Functional Insights Research Articles

WONOEP appraisal: Modeling early onset epilepsies.

Epilepsy has a peak incidence during the neonatal to early childhood period. These early onset epilepsies may be severe conditions frequently associated with comorbidities such as developmental deficits and intellectual disability and, in a significant percentage of patients, may be medication-resistant. The use of adult rodent models in the exploration of mechanisms and treatments for early life epilepsies is challenging, as it ignores significant age-specific developmental differences. More recently, models developed in immature animals, such as rodent pups, or in three-dimensional organoids may more closely model aspects of the immature brain and could result in more translatable findings. Although models are not perfect, they may offer a more controlled screening platform in studies of mechanisms and treatments, which cannot be done in pediatric patient cohorts. On the other hand, more simplified models with higher throughput capacities are required to deal with the large number of epilepsy candidate genes and the need for new treatment options. Therefore, a combination of different modeling approaches will be beneficial in addressing the unmet needs of pediatric epilepsy patients. In this review, we summarize the discussions on this topic that occurred during the XVI Workshop on Neurobiology of Epilepsy, organized in 2022 by the Neurobiology Commission of the International League Against Epilepsy. We provide an overview of selected models of early onset epilepsies, discussing their advantages and disadvantages. Heterologous expression models provide initial functional insights, and zebrafish, rodent models, and brain organoids present increasingly complex platforms for modeling and validating epilepsy-related phenomena. Together, these models offer valuable insights into early onset epilepsies and accelerate hypothesis generation and therapy discovery.

De novo genome assembly and functional insights of the first commercial pink Auricularia cornea

A pioneering pink cultivar of Auricularia cornea, first commercially cultivated in 2022, lacks genomic data, hindering research in genetic breeding, gene discovery, and product development. Here, we report the de novo assembly of the pink A. cornea Fen-A1 genome and provide a detailed functional annotation. The genome is 73.17 Mb in size, contains 86 scaffolds (N50 ∼ 5.49 Mb), 59.09% GC content and encodes 19,120 predicted genes with a BUSCO completeness of 92.60%. Comparative genomic analysis reveals the phylogenetic relatedness of Fen-A1 and remarkable gene family dynamics. Putative genes were found mapped to 3 antibiotic-related, 36 light-dependent and 25 terpene metabolites. In addition, 789 CAZymes genes were classified, revealing the dynamics of quality loss due to postharvest refrigeration. Overall, our work is the first report on a pink A. cornea genome and provides a comprehensive insight into its complex functions.

Reprogramming of normal fibroblasts into ovarian cancer-associated fibroblasts via non-vesicular paracrine signaling induces an activated fibroblast phenotype

Cancer-associated fibroblasts (CAFs) are key contributors to ovarian cancer (OC) progression and therapeutic resistance through dysregulation of the extracellular matrix (ECM). CAFs are a heterogenous population derived from different cell types through activation and reprogramming. Current studies rely on uncharacterized heterogenous primary CAFs or normal fibroblasts that fail to recapitulate CAF-like tumor behavior. Here, we present that conditioned media from ovarian cancer lines leads to an increase in the activated state of fibroblasts demonstrated by functional assays and up-regulation of known CAF-related genes and ECM pathways. Phenotypic and functional characterization demonstrated that the conditioned CAFs expressed a CAF-like phenotype, strengthened proliferation, secretory, contractility, and ECM remodeling properties when compared to resting normal fibroblasts, consistent with an activated fibroblast status. Moreover, conditioned CAFs significantly enhanced drug resistance and tumor progression. Critically, the conditioned CAFs resemble a transcriptional signature with involvement of ECM remodeling. The present study provides mechanistic and functional insights about the activation and reprogramming of CAFs in the ovarian tumor microenvironment mediated by non-vesicular paracrine signaling. Moreover, it provides a translational based approach to reprogram normal fibroblasts from both uterine and ovarian origin into CAFs using tumor-derived conditioned media. Using these resources, further development of therapeutics that possess potentiality and specificity towards CAF/ECM-mediated chemoresistance in OC are further warranted.

Schizophrenia genomics: genetic complexity and functional insights.

Determining the causes of schizophrenia has been a notoriously intractable problem, resistant to a multitude of investigative approaches over centuries. In recent decades, genomic studies have delivered hundreds of robust findings that implicate nearly 300 common genetic variants (via genome-wide association studies) and more than 20 rare variants (via whole-exome sequencing and copy number variant studies) as risk factors for schizophrenia. In parallel, functional genomic and neurobiological studies have provided exceptionally detailed information about the cellular composition of the brain and its interconnections in neurotypical individuals and, increasingly, in those with schizophrenia. Taken together, these results suggest unexpected complexity in the mechanisms that drive schizophrenia, pointing to the involvement of ensembles of genes (polygenicity) rather than single-gene causation. In this Review, we describe what we now know about the genetics of schizophrenia and consider the neurobiological implications of this information.

A spatiotemporal molecular atlas of mouse spinal cord injury identifies a distinct astrocyte subpopulation and therapeutic potential of IGFBP2

Spinal cord injury (SCI) triggers a cascade of intricate molecular and cellular changes that determine the outcome. In this study, we resolve the spatiotemporal organization of the injured mouse spinal cord and quantitatively assess in situ cell-cell communication following SCI. By analyzing existing single-cell RNA sequencing datasets alongside our spatial data, we delineate a subpopulation of Igfbp2-expressing astrocytes that migrate from the white matter (WM) to gray matter (GM) and become reactive upon SCI, termed Astro-GMii. Further, Igfbp2 upregulation promotes astrocyte migration, proliferation, and reactivity, and the secreted IGFBP2 protein fosters neurite outgrowth. Finally, we show that IGFBP2 significantly reduces neuronal loss and remarkably improves the functional recovery in a mouse model of SCI invivo. Together, this study not only provides a comprehensive molecular atlas of SCI but also exemplifies how this rich resource can be applied to endow cells and genes with functional insight and therapeutic potential.

Comparative osteology of the skull of cowbirds (Icteridae: Molothrus).

Detailed osteological descriptions of the craniomandibular complex of passerine birds are lacking for most species, limiting our understanding of their diversity and evolution. Cowbirds (genus Molothrus) are a small but widespread group of New World nine-primaried songbirds, well-known for their unique brooding parasitic behavior. However, detailed osteological data for cowbirds and other Icteridae are currently scarce and several features of their skulls remain undescribed or poorly known. To address this issue, a detailed comparative osteology of cowbird skulls is presented here for the first time based on data from x-ray microcomputed tomography, dry skeletal data, and multivariate analyses of linear morphometric data. Cowbird skulls offer some functional insights, with many finch-like features probably related to a seed-rich diet that distinguishes them from most other icterids. In addition, features previously overlooked in earlier studies might provide valuable phylogenetic information at different levels of passerine phylogeny (Passerida, Emberizoidea, Icteridae, and Agelaiinae), including some of the otic region and nasal septum. Comparisons among cowbirds show that there is substantial cranial variation within the genus, with M. oryzivorus being the most divergent cowbird species. Within the genus, distantly related species share similar overall skull morphology and proportions, but detailed osteological data allow species identification even in cases of strong convergence. Further efforts are warranted to furnish baseline data for future studies of this iconic group of Neotropical birds and to fully integrate it into phylogenetic comparative frameworks.

Molecular characterization, expression profiling, and functional analysis of prohibitin 1 in red seabream, Pagrus major

Prohibitin 1 (PHB1) is ubiquitously expressed in multiple compartments within cells and is involved in the cell cycle, cell signaling, apoptosis, transcriptional regulation, and mitochondrial biogenesis at the cellular level and in the inflammation-associated and immunological functions of B and T lymphocytes. PHB1 is an important protein that performs antioxidant regulation and immune functions inside and outside cells but has not been sufficiently studied in teleost fish. Our study aimed to elucidate the functional properties and gain new insights into the biological processes and immune system of red seabream (Pagrus major), a commercially important fish cultured in South Korea and East Asia. PHB1 mRNA was most abundantly expressed in the head kidney of healthy red seabream, and significant changes in its expression were observed after artificial infection with bacteria and viruses. On analysis, reporter gene was also significantly upregulated by polyinosinic-polycytidylic acid, lipopolysaccharides, and hydrogen peroxide. Consequent to the functional characterization of PHB1 in cells via recombinant protein preparation, the activity of leukocytes was enhanced and the reactive oxygen species-induced stress in red blood cells was reduced. The results reveal the functional characteristics of PHB1 and provide new insights into the biological processes and immune system of P. major, with beneficial implications in the study of stress responses.

Deep Learning‐Enhanced Fiber‐Coupled Laser Diode‐Based Photoacoustic Microscopy for High‐Resolution Microvasculature Imaging

AbstractBlood vessel imaging provides crucial information for physiological monitoring and disease diagnosis. Optical‐resolution photoacoustic microscopy (PAM) is gaining intense attention in high‐resolution in vivo imaging of blood vessels. However, its applicability in clinical settings is hindered by the requirement of bulky and costly pulsed lasers as well as complex alignment procedures. Here, as a novel approach to high‐quality and cost‐effective microvasculature imaging, a dual‐wavelength fiber‐coupled laser diode‐based PAM (FC‐LD‐PAM) system is demonstrated on the mouse ear and brain in vivo. Furthermore, assisted with a deep learning method, the proposed technique effectively combines the advantages when using both small and large core‐size multimode fibers—successfully enhances the resolution of blurry mouse microvascular images obtained using a large core‐size fiber, revealing fine details that are comparable to those achieved using a small core‐size fiber; significantly reduces the overall time for data acquisition up to fourfold, improving the efficiency significantly in the imaging process. In addition, the proposed approach can achieve accurate and high‐resolution mapping of oxygen saturation in vivo, providing functional insight on living tissue. Therefore, the FC‐LD‐PAM can serve as a translational tool for high‐resolution imaging with enhanced accessibility and versatility in the biomedical field.

Comprehensive genomic screening and expression profiling of trihelix family in pearl millet under abiotic stresses with emphasis on functional insights of PgTHX24

The trihelix transcription factors (THX TFs) play a crucial role in light responses and are involved in plant growth, development, and stress responses. In this study, we have identified 35 trihelix TFs in pearl millet (Pennisetum glaucum), which is one of the most widely grown C4 cereal crops in tropical semi-arid regions. Identified PgTHXs (Trihelix members of P. glaucum) were classified into 5 subgroups (GT1, GT2, GTγ, SH4, and SIP1) based on phylogenetic analysis, and these subgroup members shared similar gene structure and motif distribution pattern. Collinearity analysis exhibited gene duplication events of trihelix family members in pearl millet across the genome. Gene ontology (GO) annotation and cis-regulatory elements (CREs) analysis of PgTHXs suggested their involvement in diverse biological and molecular functions associated with plant growth, development, and stress responses. RNA sequencing data and expression profile displayed differential expression patterns of PgTHXs under abiotic stress and phytohormone treatments. The induced expression pattern of the PgTHX4, PgTHX5, PgTHX24, and PgTHX30 suggested their potential involvement in abiotic stress responses through phytohormonal signalling pathways. Among these, PgTHX24, a GT-3b member, was localized in the nucleus with self-transactivation ability. Overexpression of PgTHX24 positively regulated expression of stress-related markers in transformed pearl millet calli under drought stress conditions. Promoter activity analysis also highlighted the stress-inducible nature of PgTHX24’s promoter. Overall, our findings provide a comprehensive understanding of PgTHXs with a framework for further functional characterization to understand their regulatory role in pearl millet’s growth, development, and stress responses. Key messageThirty-five trihelix TFs were identified in pearl millet, and a comprehensive expression profile highlighted their functional diversity. Overexpression of PgTHX24 exhibited its potential involvement in abiotic stress responses.

The Future of Neurodiagnosis: Deep Learning for Earlier Intervention

This study presents an innovative deep learning framework for improved early detection of a debilitating neurodegenerative condition marked by cognitive decline and memory impairment. Timely diagnosis is crucial for effective interventions and improved patient outcomes. Our framework integrates diverse data sources, including structural and functional neuroimaging (MRI and PET) alongside clinical information, to enhance detection precision. Convolutional Neural Networks (CNNs) analyze structural MRI scans, extracting subtle changes in brain structure indicative of early disease progression. Functional insights are gleaned from PET scans, contributing to increased sensitivity. Additionally, longitudinal data is incorporated through Recurrent Neural Networks (RNNs) to capture the disease's temporal evolution. Training on a diverse dataset utilizes transfer learning, optimizing performance even with limited labeled data. Rigorous validation consistently demonstrates the model's effectiveness, achieving a 92% accuracy rate.

Structural and functional insights underlying recognition of histidine phosphotransfer protein in fungal phosphorelay systems

In human pathogenic fungi, receiver domains from hybrid histidine kinases (hHK) have to recognize one HPt. To understand the recognition mechanism, we have assessed phosphorelay from receiver domains of five hHKs of group III, IV, V, VI, and XI to HPt from Chaetomium thermophilum and obtained the structures of Ct_HPt alone and in complex with the receiver domain of hHK group VI. Our data indicate that receiver domains phosphotransfer to Ct_HPt, show a low affinity for complex formation, and prevent a Leu-Thr switch to stabilize phosphoryl groups, also derived from the structures of the receiver domains of hHK group III and Candida albicans Sln1. Moreover, we have elucidated the envelope structure of C. albicans Ypd1 using small-angle X-ray scattering which reveals an extended flexible conformation of the long loop αD–αE which is not involved in phosphotransfer. Finally, we have analyzed the role of salt bridges in the structure of Ct_HPt alone.

Mutations in LIFR rewire the JAK/STAT signaling pathway: A study unveiling mechanistic details of Stüve-Wiedemann syndrome

Stüve-Wiedemann syndrome (SWS), a rare autosomal recessive disorder, characterized by diminutive size, curvature of the elongated bones, bent fingers, episodes of heightened body temperature, respiratory distress or periods of breath-holding, and challenges with feeding, especially causes fatality in infants. SWS is an outcome of potential missense mutations in the leukemia inhibitory factor receptor gene reflected as numerous amino acid mutations at protein level. Employing in silico tools and techniques like mutational screening with Pred_MutHTP, I-Mutant2.0, PANTHER.db, PolyPhen, to classify mutations as deleterious/destabilizing, in conjunction with experimental data analysis, P136A and S279P emerged as ‘effect’-causing mutations. Pre-existing knowledge suggests, SWS progression is effectuated conformationally altered and dysfunctional LIFR, unable to bind to LIF and further form the LIF/LIFR/gp130 signalling complex. To gain functional insights into the effect of the said mutations on the wild type protein, an all-atom, explicit, solvent molecular dynamics simulation was performed following docking approaches. Consequently, referring to the RMSD, RMSF, protein dynamic network analysis, energy landscape plots and domain motion analysis, it was revealed that unbound LIFR_WT was more prone to LIF binding as usual whereas the mutants exhibited considerable domain closure to inhibit LIF binding. We conducted binding affinity analysis via MM/GBSA and dissociation constant estimation after LIFR-LIF docking and found the WT_complex to be more stable and compact as a whole when compared to the flexible mutant complexes thus being associated with SWS. Our study offers a route for understanding molecular level implications upon LIFR mutations which opens an avenue for therapeutic interventions.

International Union of Basic and Clinical Pharmacology CXV: The Class F of G Protein-Coupled Receptors.

The class F of G protein-coupled receptors (GPCRs) consists of 10 Frizzleds (FZD1-10) and Smoothened (SMO). FZDs bind and are activated by secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family, and SMO is indirectly activated by the Hedgehog (Hh) family of morphogens acting on the transmembrane protein Patched. The advance of our understanding of FZDs and SMO as dynamic transmembrane receptors and molecular machines, which emerged during the past 14 years since the first-class F GPCR IUPHAR nomenclature report, justifies an update. This article focuses on the advances in molecular pharmacology and structural biology providing new mechanistic insight into ligand recognition, receptor activation mechanisms, signal initiation, and signal specification. Furthermore, class F GPCRs continue to develop as drug targets, and novel technologies and tools such as genetically encoded biosensors and CRISP/Cas9 edited cell systems have contributed to refined functional analysis of these receptors. Also, advances in crystal structure analysis and cryogenic electron microscopy contribute to the rapid development of our knowledge about structure-function relationships, providing a great starting point for drug development. Despite the progress, questions and challenges remain to fully understand the complexity of the WNT/FZD and Hh/SMO signaling systems. SIGNIFICANCE STATEMENT: The recent years of research have brought about substantial functional and structural insight into mechanisms of activation of Frizzleds and Smoothened. While the advance furthers our mechanistic understanding of ligand recognition, receptor activation, signal specification, and initiation, broader opportunities emerge that allow targeting class F GPCRs for therapy and regenerative medicine employing both biologics and small molecule compounds.

Clusters of mammalian conserved RNA structures in UTRs associate with RBP binding sites.

RNA secondary structures play essential roles in the formation of the tertiary structure and function of a transcript. Recent genome-wide studies highlight significant potential for RNA structures in the mammalian genome. However, a major challenge is assigning functional roles to these structured RNAs. In this study, we conduct a guilt-by-association analysis of clusters of computationally predicted conserved RNA structure (CRSs) in human untranslated regions (UTRs) to associate them with gene functions. We filtered a broad pool of ∼500 000 human CRSs for UTR overlap, resulting in 4734 and 24 754 CRSs from the 5' and 3' UTR of protein-coding genes, respectively. We separately clustered these CRSs for both sets using RNAscClust, obtaining 793 and 2403 clusters, each containing an average of five CRSs per cluster. We identified overrepresented binding sites for 60 and 43 RNA-binding proteins co-localizing with the clustered CRSs. Furthermore, 104 and 441 clusters from the 5' and 3' UTRs, respectively, showed enrichment for various Gene Ontologies, including biological processes such as 'signal transduction', 'nervous system development', molecular functions like 'transferase activity'and the cellular components such as 'synapse' among others. Our study shows that significant functional insights can be gained by clustering RNA structures based on their structural characteristics.

DPP2/7 is a Potential Predictor of Prognosis and Target in Immunotherapy in Colorectal Cancer: An Integrative Multi-omics Analysis.

Colorectal cancer (CRC) ranks among the leading causes of cancerrelated deaths. This study aimed to illuminate the relationship between DPP7 (also known as DPP2) and CRC through a combination of bioinformatics and experimental methodologies. A multi-dimensional bioinformatic analysis on DPP7 was executed, covering its expression, survival implications, clinical associations, functional roles, immune interactions, and drug sensitivities. Experimental validations involved siRNA-mediated DPP7 knockdown and various cellular assays. Data from the Cancer Genome Atlas (TCGA) identified high DPP7 expression in solid CRC tumors, with elevated levels adversely affecting patient prognosis. A shift from the N0 to the N2 stage in CRC was associated with increased DPP7 expression. Functional insights indicated the involvement of DPP7 in cancer progression, particularly in extracellular matrix disassembly. Immunological analyses showed its association with immunosuppressive entities, and in vitro experiments in CRC cell lines underscored its oncogenic attributes. DPP7 could serve as a CRC prognosis marker, functioning as an oncogene and representing a potential immunotherapeutic target.

Structural and functional insights into the 2′-O-methyltransferase of SARS-CoV-2

A unique feature of coronaviruses is their utilization of self-encoded nonstructural protein 16 (nsp16), 2′-O-methyltransferase (2′-O-MTase), to cap their RNAs through ribose 2′-O-methylation modification. This process is crucial for maintaining viral genome stability, facilitating efficient translation, and enabling immune escape. Despite considerable advances in the ultrastructure of SARS-CoV-2 nsp16/nsp10, insights into its molecular mechanism have so far been limited. In this study, we systematically characterized the 2′-O-MTase activity of nsp16 in SARS-CoV-2, focusing on its dependence on nsp10 stimulation. We observed cross-reactivity between nsp16 and nsp10 in various coronaviruses due to a conserved interaction interface. However, a single residue substitution (K58T) in SARS-CoV-2 nsp10 restricted the functional activation of MERS-CoV nsp16. Furthermore, the cofactor nsp10 effectively enhanced the binding of nsp16 to the substrate RNA and the methyl donor S-adenosyl-l-methionine (SAM). Mechanistically, His-80, Lys-93, and Gly-94 of nsp10 interacted with Asp-102, Ser-105, and Asp-106 of nsp16, respectively, thereby effectively stabilizing the SAM binding pocket. Lys-43 of nsp10 interacted with Lys-38 and Gly-39 of nsp16 to dynamically regulate the RNA binding pocket and facilitate precise binding of RNA to the nsp16/nsp10 complex. By assessing the conformational epitopes of nsp16/nsp10 complex, we further determined the critical residues involved in 2′-O-MTase activity. Additionally, we utilized an in vitro biochemical platform to screen potential inhibitors targeting 2′-O-MTase activity. Overall, our results significantly enhance the understanding of viral 2′-O methylation process and mechanism, providing valuable targets for antiviral drug development.

Unveiling the Role of Direct Electron Transfer and Secondary Radicals in Electrooxidation of Tetrabromobisphenol A: Identification of Intermediates and Density Functional Theory Insights

Unveiling the Role of Direct Electron Transfer and Secondary Radicals in Electrooxidation of Tetrabromobisphenol A: Identification of Intermediates and Density Functional Theory Insights

Structural and functional insights into the helicase protein E5 of Mpox virus

Mpox virus (MPXV) can cause mpox in humans. Due to its quick and wide spread in the past two years, mpox has turned into a significant public health concern. Helicase E5 is a multi-domain protein; its primer synthesis and DNA unwinding activity are required for genome uncoating and DNA replication of MPXV. However, the in vitro DNA unwinding activity has never been demonstrated. Here, we report the structural and biochemical studies of MPXV E5, showing that the full-length protein adopts an auto-inhibited conformation. Truncation of the N-terminus can recover the in vitro unwinding activity of E5 towards the forked DNA. Further structural analysis reveals that MPXV E5 shares a conserved mechanism in DNA unwinding and primer synthesis with the homologous proteins. These findings not only advance our understanding on the function of MPXV E5, but also provide a solid basis for the development of anti-poxvirus drugs.

A symbiont fungal effector relocalizes a plastidic oxidoreductase to nuclei to induce resistance to pathogens and salt stress

The root endophytic fungus Serendipita indica establishes beneficial symbioses with a broad spectrum of plants and enhances host resilience against biotic and abiotic stresses. However, little is known about the mechanisms underlying S.indica-mediated plant protection. Here, we report S.indica effector (SIE) 141 and its host target CDSP32, a conserved thioredoxin-like protein, and underlying mechanisms for enhancing pathogen resistance and abiotic salt tolerance in Arabidopsis thaliana. SIE141 binding interfered with canonical targeting of CDSP32 to chloroplasts, leading to its re-location into the plant nucleus. This nuclear translocation is essential for both their interaction and resistance function. Furthermore, SIE141 enhanced oxidoreductase activity of CDSP32, leading to CDSP32-mediated monomerization and activation of NON-EXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1), a key regulator of systemic resistance. Our findings provide functional insights on how S.indica transfers well-known beneficial effects to host plants and indicate CDSP32 as a genetic resource to improve plant resilience to abiotic and biotic stresses.

Catalyzing precision: unraveling the diagnostic conundrum of tunisian familial hypophosphatasia case through integrative clinical and molecular approaches.

Familial Hypophosphatasia presents a complex diagnostic challenge due to its wide-ranging clinical manifestations and genetic heterogeneity. This study aims to elucidate the molecular underpinnings of familial Hypophosphatasia within a Tunisian family harboring a rare c.896 T > C mutation in the ALPL gene, offering insights into genotype-phenotype correlations and potential therapeutic avenues. The study employs a comprehensive approach, integrating biochemical examination, genetic analysis, structural modeling, and functional insights to unravel the impact of this rare mutation. Genetic investigation revealed the presence of the p.Leu299Pro mutation within the ALPL gene in affected family members. This mutation is strategically positioned in proximity to both the catalytic site and the metal-binding domain, suggesting potential functional consequences. Homology modeling techniques were employed to predict the 3D structure of TNSALP, providing insights into the structural context of the mutation. Our findings suggest that the mutation may induce conformational changes in the vicinity of the catalytic site and metal-binding domain, potentially affecting substrate recognition and catalytic efficiency. Molecular dynamics simulations were instrumental in elucidating the dynamic behavior of the tissue-nonspecific alkaline phosphatase isozyme (TNSALP) in the presence of the p.Leu299Pro mutation. The simulations indicated alterations in structural flexibility near the mutation site, with potential ramifications for the enzyme's overall stability and function. These dynamic changes may influence the catalytic efficiency of TNSALP, shedding light on the molecular underpinnings of the observed clinical manifestations within the Tunisian family. The clinical presentation of affected individuals highlighted significant phenotypic heterogeneity, underscoring the complex genotype-phenotype correlations in familial Hypophosphatasia. Variability in age of onset, severity of symptoms, and radiographic features was observed, emphasizing the need for a nuanced understanding of the clinical spectrum associated with the p.Leu299Pro mutation. This study advances our understanding of familial Hypophosphatasia by delineating the molecular consequences of the p.Leu299Pro mutation in the ALPL gene. By integrating genetic, structural, and clinical analyses, we provide insights into disease pathogenesis and lay the groundwork for personalized therapeutic strategies tailored to specific genetic profiles. Our findings underscore the importance of comprehensive genetic and clinical evaluation in guiding precision medicine approaches for familial Hypophosphatasia.