilation or repetitive behaviors. Individual 1 presented seizures starting at age seven months with epileptic spasms. That same individual developed focal impaired awareness seizures at eight years of age and was not seizure-free at age 17. Five of seven individuals received a cranial MRI. Of note, individual 3 presented with a vermal dysgenesis. Individual 4 was reported to have a period of significant regression of speech skills. Growth was found normal in all individuals. Subtle dysmorphic facial features were reported in five out of seven individuals and upslanting palpebral fissures and a prominent forehead were recurrently observed in individuals 1, 2, and 6 (Fig. 1 ). Genetic results Trio exome sequencing revealed de novo variants in NPTN in individuals 1–4, 6, and 7. A single exome test was carried out in individuals 3 and 5. The nonsense variant in NPTN in individual 3 was segregated in the parents with Sanger sequencing. The biological parents of individual 5 were not available for testing. All variants are not recurrent and are absent from gnomAD (v4 dataset). Two distinct de novo missense variants were identified in addition to five predicted loss-of-function (pLoF) variants. Multiple in silico tools predict a deleterious effect of the two NPTN missense variants (Supplementary table 2 and 4). Missense variants as well as pLoF variants are highly depleted from the gnomAD database. This indicates a selective constraint on both types of variants in a general population that lacks severe, early-onset phenotypes such as DD and ID (LOEUF = 0.25; pLI = 0.99; o/e for missense variants = 0.52; z-score = 2.68). Characterization of the NPTN variants We located in the genomic sequence of the NPTN gene the position of each of the variants identified (Fig. 2 A). NPTN consists of eight coding exons, one non-encoding 3'UTR exon, and exon 2 that can be spliced out by alternative splicing of the gene transcripts. From this process, two main glycoprotein isoforms are produced, hNp55 and hNp65. Additionally, they may display an alternative aminoacidic insert (DDEP) encoded by exon 7. The function of the DDEP insert is unknown but it is dispensable in Np for regulation of PMCA levels 31 , 33 . hNp65 is 394 or 398 amino acids long, contains a signal peptide sequence, three extracellular Ig-like domains (Ig-like I-III) encoded by exons 1–6, a single transmembrane domain (TM) encoded by exon 6, and a 34 or 38 amino acids long intracellular domain encoded by exons 6–8. Np55 is 278 or 282 amino acids long and distinctively displays the Ig-like domains II-III. NPTN missense variants affected conserved amino acids located within conserved amino acid sequences in different species (Fig. 2 B). The missense variant c.403T > A (p.W135R) is located at the hNp65-specific exon 2, and other variants affect exons 1, 2, 4, and 6 which are common for both hNp isoforms (Fig. 2 A and C). Interestingly, the missense variant c.1025C > A, p.(Pro342Leu) (hNp65: p.P342L; hNp55: p.P226L) replaces a key transmembrane amino acid in Np that interacts directly with PMCA 30 . The nonsense variant c.14C > A, p.(Ser5*) stops the transcription of the signal peptide in the mutated allele. The nonsense variants c.284C > G, p.(Ser95*) and c.342C > G, p.(Tyr114*) stop mRNA translation during the very early synthesis of the hNp65-specific Ig-like domain, probably resulting in non-functional peptides likely to be degraded (Supplementary table 1). The frameshift in the variant c.902del, p.(Asn301Thrfs*3) in exon 6 results in a stop in the mutated allele. Analysis of mRNA stability indicates that truncated mRNA from the frameshift variant c.902del (p.N301Tfs*3) is degraded and thus, not producing truncated Np65 protein (Supplementary Fig. 1). Computational analysis of the NPTN missense variants We performed a customized structural and thermodynamic analysis of NPTN missense variants using molecular dynamics and protein-protein docking modeling and calculating binding energies ( ∆G binding) (Fig. 3 and SMaterial). Based on the resolved structure of the Np65-specific Ig-like domain I 34 , our computational procedures were robust to recreate the hNp65 wt -hNp65 wt trans- homophilic binding (Fig. 3 A, top-down view). We found that W135, N130, and I133 have an important participation in the thermodynamically spontaneous attraction between hNp65 wt Ig-like domain I F-G loops (Fig. 3 A, upper frame in lateral view). In the variant hNp65 p.W135 resulting from c.403T > A, the F-G loop structure is altered and the N130 and I133 are far off from reaching effective trans- interaction positions (Fig. 3 A, middle frame in lateral view). An increased ∆G confirms the reduced binding efficiency of the hNp65 p.W135R F-G loop to form the pair hNp65 wt -hNp65 p.W135R . The interaction of the pair hNp65 p.W135R -hNp65 p.W135R was worsened by the appearance of an abnormal P122-P122 interaction with an even higher ∆G for their binding (Fig. 3 A, middle frame in lateral view). In agreement with the reported crystallographic structure of the hNp-hPMCA complex 31 , we localized P342 at the hNp transmembrane domain (TMD) facing W1043 at the hPMCA TM10 and found it participates in the stable interaction of the proteins within the cell plasma membrane (lateral view in Fig. 3 B). When the hNp65 p.P342L variant resulting from 1025C > A was projected onto the Np-PMCA interaction surface, we observed that the larger mutant residue L342 violates the effective interaction distance with hPMCA TM10 W1043, creating a thermodynamic constraint that would interfere with the intermolecular interaction (lower frame in top-down view in Fig. 3 B). Having studied other missense mutations affecting hNp Ig-like domain II structure 35 , we also analyzed the missense variant hNp65 p.A210T from c.1025C > A located at Ig-like domain II which was previously identified but not characterized 36 (Supplementary Fig. 2). Briefly, the switch from A210 to T210 resulted in replacement of stabilizing intramolecular interactions by weak CH-CH interactions with highly variable interaction distances. Furthermore, T210 adds a mutant polar OH group to a normally apolar environment that causes extra steric congestion of the domain and stabilization constraints to the Ig-like domain II structure (Supplementary Fig. 2). Expression of NPTN missense variants and effects on PMCA levels Based on our previous studies 6 , 33 , 35 , we tested the expression levels of hNp65 wt , hNp55 wt , hNp65 p.W135R , hNp65 p.P342L , and hNp55 p.P226L in transfected HEK293T cells (Supplementary Fig. 3). Other missense mutations identified 36 were also analyzed (Supplementary Fig. 3) and are described in Supplementary material. As expected 6 , 33 , 35 , hNp65 wt and hNp55 wt were efficiently detected by Western blot analysis (Supplementary Fig. 3A). Importantly, decreased expression was found for hNp65 p.W135R (p < 0.001 vs hNp65 wt ) and hNp65 p.P342L (p = 0.033 vs hNp65 wt ) whereas hNp65 p.A210T expression was only slightly decreased compared to control (p = 0.077 vs hNp65 wt , SFig. 3A,B). Levels of hNp55 p.P226L and hNp55 p.T78P were similar to hNp55 wt (SFig. 3A,B). Neuroplastin is an obligatory binding partner and post-transcriptionally promotes the expression of PMCA 6 , 12 , 13 . Therefore, we examined the effect of the missense variants on the capacity of Np to increase hPMCA2 levels 6 , 35 . hPMCA2 levels in hNp65 WT - and hNp55 WT -expressing cells were higher than the ones in non-transfected control cells (Supplementary Fig. 3B,C). Compared to PMCA2-expressing single transfected cells and to PMCA2/hNp65 WT - or PMCA2/hNp55 WT -expressing double transfected cells, all missense variants promoted hPMCA2 but some of them inefficiently. Indeed, hPMCA2 was less in cells expressing hNp65 p.P342L (p = 0.004 vs hNp65 wt ), hNp65 p.W135R (p = 0.011 vs hNp65 wt ) or Np55 p.P226L (p = 0.057 vs hNp55 WT ) (Supplementary Fig. 3B,C). Compared to controls, the ability to increase hPMCA2 (hPMCA2/hNp ratio) was reduced for hNp65 p.P342L and hNp55 p.P226L but not for hNp55 p.T78P , hNp65 p.A210T or hNp65 p.W135R (Supplementary Fig. 3D) pointing to a specific necessity of this mutated proline residue in both hNP isoforms for normal levels of hPMCA2 expression in human cells. The impact of P226L on hNp55 functionality was evaluated in Drosophila melanogaster , a classical system to study neurodevelopment and synaptic mechanism 37 , 38 (Fig. 4 ). In contrast to the three mammalian paralogs NPTN, BSG , and EMB , only a single ortholog gene encoding dBsg exists in Drosophila. DBsg shows an overall 25% amino acid sequence identity and a transmembrane domain homology of 69% (including adjacent intra- and extracellular amino acid residues) with hNp55 (Fig. 4 A, B). Deletion of dBsg expression in muscle is known to be lethal at the late embryonic to early larval (L1) stage 39 . Thus, we tested whether the co-expression of hNp55 wt or hNp55 p.P226L rescues the lethal phenotype triggered by dBsg knockdown due to mef2-Gal4-induced expression of dsRNA (Fig. 4 C,D). As expected, dBsg knockdown caused a highly penetrant lethality around the L1 stage. The larval lethality was fully rescued by hNp55 wt as virtually all progeny developed into viable adult flies, indicating tolerance to the differences between dBsg and hNp55 wt . Strikingly, hNp55 p.P226L displayed only minimal if any rescue capacity, as all progeny died before the L2 stage (Fig. 4 D). Effect of NPTN missense variants on cytosolic Ca 2+ regulation Neuroplastin-PMCA complexes are crucial for cytosolic Ca 2+ extrusion and shaping of Ca 2+ signaling in brain neurons 6 , 12 , 13 , 31 – 33 , 35 . To evaluate the functional effect of NPTN missense variants on Ca 2+ regulation, we investigate electrically-evoked cytosolic Ca 2+ transients using Ca 2+ imaging 31,34 in 14–16 days-old GCaMP5G-expressing primary hippocampal neurons (referred to as GCaMP5G-neurons) (Fig. 5 A). We quantified peak amplitude, half-width, and decay time of the evoked Ca 2+ transients, as these parameters reflect how Ca 2+ transients are shaped by the levels and activity of Np-PMCA complexes in synapses and dendrites (Fig. 5 A). In line with the previous reports showing that Np wt over-expression adds on endogenous Np to promote PMCA levels and function (gain-of-function) 6 , 12 , 31 – 33 , 35 , 40 , 41 , GCaMP5G-neurons co-expressing hNp65 wt or hNp55 wt displayed smaller evoked Ca 2+ transients with faster restoration of basal Ca 2+ levels compared to control GCaMP5G-neurons (Fig. 5 B-D). In contrast, hNp65 p.W135R , hNp65 p.P342L and hNp55 p.P226L caused abnormal or incomplete Ca 2+ transients. Indeed, whereas peak amplitudes were similarly reduced in GCaMP5G-neurons co-expressing either hNp65 p.W135R or hNp55 p.P226L compared with their wild-type expressing controls, both mutants displayed an increased half-width and decay time indicating longer Ca 2+ transients with slower recovery to baseline (Fig. 5 B-C). hNp65 p.P342L accelerated the restoration of basal Ca 2+ levels but did not sufficiently reduce the Ca 2+ peak amplitude vs hNp65 wt (Fig. 5 D). Thus, these data indicate that these missense variants act dominant negatives rather than as a complete loss-of-function. Nptn heterozygosity affects PMCA brain levels and social behavior in mice A reduction of Np expression by ~ 50% using a pan siRNA against Np mRNA resulted simultaneously in a partial reduction of PMCA1-4 in 14–16 days-old hippocampal neurons (Supplementary Fig. 4A-C) and increased half-width and decay time of electrically-evoked Ca 2+ transients, as visualized using GCaMP7s-based Ca 2+ imaging (Supplementary Fig. 4D). As anticipated, immunohistochemical evaluation in the hippocampus of 14 days-old Nptn +/− pups demonstrated that Nptn heterozygosity, resulting in ~ 50% reduction in Np levels, causes ~ 45% loss of PMCA1-4 in developing neurons (Fig. 6 A,B). Therefore, heterozygous levels of Np wt are insufficient to maintain normal endogenous PMCA levels in the rodent hippocampus and cortex. Autism is a common trait shared by the NPTN individuals (Table 1). While general stereotypic behaviors were not observed in the heterozygous Nptn +/− mice and specifically not detected in the marble burying test (not shown; Supplementary Table 5), these mice displayed reduced anxiety in the O-maze test (not shown; Supplementary Table 5). Further, we assessed autism-like behavioral features related to social interactions using the three-chamber social interaction assay (Fig. 6 C). Both Nptn +/− and Nptn +/+ mice displayed a similar preference for a novel mouse compared to an empty cup (Fig. 6 C). However, in contrast to Nptn +/+ , Nptn +/− mice did not prefer a stranger over an already familiarized mouse (Fig. 6 D). Such altered social interactions observed in Nptn +/− mice have also been reported in other mouse models of genetically caused autism 42 . Discussion We describe seven individuals with an overlapping, albeit nonspecific, phenotype. All individuals displayed de novo variants in NPTN and were diagnosed with autism and neurodevelopmental disorders, pointing to the existence of pathological mechanisms related to alterations in NPTN that affect neurodevelopment. The NPTN variants disrupted critical functions of Np on the regulation of PMCA levels and cytosolic Ca²⁺ dynamics. In particular, the missense variants exhibit dominant negative features, whereas the nonsense or frameshift variants resulted in loss of function, affecting Np expression, PMCA levels, and Ca²⁺ signal regulation. Furthermore, monoallelic Nptn condition proved insufficient to support normal PMCA function and led to autism-like social behavior in transgenic mice. This report not only confirms prior assumptions regarding the relevance of Np in human neurodevelopment derived from machine learning approaches 43 and correlative genetic studies 11 , but also provides a wider perspective to prospective pathological mechanism relevant to other affected individuals, such as those with de novo mutations in PMCA1–4-encoding genes (ATP2B1–4) 19–30 . It is confirmed that Np binds, stabilizes into protein complexes, and promotes the catalytic function of PMCA1-4. It is also shown that a significant decrease in Np levels, due to Np gene deletion or mutation or Np mRNA-interference, results in PMCA1-4 loss in neurons. Notably, in our assays, missense variants of the NPTN individuals 1 and 2 resulted in decreased production of hNp levels carrying different dysfunctional residues that replaced key conserved amino acids. While the variant of individual 1 substitutes a critical tryptophan within the amino acid sequence at the extracellular and isoform-specific Ig-I of hNp65, individual 2‘s variant replaces a proline located at the common transmembrane segment of hNp55 and hNp65 which is embedded into the plasma membrane. The identified binding instability and conformational alterations in these human missense variants indicate that they are either not effectively produced or avidly degraded, as is the case for other Np variants affecting hearing in mice 35 . Our results in HEK293T cells and Drosophila melanogaster further support that the NPTN variant of patient 2, yielding hNp55 p.P226 and hNp65 p.P342L , results in impaired biosynthesis/ degradation and is insufficient to support normal PMCA function. This impairment seems to be most detrimental during early development of the flies and is compatible with a dominant negative effect on PMCA function. Interestingly, although human variants from individuals 1 and 2 were expressed at lower levels compared to wt Np isoforms, the NPTN Ig-I variant of patient 1 hNp65 p.W135R was able to promote PMCA, but the NPTN transmembrane variant of patient 2 was inefficient in this property. Stop codon-producing nonsense or frameshift variants in individuals 3-to-7 do not produce truncated proteins and, thus, their effects may result from a diminished haploid production of hNp55/65 insufficient to maintain normal PMCA levels. Indeed, we demonstrated that either titrated Np RNAi-targeted knockdown in isolated cultured neurons or monoallelic condition in Nptn +/− mice yielded appr. 50% expression levels of Np with a significant reduction in PMCA levels. Ca 2+ imaging confirmed that a progressive decrease on Np-PMCA content is mirrored by a progressive increase in the Ca + 2 signal parameters peak amplitude, half width, and decay time. Therefore, we conclude that missense variants display loss-of-function with dominant negative features and that nonsense or frameshift variants result in hypofunctional hNp levels with diminished hNp-PMCA function. Insufficient PMCA function may not be the only mechanism underlying the phenotypes of the NPTN individuals in this cohort. The missense variant in individual 1 is located at an important extracellular module of hNp65 that mediates homophilic Np65-Np65 trans -interactions 33 . In fact, competition of this motif with high-affinity peptides or antibodies destabilizes glutamatergic synaptic contacts and impairs synaptic transmission 44 . As our thermodynamic calculations indicate that this Np65-specific variant displays weakened binding, it is possible to hypothesize the occurrence of an insufficient structural stabilization of excitatory synapses during synaptogenesis in individual 1. Additionally, as reduced synapse formation triggered by Np55 and Np65 occurs in Nptn −/− neurons due to failed TRAF6 signaling 33,44 , it is plausible that production of hNp variants in individuals 1 and 2, or haploid wild-type hNp production in individuals 3-to-7, would not trigger Np-TRAF6-dependent synapse formation sufficiently. On the other hand, insufficient hNp production could also alter the excitatory-inhibitory balance affecting development and maturation of neuronal circuits leading to epilepsy or ataxia in the NPTN individuals 1-to-3. This idea is based on the requirement of Np for the correct synaptic localization and function of AMPA receptors 45 and α1/2 subunit-containing GABA type A receptors 44,46 . Interestingly, recent studies suggest that reduced synaptic transmission involving binding of Np to GABA type A receptors sensitizes rodents to pentylenetetrazole-induced epilepsy 40,47 . Therefore, hypofunctions and/or malfunctions of other binding partners of hNp such as AMPA and/ or GABA type A receptors may play contributing role in the phenotype of the NPTN individuals. These possibilities remain to be evaluated in the future. Our results in the Nptn +/− mice shown here, in combination with our previous reports demonstrating behavioral abnormalities and cognitive deficiencies in constitutive Nptn −/− mice and inducible Np-deficient mice ¹² , provide strong evidence for the necessity of Np during brain development. Indeed, induced Nptn elimination after normal development demonstrates that Np is acutely required for associative learning and memory in adult mutant mice, while it is not required for other behaviors tested in open field, O-maze, light/dark avoidance learning, light/dark avoidance memory, and startle responses¹². In contrast, constitutive Nptn −/− mice fail to perform in all these behavioral tests ¹² . Here, we show that Nptn +/− mice with incomplete Np levels display altered social interaction behavior. This confirms our previous results showing impaired social interaction in Nptn −/− , but not in inducible Np-deficient mice, and highlights a strict requirement for normal Np function for successful cognitive development. Furthermore, the archetypical autism-model mouse behavior of reduced preference for a novel vs. a familiar mouse in the social interaction test diplayed by Nptn +/− mice strongly supports the causality of the NPTN mutations for the autism diagnozed in all NPTN individuals. The facts that all PMCA are obligatory binding partners of Np and that de novo variants in ATP2B1 and ATP2B2 are identified as causing neurodevelopmental disorders including DD/ID and autism 19–25 further strengthens the association of NPTN - ATP2B1-2 and neurodevelopmental disorders. Neuroplastin peripheral malfunctions identified in homozygous Np-deficient mutant mice are not evident in the NPTN cohort yet, and, thus they need to be confirmed. Both Nptn - and Atp2b2 -deficient mice and ATP2B2 individuals are deaf, whereas the NPTN cohort described here do not show hearing deficits. This may be attributed to the young age at diagnosis where degeneration of hair cells due to loss of Np-PMCA may not have progressed yet dramatically. Supporting this idea, outer and inner hair cells in the Nptn −/− cochlea initially develop normally before undergoing degeneration that leads to hearing loss 41 . Alternatively, the residual expression level of Np in our individuals may be sufficient to support hearing and eventually may delay hearing loss to later ages. While neurons express very high levels of Np55 and Np65, most peripheral cell types synthesize only Np55. Altered inflammatory responses by immune cells 48 or impaired pancreatic beta cells function 49 in mice associated to Np55-PMCA deficiency have not been tested nor clinically manifested in the NPTN cohort. We have provided the first evidences in mice connecting Nptn expression with normal cognitive capabilities and PMCA expression 6,12 . Consistently Np-PMCA molecular interaction and its functions in intracellular Ca 2+ regulation have been confirmed and further detailed 31–33,35,40,41 . Although Desriveres et al. using large-scale gene association identified NPTN playing a potential role for intellectual deficits in humans 11 , and Dhindsa et al. using a machine learning approach based on gene constraint, expression, and other gene-level annotations, predicted NPTN as preferably causing an autosomal dominant neurodevelopmental condition (percentiles of 90.8 for DD, 94.0 for ASD, and 97.9 for developmental and epileptic encephalopathy) 43 , more direct evidence for a critical relevance of NPTN for brain development was missing. To our knowledge, this is the first report linking NPTN to human neurodevelopment. Our clinical, animal model, cellular, and molecular data indicate that the de novo variants in NPTN are pathogenic due to dominant negative (missense variants) or as loss-of-function (nonsense/ frameshift variants) effects on PMCA function. Both types of variants lead to a clinically non-specific neurodevelopmental disorder with varying severity, albeit based on a relatively small cohort. During the writing of this report, additional individuals with variants NPTN and neurodevelopmental disorders and autism have been identified. We have, therefore, adopted NPTN at the Human Disease Genes series (www.humandiseasegenes.nl) to promote future clinical research revealing a genotype–phenotype correlation. In summary, we establish that de novo variants in NPTN as causative for a neurodevelopmental disorder and autism. Based on several lines of evidence shown here and also in the literature, we proposed that impaired or insufficient function of the Np-PMCA complexes may contribute to the NPTN disorder. Methods Recruitment of affected individuals and animal experimentation This study was approved by the ethics committee of the University of Leipzig (402/16-ek). Written informed consent for molecular genetic testing and permission for publication of the data were obtained from all individuals and/or their legal representatives by the referring physicians according to the guidelines of the ethics committees and institutional review boards of the respective institutes. The compilation of the cohort was supported by international collaboration and online matchmaking via GeneMatcher 50 . Phenotypic and genotypic information was obtained from the referring collaborators using a standardized questionnaire. Heterozygous neuroplastin-deficient mice Nptn + / − were described 12 . Mice were kept with a 12 h light/dark cycle and food and water ad libitum. Animal husbandry, behavioral tests, and tissue collection were conducted in accordance with German (Tierschutzgesetz TierSchG) and European legislations (European Communities Council Directive (2010/63/EU) for the care of laboratory animals) and with the respective legal and ethical approval by the legal authorities (Landesverwaltungsamt Halle, Sachsen-Anhalt, Germany). Identification of NPTN variants Trio exome sequencing was performed for all affected individuals and their parents, except for individuals 3 and 5 (singleton exome only). All individuals were analyzed in the context of local diagnostic protocols. As there was no causative variant identified in a known rare disease gene, and thus all individuals lacked a definite diagnosis, research evaluation of the sequencing data was conducted to potentially identify causative variants in candidate genes. The gnomAD v4 dataset served as the control population 51 . There were no significant findings, other than the described variants in NPTN , which could explain the phenotype of the respective individuals. All variants described were aligned to hg38, mapped to the NPTN MANE Select transcript NM_012428.4 and classified according to the ACMG criteria 52 , 53 (Table S1). NPTN gene sequences used for comparison are Homo sapiens , NP_001154835; Macaca fascicularis , XP_005560051; Mus musculus , NP_001392991; Rattus norvegicus , NP_001400276; Equus caballus , XP_023509800; Danio rerio , NP_991268. In silico prediction In silico predictions of the missense variants were assessed using CADD-v1.6n 52 , REVEL 53 , MutPred2 56 , VEST4 57 , and BayesDel 58 using cutoffs for deleterious predictions from Pejaver et al. 59 (Supplementary table 4). Molecular dynamics and docking Molecular dynamics simulations were performed with Gromacs 2020.3 60 and the OPLS-AA force field 61 . The models were solvated in a cubic box with the SPCE water model, and NaCl counterions were added to neutralize the system 62 . System energy was minimized with the steepest descent algorithm up to a convergence criterion of 1000 kJ mol – 1 nm – 1 . To equilibrate temperature and pressure, an equilibration step of 200 ps in the NVT ensemble and an equilibration of 200 ps in the NPT ensemble were performed. After that, a 100ns production run in the NPT ensemble was performed to obtain geometrical information on all variants considered. The LINCS algorithm was used to restrain hydrogen bonds during equilibration and production runs 63 , while the PME method was used for treating long-range electrostatic interactions with a cutoff of 10Å 64 . Temperature was kept constant at 300 K with the V-rescale thermostats, while pressure was kept constant at 1 bar with the Parrinello-Rahman barostat 65 , 66 . The atomic coordinates of hNp65 wt were taken from our previous work 34 and variants were obtained by replacing the corresponding amino acid using Pymol software. For the analysis of hNp65 p.W135R , we used the last frame obtained from the molecular dynamics simulation to compare Np65 dimers as described elsewhere 35 . Crystallographic information described elsewhere 31 was used for the docking of hNp65 p.P342L to hPMCA, which was performed directly from the structure obtained with Pymol, because this amino acid change occurs within the intermembrane space. The binding interfaces with low-energy conformations were identified using the HADDOCK docking protein-protein web server 67 , 68 . Cell cultures Human embryonic kidney cells (HEK293T) were prepared in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) with 1% penicillin/streptomycin (Gibco), 1% L-glutamine (Gibco) and 10% fetal bovine serum (Gibco) at 37°C, 5% CO 2 6 . Primary hippocampal neurons were prepared from E16-18 rat or mouse embryos following our published protocols 6 , 32 , 44 . In short, neurons were dissociated with trypsin-EGTA for 15 min at 37°C. 50,000 neurons were plated on poly-D-lysine-coated glass coverslips for 12-well plates. After one hour, when the cells were well-attached to the slips, the plating medium DMEM 1% penicillin/streptomycin, 1% L-glutamine, and 10% horse serum was replaced with 1 ml of Neurobasal medium with 2% B27 supplement (Thermo Fisher Scientific Inc.), 1% penicillin/streptomycin, and 1% L-glutamine. At day 7 in vitro , 100 µl of fresh culture medium was added. Constructs, plasmids, siRNA, and transfections Constructs of human Np wild-type (hNpwt) and human PMCA2 (hPMCA2; Addgene, ID: 47584, Cambridge, MA) have been characterized 6 . TagRFPT-GCaMP5G plasmid has been described 32 . PGP-CMV-jGCaMP7f plasmid was obtained from Addgene (ID: 104483). We used control (scrambled) siRNA duplexes and siRNA targeting all premature Np RNA (Santa Cruz Biotechnology, Inc., ID: sc-149938). NPTN missense variants were generated by PCR amplification (see Supplementary Material). DNA-fragments were inserted into linearized vector FUGW (Addgene, ID: 14883) using cold-fusion cloning (System Biosciences, Palo Alto, CA). Transfections were performed with Lipofectamine 2000 following the company’s protocol (Invitrogen, Darmstadt, Germany). Hippocampal neurons were transfected with plasmids (1 µg/well) with or without control (scrambled) siRNA or Np siRNA (0.1-1 µg) at days 10–11 in vitro . 300000 HEK293T cells were transfected with plasmids (1.5 µg/well) 24 hours after seeding in 6-well plates and harvested 24 hours later. Western blot HEK293T cells were harvested with lysis buffer (1% Triton X-100 in 50 mM Tris/HCl, pH 8.0 protease inhibitors), homogenized using an ultrasonic homogenizer, and spun down at 12.000g for 20 min. The supernatant was collected and mixed uniformly with 2X SDS loading buffer and boiled at 95°C for 5 min. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and electro-transferred to a nitrocellulose membrane (Cytiva, AmershamTM ProtranTM 0.45µm NC). After blocking with 5% nonfat milk in TBS-T solution, TBS containing 0.1% Tween 20 for 1 h, the membrane was incubated with primary antibodies (see list in SMaterial) overnight at 4°C. Afterwards, the membrane was washed three times with TBS-T and incubated with secondary antibodies (see list in SMaterial) for 1 h at room temperature. After washing, the membrane was incubated with Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore) and chemiluminescence was detected using Intas Chemocam ECL Imaging system. Immunocytochemistry Cultured neurons were fixed and stained according to established protocols 6 , 32 , 44 . Briefly, cultures were fixed with 4% paraformaldehyde (PFA) containing 2% sucrose in 1X PBS for 8–10 min, washed carefully with 1X PBS and incubated with blocking solution (10% horse serum and 0.1% Triton X-100 in PBS) two times, 20 min each. Then they were incubated for one hour with primary antibodies (SMaterial) mixed with the blocking solution. After incubation, the coverslips were washed 3 times with 1X PBS for 10 min each. Secondary antibodies (SMaterial) diluted in the blocking solution were added to the coverslips and incubated for 1 h. The coverslips were then washed 3 times with 1X PBS and mounted on microscopy slides with Mowiol. Confocal microscopy and image quantification Images of neurons were acquired using an oil immersion objective HCXAPO 63X/1.40 NA coupled to an upright confocal microscope TCS SP5 (Leica), with a pinhole value of 0.75 AU, under sequential scanning mode (200 Hz), with 1-, 3- or 6-fold digital magnification for the whole cell, cell soma, or dendrites, respectively, and digitized in a 1054 X 512 format. Calcium imaging Ca 2+ imaging was performed in transiently transfected primary hippocampal neurons at 14–16 days in vitro following our published procedures 32 . Glass coverslips were carefully placed into an imaging magnetic chamber equipped with two silver wires for field electrical stimulation (Warner Instruments, Hamden, CT) and then filled with 1 ml of Tyrode’s solution. Stimulation was 10 or 20 biphasic pulses (1 ms duration each) generated with a S48 stimulator (Astro-Med, Inc., West Warwick, RI, USA). Evoked Ca 2+ transients were recorded using an inverted microscope Observer D1 (Zeiss, Jena, Germany) with a 63X/1.20 NA objective and an EMCCD camera Evolve 512 (Delar Photometrics, Tucson, AZ) under the control of VisiView software (Visitron Systems GmbH, Puchheim, Germany). Fluorescence intensity changes were quantified using Fiji/ImageJ software and parameters were extracted using pCLAMP 10 (Molecular Devices, San Francisco, CA). Drosophila melanogaster studies UAS-hNp55 transgenic constructs were established in the vector pJFRC12 (addgene clone 26222) and used for PhiC31-mediated germline transformation into the attP40 target site on the second chromosome (cytological position 25C6) of the recipient strain (y1 w67c23; P[CarryP]attP40) 53 – 55 . This procedure was performed by BestGene (Chino Hills, CA). Transgenic flies were identified based on orange eye color and established as stocks carrying the CyO GFP balancer chromosome. For further details on the transgenic lines see Supplementary material. Social interactions of Nptn -deficient mice Social interactions of Nptn +/− and Nptn +/+ adult male matched littermate mice were analyzed by the three-chamber test as described 69 . Briefly, during 3 test phases (10 min each), the mouse could explore all compartments. In phase 1, the mouse was alone for habituation. In phase 2, an unfamiliar C57BL/6NCrl wild-type mouse (male, novel 1) was placed in one of the wire cups. In phase 3, another unfamiliar C57BL/6NCrl mouse (same sex, novel 2) was added to the other cup. Time spent in each compartment, in contact with strangers, and transitions between compartments were recorded. Statistical analysis Statistical analysis of data was performed using Prism9 software (GraphPad), with outliers from raw data screened out with the Grubbs' test (Q = 1). For Western blots and Immunocytochemistry, statistical analysis of optical density measurement data used Student’s t-test. For Ca 2+ imaging, a Mann-Whitney U test was used for group comparisons for non-parametric data with sample sizes > 20, and a Wilcoxon matched-pairs test for the inner group comparison. For the analysis of behavior, Statview (SAS Institute Inc., Cary, NC) was used for Analysis of Variance (repeated measures ANOVA) and post hoc analysis (Scheffe's test). P -value smaller than 0.05 (P < 0.05) was considered significant. Declarations Competing interests HC and MJGS are employees of and may hold stock in GeneDx, LLC. The other authors report no competing interests. Funding This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 526008379 (R.H-M. and D.M.), the China Scholarship Council (Y.L.). Work by A.M. and H.S. was partially financed by the Center for Behavioral Brain Sciences (CBBS) grant to R.H-M. R.H-M. was supported by NIH Awards NS106244 and EY022730 (to Matthew T. Colonnese). R.O.T. thanks Powered@NLHPC: This research was partially supported by the supercomputing infrastructure of the NLHPC (ECM-02). W.A. was partially supported by HPC OCÉANO (FONDEQUIP Nº EQM170214). JSC receives support from NIH National Institute of Child Health and Human Development (P50 HD103538), NIH National Institute of Neurologic Disorders and Stroke (1U24NS131172-01), and U.S. Department of Health and Human Services, Health Resources and Services Administration (MCH T7317245). B.B.A.d.V was supported by the Dutch Organisation for Health Research and Development for ZON-MW grant no. 912-12-109. Multiple authors of this publication are members of the European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability ERN-ITHACA. Author Contribution Y.L., H.S., A.M., and R.H.M. produced and analyzed cellular and molecular data. U.T. and D.M., produced and analyzed animal model data. W.A. and R.O.T. produced and analyzed in silico data. J.S.C., N.R., J.L., G.V., F.L., E.K., B.A., H.C., M.J.G.S., B.B.A.deV., R.P., G.P., K.W., Y.B., E.M.P., K.P. evaluated patients and wrote clinical reports. All authors wrote text and agreed with final manuscript version publication. K.P., D.M., and R.H.M. prepared manuscript final version. Acknowledgements We are extremely grateful to the families that participated in this study. We thank Kathrin Pohlmann for her assistance during the cell culture preparations and Karla Sowa for helping with the mouse experiments. We also thank Dr. Marnie Phillips for her helpful comments on the manuscript. A.M. and H.S. worked at and R.H-M. headed the Laboratory of Neuronal and Synaptic Signals at the Leibniz Institute for Neurobiology. Data availability Identified variants in NPTN have been uploaded to ClinVar https://www.ncbi.nlm.nih.gov/clinvar/submitters/506086/ . References Khan I, Leventhal BL Developmental Delay. StatPearls [Internet] 2024. 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Clinical and genetic details of all affected individuals with causative variants in NPTN Ind. Age (Sex) Variant (NM_012428.4) DD / ID autism, behaviour, neurological findings, Dysmorphic features Further findings 1 17y (F) c.403T>A, p.(Trp135Arg), de novo moderate (IQ 40) autism, epileptic spasms (onset 7 months); focal impaired awareness seizures (onset 8 years); still occasional seizures upslanting palpebral fissures, slight ectropion, prominent forehead with frontal upsweep and high anterior hairline no 2 7y (M) c.1025C>T, p.(Pro342Leu), de novo severe (no speech) autism, repetitive behaviour, tics (DD dystonia) mild hypertelorism, downslanting palpebral fissures, full lips, Darwin tuberculum on both ears, fetal pads, sandal gaps recurrent diarrhea, macrocephaly 3 5y (F) c.14C>A, p.(Ser5*), de novo severe autism, poor social interactions, repetitive behaviour, hypotonia, ataxic gait downslanting corners of the mouth, anteverted nares, bilateral epicanthus joint hyperlaxity, chronic obstipation, bilateral vesicoureteral reflux 4 6y (M) c.218_219del, p.(Glu73Valfs*53), de novo, mosaic NA autism, period of regression no no 5 3y (F) c.284C>G, p.(Ser95*), heterozygous mild autism, poor social interactions, ritualistic behaviour, mild cerebral palsy in lower extremities no laryngomalacia 6 2y (M) c.342C>G, p.(Tyr114*), de novo moderate autism, poor social interactions, hyperactivity, possible speech regression prominent forehead early eruption of teeth, insomnia 7 9y (M) c.902del, p.(Asn301Thrfs*3), de novo severe tentative diagnosis of autism, pica disorder, automutilation, high pain threshold flat face, full eyebrows, upward slanting palpebral fissures, epicanthal folds, long fingers with mild clinodactyly 5th fingers, normal feet with sandal gaps no Abbreviations: DD, developmental delay; F, female; Ind., Individual; ID, intellectual disability; M, male; NA, not available; y, years. Further clinical details are provided in Supplementary table 1. Additional Declarations The authors declare potential competing interests as follows: HC and MJGS are employees of and may hold stock in GeneDx, LLC. The other authors report no competing interests Supplementary Files Liangetal2025SMATERIAL.docx SMaterial Liangetal2025TABLES3S4.xlsx Table S3 and S4 Cite Share Download PDF Status: Posted Version 1 posted 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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11:13:46","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":277742,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8079156/v1/b341a140f977c5c5a91c47be.html"},{"id":95629375,"identity":"62c25ab2-bc83-4b08-a042-9c5cb3b57f27","added_by":"auto","created_at":"2025-11-11 11:13:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":677023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eNPTN\u003c/em\u003e individuals. Photographs of three individuals carrying \u003cem\u003ede novo\u003c/em\u003e missense (individuals 1 and 2) or frameshift (individual 6) mutations. These individuals are characterized along other individuals in Table 1 and described in the main text.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8079156/v1/d3b2a3837a96bda4bfb250be.png"},{"id":95657147,"identity":"f85ec469-3dce-4e1a-a0c4-a5930a42874f","added_by":"auto","created_at":"2025-11-11 16:20:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":218820,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eNPTN\u003c/em\u003e monoallelic variants. (A) Diagram of the \u003cem\u003eNPTN\u003c/em\u003egene and localization of missense (black font) and frameshift and nonsense (blue font) variants identified in the individuals. The \u003cem\u003eNPTN\u003c/em\u003e gene contains eight exons with obligatory (black lines) and alternative (blue lines) splicing points and yields two hNp isoforms (hNp55 or hNp65). In exon 1, the gray band represents the 5’ untranslated region and the light green band the signal peptide sequence. The splicing of exon 1 to exon 3 results in the elimination of exon2 encoding the hNp65-specific Ig-like domain I and leads to the synthesis of hNp55. Exons 3-5 encode Ig-like domains II and III and exon 6 encodes a single transmembrane domain (light red band) common to all hNp isoforms. The small exon 7 can be removed from hNp55 and hNp65 mRNAs producing proteins with cytoplasmic tails lacking a four amino acidic DDEP insert. (B) Missense variants substitute conserved amino acids located in highly conserved amino acidic sequences across different species. (C) The localization of the missense variants is displayed in the protein structure of hNp65. Ig: immunoglobulin.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8079156/v1/bd0590fefd91918f18ad9989.png"},{"id":95629378,"identity":"732a083c-e324-4dee-8f1f-166123f4d4bd","added_by":"auto","created_at":"2025-11-11 11:13:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":436003,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of the proteins resulting from \u003cem\u003eNPTN\u003c/em\u003e missense variants from individual 1 (A, p.W135R) and individual 2 (B, p.P342L). (A) Views and ΔG binding of each of the \u003cem\u003etrans\u003c/em\u003e-homophilic interactions were extracted from our molecular docking simulations and based on previous kinetic and crystallography studies. hNp65\u003csup\u003ewt\u003c/sup\u003e (red) and hNp65\u003csup\u003ep.W135R\u003c/sup\u003e variant (green). The sequential replacement of hNp65\u003csup\u003ewt\u003c/sup\u003e by hNp65\u003csup\u003ep.W135R\u003c/sup\u003e results in severe conformational changes with energetically less favorable and more unstable dimerization. Arrows indicate distances between the key amino acids. (B) Interactions resulting from molecular docking simulations between the transmembrane domain 10 (T10) of hPMCA (blue) with the transmembrane domain of hNp\u003csup\u003ewt\u003c/sup\u003e (red) or with hNp\u003csup\u003ep.P342L\u003c/sup\u003e (green) are based on the crystallography of the Np-PMCA complex. PM: plasma membrane. Arrows indicate that the distance necessary for the interacting amino acids P342 in hNp\u003csup\u003ewt\u003c/sup\u003e and W1043 in hPMCA is drastically reduced for the pair L342 in hNp\u003csup\u003ep.P342L\u003c/sup\u003e and W1043 in hPMCA.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8079156/v1/df1ed3c6ce891ed5095c6e48.png"},{"id":95629379,"identity":"d8478ccf-4946-49be-a992-e7746ff5ee32","added_by":"auto","created_at":"2025-11-11 11:13:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":305675,"visible":true,"origin":"","legend":"\u003cp\u003eLethality due to dBsg-KD during early muscle development is rescued by hNp55 but not hNp55\u003csup\u003eP226L\u003c/sup\u003e. (A,B) Structural homology between hNp55 and dBsg. (C) Crossing scheme for the assessment of the rescue capacity of hNp55 and hNpP226L during muscle development. Flies homozygous for the early-onset muscle Gal4 driver mef-Gal4 were crossed to effector lines homozygous for either UAS-hNp55 or UAS-hNp55P226L, and heterozygous for UAS-bsgdsRNA over the balancer chromosome TM6B carrying the dominant markers Humeral (Hu, visible on adults) and Tubby (Tb, visible on L3 larvae and pupae). Control crosses lacked the UAS-hNp55 or UAS-hNpP226L effectors. (D) Pupal progeny from the crosses in (C) on the wall of a culture vial. Note that in crosses with dbsg-KD alone or with dbsg-KD plus hNp55\u003csup\u003eP226L\u003c/sup\u003e only round-shaped Tubby pupae are detectable, whereas crosses with dbsg-KD plus hNp55 give rise to both Tubby and normally shaped pupae (stars). This clear discrepancy is confirmed by counts of adult progeny with TM6B or without TM6B.\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8079156/v1/ad20262375a1d79d4cd41af8.png"},{"id":95629381,"identity":"dc022588-62a3-4e55-b15b-15542108bf4f","added_by":"auto","created_at":"2025-11-11 11:13:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":274451,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eNPTN\u003c/em\u003e missense variants from individual 1 and 2 on cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e transients. (A) Summarized Ca\u003csup\u003e2+\u003c/sup\u003e imaging sequence (1pic/400ms) of a representative GCaMP5G-expressing secondary dendrite (100mm section) depolarized with a field electrical stimulation of 10 pulses at 20 Hz (red asterisk at t\u003csub\u003e1\u003c/sub\u003e). From these image sequences, the parameters peak amplitude (P.A.), half-width (H.W.), and 90% decay time (D.T.) were calculated to describe the evoked cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e transients\u003csup\u003e\u003cstrong\u003e31\u003c/strong\u003e\u003c/sup\u003e. (B-D) Parameters were obtained from GCaMP5G-expressing dendrites (open circles) transfected or not with one of the wt isoforms or one of the \u003cem\u003eNPTN \u003c/em\u003evariants. For data presentation clarity mean±S.E.M. are displayed, but additionally S.D. is given for each condition. (B) For P.A.: control (no label) n=81; mean=1931±S.D.=1333; hNp65\u003csup\u003ewt\u003c/sup\u003e n=71; 1408±1006, hNp65\u003csup\u003ep.W135R\u003c/sup\u003e n=57; 1335±798. **p\u0026lt;0.01 unpaired t-test \u003cem\u003evs.\u003c/em\u003e control; ns: no significant difference; unpaired t-test \u003cem\u003evs.\u003c/em\u003e hNp65\u003csup\u003ewt\u003c/sup\u003e. H.W.: control 1349±265; hNp65\u003csup\u003ewt\u003c/sup\u003e 1108±139; hNp65\u003csup\u003ep.W135R\u003c/sup\u003e 1320±306. ****p\u0026lt;0.0001 or ns \u003cem\u003evs.\u003c/em\u003e control. ###p\u0026lt;0.001 \u003cem\u003evs.\u003c/em\u003e hNp65\u003csup\u003ewt\u003c/sup\u003e. D.T.: control mean=1724±520; hNp65\u003csup\u003ewt\u003c/sup\u003e 1359±448; hNp65\u003csup\u003ep.W135R\u003c/sup\u003e 1775±570. ****p\u0026lt;0.0001 or ns \u003cem\u003evs.\u003c/em\u003e control. ####p\u0026lt;0.0001 \u003cem\u003evs.\u003c/em\u003e hNp65\u003csup\u003ewt\u003c/sup\u003e. (C) For P.A.: control (no label) n=64, mean=2116±1237; hNp55\u003csup\u003ewt\u003c/sup\u003e n=60, 1121±754; hNp55\u003csup\u003ep.P226L\u003c/sup\u003e n=82, 909±510. ***p\u0026lt;0.001 unpaired t-test \u003cem\u003evs.\u003c/em\u003e control. ##p\u0026lt;0.01 or ns unpaired t-test \u003cem\u003evs.\u003c/em\u003e hNp55\u003csup\u003ewt\u003c/sup\u003e. \u0026amp;\u0026amp;p\u0026lt;0.01 one-way ANOVA. H.W.: control 1332±467; hNp55\u003csup\u003ewt\u003c/sup\u003e 976±199; hNp55\u003csup\u003ep.P226L\u003c/sup\u003e 1079±231. **p\u0026lt;0.01 or ***p\u0026lt;0.001 \u003cem\u003evs.\u003c/em\u003e control. #p\u0026lt;0.05 \u003cem\u003evs.\u003c/em\u003e hNp55\u003csup\u003ewt\u003c/sup\u003e. D.T.: control 2034±866; hNp55\u003csup\u003ewt\u003c/sup\u003e 1123±403; hNp55\u003csup\u003ep.P226L\u003c/sup\u003e 1422±600. **p\u0026lt;0.01 or ****p\u0026lt;0.0001 \u003cem\u003evs.\u003c/em\u003e control. ##p\u0026lt;0.01 \u003cem\u003evs.\u003c/em\u003e hNp55\u003csup\u003ewt\u003c/sup\u003e. \u0026amp;\u0026amp;p\u0026lt;0.01 one-way ANOVA. (D) For P.A.: control (no label) n=64, 2597±1598; hNp65\u003csup\u003ewt \u003c/sup\u003en=67, 1102±772; hNp65\u003csup\u003ep.P342L\u003c/sup\u003e n=72, 1564±1133. ***p\u0026lt;0.001 unpaired t-test \u003cem\u003evs.\u003c/em\u003e control. ##p\u0026lt;0.01 unpaired t-test \u003cem\u003evs.\u003c/em\u003e hN65\u003csup\u003ewt\u003c/sup\u003e. \u0026amp;\u0026amp;p\u0026lt;0.01 one-way ANOVA. H.W.: control 1574±496; hNp65\u003csup\u003ewt\u003c/sup\u003e 1100±462; hNp65\u003csup\u003ep.P342L\u003c/sup\u003e 1158±552. ***p\u0026lt;0.001 \u003cem\u003evs.\u003c/em\u003e control. D.T.: control 1831±728; hNp65\u003csup\u003ewt\u003c/sup\u003e 1137±643; hNp65\u003csup\u003ep.P342L\u003c/sup\u003e 1312±830. ***p\u0026lt;0.001 or ****p\u0026lt;0.0001 \u003cem\u003evs.\u003c/em\u003e control.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8079156/v1/a946e6c189ee49e00b29cbd6.png"},{"id":95629393,"identity":"cca011ec-4578-434e-833c-669ecf0b8d0b","added_by":"auto","created_at":"2025-11-11 11:13:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":458451,"visible":true,"origin":"","legend":"\u003cp\u003eNp-PMCA hypoexpression and social behavior in \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice. (A,B) Immunoreactivity of anti-Np55/65 (red) and anti-PMCA1-4 (green) antibodies to brain slices from \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice was evaluated using confocal microscopy. Nuclei were labelled with DAPI. Maximal projections and fluorescence signal quantification in CA1 hippocampal area. \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (n=10, green) and \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(n=12, blue)\u003csup\u003e \u003c/sup\u003emice were measured after habituation (phase 1) in the 3-chamber test. (C) The pictures show the experimental phases of the three-chamber test as initiated by the habituation of either \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice (Phase 1). (D) The graph shows the time spent by the tested mouse in the compartments with an unfamiliar mouse (novel 1) or with an empty cup (empty) (Phase 2). The novel mouse is preferred over an empty cup. D. \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice prefer the novel 2 mouse over the familiar mouse, whereas \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice displayed no significant preference for an unfamiliar mouse (novel 2) versus the familiar mouse (Phase 3). Data are presented as mean±S.E.M. (One-way ANOVA with Scheffe post-hoc; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001; ns: no significant difference).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8079156/v1/756bfb9e6d7c07df88bc1970.png"},{"id":95801745,"identity":"1fa7e863-25fc-403e-8ace-f3eb9fc810e1","added_by":"auto","created_at":"2025-11-13 08:26:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3997131,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8079156/v1/8758009a-de6e-4a3d-b28e-eec5083ab919.pdf"},{"id":95656548,"identity":"ed582e46-1286-4e65-9e43-92f85b9c1f58","added_by":"auto","created_at":"2025-11-11 16:18:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1338250,"visible":true,"origin":"","legend":"\u003cp\u003eSMaterial\u003c/p\u003e","description":"","filename":"Liangetal2025SMATERIAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-8079156/v1/080b082f0f08eddaa5f6d2a0.docx"},{"id":95656570,"identity":"63f9014d-9685-4c81-b950-edef931f6e3e","added_by":"auto","created_at":"2025-11-11 16:19:04","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":31305,"visible":true,"origin":"","legend":"\u003cp\u003eTable S3 and S4\u003c/p\u003e","description":"","filename":"Liangetal2025TABLES3S4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8079156/v1/e8a13243b91a6a91240a530d.xlsx"}],"financialInterests":"The authors declare potential competing interests as follows: HC and MJGS are employees of and may hold stock in GeneDx, LLC. The other authors report no competing interests","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDe novo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e variants in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNPTN \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecause a neurodevelopmental disorder with autism and neuroplastin-PMCA hypofunction\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDevelopmental delay (DD), often linked to intellectual disability (ID) and autism spectrum disorder (ASD)\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, has a prevalence of 1\u0026ndash;3% which may vary from large international cohorts to local populations\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Due to its heterogeneity, the etiology of DD is not always diagnosed, as it remains uncertain for 60% of the affected children\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Exome and genome sequencing are being used to identify genetic variants as the plausible cause of DD, revealing that 40\u0026ndash;60% of individuals with undiagnosed DD and 30\u0026ndash;39% of the ASD cases may carry pathogenic \u003cem\u003ede novo\u003c/em\u003e variants\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Discovering genetic variants associated with ID and ASD will facilitate early diagnosis and open avenues for their treatment.\u003c/p\u003e\u003cp\u003eThe gene \u003cem\u003eNPTN\u003c/em\u003e encodes two isoforms of the type I transmembrane glycoprotein neuroplastin expressed in the brain, human neuroplastin55 (hNp55) and neuron-specific human neuroplastin65 (hNp65)\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. High levels of neuroplastin mRNAs are detected at 19\u0026ndash;24 post-coital weeks (PCW) in neurons across fetal brain regions\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, with peak levels in the prefrontal cortex of 18-year-old individuals\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The isoforms hNp55 and hNp65 are enriched in human synapses\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. \u003cem\u003eNPTN\u003c/em\u003e is often deleted or duplicated in individuals with 15q24 microdeletion syndrome\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Also, a single nucleotide polymorphism in the \u003cem\u003eNPTN\u003c/em\u003e promoter is associated with thinner frontal and temporal lobes in the left hemisphere of the brain, correlating with verbal and non-verbal abilities in adolescents\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Studies in \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mutant mice provide direct evidence for the necessity of normal Np55/65 expression for multiple cognitive functions\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/b\u003e,\u003cb\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNeuroplastin55/65 are obligatory binding partners in protein complexes for more than 95% of the four plasma membrane Ca\u003csup\u003e2+\u003c/sup\u003e-ATPases (PMCA1-4)\u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e. The abundance and importance of these associations remain unexplored for human brain. PMCA1-4 are ATP-fed Ca\u003csup\u003e2+\u003c/sup\u003e-H\u003csup\u003e+\u003c/sup\u003e co-transporters that pump Ca\u003csup\u003e2+\u003c/sup\u003e towards the extracellular space to reinstate resting cytosolic levels and regulate intracellular Ca\u003csup\u003e2+\u003c/sup\u003e signaling while setting the perisynaptic alkaline pH necessary for the activation of ionotropic glutamate receptors of the NMDA type (iGluNRs)\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Mutations in the PMCA1-4 genes (\u003cem\u003eATP2B1-4\u003c/em\u003e) in individuals with DD, ASD, and other neurodevelopmental disorders decrease the expression and/or activity of these Ca\u003csup\u003e2+\u003c/sup\u003e pumps, leading to defective restoration of cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e levels\u003csup\u003e\u003cb\u003e19\u0026ndash;30\u003c/b\u003e\u003c/sup\u003e. Importantly, the expression, stabilization, and activity of PMCA1-4 are strongly dependent on Np55/65 binding\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and constitutive and inducible \u003cem\u003eNptn\u003c/em\u003e-deficient mice display massive PMCA1-4 loss associated with cognitive impairments and deficits in social and affective behaviours\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHere, we report mild to severe DD/ID and autism in seven individuals with \u003cem\u003ede novo\u003c/em\u003e missense, nonsense or frameshift in \u003cem\u003eNPTN\u003c/em\u003e. The missense variants cause structural abnormalities in hNp as evaluated \u003cem\u003ein silico\u003c/em\u003e through protein modelling and molecular dynamics. We confirmed that, in a human cell line, cultured primary rodent neurons, and \u003cem\u003ein vivo\u003c/em\u003e in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e, the missense variants are inefficiently expressed and exert dominant negative effects on PMCA levels resulting in failed cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e regulation. \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice express reduced amounts of both Np and PMCA. In a social behavior test, \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice display loss of preference for a novel mouse representing an endophenotype analog to social deficits that characterize autism. We conclude that these \u003cem\u003ede novo\u003c/em\u003e variants in \u003cem\u003eNPTN\u003c/em\u003e cause a neurodevelopmental disorder likely through direct Np-PMCA hypofunction and Ca\u003csup\u003e2+\u003c/sup\u003e-deregulation in central neurons.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eClinical and genetic analysis\u003c/h2\u003e\u003cp\u003eWe describe a cohort of seven individuals with heterozygous variants in \u003cem\u003eNPTN\u003c/em\u003e, six of which are of \u003cem\u003ede novo\u003c/em\u003e origin. An overview of the clinical evaluation of all individuals is presented in table 1. Additional descriptions are provided as case reports in the supplementary data and in supplementary table 3 and 4. All seven individuals presented with developmental delay (DD) and/or intellectual disability (ID) ranging from mild to severe. Four individuals displayed severe, two moderate, and one mild DD/ID, respectively. All individuals were diagnosed with autism spectrum disorder. Other behavioral findings included poor social interactions, high pain threshold, automutilation or repetitive behaviors. Individual 1 presented seizures starting at age seven months with epileptic spasms. That same individual developed focal impaired awareness seizures at eight years of age and was not seizure-free at age 17. Five of seven individuals received a cranial MRI. Of note, individual 3 presented with a vermal dysgenesis. Individual 4 was reported to have a period of significant regression of speech skills. Growth was found normal in all individuals. Subtle dysmorphic facial features were reported in five out of seven individuals and upslanting palpebral fissures and a prominent forehead were recurrently observed in individuals 1, 2, and 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGenetic results\u003c/h3\u003e\n\u003cp\u003eTrio exome sequencing revealed \u003cem\u003ede novo\u003c/em\u003e variants in \u003cem\u003eNPTN\u003c/em\u003e in individuals 1\u0026ndash;4, 6, and 7. A single exome test was carried out in individuals 3 and 5. The nonsense variant in \u003cem\u003eNPTN\u003c/em\u003e in individual 3 was segregated in the parents with Sanger sequencing. The biological parents of individual 5 were not available for testing. All variants are not recurrent and are absent from gnomAD (v4 dataset). Two distinct \u003cem\u003ede novo\u003c/em\u003e missense variants were identified in addition to five predicted loss-of-function (pLoF) variants. Multiple \u003cem\u003ein silico\u003c/em\u003e tools predict a deleterious effect of the two \u003cem\u003eNPTN\u003c/em\u003e missense variants (Supplementary table 2 and 4). Missense variants as well as pLoF variants are highly depleted from the gnomAD database. This indicates a selective constraint on both types of variants in a general population that lacks severe, early-onset phenotypes such as DD and ID (LOEUF\u0026thinsp;=\u0026thinsp;0.25; pLI\u0026thinsp;=\u0026thinsp;0.99; o/e for missense variants\u0026thinsp;=\u0026thinsp;0.52; z-score\u0026thinsp;=\u0026thinsp;2.68).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCharacterization of the\u003c/b\u003e \u003cb\u003eNPTN\u003c/b\u003e \u003cb\u003evariants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe located in the genomic sequence of the \u003cem\u003eNPTN\u003c/em\u003e gene the position of each of the variants identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). \u003cem\u003eNPTN\u003c/em\u003e consists of eight coding exons, one non-encoding 3'UTR exon, and exon 2 that can be spliced out by alternative splicing of the gene transcripts. From this process, two main glycoprotein isoforms are produced, hNp55 and hNp65. Additionally, they may display an alternative aminoacidic insert (DDEP) encoded by exon 7. The function of the DDEP insert is unknown but it is dispensable in Np for regulation of PMCA levels\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. hNp65 is 394 or 398 amino acids long, contains a signal peptide sequence, three extracellular Ig-like domains (Ig-like I-III) encoded by exons 1\u0026ndash;6, a single transmembrane domain (TM) encoded by exon 6, and a 34 or 38 amino acids long intracellular domain encoded by exons 6\u0026ndash;8. Np55 is 278 or 282 amino acids long and distinctively displays the Ig-like domains II-III. \u003cem\u003eNPTN\u003c/em\u003e missense variants affected conserved amino acids located within conserved amino acid sequences in different species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The missense variant c.403T\u0026thinsp;\u0026gt;\u0026thinsp;A (p.W135R) is located at the hNp65-specific exon 2, and other variants affect exons 1, 2, 4, and 6 which are common for both hNp isoforms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and C). Interestingly, the missense variant c.1025C\u0026thinsp;\u0026gt;\u0026thinsp;A, p.(Pro342Leu) (hNp65: p.P342L; hNp55: p.P226L) replaces a key transmembrane amino acid in Np that interacts directly with PMCA\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The nonsense variant c.14C\u0026thinsp;\u0026gt;\u0026thinsp;A, p.(Ser5*) stops the transcription of the signal peptide in the mutated allele. The nonsense variants c.284C\u0026thinsp;\u0026gt;\u0026thinsp;G, p.(Ser95*) and c.342C\u0026thinsp;\u0026gt;\u0026thinsp;G, p.(Tyr114*) stop mRNA translation during the very early synthesis of the hNp65-specific Ig-like domain, probably resulting in non-functional peptides likely to be degraded (Supplementary table 1). The frameshift in the variant c.902del, p.(Asn301Thrfs*3) in exon 6 results in a stop in the mutated allele. Analysis of mRNA stability indicates that truncated mRNA from the frameshift variant c.902del (p.N301Tfs*3) is degraded and thus, not producing truncated Np65 protein (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eComputational analysis of the\u003c/b\u003e \u003cb\u003eNPTN\u003c/b\u003e \u003cb\u003emissense variants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe performed a customized structural and thermodynamic analysis of \u003cem\u003eNPTN\u003c/em\u003e missense variants using molecular dynamics and protein-protein docking modeling and calculating binding energies (\u003cem\u003e∆G\u003c/em\u003e binding) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and SMaterial). Based on the resolved structure of the Np65-specific Ig-like domain I\u003csup\u003e\u003cb\u003e34\u003c/b\u003e\u003c/sup\u003e, our computational procedures were robust to recreate the hNp65\u003csup\u003ewt\u003c/sup\u003e-hNp65\u003csup\u003ewt\u003c/sup\u003e \u003cem\u003etrans-\u003c/em\u003ehomophilic binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, top-down view). We found that W135, N130, and I133 have an important participation in the thermodynamically spontaneous attraction between hNp65\u003csup\u003ewt\u003c/sup\u003e Ig-like domain I F-G loops (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, upper frame in lateral view). In the variant hNp65\u003csup\u003ep.W135\u003c/sup\u003e resulting from c.403T\u0026thinsp;\u0026gt;\u0026thinsp;A, the F-G loop structure is altered and the N130 and I133 are far off from reaching effective \u003cem\u003etrans-\u003c/em\u003einteraction positions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, middle frame in lateral view). An increased \u003cem\u003e∆G\u003c/em\u003e confirms the reduced binding efficiency of the hNp65\u003csup\u003ep.W135R\u003c/sup\u003e F-G loop to form the pair hNp65\u003csup\u003ewt\u003c/sup\u003e-hNp65\u003csup\u003ep.W135R\u003c/sup\u003e. The interaction of the pair hNp65\u003csup\u003ep.W135R\u003c/sup\u003e-hNp65\u003csup\u003ep.W135R\u003c/sup\u003e was worsened by the appearance of an abnormal P122-P122 interaction with an even higher \u003cem\u003e∆G\u003c/em\u003e for their binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, middle frame in lateral view).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn agreement with the reported crystallographic structure of the hNp-hPMCA complex\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, we localized P342 at the hNp transmembrane domain (TMD) facing W1043 at the hPMCA TM10 and found it participates in the stable interaction of the proteins within the cell plasma membrane (lateral view in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). When the hNp65\u003csup\u003ep.P342L\u003c/sup\u003e variant resulting from 1025C\u0026thinsp;\u0026gt;\u0026thinsp;A was projected onto the Np-PMCA interaction surface, we observed that the larger mutant residue L342 violates the effective interaction distance with hPMCA TM10 W1043, creating a thermodynamic constraint that would interfere with the intermolecular interaction (lower frame in top-down view in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Having studied other missense mutations affecting hNp Ig-like domain II structure\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, we also analyzed the missense variant hNp65\u003csup\u003ep.A210T\u003c/sup\u003e from c.1025C\u0026thinsp;\u0026gt;\u0026thinsp;A located at Ig-like domain II which was previously identified but not characterized\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;2). Briefly, the switch from A210 to T210 resulted in replacement of stabilizing intramolecular interactions by weak CH-CH interactions with highly variable interaction distances. Furthermore, T210 adds a mutant polar OH group to a normally apolar environment that causes extra steric congestion of the domain and stabilization constraints to the Ig-like domain II structure (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of\u003c/b\u003e \u003cb\u003eNPTN\u003c/b\u003e \u003cb\u003emissense variants and effects on PMCA levels\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBased on our previous studies\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, we tested the expression levels of hNp65\u003csup\u003ewt\u003c/sup\u003e, hNp55\u003csup\u003ewt\u003c/sup\u003e, hNp65\u003csup\u003ep.W135R\u003c/sup\u003e, hNp65\u003csup\u003ep.P342L\u003c/sup\u003e, and hNp55\u003csup\u003ep.P226L\u003c/sup\u003e in transfected HEK293T cells (Supplementary Fig.\u0026nbsp;3). Other missense mutations identified\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e were also analyzed (Supplementary Fig.\u0026nbsp;3) and are described in Supplementary material. As expected\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, hNp65\u003csup\u003ewt\u003c/sup\u003e and hNp55\u003csup\u003ewt\u003c/sup\u003e were efficiently detected by Western blot analysis (Supplementary Fig.\u0026nbsp;3A). Importantly, decreased expression was found for hNp65\u003csup\u003ep.W135R\u003c/sup\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 \u003cem\u003evs\u003c/em\u003e hNp65\u003csup\u003ewt\u003c/sup\u003e) and hNp65\u003csup\u003ep.P342L\u003c/sup\u003e (p\u0026thinsp;=\u0026thinsp;0.033 \u003cem\u003evs\u003c/em\u003e hNp65\u003csup\u003ewt\u003c/sup\u003e) whereas hNp65\u003csup\u003ep.A210T\u003c/sup\u003e expression was only slightly decreased compared to control (p\u0026thinsp;=\u0026thinsp;0.077 \u003cem\u003evs\u003c/em\u003e hNp65\u003csup\u003ewt\u003c/sup\u003e, SFig. 3A,B). Levels of hNp55\u003csup\u003ep.P226L\u003c/sup\u003e and hNp55\u003csup\u003ep.T78P\u003c/sup\u003e were similar to hNp55\u003csup\u003ewt\u003c/sup\u003e (SFig. 3A,B). Neuroplastin is an obligatory binding partner and post-transcriptionally promotes the expression of PMCA\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Therefore, we examined the effect of the missense variants on the capacity of Np to increase hPMCA2 levels\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. hPMCA2 levels in hNp65\u003csup\u003eWT\u003c/sup\u003e- and hNp55\u003csup\u003eWT\u003c/sup\u003e-expressing cells were higher than the ones in non-transfected control cells (Supplementary Fig.\u0026nbsp;3B,C). Compared to PMCA2-expressing single transfected cells and to PMCA2/hNp65\u003csup\u003eWT\u003c/sup\u003e- or PMCA2/hNp55\u003csup\u003eWT\u003c/sup\u003e-expressing double transfected cells, all missense variants promoted hPMCA2 but some of them inefficiently. Indeed, hPMCA2 was less in cells expressing hNp65\u003csup\u003ep.P342L\u003c/sup\u003e (p\u0026thinsp;=\u0026thinsp;0.004 \u003cem\u003evs\u003c/em\u003e hNp65\u003csup\u003ewt\u003c/sup\u003e), hNp65\u003csup\u003ep.W135R\u003c/sup\u003e (p\u0026thinsp;=\u0026thinsp;0.011 \u003cem\u003evs\u003c/em\u003e hNp65\u003csup\u003ewt\u003c/sup\u003e) or Np55\u003csup\u003ep.P226L\u003c/sup\u003e (p\u0026thinsp;=\u0026thinsp;0.057 \u003cem\u003evs\u003c/em\u003e hNp55\u003csup\u003eWT\u003c/sup\u003e) (Supplementary Fig.\u0026nbsp;3B,C). Compared to controls, the ability to increase hPMCA2 (hPMCA2/hNp ratio) was reduced for hNp65\u003csup\u003ep.P342L\u003c/sup\u003e and hNp55\u003csup\u003ep.P226L\u003c/sup\u003e but not for hNp55\u003csup\u003ep.T78P\u003c/sup\u003e, hNp65\u003csup\u003ep.A210T\u003c/sup\u003e or hNp65\u003csup\u003ep.W135R\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;3D) pointing to a specific necessity of this mutated proline residue in both hNP isoforms for normal levels of hPMCA2 expression in human cells.\u003c/p\u003e\u003cp\u003eThe impact of P226L on hNp55 functionality was evaluated in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e, a classical system to study neurodevelopment and synaptic mechanism\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast to the three mammalian paralogs \u003cem\u003eNPTN, BSG\u003c/em\u003e, and \u003cem\u003eEMB\u003c/em\u003e, only a single ortholog gene encoding dBsg exists in \u003cem\u003eDrosophila.\u003c/em\u003e DBsg shows an overall 25% amino acid sequence identity and a transmembrane domain homology of 69% (including adjacent intra- and extracellular amino acid residues) with hNp55 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Deletion of dBsg expression in muscle is known to be lethal at the late embryonic to early larval (L1) stage\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Thus, we tested whether the co-expression of hNp55\u003csup\u003ewt\u003c/sup\u003e or hNp55\u003csup\u003ep.P226L\u003c/sup\u003e rescues the lethal phenotype triggered by dBsg knockdown due to mef2-Gal4-induced expression of dsRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,D). As expected, dBsg knockdown caused a highly penetrant lethality around the L1 stage. The larval lethality was fully rescued by hNp55\u003csup\u003ewt\u003c/sup\u003e as virtually all progeny developed into viable adult flies, indicating tolerance to the differences between dBsg and hNp55\u003csup\u003ewt\u003c/sup\u003e. Strikingly, hNp55\u003csup\u003ep.P226L\u003c/sup\u003e displayed only minimal if any rescue capacity, as all progeny died before the L2 stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003eNPTN\u003c/b\u003e \u003cb\u003emissense variants on cytosolic Ca\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eregulation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNeuroplastin-PMCA complexes are crucial for cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e extrusion and shaping of Ca\u003csup\u003e2+\u003c/sup\u003e signaling in brain neurons\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. To evaluate the functional effect of \u003cem\u003eNPTN\u003c/em\u003e missense variants on Ca\u003csup\u003e2+\u003c/sup\u003e regulation, we investigate electrically-evoked cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e transients using Ca\u003csup\u003e2+\u003c/sup\u003e imaging\u003csup\u003e\u003cb\u003e31,34\u003c/b\u003e\u003c/sup\u003e in 14\u0026ndash;16 days-old GCaMP5G-expressing primary hippocampal neurons (referred to as GCaMP5G-neurons) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We quantified peak amplitude, half-width, and decay time of the evoked Ca\u003csup\u003e2+\u003c/sup\u003e transients, as these parameters reflect how Ca\u003csup\u003e2+\u003c/sup\u003e transients are shaped by the levels and activity of Np-PMCA complexes in synapses and dendrites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In line with the previous reports showing that Np\u003csup\u003ewt\u003c/sup\u003e over-expression adds on endogenous Np to promote PMCA levels and function (gain-of-function)\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, GCaMP5G-neurons co-expressing hNp65\u003csup\u003ewt\u003c/sup\u003e or hNp55\u003csup\u003ewt\u003c/sup\u003e displayed smaller evoked Ca\u003csup\u003e2+\u003c/sup\u003e transients with faster restoration of basal Ca\u003csup\u003e2+\u003c/sup\u003e levels compared to control GCaMP5G-neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D). In contrast, hNp65\u003csup\u003ep.W135R\u003c/sup\u003e, hNp65\u003csup\u003ep.P342L\u003c/sup\u003e and hNp55\u003csup\u003ep.P226L\u003c/sup\u003e caused abnormal or incomplete Ca\u003csup\u003e2+\u003c/sup\u003e transients. Indeed, whereas peak amplitudes were similarly reduced in GCaMP5G-neurons co-expressing either hNp65\u003csup\u003ep.W135R\u003c/sup\u003e or hNp55\u003csup\u003ep.P226L\u003c/sup\u003e compared with their wild-type expressing controls, both mutants displayed an increased half-width and decay time indicating longer Ca\u003csup\u003e2+\u003c/sup\u003e transients with slower recovery to baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). hNp65\u003csup\u003ep.P342L\u003c/sup\u003e accelerated the restoration of basal Ca\u003csup\u003e2+\u003c/sup\u003e levels but did not sufficiently reduce the Ca\u003csup\u003e2+\u003c/sup\u003e peak amplitude \u003cem\u003evs\u003c/em\u003e hNp65\u003csup\u003ewt\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Thus, these data indicate that these missense variants act dominant negatives rather than as a complete loss-of-function.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eNptn\u003c/b\u003e \u003cb\u003eheterozygosity affects PMCA brain levels and social behavior in mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA reduction of Np expression by ~\u0026thinsp;50% using a pan siRNA against Np mRNA resulted simultaneously in a partial reduction of PMCA1-4 in 14\u0026ndash;16 days-old hippocampal neurons (Supplementary Fig.\u0026nbsp;4A-C) and increased half-width and decay time of electrically-evoked Ca\u003csup\u003e2+\u003c/sup\u003e transients, as visualized using GCaMP7s-based Ca\u003csup\u003e2+\u003c/sup\u003e imaging (Supplementary Fig.\u0026nbsp;4D). As anticipated, immunohistochemical evaluation in the hippocampus of 14 days-old \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e pups demonstrated that \u003cem\u003eNptn\u003c/em\u003e heterozygosity, resulting in ~\u0026thinsp;50% reduction in Np levels, causes\u0026thinsp;~\u0026thinsp;45% loss of PMCA1-4 in developing neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,B). Therefore, heterozygous levels of Np\u003csup\u003ewt\u003c/sup\u003e are insufficient to maintain normal endogenous PMCA levels in the rodent hippocampus and cortex.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAutism is a common trait shared by the \u003cem\u003eNPTN\u003c/em\u003e individuals (Table\u0026nbsp;1). While general stereotypic behaviors were not observed in the heterozygous \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and specifically not detected in the marble burying test (not shown; Supplementary Table\u0026nbsp;5), these mice displayed reduced anxiety in the O-maze test (not shown; Supplementary Table\u0026nbsp;5). Further, we assessed autism-like behavioral features related to social interactions using the three-chamber social interaction assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Both \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice displayed a similar preference for a novel mouse compared to an empty cup (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). However, in contrast to \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice did not prefer a stranger over an already familiarized mouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Such altered social interactions observed in \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice have also been reported in other mouse models of genetically caused autism\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe describe seven individuals with an overlapping, albeit nonspecific, phenotype. All individuals displayed \u003cem\u003ede novo\u003c/em\u003e variants in \u003cem\u003eNPTN\u003c/em\u003e and were diagnosed with autism and neurodevelopmental disorders, pointing to the existence of pathological mechanisms related to alterations in \u003cem\u003eNPTN\u003c/em\u003e that affect neurodevelopment. The \u003cem\u003eNPTN\u003c/em\u003e variants disrupted critical functions of Np on the regulation of PMCA levels and cytosolic Ca²⁺ dynamics. In particular, the missense variants exhibit dominant negative features, whereas the nonsense or frameshift variants resulted in loss of function, affecting Np expression, PMCA levels, and Ca²⁺ signal regulation. Furthermore, monoallelic \u003cem\u003eNptn\u003c/em\u003e condition proved insufficient to support normal PMCA function and led to autism-like social behavior in transgenic mice. This report not only confirms prior assumptions regarding the relevance of Np in human neurodevelopment derived from machine learning approaches\u003csup\u003e\u003cstrong\u003e43\u003c/strong\u003e\u003c/sup\u003e and correlative genetic studies\u003csup\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/sup\u003e, but also provides a wider perspective to prospective pathological mechanism relevant to other affected individuals, such as those with \u003cem\u003ede novo\u003c/em\u003e mutations in PMCA1–4-encoding genes (ATP2B1–4)\u003csup\u003e\u003cstrong\u003e19–30\u003c/strong\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIt is confirmed that Np binds, stabilizes into protein complexes, and promotes the catalytic function of PMCA1-4. It is also shown that a significant decrease in Np levels, due to Np gene deletion or mutation or Np mRNA-interference, results in PMCA1-4 loss in neurons. Notably, in our assays, missense variants of the \u003cem\u003eNPTN\u003c/em\u003e individuals 1 and 2 resulted in decreased production of hNp levels carrying different dysfunctional residues that replaced key conserved amino acids. While the variant of individual 1 substitutes a critical tryptophan within the amino acid sequence at the extracellular and isoform-specific Ig-I of hNp65, individual 2‘s variant replaces a proline located at the common transmembrane segment of hNp55 and hNp65 which is embedded into the plasma membrane. The identified binding instability and conformational alterations in these human missense variants indicate that they are either not effectively produced or avidly degraded, as is the case for other Np variants affecting hearing in mice\u003csup\u003e\u003cstrong\u003e35\u003c/strong\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur results in HEK293T cells and \u003cem\u003eDrosophila melanogaster\u003c/em\u003e further support that the \u003cem\u003eNPTN\u003c/em\u003e variant of patient 2, yielding hNp55\u003csup\u003ep.P226\u003c/sup\u003e and hNp65\u003csup\u003ep.P342L\u003c/sup\u003e, results in impaired biosynthesis/ degradation and is insufficient to support normal PMCA function. This impairment seems to be most detrimental during early development of the flies and is compatible with a dominant negative effect on PMCA function. Interestingly, although human variants from individuals 1 and 2 were expressed at lower levels compared to wt Np isoforms, the \u003cem\u003eNPTN\u003c/em\u003e Ig-I variant of patient 1 hNp65\u003csup\u003ep.W135R\u003c/sup\u003e was able to promote PMCA, but the \u003cem\u003eNPTN\u003c/em\u003e transmembrane variant of patient 2 was inefficient in this property. Stop codon-producing nonsense or frameshift variants in individuals 3-to-7 do not produce truncated proteins and, thus, their effects may result from a diminished haploid production of hNp55/65 insufficient to maintain normal PMCA levels. Indeed, we demonstrated that either titrated Np RNAi-targeted knockdown in isolated cultured neurons or monoallelic condition in \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/−\u003c/em\u003e\u003c/sup\u003e mice yielded appr. 50% expression levels of Np with a significant reduction in PMCA levels. Ca\u003csup\u003e2+\u003c/sup\u003e imaging confirmed that a progressive decrease on Np-PMCA content is mirrored by a progressive increase in the Ca\u003csup\u003e+ 2\u003c/sup\u003e signal parameters peak amplitude, half width, and decay time. Therefore, we conclude that missense variants display loss-of-function with dominant negative features and that nonsense or frameshift variants result in hypofunctional hNp levels with diminished hNp-PMCA function.\u003c/p\u003e\n\u003cp\u003eInsufficient PMCA function may not be the only mechanism underlying the phenotypes of the \u003cem\u003eNPTN\u003c/em\u003e individuals in this cohort. The missense variant in individual 1 is located at an important extracellular module of hNp65 that mediates homophilic Np65-Np65 \u003cem\u003etrans\u003c/em\u003e-interactions\u003csup\u003e\u003cstrong\u003e33\u003c/strong\u003e\u003c/sup\u003e. In fact, competition of this motif with high-affinity peptides or antibodies destabilizes glutamatergic synaptic contacts and impairs synaptic transmission\u003csup\u003e\u003cstrong\u003e44\u003c/strong\u003e\u003c/sup\u003e. As our thermodynamic calculations indicate that this Np65-specific variant displays weakened binding, it is possible to hypothesize the occurrence of an insufficient structural stabilization of excitatory synapses during synaptogenesis in individual 1. Additionally, as reduced synapse formation triggered by Np55 and Np65 occurs in \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e neurons due to failed TRAF6 signaling\u003csup\u003e\u003cstrong\u003e33,44\u003c/strong\u003e\u003c/sup\u003e, it is plausible that production of hNp variants in individuals 1 and 2, or haploid wild-type hNp production in individuals 3-to-7, would not trigger Np-TRAF6-dependent synapse formation sufficiently. On the other hand, insufficient hNp production could also alter the excitatory-inhibitory balance affecting development and maturation of neuronal circuits leading to epilepsy or ataxia in the \u003cem\u003eNPTN\u003c/em\u003e individuals 1-to-3. This idea is based on the requirement of Np for the correct synaptic localization and function of AMPA receptors\u003csup\u003e\u003cstrong\u003e45\u003c/strong\u003e\u003c/sup\u003e and α1/2 subunit-containing GABA type A receptors\u003csup\u003e\u003cstrong\u003e44,46\u003c/strong\u003e\u003c/sup\u003e. Interestingly, recent studies suggest that reduced synaptic transmission involving binding of Np to GABA type A receptors sensitizes rodents to pentylenetetrazole-induced epilepsy\u003csup\u003e\u003cstrong\u003e40,47\u003c/strong\u003e\u003c/sup\u003e. Therefore, hypofunctions and/or malfunctions of other binding partners of hNp such as AMPA and/ or GABA type A receptors may play contributing role in the phenotype of the \u003cem\u003eNPTN\u003c/em\u003e individuals. These possibilities remain to be evaluated in the future.\u003c/p\u003e\n\u003cp\u003eOur results in the \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/−\u003c/em\u003e\u003c/sup\u003e mice shown here, in combination with our previous reports demonstrating behavioral abnormalities and cognitive deficiencies in constitutive \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e mice and inducible Np-deficient mice\u003cstrong\u003e¹²\u003c/strong\u003e, provide strong evidence for the necessity of Np during brain development. Indeed, induced \u003cem\u003eNptn\u003c/em\u003e elimination after normal development demonstrates that Np is acutely required for associative learning and memory in adult mutant mice, while it is not required for other behaviors tested in open field, O-maze, light/dark avoidance learning, light/dark avoidance memory, and startle responses¹². In contrast, constitutive \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e mice fail to perform in all these behavioral tests\u003cstrong\u003e¹²\u003c/strong\u003e. Here, we show that \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/−\u003c/em\u003e\u003c/sup\u003e mice with incomplete Np levels display altered social interaction behavior. This confirms our previous results showing impaired social interaction in \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e, but not in inducible Np-deficient mice, and highlights a strict requirement for normal Np function for successful cognitive development. Furthermore, the archetypical autism-model mouse behavior of reduced preference for a novel \u003cem\u003evs.\u003c/em\u003e a familiar mouse in the social interaction test diplayed by \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/−\u003c/em\u003e\u003c/sup\u003e mice strongly supports the causality of the \u003cem\u003eNPTN\u003c/em\u003e mutations for the autism diagnozed in all \u003cem\u003eNPTN\u003c/em\u003e individuals.\u003c/p\u003e\n\u003cp\u003eThe facts that all PMCA are obligatory binding partners of Np and that \u003cem\u003ede novo\u003c/em\u003e variants in \u003cem\u003eATP2B1\u003c/em\u003e and \u003cem\u003eATP2B2\u003c/em\u003e are identified as causing neurodevelopmental disorders including DD/ID and autism\u003csup\u003e\u003cstrong\u003e19–25\u003c/strong\u003e\u003c/sup\u003e further strengthens the association of \u003cem\u003eNPTN\u003c/em\u003e-\u003cem\u003eATP2B1-2\u003c/em\u003e and neurodevelopmental disorders. Neuroplastin peripheral malfunctions identified in homozygous Np-deficient mutant mice are not evident in the \u003cem\u003eNPTN\u003c/em\u003e cohort yet, and, thus they need to be confirmed. Both \u003cem\u003eNptn\u003c/em\u003e- and \u003cem\u003eAtp2b2\u003c/em\u003e-deficient mice and \u003cem\u003eATP2B2\u003c/em\u003e individuals are deaf, whereas the \u003cem\u003eNPTN\u003c/em\u003e cohort described here do not show hearing deficits. This may be attributed to the young age at diagnosis where degeneration of hair cells due to loss of Np-PMCA may not have progressed yet dramatically. Supporting this idea, outer and inner hair cells in the \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e cochlea initially develop normally before undergoing degeneration that leads to hearing loss\u003csup\u003e\u003cstrong\u003e41\u003c/strong\u003e\u003c/sup\u003e. Alternatively, the residual expression level of Np in our individuals may be sufficient to support hearing and eventually may delay hearing loss to later ages. While neurons express very high levels of Np55 and Np65, most peripheral cell types synthesize only Np55. Altered inflammatory responses by immune cells\u003csup\u003e\u003cstrong\u003e48\u003c/strong\u003e\u003c/sup\u003e or impaired pancreatic beta cells function\u003csup\u003e\u003cstrong\u003e49\u003c/strong\u003e\u003c/sup\u003e in mice associated to Np55-PMCA deficiency have not been tested nor clinically manifested in the \u003cem\u003eNPTN\u003c/em\u003e cohort.\u003c/p\u003e\n\u003cp\u003eWe have provided the first evidences in mice connecting \u003cem\u003eNptn\u003c/em\u003e expression with normal cognitive capabilities and PMCA expression\u003csup\u003e\u003cstrong\u003e6,12\u003c/strong\u003e\u003c/sup\u003e. Consistently Np-PMCA molecular interaction and its functions in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e regulation have been confirmed and further detailed\u003csup\u003e\u003cstrong\u003e31–33,35,40,41\u003c/strong\u003e\u003c/sup\u003e. Although Desriveres \u003cem\u003eet al.\u003c/em\u003e using large-scale gene association identified \u003cem\u003eNPTN\u003c/em\u003e playing a potential role for intellectual deficits in humans\u003csup\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/sup\u003e, and Dhindsa \u003cem\u003eet al.\u003c/em\u003e using a machine learning approach based on gene constraint, expression, and other gene-level annotations, predicted \u003cem\u003eNPTN\u003c/em\u003e as preferably causing an autosomal dominant neurodevelopmental condition (percentiles of 90.8 for DD, 94.0 for ASD, and 97.9 for developmental and epileptic encephalopathy)\u003csup\u003e\u003cstrong\u003e43\u003c/strong\u003e\u003c/sup\u003e, more direct evidence for a critical relevance of \u003cem\u003eNPTN\u003c/em\u003e for brain development was missing. To our knowledge, this is the first report linking \u003cem\u003eNPTN\u003c/em\u003e to human neurodevelopment. Our clinical, animal model, cellular, and molecular data indicate that the \u003cem\u003ede novo\u003c/em\u003e variants in \u003cem\u003eNPTN\u003c/em\u003e are pathogenic due to dominant negative (missense variants) or as loss-of-function (nonsense/ frameshift variants) effects on PMCA function. Both types of variants lead to a clinically non-specific neurodevelopmental disorder with varying severity, albeit based on a relatively small cohort. During the writing of this report, additional individuals with variants \u003cem\u003eNPTN\u003c/em\u003e and neurodevelopmental disorders and autism have been identified. We have, therefore, adopted \u003cem\u003eNPTN\u003c/em\u003e at the Human Disease Genes series (www.humandiseasegenes.nl) to promote future clinical research revealing a genotype–phenotype correlation.\u003c/p\u003e\n\u003cp\u003eIn summary, we establish that \u003cem\u003ede novo\u003c/em\u003e variants in \u003cem\u003eNPTN\u003c/em\u003e as causative for a neurodevelopmental disorder and autism. Based on several lines of evidence shown here and also in the literature, we proposed that impaired or insufficient function of the Np-PMCA complexes may contribute to the \u003cem\u003eNPTN\u003c/em\u003e disorder.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eRecruitment of affected individuals and animal experimentation\u003c/h2\u003e\u003cp\u003e This study was approved by the ethics committee of the University of Leipzig (402/16-ek). Written informed consent for molecular genetic testing and permission for publication of the data were obtained from all individuals and/or their legal representatives by the referring physicians according to the guidelines of the ethics committees and institutional review boards of the respective institutes. The compilation of the cohort was supported by international collaboration and online matchmaking \u003cem\u003evia\u003c/em\u003e GeneMatcher\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Phenotypic and genotypic information was obtained from the referring collaborators using a standardized questionnaire. Heterozygous neuroplastin-deficient mice \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e+\u003cem\u003e/\u003c/em\u003e\u0026minus;\u003c/sup\u003e were described\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Mice were kept with a 12 h light/dark cycle and food and water ad libitum. Animal husbandry, behavioral tests, and tissue collection were conducted in accordance with German (Tierschutzgesetz TierSchG) and European legislations (European Communities Council Directive (2010/63/EU) for the care of laboratory animals) and with the respective legal and ethical approval by the legal authorities (Landesverwaltungsamt Halle, Sachsen-Anhalt, Germany).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of\u003c/b\u003e \u003cb\u003eNPTN\u003c/b\u003e \u003cb\u003evariants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTrio exome sequencing was performed for all affected individuals and their parents, except for individuals 3 and 5 (singleton exome only). All individuals were analyzed in the context of local diagnostic protocols. As there was no causative variant identified in a known rare disease gene, and thus all individuals lacked a definite diagnosis, research evaluation of the sequencing data was conducted to potentially identify causative variants in candidate genes. The gnomAD v4 dataset served as the control population\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. There were no significant findings, other than the described variants in \u003cem\u003eNPTN\u003c/em\u003e, which could explain the phenotype of the respective individuals. All variants described were aligned to hg38, mapped to the \u003cem\u003eNPTN\u003c/em\u003e MANE Select transcript NM_012428.4 and classified according to the ACMG criteria\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e (Table S1). \u003cem\u003eNPTN\u003c/em\u003e gene sequences used for comparison are \u003cem\u003eHomo sapiens\u003c/em\u003e, NP_001154835; \u003cem\u003eMacaca fascicularis\u003c/em\u003e, XP_005560051; \u003cem\u003eMus musculus\u003c/em\u003e, NP_001392991; \u003cem\u003eRattus norvegicus\u003c/em\u003e, NP_001400276; \u003cem\u003eEquus caballus\u003c/em\u003e, XP_023509800; \u003cem\u003eDanio rerio\u003c/em\u003e, NP_991268.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn silico\u003c/b\u003e \u003cb\u003eprediction\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn silico\u003c/em\u003e predictions of the missense variants were assessed using CADD-v1.6n\u003csup\u003e\u003cb\u003e52\u003c/b\u003e\u003c/sup\u003e, REVEL\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, MutPred2\u003csup\u003e\u003cb\u003e56\u003c/b\u003e\u003c/sup\u003e, VEST4\u003csup\u003e\u003cb\u003e57\u003c/b\u003e\u003c/sup\u003e, and BayesDel\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e using cutoffs for deleterious predictions from Pejaver et al.\u003csup\u003e\u003cb\u003e59\u003c/b\u003e\u003c/sup\u003e (Supplementary table 4).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMolecular dynamics and docking\u003c/h2\u003e\u003cp\u003eMolecular dynamics simulations were performed with Gromacs 2020.3\u003csup\u003e\u003cb\u003e60\u003c/b\u003e\u003c/sup\u003e and the OPLS-AA force field\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The models were solvated in a cubic box with the SPCE water model, and NaCl counterions were added to neutralize the system\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. System energy was minimized with the steepest descent algorithm up to a convergence criterion of 1000 kJ mol\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003enm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. To equilibrate temperature and pressure, an equilibration step of 200 ps in the NVT ensemble and an equilibration of 200 ps in the NPT ensemble were performed. After that, a 100ns production run in the NPT ensemble was performed to obtain geometrical information on all variants considered. The LINCS algorithm was used to restrain hydrogen bonds during equilibration and production runs\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, while the PME method was used for treating long-range electrostatic interactions with a cutoff of 10\u0026Aring;\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Temperature was kept constant at 300 K with the V-rescale thermostats, while pressure was kept constant at 1 bar with the Parrinello-Rahman barostat\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The atomic coordinates of hNp65\u003csup\u003ewt\u003c/sup\u003e were taken from our previous work\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and variants were obtained by replacing the corresponding amino acid using Pymol software. For the analysis of hNp65\u003csup\u003ep.W135R\u003c/sup\u003e, we used the last frame obtained from the molecular dynamics simulation to compare Np65 dimers as described elsewhere\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Crystallographic information described elsewhere\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e was used for the docking of hNp65\u003csup\u003ep.P342L\u003c/sup\u003e to hPMCA, which was performed directly from the structure obtained with Pymol, because this amino acid change occurs within the intermembrane space. The binding interfaces with low-energy conformations were identified using the HADDOCK docking protein-protein web server\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell cultures\u003c/h3\u003e\n\u003cp\u003eHuman embryonic kidney cells (HEK293T) were prepared in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, Gibco) with 1% penicillin/streptomycin (Gibco), 1% L-glutamine (Gibco) and 10% fetal bovine serum (Gibco) at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u003cb\u003e6\u003c/b\u003e\u003c/sup\u003e. Primary hippocampal neurons were prepared from E16-18 rat or mouse embryos following our published protocols\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. In short, neurons were dissociated with trypsin-EGTA for 15 min at 37\u0026deg;C. 50,000 neurons were plated on poly-D-lysine-coated glass coverslips for 12-well plates. After one hour, when the cells were well-attached to the slips, the plating medium DMEM 1% penicillin/streptomycin, 1% L-glutamine, and 10% horse serum was replaced with 1 ml of Neurobasal medium with 2% B27 supplement (Thermo Fisher Scientific Inc.), 1% penicillin/streptomycin, and 1% L-glutamine. At day 7 \u003cem\u003ein vitro\u003c/em\u003e, 100 \u0026micro;l of fresh culture medium was added.\u003c/p\u003e\n\u003ch3\u003eConstructs, plasmids, siRNA, and transfections\u003c/h3\u003e\n\u003cp\u003eConstructs of human Np wild-type (hNpwt) and human PMCA2 (hPMCA2; Addgene, ID: 47584, Cambridge, MA) have been characterized\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. TagRFPT-GCaMP5G plasmid has been described\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. PGP-CMV-jGCaMP7f plasmid was obtained from Addgene (ID: 104483). We used control (scrambled) siRNA duplexes and siRNA targeting all premature Np RNA (Santa Cruz Biotechnology, Inc., ID: sc-149938). \u003cem\u003eNPTN\u003c/em\u003e missense variants were generated by PCR amplification (see Supplementary Material). DNA-fragments were inserted into linearized vector FUGW (Addgene, ID: 14883) using cold-fusion cloning (System Biosciences, Palo Alto, CA). Transfections were performed with Lipofectamine 2000 following the company\u0026rsquo;s protocol (Invitrogen, Darmstadt, Germany). Hippocampal neurons were transfected with plasmids (1 \u0026micro;g/well) with or without control (scrambled) siRNA or Np siRNA (0.1-1 \u0026micro;g) at days 10\u0026ndash;11 \u003cem\u003ein vitro\u003c/em\u003e. 300000 HEK293T cells were transfected with plasmids (1.5 \u0026micro;g/well) 24 hours after seeding in 6-well plates and harvested 24 hours later.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot\u003c/h2\u003e\u003cp\u003eHEK293T cells were harvested with lysis buffer (1% Triton X-100 in 50 mM Tris/HCl, pH 8.0 protease inhibitors), homogenized using an ultrasonic homogenizer, and spun down at 12.000g for 20 min. The supernatant was collected and mixed uniformly with 2X SDS loading buffer and boiled at 95\u0026deg;C for 5 min. Proteins were separated by sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and electro-transferred to a nitrocellulose membrane (Cytiva, AmershamTM ProtranTM 0.45\u0026micro;m NC). After blocking with 5% nonfat milk in TBS-T solution, TBS containing 0.1% Tween 20 for 1 h, the membrane was incubated with primary antibodies (see list in SMaterial) overnight at 4\u0026deg;C. Afterwards, the membrane was washed three times with TBS-T and incubated with secondary antibodies (see list in SMaterial) for 1 h at room temperature. After washing, the membrane was incubated with Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore) and chemiluminescence was detected using Intas Chemocam ECL Imaging system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eImmunocytochemistry\u003c/h2\u003e\u003cp\u003eCultured neurons were fixed and stained according to established protocols\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Briefly, cultures were fixed with 4% paraformaldehyde (PFA) containing 2% sucrose in 1X PBS for 8\u0026ndash;10 min, washed carefully with 1X PBS and incubated with blocking solution (10% horse serum and 0.1% Triton X-100 in PBS) two times, 20 min each. Then they were incubated for one hour with primary antibodies (SMaterial) mixed with the blocking solution. After incubation, the coverslips were washed 3 times with 1X PBS for 10 min each. Secondary antibodies (SMaterial) diluted in the blocking solution were added to the coverslips and incubated for 1 h. The coverslips were then washed 3 times with 1X PBS and mounted on microscopy slides with Mowiol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eConfocal microscopy and image quantification\u003c/h2\u003e\u003cp\u003eImages of neurons were acquired using an oil immersion objective HCXAPO 63X/1.40 NA coupled to an upright confocal microscope TCS SP5 (Leica), with a pinhole value of 0.75 AU, under sequential scanning mode (200 Hz), with 1-, 3- or 6-fold digital magnification for the whole cell, cell soma, or dendrites, respectively, and digitized in a 1054 X 512 format.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCalcium imaging\u003c/h2\u003e\u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e imaging was performed in transiently transfected primary hippocampal neurons at 14\u0026ndash;16 days \u003cem\u003ein vitro\u003c/em\u003e following our published procedures\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Glass coverslips were carefully placed into an imaging magnetic chamber equipped with two silver wires for field electrical stimulation (Warner Instruments, Hamden, CT) and then filled with 1 ml of Tyrode\u0026rsquo;s solution. Stimulation was 10 or 20 biphasic pulses (1 ms duration each) generated with a S48 stimulator (Astro-Med, Inc., West Warwick, RI, USA). Evoked Ca\u003csup\u003e2+\u003c/sup\u003e transients were recorded using an inverted microscope Observer D1 (Zeiss, Jena, Germany) with a 63X/1.20 NA objective and an EMCCD camera Evolve 512 (Delar Photometrics, Tucson, AZ) under the control of VisiView software (Visitron Systems GmbH, Puchheim, Germany). Fluorescence intensity changes were quantified using Fiji/ImageJ software and parameters were extracted using pCLAMP 10 (Molecular Devices, San Francisco, CA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDrosophila melanogaster\u003c/b\u003e \u003cb\u003estudies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUAS-hNp55 transgenic constructs were established in the vector pJFRC12 (addgene clone 26222) and used for PhiC31-mediated germline transformation into the attP40 target site on the second chromosome (cytological position 25C6) of the recipient strain (y1 w67c23; P[CarryP]attP40)\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. This procedure was performed by BestGene (Chino Hills, CA). Transgenic flies were identified based on orange eye color and established as stocks carrying the CyO\u003csup\u003eGFP\u003c/sup\u003e balancer chromosome. For further details on the transgenic lines see Supplementary material.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSocial interactions of Nptn\u003c/b\u003e\u003cb\u003e-deficient mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSocial interactions of \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e adult male matched littermate mice were analyzed by the three-chamber test as described\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Briefly, during 3 test phases (10 min each), the mouse could explore all compartments. In phase 1, the mouse was alone for habituation. In phase 2, an unfamiliar C57BL/6NCrl wild-type mouse (male, novel 1) was placed in one of the wire cups. In phase 3, another unfamiliar C57BL/6NCrl mouse (same sex, novel 2) was added to the other cup. Time spent in each compartment, in contact with strangers, and transitions between compartments were recorded.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis of data was performed using Prism9 software (GraphPad), with outliers from raw data screened out with the Grubbs' test (Q\u0026thinsp;=\u0026thinsp;1). For Western blots and Immunocytochemistry, statistical analysis of optical density measurement data used Student\u0026rsquo;s t-test. For Ca\u003csup\u003e2+\u003c/sup\u003e imaging, a Mann-Whitney U test was used for group comparisons for non-parametric data with sample sizes\u0026thinsp;\u0026gt;\u0026thinsp;20, and a Wilcoxon matched-pairs test for the inner group comparison. For the analysis of behavior, Statview (SAS Institute Inc., Cary, NC) was used for Analysis of Variance (repeated measures ANOVA) and post hoc analysis (Scheffe's test). \u003cem\u003eP\u003c/em\u003e-value smaller than 0.05 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was considered significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eHC and MJGS are employees of and may hold stock in GeneDx, LLC. The other authors report no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) \u0026ndash; 526008379 (R.H-M. and D.M.), the China Scholarship Council (Y.L.). Work by A.M. and H.S. was partially financed by the Center for Behavioral Brain Sciences (CBBS) grant to R.H-M. R.H-M. was supported by NIH Awards NS106244 and EY022730 (to Matthew T. Colonnese). R.O.T. thanks Powered@NLHPC: This research was partially supported by the supercomputing infrastructure of the NLHPC (ECM-02). W.A. was partially supported by HPC OC\u0026Eacute;ANO (FONDEQUIP N\u0026ordm; EQM170214). JSC receives support from NIH National Institute of Child Health and Human Development (P50 HD103538), NIH National Institute of Neurologic Disorders and Stroke (1U24NS131172-01), and U.S. Department of Health and Human Services, Health Resources and Services Administration (MCH T7317245). B.B.A.d.V was supported by the Dutch Organisation for Health Research and Development for ZON-MW grant no. 912-12-109. Multiple authors of this publication are members of the European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability ERN-ITHACA.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.L., H.S., A.M., and R.H.M. produced and analyzed cellular and molecular data. U.T. and D.M., produced and analyzed animal model data. W.A. and R.O.T. produced and analyzed in silico data. J.S.C., N.R., J.L., G.V., F.L., E.K., B.A., H.C., M.J.G.S., B.B.A.deV., R.P., G.P., K.W., Y.B., E.M.P., K.P. evaluated patients and wrote clinical reports. All authors wrote text and agreed with final manuscript version publication. K.P., D.M., and R.H.M. prepared manuscript final version.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eWe are extremely grateful to the families that participated in this study. We thank Kathrin Pohlmann for her assistance during the cell culture preparations and Karla Sowa for helping with the mouse experiments. We also thank Dr. Marnie Phillips for her helpful comments on the manuscript. A.M. and H.S. worked at and R.H-M. headed the Laboratory of Neuronal and Synaptic Signals at the Leibniz Institute for Neurobiology.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eIdentified variants in \u003cem\u003eNPTN\u003c/em\u003e have been uploaded to ClinVar \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/clinvar/submitters/506086/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/clinvar/submitters/506086/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKhan I, Leventhal BL Developmental Delay. StatPearls [Internet] 2024. 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Nature 486(7402):256\u0026ndash;260\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"954\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 954px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1. Clinical and genetic details of all affected individuals with causative variants in \u003cem\u003eNPTN\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInd.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (Sex)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariant (NM_012428.4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDD / ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eautism, behaviour, neurological findings,\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDysmorphic features\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFurther findings\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e17y (F)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003ec.403T\u0026gt;A, p.(Trp135Arg),\u003c/p\u003e\n \u003cp\u003ede novo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003emoderate (IQ 40)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eautism, epileptic spasms (onset 7 months); focal impaired awareness seizures (onset 8 years); still occasional seizures\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eupslanting palpebral fissures, slight ectropion, prominent forehead with frontal upsweep and high anterior hairline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e7y (M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003ec.1025C\u0026gt;T, p.(Pro342Leu),\u003c/p\u003e\n \u003cp\u003ede novo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003esevere (no speech)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eautism, repetitive behaviour, tics (DD dystonia)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003emild hypertelorism, downslanting palpebral fissures, full lips, Darwin tuberculum on both ears, fetal pads, sandal gaps\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003erecurrent diarrhea, macrocephaly\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e5y (F)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003ec.14C\u0026gt;A,\u003c/p\u003e\n \u003cp\u003ep.(Ser5*),\u003c/p\u003e\n \u003cp\u003ede novo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003esevere\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eautism, poor social interactions, repetitive behaviour, hypotonia, ataxic gait\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003edownslanting corners of the mouth, anteverted nares, bilateral epicanthus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003ejoint hyperlaxity, chronic obstipation, bilateral vesicoureteral reflux\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e6y (M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003ec.218_219del, p.(Glu73Valfs*53), de novo, mosaic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eautism, period of regression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e3y (F)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003ec.284C\u0026gt;G, p.(Ser95*),\u003c/p\u003e\n \u003cp\u003eheterozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003emild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eautism, poor social interactions, ritualistic behaviour, mild cerebral palsy in lower extremities\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003elaryngomalacia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2y (M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003ec.342C\u0026gt;G, p.(Tyr114*),\u003c/p\u003e\n \u003cp\u003ede novo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003emoderate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eautism, poor social interactions, hyperactivity, possible speech regression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eprominent forehead\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eearly eruption of teeth, insomnia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e9y (M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003ec.902del, p.(Asn301Thrfs*3),\u003c/p\u003e\n \u003cp\u003ede novo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003esevere\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003etentative diagnosis of autism, pica disorder, automutilation, high pain threshold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 284px;\"\u003e\n \u003cp\u003eflat face, full eyebrows, upward slanting palpebral fissures, epicanthal folds, long fingers with mild clinodactyly 5th fingers, normal feet with sandal gaps\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: DD, developmental delay; F, female; Ind., Individual; ID, intellectual disability; M, male; NA, not available; y, years.\u003c/p\u003e\n\u003cp\u003eFurther clinical details are provided in Supplementary table 1.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"autism, epilepsy, speech delay, neuroplastin, PMCA, calcium homeostasis","lastPublishedDoi":"10.21203/rs.3.rs-8079156/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8079156/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eNPTN\u003c/em\u003e encodes human neuroplastin (hNp), a subunit of plasma membrane Ca\u003csup\u003e2+\u003c/sup\u003e-ATPases (PMCA). The critical importance of hNp and its associations with PMCA are unknown for the human brain. Here, we describe \u003cem\u003ede novo NPTN\u003c/em\u003e variants in seven individuals with autism and mild-to-severe DD/ID and evaluate them using animal models and \u003cem\u003ein silico\u003c/em\u003e, molecular and cellular approaches. We identified \u003cem\u003eNPTN\u003c/em\u003e variants with dominant-negative (missense) or loss-of-function (nonsense/ frameshift) effect on hNp-PMCA expression and function. The missense variants caused structural and thermodynamic molecular abnormalities and lower expression of hNp in HEK cells. In neurons, hNp missense variants affected PMCA levels and cytosolic Ca\u0026sup2;⁺ regulation. In \u003cem\u003eDrosophila\u003c/em\u003e, a missense mutation with affected PMCA interaction failed to prevent a lethal phenotype caused by hNp ortholog elimination. In \u003cem\u003eNptn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, levels of Np and PMCA were reduced and insufficient for normal social behavior. Therefore, we show that \u003cem\u003ede novo\u003c/em\u003e variants in \u003cem\u003eNPTN\u003c/em\u003e cause a neurodevelopmental disorder with intellectual disability and autism, likely linked to PMCA dysfunction.\u003c/p\u003e","manuscriptTitle":"De novo variants in NPTN cause a neurodevelopmental disorder with autism and neuroplastin-PMCA hypofunction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-11 11:13:41","doi":"10.21203/rs.3.rs-8079156/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6f568ff-a654-49fb-ae04-698305a2740f","owner":[],"postedDate":"November 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57748333,"name":"Medical Genetics"},{"id":57748334,"name":"General Cell Biology \u0026 Physiology"},{"id":57748335,"name":"Neurology"},{"id":57748336,"name":"Cellular \u0026 Molecular Neuroscience"}],"tags":[],"updatedAt":"2025-11-12T17:32:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-11 11:13:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8079156","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8079156","identity":"rs-8079156","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}