Expanding the Genotypic and Phenotypic Spectrum of PGAP1 Deficiency: Clinical and Functional Insights from 15 Patients

Expanding the Genotypic and Phenotypic Spectrum of PGAP1 Deficiency: Clinical and Functional Insights from 15 Patients | 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 Article Expanding the Genotypic and Phenotypic Spectrum of PGAP1 Deficiency: Clinical and Functional Insights from 15 Patients Leszek Lisowski, Florencia Haase, Oselyne Ong, Irene Valenzuela, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8463184/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Glycosylphosphatidylinositol-anchored proteins (GPI-APs) play essential roles in neuronal development, synaptic organisation, and signal transduction. Defects in GPI-anchor biosynthesis and remodelling cause rare multisystem neurodevelopmental disorders, among which post-GPI attachment to proteins 1 (PGAP1) deficiency is particularly uncommon. PGAP1 encodes an inositol deacylase that removes an acyl chain from the inositol ring of the GPI anchor, an early remodelling step required for proper trafficking and membrane localisation of GPI-APs. Loss of PGAP1 function leads to structurally abnormal GPI-APs and impaired cellular processes. Fewer than 40 affected individuals have been described, limiting understanding of the clinical spectrum. We systematically characterised 15 individuals with PGAP1 deficiency from 11 unrelated families across 11 countries using standardised phenotyping, molecular analysis, and functional assays. Eight previously unreported pathogenic variants were identified across six exons, including splice-site, truncating, and missense variants, with exon 2 containing the largest cluster. Individuals ranged from 8 months to 23 years. Intellectual disability and global motor delay were the most common features (93.3%), and only 40.0% achieved independent ambulation. Seizures occurred in 66.7% of cases. Scoliosis was observed in 40.0%, and peripheral neuropathy in 33.3%, the latter representing an under-recognised aspect of the disorder. Functional studies showed resistance to phosphatidylinositol-specific phospholipase C cleavage of GPI-APs, confirming impaired GPI-anchor remodelling. These findings expand the genotypic and phenotypic landscape of PGAP1 deficiency, highlight neuropathy as part of the disease spectrum, and emphasise the need for genetic testing and functional validation for accurate diagnosis and management. Health sciences/Diseases/Neurological disorders/Neurodevelopmental disorders Biological sciences/Genetics/Genotype/Genetic predisposition to disease Health sciences/Medical research/Genetics research PGAP1 GPI-anchor Genetic mutations Intellectual disability Developmental delay Neuropathy Figures Figure 1 Figure 2 Introduction Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are essential for numerous cellular functions, particularly within the central nervous system (CNS), where they influence neuronal activity and synaptic organization. These proteins are tethered to the cell membrane via GPI anchors; complex glycolipids synthesized in the endoplasmic reticulum. (1) Beyond the CNS, GPI-APs also play key roles in physiological processes such as reproduction, immune response and metabolism underscoring their broader systemic importance. (2)˒(3) Mutations in genes responsible for GPI anchor biosynthesis are frequently implicated in CNS disorders. The phosphatidylinositol glycan (PIG) and post-GPI attachment to proteins (PGAP) gene families are central to this biosynthetic pathway. Variants in genes such as PIGV , PIGO , PGAP2 , PIGW , and PGAP3 have been linked to hypophosphatasia with mental retardation syndrome (HPMRS),(4-8) emphasizing the role of GPI-APs in neurodevelopment. Disruptions in this pathway have also been associated with psychiatric conditions including schizophrenia, bipolar disorder, depression, and panic disorder, likely due to their impact on neuronal connectivity and synaptic function.(9-11) Among these genes, post-GPI attachment to proteins 1 ( PGAP1 ) encodes a critical inositol deacylase involved in GPI anchor remodelling. This enzyme removes an acyl chain from the inositol ring, a necessary step for the correct structural conformation, membrane localization, and function of GPI-APs (Figure 1).(12-15) Mutations in PGAP1 (OMIM *611655) lead to defective GPI-APs that are improperly anchored to the cell surface, resulting in a range of neurodevelopmental abnormalities (“Neurodevelopmental disorder with dysmorphic features, spasticity, and brain abnormalities”, OMIM #615802). Individuals with PGAP1 mutations commonly exhibit intellectual disability, seizures, and delayed motor development. (16-22) These phenotypes highlight the essential role of PGAP1 in maintaining neuronal function and synaptic integrity. Diagnosing PGAP1 -related disorders in clinical settings is often challenging due to their heterogeneous and non-specific presentation and can be confused with other disorders due to overlapping symptoms, especially within the scope of Congenital Disorders of Glycosylation (CDG). Affected individuals typically present in early infancy with developmental delays, varying levels of intellectual disability, and neurological features such as hypotonia and seizures. Clinical examination may suggest CNS involvement, but findings are often non-localizing. Neuroimaging, particularly magnetic resonance imaging (MRI), may reveal nonspecific anomalies such as delayed myelination or corpus callosum thinning, indicative of diffuse neurodevelopmental disturbance.(18) Genetic testing, particularly whole exome sequencing (WES), plays a pivotal role in establishing a definitive diagnosis. Identifying pathogenic PGAP1 variants confirms the diagnosis and delineates the molecular aetiology. Advances in high-throughput sequencing have facilitated the detection of GPI-pathway gene mutations, including PGAP1 , in individuals with unexplained neurological syndromes. Nevertheless, PGAP1 variants remain exceedingly rare, with only seven studies, as of the time of writing, reporting a total of 11 affected individuals. Most reported variants are unique to individual families, with recurrence observed primarily in siblings. This study aims to enhance our understanding of PGAP1 -related disorders by reporting novel pathogenic variants and systematically correlating these with detailed clinical phenotypes. By analysing 15 individuals from eleven unrelated families diagnosed through combined clinical, radiological, and molecular assessment, we expand the phenotypic and genotypic spectrum of PGAP1 mutations. Our findings aim to support improved diagnostic precision and inform future management strategies for affected individuals, grounded in a deeper comprehension of the pathology's natural history. Case Description 2.1 Subjects This study characterizes a cohort of individuals with PGAP1 -related disorders from multiple familial lines across eleven countries, highlighting the diverse clinical and genetic manifestations associated with the condition. The cohort comprised 15 individuals, aged 8 months to 23 years, diagnosed based on clinical presentation, physical examination, laboratory findings, and confirmed genetic analysis. The participants, representing both sexes and various ethnic backgrounds, exhibited a spectrum of symptoms consistent with GPI-anchor deficiencies, with intellectual disability and motor developmental delay being the most common features. In all cases, parents reported no prior family history of similar disorders, suggesting that PGAP1 mutations occurred de novo or were inherited in an autosomal recessive manner. Detailed family histories and medical records were reviewed to identify any recurring phenotypes or related conditions, and pedigrees were constructed to illustrate familial relationships and inheritance patterns. Each patient’s clinical records were reviewed and findings on clinical examination including sensory and motor function, muscle tone, and reflexes were recorded. The results of neuroimaging and EEG recordings (where available) were also reviewed. Diagnostic neuroimaging, specifically brain MRI, was performed in several cases and frequently revealed structural abnormalities such as thinning of the corpus callosum. Electroencephalography (EEG) were used to look for epileptiform activity. Results of developmental assessments were reviewed to quantify the degree of intellectual disability and characterize developmental delay patterns. These evaluations provided critical insights into the functional impairments associated with PGAP1 mutations. Together with the clinical and genetic data from each individual, they contribute to a more comprehensive understanding of the phenotypic spectrum and natural history of this ultra-rare disorder. Methods Clinical To assess the global burden and aiming to find genotype–phenotype correlations associated with PGAP1 mutations, 17 new individuals were identified through international research collaborations, of whom 15 were included in this study. A dedicated registry was established, and data was collected on confirmed pathogenic or likely pathogenic variants in the PGAP1 gene, provided by contributing clinicians, geneticists and families. Molecular data included variant type and copy number information. Parents/legal guardians provided informed consent, and participating clinicians or patient families completed a standardized phenotyping questionnaire. This included information on facial dysmorphism, microcephaly, intellectual disability, speech development, motor milestones (e.g., independent walking), hypotonia, neuropathies, feeding and respiratory issues, stereotypic movements, ophthalmologic findings, seizures, EEG abnormalities, neuroimaging, and other relevant clinical features. Genetic and Sequencing Analyses Genetic diagnoses were established using clinical exome sequencing and, where available, RNA sequencing. Exome sequencing was performed at accredited diagnostic sequencing facilities following standard capture-based library preparation and next-generation sequencing protocols, as previously described(23). Variant calling and annotation were conducted using validated clinical bioinformatics pipelines at the respective laboratories. For selected individuals, transcriptome analysis was performed using RNA sequencing from patient-derived samples to assess the impact of PGAP1 variants on transcript structure and expression, following established protocols(24). RNA-seq data were analysed using standard alignment, quantification, and splice-aware methods at the contributing sequencing facilities. Identified variants were subsequently integrated with clinical data and in silico predictions for downstream interpretation. Molecular Studies Functional assessments of PGAP1 activity were performed. These analyses followed previously established methodologies. 16 The functional status of GPI-anchored proteins (GPI-APs) was evaluated by assessing cellular sensitivity to bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), which normally cleaves the protein portion of GPI-APs (e.g., CD55, CD59, CD73, or uPAR) from the cell surface. In PGAP1 -deficient cells, this cleavage is impaired, resulting in resistance to PI-PLC treatment, a key diagnostic indicator due to the lack of specific PGAP1 antibodies. To quantify PGAP1 activity, fibroblasts were incubated with 50 µL of 1 U/mL PI-PLC (P6466 Invitrogen) in Opti-MEM I solution (Invitrogen) at 37°C for 1 to 1.5 hours. Post-treatment, cells were stained for GPI-AP markers (CD55, CD59, CD73) and analysed by flow cytometry. This enabled quantitative comparison of GPI-AP surface expression in patient versus control cells following PI-PLC treatment, providing a robust assessment of PGAP1 functionality. PGAP1 deficient CHO cells (C10)(25) were transfected with the strong promoter SRα (pME) driven wild-type or mutant PGAP1 and and five days later, cells were treated with PIPLC and stained for GPI-AP markers (CD55, CD59, uPAR) and analysed by flow cytometry. In silico Variant-Level Analysis In addition to clinical characterization and registry data collection, molecular analyses were performed using in silico prediction tools and functional annotation software to assess the pathogenicity of each PGAP1 variant. Annotated genomic coordinates were aligned to transcript NM_024989.3. All reported variants were evaluated using Alamut Visual Plus v1.12 (SOPHiA GENETICS), which integrates multiple pathogenicity predictors (CADD, REVEL, MutationTaster, PolyPhen-2, SIFT), conservation metrics (phyloP), splice-site algorithms (MaxEnt, NNSPLICE, SSF), and ClinVar classification. Variants were categorized into missense, nonsense, splice-site, or frameshift changes. For splice-site variants, disruptions to canonical donor/acceptor motifs and predicted changes in splice efficiency were examined. Frameshift and nonsense variants were assessed for potential to induce premature termination codons (PTCs) upstream of the nonsense-mediated decay (NMD) threshold. Variants were classified per ACMG guidelines and supported with transcript-level predictions when available. This approach enabled the mechanistic interpretation of loss-of-function consequences for the majority of detected PGAP1 variants. Results Genetic Analysis of PGAP1 Variants Sequencing of the PGAP1 gene in 15 individuals revealed that 9/15 (60.0%) carried homozygous pathogenic variants, while 6/15 (37.5%) had compound heterozygous mutations. Most variants were private to a single family; however, the truncating combinations c.2199del p.(Leu735*) and c.2357_2358insTA p.(Ser787*) were each observed in two unrelated families (D‑II and J‑II). Siblings within the same family shared zygosity, consistent with autosomal recessive inheritance. In total, 11 unrelated families were represented in this cohort. Among the detected variants, six were splice site mutations (see Supplementary 1 - Family A), two were missense and four were a frameshift or truncating variant. Four patients carried compound heterozygous mutations. Only one variant was shared between unrelated families (c.2199del p.(Leu735*) and c.2357_2358insTA p.(Ser787*)), underscoring the allelic heterogeneity of PGAP1 -related disorders. Pedigree analyses (see Supplementary Figure 2 - Families A, B and C) demonstrated autosomal recessive inheritance patterns. Two families reported to be consanguineous. Clinical Features and Summary The clinical characteristics of all 15 newly identified individuals are summarized in Table 1, which also incorporates data from 11 previously published cases to support comparative analysis. The ages of the individuals at evaluation ranged from 8 months to 23 years, with nine females and six males included in the cohort. This expanded case series integrates genotypic and phenotypic data from 15 new patients with PGAP1 mutations and previously reported cases, bringing the total number of recorded cases to 27. Although the small sample size limits statistical power, this section discusses the observed phenotypic range, highlighting features that appear recurrent, rare, or previously unreported. Physical abnormalities Physical abnormalities, notably facial dysmorphisms, were observed in 11/15 (73.3%) of the newly reported PGAP1 deficient patients under evaluation. These included distinctive features, such as arched eyebrow, synophrys (joining of the eyebrows), hypertelorism (widely spaced eyes), and prognathism (protruding jaw), prominent forehead, pronounced nose with characteristics such as a bulbous nasal tip and a high nasal bridge. Club foot was additionally noted in one PGAP1 -related disorder patient, with a single patient also reporting shortened phalanges and clinodactyly of their fifth digit. Beyond facial dysmorphisms, microcephaly was reported in 6/15 (42.9%) of the assessed PGAP1 -related disorder patients, indicating incomplete brain development. For the first time, scoliosis was noted in 6/15 (40.0%), strabismus (and history of strabismus) in 5/15 (33.3%) and nystagmus in one of the PGAP1-deficient patients. It should be noted that scoliosis was often not assessed, likely as it has not been previously associated with PGAP1 deficiencies (however, it has been associated with other GPI anchor protein disorders, such as PIGT disorder(26)). Cognitive Function Intellectual disability was diagnosed in 14/15 (93.3%) individuals and represents a core clinical feature of PGAP1 deficiency. Intellectual disability is typically defined as an IQ < 70, with impairments in reasoning, problem-solving, learning, and adaptive functioning(27). Speech impairments were also reported in 13/15 (86.6%), ranging from delayed to absent verbal communication. Motor Development and Function Motor developmental delays were prominent symptoms observed in 14/15 (93.3%) of the patient cohort. In addition to the delays in motor development, the overall motor function was significantly compromised across the PGAP1-deficiency cohort. Self-feeding was documented in only 4 of 15 individuals (26.7 %), suggesting that most either lacked this ability or it was not reported. Of those four, three were able to eat semi-solid and solid foods, while one self-fed only with milk bottles. Difficulty biting hard foods was reported in 4/15 (26.7%) participants. Furthermore, only 5/15 (33.3%) of the patients were able to walk independently, and 3/15 (20.0%) required some form of external support. This highlights the extensive impact of PGAP1 gene mutations on basic motor functions and independence in daily activities. Neuromuscular Findings PGAP1 deficiency is known to cause spastic paraplegia type 47 (SPG47), which is classified as a neurodegenerative and neuromuscular disorder due to its prominent motor involvement. In our cohort, hypotonia and peripheral neuropathy were observed in 10/15 (66.7%) and 5/15 (33.3%) individuals, respectively. The presence of hypotonia and delayed gross motor milestones is consistent with previously reported cases of SPG47. The finding of neuropathy in several patients represents a potentially novel observation within PGAP1-related disease, although similar features have been described in other GPI-anchor biosynthesis disorders. Where available, nerve conduction studies are being reviewed to further characterize this finding and confirm whether it reflects a true peripheral neuropathy or secondary motor impairment. Sleep apnea was documented in one individual and may also reflect neuromuscular dysfunction within the spectrum of this disorder. Neurological Manifestations Seizures were observed in 10/15 (66.7%) individuals, consistent with the high prevalence of epilepsy previously reported in PGAP1 deficiency. EEG data were available for seven patients, with abnormal findings in five (5/7). The EEG abnormalities were described as non-specific background slowing and multifocal epileptiform discharges, although detailed reports were not available for all cases. Stereotypic movements, including repetitive hand or body movements, were noted in 8/15 (53.3%) patients. While these movements are not considered neuromuscular in origin, they are frequently reported among individuals with neurodevelopmental disorders and may reflect cortico-striatal circuit dysfunction. Together, these findings highlight the broad neurological spectrum associated with PGAP1 deficiency, encompassing epileptic activity, motor stereotypies, and global developmental impairment. Brain Anatomy Assessment (MRI) MRI findings were available for seven individuals. Among them, 3/7 (42.8%) exhibited abnormalities such as a thin or partially absent corpus callosum, translating to 13.3% (2/15) across the entire cohort. These findings are consistent with global neurodevelopmental impairment and defective interhemispheric connectivity. In silico Characterization In our cohort of fourteen individuals, we identified a diverse set of PGAP1 pathogenic variants (Figure 2), spanning splice‑site, frameshift/truncating, in‑frame deletion, and missense classes, observed in both homozygous and compound‑heterozygous configurations. Six individuals carried canonical splice‑site variants: c.1221–3A>G (A-II:1), c.2286+5G>A (B-II:1-3) and c.2286+5G>T (I-II). Splice prediction (MaxEnt/NNSPLICE/SSF) indicates substantial weakening or loss of canonical donor/acceptor recognition (see Supplementary Table 1). Experimental confirmation of splicing disruption caused by the c.1221–3A>G variant is shown in Supplementary Figure 1, using blood- and fibroblast-derived mRNA from the proband in Family A (published as case A034- PGAP1 in Bournazos et al 2022). In addition, one homozygous junctional deletion, c.1081_1089+3del (G-II), spans the exon–intron boundary and is predicted to abolish donor recognition across algorithms; the submitted protein annotation for this allele is p.(Val360_Asn363del), consistent with exon skipping/in‑frame loss. We also identified three individuals with homozygous frameshift or truncating variants: c.289del (p.Ser97Valfs30) (F-II) , c.2024delT (p.Leu681Argfs4) (H-II), and C.1367A>G (p.Glu456Gly) (E-II). Each of these introduced premature stop codons well upstream of the nonsense-mediated decay (NMD) threshold, supporting a mechanism of transcript degradation. The variant, c.1367A>G affects a highly conserved residue and is predicted to be damaging by PolyPhen (0.999), SIFT, and MutationTaster. Compound heterozygosity was observed in four families. Two siblings (C-II:1 and C-II:2) carried splice-donor variants c.1089+1G>A and c.1089+5delG, both affecting the intron-9 donor and consistent with exon skipping. Consistent with loss of function, bulk RNA-seq from Family C showed reduced PGAP1 transcript abundance in both siblings, with junction reads dominated by canonical splice sites at the coverage displayed (Supplementary Figure 4). Two unrelated individuals (D-II and J-II) harboured the recurrent truncating combination c.2199del (p.Leu735*) and c.2357_2358insTA (p.Ser787*/p.Arg786Serfs*35), which introduces premature termination codons. Additionally, one individual from Family K (K-II) carried a truncating–missense combination in trans: c.2357_2358insTA (p.Ser787*) and c.2241T>G (p.Cys747Trp). Collectively, these variants span multiple pathogenic mechanisms, including splice disruption, loss-of-function through premature truncation, and potential structural compromise of PGAP1 enzymatic activity. The intolerance of PGAP1 to loss-of-function alleles (gnomAD LOEUF 0.42; pLI ~0) further supports the pathogenic nature of the identified variants. Validation in tested individuals revealed resistance to PI-PLC cleavage of GPI-anchored markers, confirming disrupted GPI-anchor remodelling and providing cellular evidence for PGAP1 deficiency. A detailed breakdown of each variant’s predicted impact, conservation, and functional annotation is provided in Supplementary Table 1. In terms of reported pathogenicity, as of 31 Oct 2025, only a subset of the variants observed in our cohort have ClinVar entries. The splice-donor variant c.1089+1G>A is catalogued as Likely pathogenic (VCV000986862; single-submitter; criteria provided). The intronic c.2286+5G>A is listed in ClinVar as a variant of uncertain significance in the context of PGAP1 -related neurodevelopmental disorder, per ClinVar Miner’s index of ClinVar submissions. The truncating insertion c.2357_2358insTA appears among pathogenic PGAP1 variants in ClinVar-derived listings (reported as p.Arg786fs), indicating at least one pathogenic ClinVar submission for this allele. For the remaining cohort alleles: c.1221–3A>G, c.2286+5G>T, c.1089+5delG, c.2199del, c.1367A>G (p.Glu456Gly), c.289del (p.Ser97Valfs*30), c.1081_1089+3del (p.Val360_Asn363del) and c.2024delT (p.Leu681Argfs*4), we found no dedicated ClinVar records at the time of review; where ClinVar is silent or uncertain, our classification rests on RNA/functional data (e.g., exon-23 skipping confirmed for c.2286+5G>T), predicted loss-of-function, and segregation within families, consistent with prior PGAP1 literature. PGAP1 functional analysis In the current report we show functional analysis of PGAP1 activity of two patients. As previously described, bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) typically cleaves GPI-anchored proteins such as CD59, CD55, CD73, or uPAR from the cell surface. In PGAP1 -deficient cells, this cleavage is impaired due to abnormal GPI structure, rendering cells resistant to PI-PLC. In our assays, fibroblasts from affected individual were treated with PI-PLC, followed by flow cytometry using antibodies against CD59, CD55, CD73. In the tested patients, cell surface levels of GPI-APs remained high post-treatment, resembling untreated control cells, whereas healthy control cells exhibited marked reduction in GPI-AP expression after PI-PLC treatment (Supplementary Figure 3, Family A). These results confirm impaired PGAP1 function in the affected individual. As fibroblasts from the Family E patient were not available, a functional assay was performed using PGAP1-deficient CHO cells. Cells transfected with Glu456Gly-mutant PGAP1 cDNA remained resistant to PI-PLC treatment, whereas cells transfected with wild-type PGAP1 were sensitive, indicating that the mutant PGAP1 protein exhibits severely reduced enzymatic activity (Supplementary Figure 3, Family E). Discussion This study significantly expands the clinical and molecular understanding of PGAP1 -related disorders by characterizing 15 new individuals carrying twelve unique pathogenic variants, including both novel and previously unreported combinations of splice-site, frameshift, and missense changes(16-18, 20-22). The phenotypic features observed in this cohort, such as global developmental delay, intellectual disability, epilepsy, and facial dysmorphism, are consistent with previously reported presentations of PGAP1 -related disease. Notably, peripheral neuropathy was identified in several individuals, representing a potentially novel feature of PGAP1 deficiency. While neuropathy has not been reported in prior PGAP1 cases, it has been observed in other GPI-anchor biosynthesis disorders (e.g., PIGL, PIGO, PIGT, PIGA,PIGB), suggesting that peripheral nerve involvement may occur within this broader disease spectrum(28-31). These observations broaden the clinical spectrum of PGAP1 deficiency. Our findings also support prior reports of intra-familial variability in clinical expression, likely influenced by additional genetic or epigenetic modifiers 15 . Neuroimaging in several patients revealed corpus callosum thinning and reduced white matter volume, consistent with earlier studies and reinforcing the role of PGAP1 in CNS myelination and interhemispheric connectivity(16, 17, 32). Additionally, homozygous PGAP1 variants have been implicated in hereditary spastic paraplegia and non-syndromic intellectual disability(20, 33), suggesting a broader phenotypic continuum than previously recognized. However, the pathogenic relevance of some reported alleles remains to be validated. From a molecular standpoint, the variants identified in our cohort overwhelmingly support a loss-of-function mechanism. The most informative were the homozygous or compound-heterozygous splice-disrupting alleles. For c.1221–3A>G (acceptor −3) and c.2286+5G>A / c.2286+5G>T (donor +5), in silico predictions indicate marked weakening of splice-site strength, and exon 23 skipping was experimentally confirmed for c.2286+5G>T. The compound-heterozygous pair c.1089+1G>A and c.1089+5delG targets the same donor region in intron 9 and is expected to ablate proper splicing. The homozygous c.1081_1089+3del, spanning an exon–intron boundary, was predicted to eliminate canonical donor recognition by all tested algorithms, further implicating aberrant splicing. Loss-of-function coding variants included homozygous c.289del (p.Ser97Valfs*30) and c.2024delT (p.Leu681Argfs*4), as well as the compound-heterozygous truncating combination c.2199del with c.2357_2358insTA (identified in two unrelated individuals, D-II and J-II); each introduces a premature termination codon well upstream of the final exon–exon junction (c.2580), consistent with nonsense-mediated decay. The additional compound-heterozygous genotype in Family K combined truncating c.2357_2358insTA with missense c.2241T>G (p.Cys747Trp). The sole homozygous missense variant in the cohort was c.1367A>G (p.Tyr456Cys), alters a highly conserved tyrosine and was uniformly predicted to be damaging by PolyPhen-2, SIFT, MutationTaster, and phyloP. Functional validation of the variants was achieved through flow cytometry in all available patient-derived samples. Cells displayed resistance to PI-PLC-mediated cleavage of GPI-anchored proteins (e.g., CD55, CD59), confirming defective inositol deacylation and disruption of GPI-anchor remodeling, hallmarks of PGAP1 deficiency 15 . Studies on B lymphoblastoid and CHO cells expressing PGAP1 variants have previously demonstrated complete loss of enzymatic activity 15 , consistent with our findings. No recurrent positional mutational hotspots were apparent. Variants were largely private; however, we observed a recurrent truncating combination (c.2199del + c.2357_2358insTA) in two unrelated individuals, and the insertion c.2357_2358insTA occurred in three individuals from three families (two in trans with c.2199del, one with c.2241T>G), underscoring allelic heterogeneity with occasional recurrence. The predominance of private variants likely reflects the ultra-rare nature of PGAP1-related disease and/or broad vulnerability of PGAP1 to deleterious missense and truncating changes. This is consistent with the gene’s high evolutionary conservation (≈>98% identity across primates and strong conservation in vertebrates), underscoring its essential biological function. From a therapeutic perspective, PGAP1 -related disorders represent strong candidates for gene replacement strategies, given their autosomal recessive inheritance and primary LoF etiology. The compact size of PGAP1 cDNA is compatible with AAV-mediated delivery, and the disease’s cellular signature (PI-PLC resistance) offers a quantifiable biomarker for preclinical optimization. In addition to gene therapy, pharmacological chaperones, ER stress modulators, or small molecules enhancing GPI-anchor trafficking may provide adjunctive or standalone treatments, particularly in cases involving hypomorphic missense variants. In summary, although the marked phenotypic variability among reported cases precluded definitive genotype–phenotype correlations, this study advances the clinical and mechanistic understanding of PGAP1 deficiency by integrating detailed phenotypic analysis with computational and functional validation. These findings help lay the groundwork for better diagnostics and targeted therapies for this rare neurodevelopmental disorder. Declarations Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethical Approval This study was approved by the Sydney Children’s Hospitals Network Human Research Ethics Committee (SCHN HREC), Sydney, Australia (protocol 2024/ETH02025). Author Contributions FH, OTWO, WG and LL conceived and designed the study. FH and OTWO collated collaborator datasets, FH carried out the in silico variant analysis and wrote the manuscript. Molecular and functional experiments were performed by OTWO, AB, IV, DH, TK and YM. Clinical data and patient management details were contributed by ZA, RW, FS, JS, HS, IV, JA, TL, ALK, RB and TBH. JM assisted FH with manuscript revisions. WG and LL provided supervision. All authors critically reviewed the manuscript, contributed to subsequent revisions and approved the final version. Funding This work is partly supported by Gujarat State Biotechnology Mission (GSBTM) [grant no.: GSBTM/MD/PROJECTS/SSA/505/4865/2016-17] and philanthropic donation from PGAP1 families. Acknowledgments We extend our deepest gratitude to the families whose participation made this work possible, and to the many colleagues who generously shared detailed clinical information including clinician Dr. Mike Fields for providing data on Family A. References Kinoshita T, Maeda Y, Fujita M. Transport of glycosylphosphatidylinositol-anchored proteins from the endoplasmic reticulum. Biochim Biophys Acta. 2013;1833(11):2473-8. Lee GH, Fujita M, Takaoka K, Murakami Y, Fujihara Y, Kanzawa N, et al. A GPI processing phospholipase A2, PGAP6, modulates Nodal signaling in embryos by shedding CRIPTO. J Cell Biol. 2016;215(5):705-18. Fujihara Y, Ikawa M. GPI-AP release in cellular, developmental, and reproductive biology. J Lipid Res. 2016;57(4):538-45. Chiyonobu T, Inoue N, Morimoto M, Kinoshita T, Murakami Y. Glycosylphosphatidylinositol (GPI) anchor deficiency caused by mutations in PIGW is associated with West syndrome and hyperphosphatasia with mental retardation syndrome. J Med Genet. 2014;51(3):203-7. Hansen L, Tawamie H, Murakami Y, Mang Y, ur Rehman S, Buchert R, et al. Hypomorphic mutations in PGAP2, encoding a GPI-anchor-remodeling protein, cause autosomal-recessive intellectual disability. Am J Hum Genet. 2013;92(4):575-83. Howard MF, Murakami Y, Pagnamenta AT, Daumer-Haas C, Fischer B, Hecht J, et al. Mutations in PGAP3 impair GPI-anchor maturation, causing a subtype of hyperphosphatasia with mental retardation. Am J Hum Genet. 2014;94(2):278-87. Krawitz PM, Murakami Y, Rieß A, Hietala M, Krüger U, Zhu N, et al. PGAP2 mutations, affecting the GPI-anchor-synthesis pathway, cause hyperphosphatasia with mental retardation syndrome. Am J Hum Genet. 2013;92(4):584-9. Perez Y, Wormser O, Sadaka Y, Birk R, Narkis G, Birk OS. A Rare Variant in PGAP2 Causes Autosomal Recessive Hyperphosphatasia with Mental Retardation Syndrome, with a Mild Phenotype in Heterozygous Carriers. Biomed Res Int. 2017;2017:3470234. Kinoshita T, Fujita M, Maeda Y. Biosynthesis, remodelling and functions of mammalian GPI-anchored proteins: recent progress. J Biochem. 2008;144(3):287-94. Maydan G, Noyman I, Har-Zahav A, Neriah ZB, Pasmanik-Chor M, Yeheskel A, et al. Multiple congenital anomalies-hypotonia-seizures syndrome is caused by a mutation in PIGN. J Med Genet. 2011;48(6):383-9. Freeze HH, Eklund EA, Ng BG, Patterson MC. Neurological aspects of human glycosylation disorders. Annu Rev Neurosci. 2015;38:105-25. Bütikofer P, Boschung M, Brodbeck U, Menon AK. Phosphatidylinositol Hydrolysis by Trypanosoma brucei Glycosylphosphatidylinositol Phospholipase C*. Journal of Biological Chemistry. 1996;271(26):15533-41. Ikezawa H, Yamanegi M, Taguchi R, Miyashita T, Ohyabu T. Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. I. purification, properties and phosphatase-releasing activity. Biochim Biophys Acta. 1976;450(2):154-64. Mortara RA, Minelli LM, Vandekerckhove F, Nussenzweig V, Ramalho-Pinto FJ. Phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of GPI-anchored surface molecules of Trypanosoma cruzi triggers in vitro morphological reorganization of trypomastigotes. J Eukaryot Microbiol. 2001;48(1):27-37. Tanaka S, Maeda Y, Tashima Y, Kinoshita T. Inositol deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p. J Biol Chem. 2004;279(14):14256-63. Bosch DG, Boonstra FN, Kinoshita T, Jhangiani S, de Ligt J, Cremers FP, et al. Cerebral visual impairment and intellectual disability caused by PGAP1 variants. Eur J Hum Genet. 2015;23(12):1689-93. Granzow M, Paramasivam N, Hinderhofer K, Fischer C, Chotewutmontri S, Kaufmann L, et al. Loss of function of PGAP1 as a cause of severe encephalopathy identified by Whole Exome Sequencing: Lessons of the bioinformatics pipeline. Mol Cell Probes. 2015;29(5):323-9. Kettwig M, Elpeleg O, Wegener E, Dreha-Kulaczewski S, Henneke M, Gärtner J, et al. Compound heterozygous variants in PGAP1 causing severe psychomotor retardation, brain atrophy, recurrent apneas and delayed myelination: a case report and literature review. BMC Neurol. 2016;16:74. Murakami Y, Tawamie H, Maeda Y, Büttner C, Buchert R, Radwan F, et al. Null mutation in PGAP1 impairing Gpi-anchor maturation in patients with intellectual disability and encephalopathy. PLoS Genet. 2014;10(5):e1004320. Novarino G, Fenstermaker AG, Zaki MS, Hofree M, Silhavy JL, Heiberg AD, et al. Exome sequencing links corticospinal motor neuron disease to common neurodegenerative disorders. Science. 2014;343(6170):506-11. Trujillano D, Bertoli-Avella AM, Kumar Kandaswamy K, Weiss ME, Köster J, Marais A, et al. Clinical exome sequencing: results from 2819 samples reflecting 1000 families. Eur J Hum Genet. 2017;25(2):176-82. Williams C, Jiang YH, Shashi V, Crimian R, Schoch K, Harper A, et al. Additional evidence that PGAP1 loss of function causes autosomal recessive global developmental delay and encephalopathy. Clin Genet. 2015;88(6):597-9. Froukh T, Nafie O, Al Hait SAS, Laugwitz L, Sommerfeld J, Sturm M, et al. Genetic basis of neurodevelopmental disorders in 103 Jordanian families. Clin Genet. 2020;97(4):621-7. Witt D, Faust U, Strobl-Wildemann G, Sturm M, Buchert R, Zuleger T, et al. Genome sequencing identifies complex structural MLH1 variant in unsolved Lynch syndrome. Mol Genet Genomic Med. 2023;11(6):e2151. Shinohara T, Harada M, Ogi K, Maruyama M, Fujii R, Tanaka H, et al. Identification of a G protein-coupled receptor specifically responsive to beta-alanine. J Biol Chem. 2004;279(22):23559-64. Bayat A, Knaus A, Juul AW, Dukic D, Gardella E, Charzewska A, et al. PIGT-CDG, a disorder of the glycosylphosphatidylinositol anchor: description of 13 novel patients and expansion of the clinical characteristics. Genet Med. 2019;21(10):2216-23. Committee to Evaluate the Supplemental Security Income Disability Program for Children with Mental Disorders; Board on the Health of Select Populations; Board on Children Y, and Families; Institute of Medicine; Division of Behavioral and Social Sciences and Education; The National Academies of Sciences, Engineering, and Medicine; Boat TF, Wu JT, editors. Mental Disorders and Disabilities Among Low-Income Children. Washington (DC): National Academies Press (US); 2015 Oct 28. Available from: https://www.ncbi.nlm.nih.gov/books/NBK332882/ doi: 10.17226/21780. Murakami Y, Nguyen TTM, Baratang N, Raju PK, Knaus A, Ellard S, et al. Mutations in PIGB Cause an Inherited GPI Biosynthesis Defect with an Axonal Neuropathy and Metabolic Abnormality in Severe Cases. Am J Hum Genet. 2019;105(2):384-94. Horn D, Krawitz P, Mannhardt A, Korenke GC, Meinecke P. Hyperphosphatasia-mental retardation syndrome due to PIGV mutations: expanded clinical spectrum. Am J Med Genet A. 2011;155a(8):1917-22. Record CJ, O'Connor A, Verbeek NE, van Rheenen W, Zamba Papanicolaou E, Peric S, et al. Recessive Variants in PIGG Cause a Motor Neuropathy with Variable Conduction Block, Childhood Tremor, and Febrile Seizures: Expanding the Phenotype. Annals of Neurology. 2025;97(2):388-96. Kato M, Saitsu H, Murakami Y, Kikuchi K, Watanabe S, Iai M, et al. PIGA mutations cause early-onset epileptic encephalopathies and distinctive features. Neurology. 2014;82(18):1587-96. Um JW, Ko J. Neural Glycosylphosphatidylinositol-Anchored Proteins in Synaptic Specification. Trends Cell Biol. 2017;27(12):931-45. Riazuddin S, Hussain M, Razzaq A, Iqbal Z, Shahzad M, Polla DL, et al. Exome sequencing of Pakistani consanguineous families identifies 30 novel candidate genes for recessive intellectual disability. Mol Psychiatry. 2017;22(11):1604-14. Table Table 1 is available in the Supplementary Files section. Additional Declarations There is no duality of interest Supplementary Files SupplementaryMaterialPGAP1.docx Supplementary Material Table1.xlsx Table 1. Genotypic and phenotypic summary of individuals with PGAP1 deficiency included in the present cohort and previously published cases. Columns labelled A–K correspond to individuals/families reported in the present study (started in 2021), including affected siblings where applicable (e.g., A-II:1, A-II:2). Columns to the right summarizes previously published individuals, grouped by publication. “+” indicates feature present; “–” indicates absent; “NR” indicates not reported/not assessed. Abbreviations: M, months; IQ, intelligence quotient. Notes and brief qualifiers within cells (e.g., “supported steps,” “ataxia,” “truncal hypotonia”) reflect the wording provided by contributing clinicians and/or parent/guardians. Cite Share Download PDF Status: Under Review Version 1 posted Reviewer # 2 agreed at journal 27 Apr, 2026 Review # 1 received at journal 08 Feb, 2026 Reviewer # 1 agreed at journal 04 Feb, 2026 Reviewers invited by journal 15 Jan, 2026 Submission checks completed at journal 14 Jan, 2026 First submitted to journal 27 Dec, 2025 Editor assigned by journal 27 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8463184","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":575222700,"identity":"1f817859-c2c2-4add-b01c-856f032e2864","order_by":0,"name":"Leszek Lisowski","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACPnbGBiCVwMMP4iUQo4WNGapFsoF4LQwQ4w0OEOswNmbm5g8f29JkjI+fTvvwgMEmX96B+ZkEAYe1Sc5sy+ExO5O7eUYCQ5rlxgNsZgS1MPNuq+Axu8G7Gei+wwaGDQwEtTR/BmkxngHXwv6NkJYGad5tOTwGElAt8gw8hB0mOfNfGo8E0C8MCQZpBgbMPMUW+LTws7c//vDhTLI9f/vZzYw/KmwM5NvbN97ApwUNGADRYRLUQ4B8A8laRsEoGAWjYJgDAIv2PA5sg3N1AAAAAElFTkSuQmCC","orcid":"","institution":"Children's Medical Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Leszek","middleName":"","lastName":"Lisowski","suffix":""},{"id":575222701,"identity":"451ceeeb-62e6-46e9-b3fd-a1bd4c7089ba","order_by":1,"name":"Florencia Haase","email":"","orcid":"https://orcid.org/0000-0003-4191-9112","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Florencia","middleName":"","lastName":"Haase","suffix":""},{"id":575222702,"identity":"56016c6b-5121-456f-96bb-ba25a984c9d1","order_by":2,"name":"Oselyne Ong","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Oselyne","middleName":"","lastName":"Ong","suffix":""},{"id":575222703,"identity":"a5d014d6-dafc-4bd9-a41a-9bb99359474d","order_by":3,"name":"Irene Valenzuela","email":"","orcid":"https://orcid.org/0000-0003-2350-6058","institution":"University Hospital Vall d´Hebron; Medicine Genetics Group, Valle Hebron Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Irene","middleName":"","lastName":"Valenzuela","suffix":""},{"id":575222704,"identity":"46df9c7c-20dd-4e33-aa91-761bacfbebb9","order_by":4,"name":"Adam Bournazos","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Adam","middleName":"","lastName":"Bournazos","suffix":""},{"id":575222705,"identity":"87425ebf-8e8f-413f-ad53-ce04caef92c7","order_by":5,"name":"Zainab Al Masseri","email":"","orcid":"https://orcid.org/0000-0002-6202-3721","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zainab","middleName":"Al","lastName":"Masseri","suffix":""},{"id":575222706,"identity":"a6a5e6c7-9aab-452f-a411-6908a4b4732d","order_by":6,"name":"Frenny Sheth","email":"","orcid":"https://orcid.org/0000-0001-8315-9436","institution":"FRIGE's Institute of Human Genetics","correspondingAuthor":false,"prefix":"","firstName":"Frenny","middleName":"","lastName":"Sheth","suffix":""},{"id":575222707,"identity":"d75ff020-d530-44aa-9361-a00541c0fab9","order_by":7,"name":"Jayesh Sheth","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jayesh","middleName":"","lastName":"Sheth","suffix":""},{"id":575222708,"identity":"010db7d5-506c-4cfe-9440-8d1c94c71fd3","order_by":8,"name":"Harsh Sheth","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Harsh","middleName":"","lastName":"Sheth","suffix":""},{"id":575222709,"identity":"04c7d25c-ee39-4e59-a745-84c9339ce61f","order_by":9,"name":"Rebecca Buchert","email":"","orcid":"https://orcid.org/0000-0001-7576-3326","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"","lastName":"Buchert","suffix":""},{"id":575222710,"identity":"0b8d355f-7c70-4bae-a553-c4d4a8ef6e35","order_by":10,"name":"Tobias Haack","email":"","orcid":"https://orcid.org/0000-0001-6033-4836","institution":"University of Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Tobias","middleName":"","lastName":"Haack","suffix":""},{"id":575222711,"identity":"d903bfc2-cd0a-4bdf-bc7d-977976e3a8dc","order_by":11,"name":"Joris Andrieux","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Joris","middleName":"","lastName":"Andrieux","suffix":""},{"id":575222712,"identity":"e9e4d0ed-e8ed-448e-a388-3ff77f4ade75","order_by":12,"name":"Thomas Liehr","email":"","orcid":"https://orcid.org/0000-0003-1672-3054","institution":"Jena University Hospital, Friedrich Schiller University","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Liehr","suffix":""},{"id":575222713,"identity":"18c951c7-9109-43e5-a6a6-029c949f9930","order_by":13,"name":"Alma Kuechler","email":"","orcid":"","institution":"Universitätsklinikum Essen, Universität Duisburg-Essen","correspondingAuthor":false,"prefix":"","firstName":"Alma","middleName":"","lastName":"Kuechler","suffix":""},{"id":575222714,"identity":"2ff6cf2f-8324-485d-8808-bb243c80eb33","order_by":14,"name":"Daniel De Los Reyes Helices","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"De Los Reyes","lastName":"Helices","suffix":""},{"id":575222715,"identity":"30de37f2-d8e7-4b5d-afa6-a16840e0b21c","order_by":15,"name":"Jessica Merjane","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Merjane","suffix":""},{"id":575222716,"identity":"257b843c-3fc0-4990-9d01-2886384da112","order_by":16,"name":"Sandra Cooper","email":"","orcid":"https://orcid.org/0000-0002-7314-5097","institution":"The University of Sydney, The Children's Hospital at Westmead, The Children's Medical Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Sandra","middleName":"","lastName":"Cooper","suffix":""},{"id":575222717,"identity":"0f33df21-d2da-4ebb-a8a8-33d45f3b85f7","order_by":17,"name":"Taroh Kinoshita","email":"","orcid":"https://orcid.org/0000-0001-7166-7257","institution":"Center for Infectious Disease Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Taroh","middleName":"","lastName":"Kinoshita","suffix":""},{"id":575222718,"identity":"08d74a11-6189-4edf-94bb-e18c76270efe","order_by":18,"name":"Yoshiko Murakami","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yoshiko","middleName":"","lastName":"Murakami","suffix":""},{"id":575222719,"identity":"68adbf5b-0135-4da2-b067-744b7f7a5cb2","order_by":19,"name":"Richard Webste Webster","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"Webste","lastName":"Webster","suffix":""},{"id":575222720,"identity":"13dbb1ac-937e-408b-a811-02718fb0df27","order_by":20,"name":"Wendy Gold","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wendy","middleName":"","lastName":"Gold","suffix":""}],"badges":[],"createdAt":"2025-12-27 21:50:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8463184/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8463184/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100749485,"identity":"fc56b467-5f30-4c9b-bb80-49b5d0ef4dcf","added_by":"auto","created_at":"2026-01-21 04:21:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":152517,"visible":true,"origin":"","legend":"","description":"","filename":"ClinicalReportPGAP1Dec2025v1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/cd32b3ca7c8a843201bc25f2.docx"},{"id":100857712,"identity":"7e9334d3-ba47-46c6-9fde-78a2e2aab72a","added_by":"auto","created_at":"2026-01-22 07:20:59","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17885,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/4ea9be0cf4cc654e2589315b.xlsx"},{"id":100796562,"identity":"9d701feb-c3fd-4c57-b95e-bcf48d4b7dc1","added_by":"auto","created_at":"2026-01-21 13:44:13","extension":"json","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":19615,"visible":true,"origin":"","legend":"","description":"","filename":"159325EJHG.json","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/f7398156ac29eb2591f40e8b.json"},{"id":100749486,"identity":"6122d0a3-c2c4-4bcd-96e9-1973211d2b45","added_by":"auto","created_at":"2026-01-21 04:21:54","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1687387,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialPGAP1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/e3bb95a29588e9e1d9a0044f.docx"},{"id":100796594,"identity":"a3737c68-1d6e-41b1-b7f4-45fe00355c0f","added_by":"auto","created_at":"2026-01-21 13:44:23","extension":"xml","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":111097,"visible":true,"origin":"","legend":"","description":"","filename":"159325EJHG0enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/e68c1ae9934d7879195a74f3.xml"},{"id":100749490,"identity":"0d8bd4da-52a9-4b8f-8ac6-10c7e1a1a221","added_by":"auto","created_at":"2026-01-21 04:21:54","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":307686,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/d722f89b36d862d0a7d0bea0.pdf"},{"id":100796541,"identity":"58932bff-d9f5-4f0a-9414-342a390734a5","added_by":"auto","created_at":"2026-01-21 13:44:02","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78143,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/0840da94f6bc91f3ae23b117.pdf"},{"id":100804061,"identity":"7820133f-21ab-4b6e-99f6-e8544527bff5","added_by":"auto","created_at":"2026-01-21 14:35:42","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":108822,"visible":true,"origin":"","legend":"","description":"","filename":"159325EJHG0structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/95b87f17109dafefbdb13567.xml"},{"id":100804083,"identity":"60baaec7-8752-46a6-8093-169573506a4d","added_by":"auto","created_at":"2026-01-21 14:36:16","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124733,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/7a279903508dbf6d15e69591.html"},{"id":100749483,"identity":"87fba594-a4d6-4293-a1ef-25111f5da106","added_by":"auto","created_at":"2026-01-21 04:21:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":452374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of GPI anchor biosynthesis and remodeling of GPI-APs. \u003c/strong\u003eGPI anchor synthesis is initiated in the endoplasmic reticulum (ER) through the stepwise assembly of the GPI precursor, followed by attachment of the protein via the GPI transamidase complex. After protein attachment, PGAP1 catalyzes deacylation of the inositol-linked acyl chain in the ER, representing the first remodeling step required for proper GPI maturation. In the absence of PGAP1 activity, inositol-acylated GPI-AP intermediates accumulate, impairing subsequent remodeling and intracellular trafficking. During passage through the Golgi apparatus, PGAP5 removes the ethanolamine phosphate side chain from the second mannose, and PGAP3 catalyzes deacylation of the unsaturated fatty acid at the sn-2 position of the phosphatidylinositol moiety. This is followed by PGAP2-mediated reacylation, generating a mature, stable GPI anchor competent for efficient plasma membrane expression. Fully remodeled GPI-APs are transported to the cell surface, where they may be released by phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage under experimental conditions.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/8d8b2ae7d709f7f0aae76e4f.png"},{"id":102294946,"identity":"3231c441-1e7f-491c-98fd-e9198d9445da","added_by":"auto","created_at":"2026-02-10 10:05:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":249123,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic distribution of PGAP1 variants across the 27 coding exons of transcript NM_024989.3. This diagram maps both novel (bolded) and previously reported PGAP1 variants along the coding exons of the canonical transcript NM_024989.3. Variants include homozygous and compound heterozygous splice site mutations, frameshift deletions/insertions, nonsense, and missense changes. The figure illustrates the broad distribution of pathogenic variants across the 27 exons, without evidence of mutational clustering, consistent with the gene’s intolerance to loss-of-function mutations across its entire coding region.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/2cb58890da64f92400d84e28.png"},{"id":102298398,"identity":"a9524d6e-9e81-4fc2-9fd2-6ae2b4a7a067","added_by":"auto","created_at":"2026-02-10 10:38:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1298575,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/e7b81a41-2684-41bb-9aea-a273b1f1ab0c.pdf"},{"id":100749480,"identity":"dc83425f-ccae-40f0-9cb9-d1593c3a1b92","added_by":"auto","created_at":"2026-01-21 04:21:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1687387,"visible":true,"origin":"","legend":"Supplementary Material","description":"","filename":"SupplementaryMaterialPGAP1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/f2e8cf7697ffa597eb64cdcc.docx"},{"id":100749478,"identity":"90bf5e38-a31c-4955-a204-fe75153e009d","added_by":"auto","created_at":"2026-01-21 04:21:54","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17885,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1. Genotypic and phenotypic summary of individuals with PGAP1 deficiency included in the present cohort and previously published cases.\u003c/strong\u003e Columns labelled A–K correspond to individuals/families reported in the present study (started in 2021), including affected siblings where applicable (e.g., A-II:1, A-II:2). Columns to the right summarizes previously published individuals, grouped by publication. “+” indicates feature present; “–” indicates absent; “NR” indicates not reported/not assessed. Abbreviations: M, months; IQ, intelligence quotient. Notes and brief qualifiers within cells (e.g., “supported steps,” “ataxia,” “truncal hypotonia”) reflect the wording provided by contributing clinicians and/or parent/guardians.\u003c/p\u003e","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8463184/v1/0b91a50107384eeea5c17c2a.xlsx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Expanding the Genotypic and Phenotypic Spectrum of PGAP1 Deficiency: Clinical and Functional Insights from 15 Patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlycosylphosphatidylinositol-anchored proteins (GPI-APs) are essential for numerous cellular functions, particularly within the central nervous system (CNS), where they influence neuronal activity and synaptic organization. These proteins are tethered to the cell membrane via GPI anchors; complex glycolipids synthesized in the endoplasmic reticulum. (1) Beyond the CNS, GPI-APs also play key roles in physiological processes such as reproduction, immune response and metabolism underscoring their broader systemic importance. (2)˒(3)\u003c/p\u003e\n\u003cp\u003eMutations in genes responsible for GPI anchor biosynthesis are frequently implicated in CNS disorders. The phosphatidylinositol glycan (PIG) and post-GPI attachment to proteins (PGAP) gene families are central to this biosynthetic pathway. Variants in genes such as \u003cem\u003ePIGV\u003c/em\u003e, \u003cem\u003ePIGO\u003c/em\u003e, \u003cem\u003ePGAP2\u003c/em\u003e, \u003cem\u003ePIGW\u003c/em\u003e, and \u003cem\u003ePGAP3\u003c/em\u003e have been linked to hypophosphatasia with mental retardation syndrome (HPMRS),(4-8) emphasizing the role of GPI-APs in neurodevelopment. Disruptions in this pathway have also been associated with psychiatric conditions including schizophrenia, bipolar disorder, depression, and panic disorder, likely due to their impact on neuronal connectivity and synaptic function.(9-11)\u003c/p\u003e\n\u003cp\u003eAmong these genes, \u003cem\u003epost-GPI attachment to proteins 1\u003c/em\u003e (\u003cem\u003ePGAP1\u003c/em\u003e) encodes a critical inositol deacylase involved in GPI anchor remodelling. This enzyme removes an acyl chain from the inositol ring, a necessary step for the correct structural conformation, membrane localization, and function of GPI-APs (Figure 1).(12-15) Mutations in \u003cem\u003ePGAP1\u003c/em\u003e (OMIM *611655) lead to defective GPI-APs that are improperly anchored to the cell surface, resulting in a range of neurodevelopmental abnormalities (\u0026ldquo;Neurodevelopmental disorder with dysmorphic features, spasticity, and brain abnormalities\u0026rdquo;, OMIM #615802). Individuals with \u003cem\u003ePGAP1\u003c/em\u003e mutations commonly exhibit intellectual disability, seizures, and delayed motor development. (16-22) These phenotypes highlight the essential role of \u003cem\u003ePGAP1\u003c/em\u003e in maintaining neuronal function and synaptic integrity.\u003c/p\u003e\n\u003cp\u003eDiagnosing \u003cem\u003ePGAP1\u003c/em\u003e-related disorders in clinical settings is often challenging due to their heterogeneous and non-specific presentation and can be confused with other disorders due to overlapping symptoms, especially within the scope of Congenital Disorders of Glycosylation (CDG). Affected individuals typically present in early infancy with developmental delays, varying levels of intellectual disability, and neurological features such as hypotonia and seizures. Clinical examination may suggest CNS involvement, but findings are often non-localizing. Neuroimaging, particularly magnetic resonance imaging (MRI), may reveal nonspecific anomalies such as delayed myelination or corpus callosum thinning, indicative of diffuse neurodevelopmental disturbance.(18)\u003c/p\u003e\n\u003cp\u003eGenetic testing, particularly whole exome sequencing (WES), plays a pivotal role in establishing a definitive diagnosis. Identifying pathogenic \u003cem\u003ePGAP1\u003c/em\u003e variants confirms the diagnosis and delineates the molecular aetiology. Advances in high-throughput sequencing have facilitated the detection of GPI-pathway gene mutations, including \u003cem\u003ePGAP1\u003c/em\u003e, in individuals with unexplained neurological syndromes.\u0026nbsp;Nevertheless, \u003cem\u003ePGAP1\u003c/em\u003e variants remain exceedingly rare, with only seven studies, as of the time of writing, reporting a total of 11 affected individuals.\u0026nbsp;Most reported variants are unique to individual families, with recurrence observed primarily in siblings.\u003c/p\u003e\n\u003cp\u003eThis study aims to enhance our understanding of \u003cem\u003ePGAP1\u003c/em\u003e-related disorders by reporting novel pathogenic variants and systematically correlating these with detailed clinical phenotypes. By analysing 15 individuals from eleven unrelated families diagnosed through combined clinical, radiological, and molecular assessment, we expand the phenotypic and genotypic spectrum of \u003cem\u003ePGAP1\u003c/em\u003e mutations. Our findings aim to support improved diagnostic precision and inform future management strategies for affected individuals, grounded in a deeper comprehension of the pathology\u0026apos;s natural history.\u0026nbsp;\u003c/p\u003e"},{"header":"Case Description","content":"\u003ch2\u003e2.1 Subjects\u003c/h2\u003e\n\u003cp\u003eThis study characterizes a cohort of individuals with \u003cem\u003ePGAP1\u003c/em\u003e-related disorders from multiple familial lines across eleven countries, highlighting the diverse clinical and genetic manifestations associated with the condition. The cohort comprised 15 individuals, aged 8 months to 23 years, diagnosed based on clinical presentation, physical examination, laboratory findings, and confirmed genetic analysis. The participants, representing both sexes and various ethnic backgrounds, exhibited a spectrum of symptoms consistent with GPI-anchor deficiencies, with intellectual disability and motor developmental delay being the most common features.\u003c/p\u003e\n\u003cp\u003eIn all cases, parents reported no prior family history of similar disorders, suggesting that \u003cem\u003ePGAP1\u003c/em\u003e mutations occurred de novo or were inherited in an autosomal recessive manner. Detailed family histories and medical records were reviewed to identify any recurring phenotypes or related conditions, and pedigrees were constructed to illustrate familial relationships and inheritance patterns.\u003c/p\u003e\n\u003cp\u003eEach patient\u0026rsquo;s clinical records were reviewed and findings on clinical examination including sensory and motor function, muscle tone, and reflexes were recorded. The results of neuroimaging and EEG recordings (where available) were also reviewed. Diagnostic neuroimaging, specifically brain MRI, was performed in several cases and frequently revealed structural abnormalities such as thinning of the corpus callosum. Electroencephalography (EEG) were used to look for epileptiform activity.\u003c/p\u003e\n\u003cp\u003eResults of developmental assessments were reviewed to quantify the degree of intellectual disability and characterize developmental delay patterns. These evaluations provided critical insights into the functional impairments associated with PGAP1 mutations. Together with the clinical and genetic data from each individual, they contribute to a more comprehensive understanding of the phenotypic spectrum and natural history of this ultra-rare disorder.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eClinical\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the global burden and aiming to find genotype\u0026ndash;phenotype correlations associated with \u003cem\u003ePGAP1\u003c/em\u003e mutations, 17 new individuals were identified through international research collaborations, of whom 15 were included in this study. A dedicated registry was established, and data was collected on confirmed pathogenic or likely pathogenic variants in the \u003cem\u003ePGAP1\u003c/em\u003e gene, provided by contributing clinicians, geneticists and families. Molecular data included variant type and copy number information.\u003c/p\u003e\n\u003cp\u003eParents/legal guardians provided informed consent, and participating clinicians or patient families completed a standardized phenotyping questionnaire. This included information on facial dysmorphism, microcephaly, intellectual disability, speech development, motor milestones (e.g., independent walking), hypotonia, neuropathies, feeding and respiratory issues, stereotypic movements, ophthalmologic findings, seizures, EEG abnormalities, neuroimaging, and other relevant clinical features.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGenetic and Sequencing Analyses\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenetic diagnoses were established using clinical exome sequencing and, where available, RNA sequencing. Exome sequencing was performed at accredited diagnostic sequencing facilities following standard capture-based library preparation and next-generation sequencing protocols, as previously described(23). Variant calling and annotation were conducted using validated clinical bioinformatics pipelines at the respective laboratories.\u003c/p\u003e\n\u003cp\u003eFor selected individuals, transcriptome analysis was performed using RNA sequencing from patient-derived samples to assess the impact of PGAP1 variants on transcript structure and expression, following established protocols(24). RNA-seq data were analysed using standard alignment, quantification, and splice-aware methods at the contributing sequencing facilities. Identified variants were subsequently integrated with clinical data and in silico predictions for downstream interpretation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMolecular Studies\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunctional assessments of \u003cem\u003ePGAP1\u003c/em\u003e activity were performed. These analyses followed previously established methodologies.\u003csup\u003e16\u003c/sup\u003e The functional status of GPI-anchored proteins (GPI-APs) was evaluated by assessing cellular sensitivity to bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), which normally cleaves the protein portion of GPI-APs (e.g., CD55, CD59, CD73, or uPAR) from the cell surface.\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003ePGAP1\u003c/em\u003e-deficient cells, this cleavage is impaired, resulting in resistance to PI-PLC treatment, a key diagnostic indicator due to the lack of specific PGAP1 antibodies. To quantify PGAP1 activity, fibroblasts were incubated with 50 \u0026micro;L of 1 U/mL PI-PLC (P6466 Invitrogen) in Opti-MEM I solution (Invitrogen) at 37\u0026deg;C for 1 to 1.5 hours. Post-treatment, cells were stained for GPI-AP markers (CD55, CD59, CD73) and analysed by flow cytometry. This enabled quantitative comparison of GPI-AP surface expression in patient versus control cells following PI-PLC treatment, providing a robust assessment of PGAP1 functionality.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePGAP1\u003c/em\u003e deficient CHO cells (C10)(25) were transfected with the strong promoter SR\u0026alpha; (pME)\u0026nbsp;driven wild-type or mutant\u003cem\u003e\u0026nbsp;PGAP1\u003c/em\u003e and and five days later, cells were treated with PIPLC and\u0026nbsp;stained for GPI-AP markers (CD55, CD59, uPAR) and analysed by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn silico Variant-Level Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to clinical characterization and registry data collection, molecular analyses were performed using \u003cem\u003ein silico\u003c/em\u003e prediction tools and functional annotation software to assess the pathogenicity of each PGAP1 variant. Annotated genomic coordinates were aligned to transcript NM_024989.3. All reported variants were evaluated using Alamut Visual Plus v1.12 (SOPHiA GENETICS), which integrates multiple pathogenicity predictors (CADD, REVEL, MutationTaster, PolyPhen-2, SIFT), conservation metrics (phyloP), splice-site algorithms (MaxEnt, NNSPLICE, SSF), and ClinVar classification.\u003c/p\u003e\n\u003cp\u003eVariants were categorized into missense, nonsense, splice-site, or frameshift changes. For splice-site variants, disruptions to canonical donor/acceptor motifs and predicted changes in splice efficiency were examined. Frameshift and nonsense variants were assessed for potential to induce premature termination codons (PTCs) upstream of the nonsense-mediated decay (NMD) threshold. Variants were classified per ACMG guidelines and supported with transcript-level predictions when available. This approach enabled the mechanistic interpretation of loss-of-function consequences for the majority of detected PGAP1 variants.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGenetic Analysis of \u003cem\u003ePGAP1\u003c/em\u003e Variants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequencing of the \u003cem\u003ePGAP1\u003c/em\u003e gene in 15 individuals revealed that 9/15 (60.0%) carried homozygous pathogenic variants, while 6/15 (37.5%) had compound heterozygous mutations. Most variants were private to a single family; however, the truncating combinations c.2199del p.(Leu735*) and c.2357_2358insTA p.(Ser787*) were each observed in two unrelated families (D‑II and J‑II). Siblings within the same family shared zygosity, consistent with autosomal recessive inheritance. In total, 11 unrelated families were represented in this cohort.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the detected variants, six were splice site mutations (see Supplementary 1 - Family A), two were missense and four were a frameshift or truncating variant. Four patients carried compound heterozygous mutations. Only one variant was shared between unrelated families (c.2199del p.(Leu735*) and c.2357_2358insTA p.(Ser787*)), underscoring the allelic heterogeneity of \u003cem\u003ePGAP1\u003c/em\u003e-related disorders. Pedigree analyses (see Supplementary Figure 2 - Families A, B and C) demonstrated autosomal recessive inheritance patterns.\u0026nbsp;Two families reported to be consanguineous.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Features and Summary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe clinical characteristics of all 15 newly identified individuals are summarized in Table 1, which also incorporates data from 11 previously published cases to support comparative analysis. The ages of the individuals at evaluation ranged from 8 months to 23 years, with nine females and six males included in the cohort.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis expanded case series integrates genotypic and phenotypic data from 15 new patients with \u003cem\u003ePGAP1\u003c/em\u003e mutations and previously reported cases, bringing the total number of recorded cases to 27. Although the small sample size limits statistical power, this section discusses the observed phenotypic range, highlighting features that appear recurrent, rare, or previously unreported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysical abnormalities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhysical abnormalities, notably facial dysmorphisms, were observed in 11/15 (73.3%) of the newly reported PGAP1 deficient patients under evaluation. These included distinctive features, such as arched eyebrow, synophrys (joining of the eyebrows), hypertelorism (widely spaced eyes), and prognathism (protruding jaw), prominent forehead, pronounced nose with characteristics such as a bulbous nasal tip and a high nasal bridge. Club foot was additionally noted in one \u003cem\u003ePGAP1\u003c/em\u003e-related disorder patient, with a single patient also reporting shortened phalanges and clinodactyly of their fifth digit.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBeyond facial dysmorphisms, microcephaly was reported in 6/15 (42.9%) of the assessed \u003cem\u003ePGAP1\u003c/em\u003e-related disorder patients, indicating incomplete brain development. For the first time, scoliosis was noted in 6/15 (40.0%), strabismus (and history of strabismus) in 5/15 (33.3%) and nystagmus in one of the PGAP1-deficient patients. It should be noted that scoliosis was often not assessed, likely as it has not been previously associated with PGAP1 deficiencies (however, it has been associated with other GPI anchor protein disorders, such as \u003cem\u003ePIGT\u0026nbsp;\u003c/em\u003edisorder(26)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCognitive Function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntellectual disability was diagnosed in 14/15 (93.3%) individuals and represents a core clinical feature of \u003cem\u003ePGAP1\u003c/em\u003e deficiency. Intellectual disability is typically defined as an IQ \u0026lt; 70, with impairments in reasoning, problem-solving, learning, and adaptive functioning(27). Speech impairments were also reported in 13/15 (86.6%), ranging from delayed to absent verbal communication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMotor Development and Function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMotor developmental delays were prominent symptoms observed in 14/15 (93.3%) of the patient cohort. In addition to the delays in motor development, the overall motor function was significantly compromised across the PGAP1-deficiency\u003cem\u003e\u0026nbsp;\u003c/em\u003ecohort. Self-feeding was documented in only 4 of 15 individuals (26.7 %), suggesting that most either lacked this ability or it was not reported. Of those four, three were able to eat semi-solid and solid foods, while one self-fed only with milk bottles. Difficulty biting hard foods was reported in 4/15 (26.7%) participants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, only 5/15 (33.3%) of the patients were able to walk independently, and 3/15 (20.0%) required some form of external support. This highlights the extensive impact of \u003cem\u003ePGAP1\u003c/em\u003e gene mutations on basic motor functions and independence in daily activities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeuromuscular Findings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePGAP1\u0026nbsp;\u003c/em\u003edeficiency is known to cause spastic paraplegia type 47 (SPG47), which is classified as a neurodegenerative and neuromuscular disorder due to its prominent motor involvement. In our cohort, hypotonia and peripheral neuropathy were observed in 10/15 (66.7%) and 5/15 (33.3%) individuals, respectively. The presence of hypotonia and delayed gross motor milestones is consistent with previously reported cases of SPG47.\u003c/p\u003e\n\u003cp\u003eThe finding of neuropathy in several patients represents a potentially novel observation within PGAP1-related disease, although similar features have been described in other GPI-anchor biosynthesis disorders. Where available, nerve conduction studies are being reviewed to further characterize this finding and confirm whether it reflects a true peripheral neuropathy or secondary motor impairment.\u003c/p\u003e\n\u003cp\u003eSleep apnea was documented in one individual and may also reflect neuromuscular dysfunction within the spectrum of this disorder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeurological Manifestations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeizures were observed in 10/15 (66.7%) individuals, consistent with the high prevalence of epilepsy previously reported in \u003cem\u003ePGAP1\u0026nbsp;\u003c/em\u003edeficiency. EEG data were available for seven patients, with abnormal findings in five (5/7). The EEG abnormalities were described as non-specific background slowing and multifocal epileptiform discharges, although detailed reports were not available for all cases.\u003c/p\u003e\n\u003cp\u003eStereotypic movements, including repetitive hand or body movements, were noted in 8/15 (53.3%) patients. While these movements are not considered neuromuscular in origin, they are frequently reported among individuals with neurodevelopmental disorders and may reflect cortico-striatal circuit dysfunction.\u003c/p\u003e\n\u003cp\u003eTogether, these findings highlight the broad neurological spectrum associated with \u003cem\u003ePGAP1\u003c/em\u003e deficiency, encompassing epileptic activity, motor stereotypies, and global developmental impairment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBrain Anatomy Assessment (MRI)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMRI findings were available for seven individuals. Among them, 3/7 (42.8%) exhibited abnormalities such as a thin or partially absent corpus callosum, translating to 13.3% (2/15) across the entire cohort. These findings are consistent with global neurodevelopmental impairment and defective interhemispheric connectivity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn silico\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our cohort of fourteen individuals, we identified a diverse set of \u003cem\u003ePGAP1\u003c/em\u003e pathogenic variants (Figure 2), spanning splice‑site, frameshift/truncating, in‑frame deletion, and missense classes, observed in both homozygous and compound‑heterozygous configurations. Six individuals carried canonical splice‑site variants: c.1221\u0026ndash;3A\u0026gt;G (A-II:1), c.2286+5G\u0026gt;A (B-II:1-3) and c.2286+5G\u0026gt;T (I-II). Splice prediction (MaxEnt/NNSPLICE/SSF) indicates substantial weakening or loss of canonical donor/acceptor recognition (see Supplementary Table 1). Experimental confirmation of splicing disruption caused by the c.1221\u0026ndash;3A\u0026gt;G variant is shown in Supplementary Figure 1, using blood- and fibroblast-derived mRNA from the proband in Family A (published as case A034-\u003cem\u003ePGAP1\u003c/em\u003e in Bournazos et al 2022). In addition, one homozygous junctional deletion, c.1081_1089+3del (G-II), spans the exon\u0026ndash;intron boundary and is predicted to abolish donor recognition across algorithms; the submitted protein annotation for this allele is p.(Val360_Asn363del), consistent with exon skipping/in‑frame loss.\u003c/p\u003e\n\u003cp\u003eWe also identified three individuals with homozygous frameshift or truncating variants: c.289del (p.Ser97Valfs30)\u003cem\u003e\u0026nbsp;\u003c/em\u003e(F-II)\u003cem\u003e,\u0026nbsp;\u003c/em\u003ec.2024delT (p.Leu681Argfs4) (H-II), and C.1367A\u0026gt;G (p.Glu456Gly) (E-II). Each of these introduced premature stop codons well upstream of the nonsense-mediated decay (NMD) threshold, supporting a mechanism of transcript degradation. The variant, c.1367A\u0026gt;G affects a highly conserved residue and is predicted to be damaging by PolyPhen (0.999), SIFT, and MutationTaster.\u003c/p\u003e\n\u003cp\u003eCompound heterozygosity was observed in four families. Two siblings (C-II:1 and C-II:2) carried splice-donor variants c.1089+1G\u0026gt;A and c.1089+5delG, both affecting the intron-9 donor and consistent with exon skipping. Consistent with loss of function, bulk RNA-seq from Family C showed reduced PGAP1 transcript abundance in both siblings, with junction reads dominated by canonical splice sites at the coverage displayed (Supplementary Figure 4). Two unrelated individuals (D-II and J-II) harboured the recurrent truncating combination c.2199del (p.Leu735*) and c.2357_2358insTA (p.Ser787*/p.Arg786Serfs*35), which introduces premature termination codons. Additionally, one individual from Family K (K-II) carried a truncating\u0026ndash;missense combination in trans: c.2357_2358insTA (p.Ser787*) and c.2241T\u0026gt;G (p.Cys747Trp).\u003c/p\u003e\n\u003cp\u003eCollectively, these variants span multiple pathogenic mechanisms, including splice disruption, loss-of-function through premature truncation, and potential structural compromise of PGAP1 enzymatic activity. The intolerance of \u003cem\u003ePGAP1\u003c/em\u003e to loss-of-function alleles (gnomAD LOEUF 0.42; pLI ~0) further supports the pathogenic nature of the identified variants. Validation in tested individuals revealed resistance to PI-PLC cleavage of GPI-anchored markers, confirming disrupted GPI-anchor remodelling and providing cellular evidence for PGAP1 deficiency.\u003c/p\u003e\n\u003cp\u003eA detailed breakdown of each variant\u0026rsquo;s predicted impact, conservation, and functional annotation is provided in Supplementary Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn terms of reported pathogenicity, as of 31 Oct 2025, only a subset of the variants observed in our cohort have ClinVar entries. The splice-donor variant c.1089+1G\u0026gt;A is catalogued as Likely pathogenic (VCV000986862; single-submitter; criteria provided). The intronic c.2286+5G\u0026gt;A is listed in ClinVar as a variant of uncertain significance in the context of \u003cem\u003ePGAP1\u003c/em\u003e-related neurodevelopmental disorder, per ClinVar Miner\u0026rsquo;s index of ClinVar submissions. The truncating insertion c.2357_2358insTA appears among pathogenic PGAP1 variants in ClinVar-derived listings (reported as p.Arg786fs), indicating at least one pathogenic ClinVar submission for this allele. For the remaining cohort alleles: c.1221\u0026ndash;3A\u0026gt;G, c.2286+5G\u0026gt;T, c.1089+5delG, c.2199del, c.1367A\u0026gt;G (p.Glu456Gly), c.289del (p.Ser97Valfs*30), c.1081_1089+3del (p.Val360_Asn363del) and c.2024delT (p.Leu681Argfs*4), we found no dedicated ClinVar records at the time of review; where ClinVar is silent or uncertain, our classification rests on RNA/functional data (e.g., exon-23 skipping confirmed for c.2286+5G\u0026gt;T), predicted loss-of-function, and segregation within families, consistent with prior PGAP1 literature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePGAP1 functional analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the current report we show functional analysis of PGAP1 activity of two patients. As previously described, bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) typically cleaves GPI-anchored proteins such as CD59, CD55, CD73, or uPAR from the cell surface. In \u003cem\u003ePGAP1\u003c/em\u003e-deficient cells, this cleavage is impaired due to abnormal GPI structure, rendering cells resistant to PI-PLC.\u003c/p\u003e\n\u003cp\u003eIn our assays, fibroblasts from affected individual were treated with PI-PLC, followed by flow cytometry using antibodies against CD59, CD55, CD73. In the tested patients, cell surface levels of GPI-APs remained high post-treatment, resembling untreated control cells, whereas healthy control cells exhibited marked reduction in GPI-AP expression after PI-PLC treatment (Supplementary Figure 3, Family A). These results confirm impaired PGAP1 function in the affected individual. As fibroblasts from the Family E patient were not available, a functional assay was performed using PGAP1-deficient CHO cells. Cells transfected with Glu456Gly-mutant PGAP1 cDNA remained resistant to PI-PLC treatment, whereas cells transfected with wild-type PGAP1 were sensitive, indicating that the mutant PGAP1 protein exhibits severely reduced enzymatic activity (Supplementary Figure 3, Family E).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study significantly expands the clinical and molecular understanding of \u003cem\u003ePGAP1\u003c/em\u003e-related disorders by characterizing 15 new individuals carrying twelve unique pathogenic variants, including both novel and previously unreported combinations of splice-site, frameshift, and missense changes(16-18, 20-22). The phenotypic features observed in this cohort, such as global developmental delay, intellectual disability, epilepsy, and facial dysmorphism, are consistent with previously reported presentations of \u003cem\u003ePGAP1\u003c/em\u003e-related disease. Notably, peripheral neuropathy was identified in several individuals, representing a potentially novel feature of \u003cem\u003ePGAP1\u003c/em\u003e deficiency. While neuropathy has not been reported in prior PGAP1 cases, it has been observed in other GPI-anchor biosynthesis disorders (e.g., PIGL, PIGO, PIGT, PIGA,PIGB), suggesting that peripheral nerve involvement may occur within this broader disease spectrum(28-31). These observations broaden the clinical spectrum of \u003cem\u003ePGAP1\u003c/em\u003e deficiency.\u003c/p\u003e\n\u003cp\u003eOur findings also support prior reports of intra-familial variability in clinical expression, likely influenced by additional genetic or epigenetic modifiers\u003csup\u003e15\u003c/sup\u003e. Neuroimaging in several patients revealed corpus callosum thinning and reduced white matter volume, consistent with earlier studies and reinforcing the role of PGAP1 in CNS myelination and interhemispheric connectivity(16, 17, 32). Additionally, homozygous \u003cem\u003ePGAP1\u003c/em\u003e variants have been implicated in hereditary spastic paraplegia and non-syndromic intellectual disability(20, 33), suggesting a broader phenotypic continuum than previously recognized. However, the pathogenic relevance of some reported alleles remains to be validated.\u003c/p\u003e\n\u003cp\u003eFrom a molecular standpoint, the variants identified in our cohort overwhelmingly support a loss-of-function mechanism. The most informative were the homozygous or compound-heterozygous splice-disrupting alleles. For c.1221\u0026ndash;3A\u0026gt;G (acceptor \u0026minus;3) and c.2286+5G\u0026gt;A / c.2286+5G\u0026gt;T (donor +5), \u003cem\u003ein silico\u003c/em\u003e predictions indicate marked weakening of splice-site strength, and exon 23 skipping was experimentally confirmed for c.2286+5G\u0026gt;T. The compound-heterozygous pair c.1089+1G\u0026gt;A and c.1089+5delG targets the same donor region in intron 9 and is expected to ablate proper splicing. The homozygous c.1081_1089+3del, spanning an exon\u0026ndash;intron boundary, was predicted to eliminate canonical donor recognition by all tested algorithms, further implicating aberrant splicing.\u003c/p\u003e\n\u003cp\u003eLoss-of-function coding variants included homozygous c.289del (p.Ser97Valfs*30) and c.2024delT (p.Leu681Argfs*4), as well as the compound-heterozygous truncating combination c.2199del with c.2357_2358insTA (identified in two unrelated individuals, D-II and J-II); each introduces a premature termination codon well upstream of the final exon\u0026ndash;exon junction (c.2580), consistent with nonsense-mediated decay. The additional compound-heterozygous genotype in Family K combined truncating c.2357_2358insTA with missense c.2241T\u0026gt;G (p.Cys747Trp). The sole homozygous missense variant in the cohort was c.1367A\u0026gt;G (p.Tyr456Cys), alters a highly conserved tyrosine and was uniformly predicted to be damaging by PolyPhen-2, SIFT, MutationTaster, and phyloP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunctional validation of the variants was achieved through flow cytometry in all available patient-derived samples. Cells displayed resistance to PI-PLC-mediated cleavage of GPI-anchored proteins (e.g., CD55, CD59), confirming defective inositol deacylation and disruption of GPI-anchor remodeling, hallmarks of PGAP1 deficiency \u003csup\u003e15\u003c/sup\u003e. Studies on B lymphoblastoid and CHO cells expressing PGAP1 variants have previously demonstrated complete loss of enzymatic activity \u003csup\u003e15\u003c/sup\u003e, consistent with our findings.\u003c/p\u003e\n\u003cp\u003eNo recurrent positional mutational hotspots were apparent. Variants were largely private; however, we observed a recurrent truncating combination (c.2199del + c.2357_2358insTA) in two unrelated individuals, and the insertion c.2357_2358insTA occurred in three individuals from three families (two in trans with c.2199del, one with c.2241T\u0026gt;G), underscoring allelic heterogeneity with occasional recurrence. The predominance of private variants likely reflects the ultra-rare nature of PGAP1-related disease and/or broad vulnerability of \u003cem\u003ePGAP1\u003c/em\u003e to deleterious missense and truncating changes. This is consistent with the gene\u0026rsquo;s high evolutionary conservation (\u0026asymp;\u0026gt;98% identity across primates and strong conservation in vertebrates), underscoring its essential biological function.\u003c/p\u003e\n\u003cp\u003eFrom a therapeutic perspective, \u003cem\u003ePGAP1\u003c/em\u003e-related disorders represent strong candidates for gene replacement strategies, given their autosomal recessive inheritance and primary LoF etiology. The compact size of PGAP1 cDNA is compatible with AAV-mediated delivery, and the disease\u0026rsquo;s cellular signature (PI-PLC resistance) offers a quantifiable biomarker for preclinical optimization. In addition to gene therapy, pharmacological chaperones, ER stress modulators, or small molecules enhancing GPI-anchor trafficking may provide adjunctive or standalone treatments, particularly in cases involving hypomorphic missense variants.\u003c/p\u003e\n\u003cp\u003eIn summary, although the marked phenotypic variability among reported cases precluded definitive genotype\u0026ndash;phenotype correlations, this study advances the clinical and mechanistic understanding of \u003cem\u003ePGAP1\u003c/em\u003e deficiency by integrating detailed phenotypic analysis with computational and functional validation. These findings help lay the groundwork for better diagnostics and targeted therapies for this rare neurodevelopmental disorder.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Sydney Children\u0026rsquo;s Hospitals Network Human Research Ethics Committee (SCHN HREC), Sydney, Australia (protocol 2024/ETH02025).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFH, OTWO, WG and LL conceived and designed the study. FH and OTWO collated collaborator datasets, FH carried out the in silico\u0026nbsp;variant analysis and wrote the manuscript. Molecular and functional experiments were performed by OTWO, AB, IV, DH, TK and YM. Clinical data and patient management details were contributed by ZA, RW, FS, JS, HS, IV, JA, TL, ALK, RB and TBH. JM assisted FH with manuscript revisions. WG and LL provided supervision. All authors critically reviewed the manuscript, contributed to subsequent revisions and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is partly supported by Gujarat State Biotechnology Mission (GSBTM) [grant no.: GSBTM/MD/PROJECTS/SSA/505/4865/2016-17] and philanthropic donation from PGAP1 families.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our deepest gratitude to the families whose participation made this work possible, and to the many colleagues who generously shared detailed clinical information including clinician Dr. Mike Fields for providing data on Family A.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKinoshita T, Maeda Y, Fujita M. Transport of glycosylphosphatidylinositol-anchored proteins from the endoplasmic reticulum. Biochim Biophys Acta. 2013;1833(11):2473-8.\u003c/li\u003e\n\u003cli\u003eLee GH, Fujita M, Takaoka K, Murakami Y, Fujihara Y, Kanzawa N, et al. A GPI processing phospholipase A2, PGAP6, modulates Nodal signaling in embryos by shedding CRIPTO. J Cell Biol. 2016;215(5):705-18.\u003c/li\u003e\n\u003cli\u003eFujihara Y, Ikawa M. GPI-AP release in cellular, developmental, and reproductive biology. J Lipid Res. 2016;57(4):538-45.\u003c/li\u003e\n\u003cli\u003eChiyonobu T, Inoue N, Morimoto M, Kinoshita T, Murakami Y. Glycosylphosphatidylinositol (GPI) anchor deficiency caused by mutations in PIGW is associated with West syndrome and hyperphosphatasia with mental retardation syndrome. J Med Genet. 2014;51(3):203-7.\u003c/li\u003e\n\u003cli\u003eHansen L, Tawamie H, Murakami Y, Mang Y, ur Rehman S, Buchert R, et al. Hypomorphic mutations in PGAP2, encoding a GPI-anchor-remodeling protein, cause autosomal-recessive intellectual disability. Am J Hum Genet. 2013;92(4):575-83.\u003c/li\u003e\n\u003cli\u003eHoward MF, Murakami Y, Pagnamenta AT, Daumer-Haas C, Fischer B, Hecht J, et al. Mutations in PGAP3 impair GPI-anchor maturation, causing a subtype of hyperphosphatasia with mental retardation. Am J Hum Genet. 2014;94(2):278-87.\u003c/li\u003e\n\u003cli\u003eKrawitz PM, Murakami Y, Rie\u0026szlig; A, Hietala M, Kr\u0026uuml;ger U, Zhu N, et al. PGAP2 mutations, affecting the GPI-anchor-synthesis pathway, cause hyperphosphatasia with mental retardation syndrome. Am J Hum Genet. 2013;92(4):584-9.\u003c/li\u003e\n\u003cli\u003ePerez Y, Wormser O, Sadaka Y, Birk R, Narkis G, Birk OS. A Rare Variant in PGAP2 Causes Autosomal Recessive Hyperphosphatasia with Mental Retardation Syndrome, with a Mild Phenotype in Heterozygous Carriers. Biomed Res Int. 2017;2017:3470234.\u003c/li\u003e\n\u003cli\u003eKinoshita T, Fujita M, Maeda Y. Biosynthesis, remodelling and functions of mammalian GPI-anchored proteins: recent progress. J Biochem. 2008;144(3):287-94.\u003c/li\u003e\n\u003cli\u003eMaydan G, Noyman I, Har-Zahav A, Neriah ZB, Pasmanik-Chor M, Yeheskel A, et al. Multiple congenital anomalies-hypotonia-seizures syndrome is caused by a mutation in PIGN. J Med Genet. 2011;48(6):383-9.\u003c/li\u003e\n\u003cli\u003eFreeze HH, Eklund EA, Ng BG, Patterson MC. Neurological aspects of human glycosylation disorders. Annu Rev Neurosci. 2015;38:105-25.\u003c/li\u003e\n\u003cli\u003eB\u0026uuml;tikofer P, Boschung M, Brodbeck U, Menon AK. Phosphatidylinositol Hydrolysis by Trypanosoma brucei Glycosylphosphatidylinositol Phospholipase C*. Journal of Biological Chemistry. 1996;271(26):15533-41.\u003c/li\u003e\n\u003cli\u003eIkezawa H, Yamanegi M, Taguchi R, Miyashita T, Ohyabu T. Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. I. purification, properties and phosphatase-releasing activity. Biochim Biophys Acta. 1976;450(2):154-64.\u003c/li\u003e\n\u003cli\u003eMortara RA, Minelli LM, Vandekerckhove F, Nussenzweig V, Ramalho-Pinto FJ. Phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of GPI-anchored surface molecules of Trypanosoma cruzi triggers in vitro morphological reorganization of trypomastigotes. J Eukaryot Microbiol. 2001;48(1):27-37.\u003c/li\u003e\n\u003cli\u003eTanaka S, Maeda Y, Tashima Y, Kinoshita T. Inositol deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p. J Biol Chem. 2004;279(14):14256-63.\u003c/li\u003e\n\u003cli\u003eBosch DG, Boonstra FN, Kinoshita T, Jhangiani S, de Ligt J, Cremers FP, et al. Cerebral visual impairment and intellectual disability caused by PGAP1 variants. Eur J Hum Genet. 2015;23(12):1689-93.\u003c/li\u003e\n\u003cli\u003eGranzow M, Paramasivam N, Hinderhofer K, Fischer C, Chotewutmontri S, Kaufmann L, et al. Loss of function of PGAP1 as a cause of severe encephalopathy identified by Whole Exome Sequencing: Lessons of the bioinformatics pipeline. Mol Cell Probes. 2015;29(5):323-9.\u003c/li\u003e\n\u003cli\u003eKettwig M, Elpeleg O, Wegener E, Dreha-Kulaczewski S, Henneke M, G\u0026auml;rtner J, et al. Compound heterozygous variants in PGAP1 causing severe psychomotor retardation, brain atrophy, recurrent apneas and delayed myelination: a case report and literature review. BMC Neurol. 2016;16:74.\u003c/li\u003e\n\u003cli\u003eMurakami Y, Tawamie H, Maeda Y, B\u0026uuml;ttner C, Buchert R, Radwan F, et al. Null mutation in PGAP1 impairing Gpi-anchor maturation in patients with intellectual disability and encephalopathy. PLoS Genet. 2014;10(5):e1004320.\u003c/li\u003e\n\u003cli\u003eNovarino G, Fenstermaker AG, Zaki MS, Hofree M, Silhavy JL, Heiberg AD, et al. Exome sequencing links corticospinal motor neuron disease to common neurodegenerative disorders. Science. 2014;343(6170):506-11.\u003c/li\u003e\n\u003cli\u003eTrujillano D, Bertoli-Avella AM, Kumar Kandaswamy K, Weiss ME, K\u0026ouml;ster J, Marais A, et al. Clinical exome sequencing: results from 2819 samples reflecting 1000 families. Eur J Hum Genet. 2017;25(2):176-82.\u003c/li\u003e\n\u003cli\u003eWilliams C, Jiang YH, Shashi V, Crimian R, Schoch K, Harper A, et al. Additional evidence that PGAP1 loss of function causes autosomal recessive global developmental delay and encephalopathy. Clin Genet. 2015;88(6):597-9.\u003c/li\u003e\n\u003cli\u003eFroukh T, Nafie O, Al Hait SAS, Laugwitz L, Sommerfeld J, Sturm M, et al. Genetic basis of neurodevelopmental disorders in 103 Jordanian families. Clin Genet. 2020;97(4):621-7.\u003c/li\u003e\n\u003cli\u003eWitt D, Faust U, Strobl-Wildemann G, Sturm M, Buchert R, Zuleger T, et al. Genome sequencing identifies complex structural MLH1 variant in unsolved Lynch syndrome. Mol Genet Genomic Med. 2023;11(6):e2151.\u003c/li\u003e\n\u003cli\u003eShinohara T, Harada M, Ogi K, Maruyama M, Fujii R, Tanaka H, et al. Identification of a G protein-coupled receptor specifically responsive to beta-alanine. J Biol Chem. 2004;279(22):23559-64.\u003c/li\u003e\n\u003cli\u003eBayat A, Knaus A, Juul AW, Dukic D, Gardella E, Charzewska A, et al. PIGT-CDG, a disorder of the glycosylphosphatidylinositol anchor: description of 13 novel patients and expansion of the clinical characteristics. Genet Med. 2019;21(10):2216-23.\u003c/li\u003e\n\u003cli\u003eCommittee to Evaluate the Supplemental Security Income Disability Program for Children with Mental Disorders; Board on the Health of Select Populations; Board on Children Y, and Families; Institute of Medicine; Division of Behavioral and Social Sciences and Education; The National Academies of Sciences, Engineering, and Medicine; Boat TF, Wu JT, editors. Mental Disorders and Disabilities Among Low-Income Children. Washington (DC): National Academies Press (US); 2015 Oct 28. Available from: https://www.ncbi.nlm.nih.gov/books/NBK332882/ doi: 10.17226/21780.\u003c/li\u003e\n\u003cli\u003eMurakami Y, Nguyen TTM, Baratang N, Raju PK, Knaus A, Ellard S, et al. Mutations in PIGB Cause an Inherited GPI Biosynthesis Defect with an Axonal Neuropathy and Metabolic Abnormality in Severe Cases. Am J Hum Genet. 2019;105(2):384-94.\u003c/li\u003e\n\u003cli\u003eHorn D, Krawitz P, Mannhardt A, Korenke GC, Meinecke P. Hyperphosphatasia-mental retardation syndrome due to PIGV mutations: expanded clinical spectrum. Am J Med Genet A. 2011;155a(8):1917-22.\u003c/li\u003e\n\u003cli\u003eRecord CJ, O\u0026apos;Connor A, Verbeek NE, van Rheenen W, Zamba Papanicolaou E, Peric S, et al. Recessive Variants in PIGG Cause a Motor Neuropathy with Variable Conduction Block, Childhood Tremor, and Febrile Seizures: Expanding the Phenotype. Annals of Neurology. 2025;97(2):388-96.\u003c/li\u003e\n\u003cli\u003eKato M, Saitsu H, Murakami Y, Kikuchi K, Watanabe S, Iai M, et al. PIGA mutations cause early-onset epileptic encephalopathies and distinctive features. Neurology. 2014;82(18):1587-96.\u003c/li\u003e\n\u003cli\u003eUm JW, Ko J. Neural Glycosylphosphatidylinositol-Anchored Proteins in Synaptic Specification. Trends Cell Biol. 2017;27(12):931-45.\u003c/li\u003e\n\u003cli\u003eRiazuddin S, Hussain M, Razzaq A, Iqbal Z, Shahzad M, Polla DL, et al. Exome sequencing of Pakistani consanguineous families identifies 30 novel candidate genes for recessive intellectual disability. Mol Psychiatry. 2017;22(11):1604-14.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"PGAP1, GPI-anchor, Genetic mutations, Intellectual disability, Developmental delay, Neuropathy","lastPublishedDoi":"10.21203/rs.3.rs-8463184/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8463184/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlycosylphosphatidylinositol-anchored proteins (GPI-APs) play essential roles in neuronal development, synaptic organisation, and signal transduction. Defects in GPI-anchor biosynthesis and remodelling cause rare multisystem neurodevelopmental disorders, among which post-GPI attachment to proteins 1 (PGAP1) deficiency is particularly uncommon. PGAP1 encodes an inositol deacylase that removes an acyl chain from the inositol ring of the GPI anchor, an early remodelling step required for proper trafficking and membrane localisation of GPI-APs. Loss of PGAP1 function leads to structurally abnormal GPI-APs and impaired cellular processes. Fewer than 40 affected individuals have been described, limiting understanding of the clinical spectrum.\u003c/p\u003e \u003cp\u003eWe systematically characterised 15 individuals with PGAP1 deficiency from 11 unrelated families across 11 countries using standardised phenotyping, molecular analysis, and functional assays. Eight previously unreported pathogenic variants were identified across six exons, including splice-site, truncating, and missense variants, with exon 2 containing the largest cluster. Individuals ranged from 8 months to 23 years. Intellectual disability and global motor delay were the most common features (93.3%), and only 40.0% achieved independent ambulation. Seizures occurred in 66.7% of cases. Scoliosis was observed in 40.0%, and peripheral neuropathy in 33.3%, the latter representing an under-recognised aspect of the disorder. Functional studies showed resistance to phosphatidylinositol-specific phospholipase C cleavage of GPI-APs, confirming impaired GPI-anchor remodelling.\u003c/p\u003e \u003cp\u003eThese findings expand the genotypic and phenotypic landscape of PGAP1 deficiency, highlight neuropathy as part of the disease spectrum, and emphasise the need for genetic testing and functional validation for accurate diagnosis and management.\u003c/p\u003e","manuscriptTitle":"Expanding the Genotypic and Phenotypic Spectrum of PGAP1 Deficiency: Clinical and Functional Insights from 15 Patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 04:21:49","doi":"10.21203/rs.3.rs-8463184/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-27T17:35:21+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-08T11:21:41+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-04T22:31:34+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-01-15T18:25:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-14T12:17:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Human Genetics","date":"2025-12-27T21:47:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-27T21:47:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3d8c8fc0-f3a9-45da-97bf-86c34db13297","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61206861,"name":"Health sciences/Diseases/Neurological disorders/Neurodevelopmental disorders"},{"id":61206862,"name":"Biological sciences/Genetics/Genotype/Genetic predisposition to disease"},{"id":61206863,"name":"Health sciences/Medical research/Genetics research"}],"tags":[],"updatedAt":"2026-01-21T04:21:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 04:21:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8463184","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8463184","identity":"rs-8463184","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}