TP53 gene is mutated in approximately half of all human malignancies, including those of the breast, colon, lung, liver, prostate, bladder, and skin. When DNA damage occurs, the TP53 gene on human chromosome 17 stops the cell cycle. If p53 protein is mutated, the cell cycle is unrestricted and the damaged DNA is replicated, resulting in uncontrolled cell proliferation and cancer tumours. Tumor-associated p53 mutations are usually associated with phenotypes distinct from those caused by the loss of the tumor-suppressing function exerted by wild-type p53protein. Many of these mutant p53 proteins have oncogenic characteristics, and therefore modulate the ability of cancer cells to proliferate, escape apoptosis, invade and metastasize. Because p53 deficiency is so common in human cancer, this protein is an excellent option for cancer treatment. In this review, we will discuss some of the molecular pathways by which mutant p53 proteins might perform their oncogenic activities, as well as prospective treatment methods based on restoring tumor suppressive p53 functions.The p53 protein is a transcription factor known as the "guardian of the genome" because of its critical function in preserving genomic integrity. The  TP53 gene on human chromosome 17. The p53 protein is posited to inhibit the phenotypic and genomic alterations associated with cancer development through a complex interplay with several signaling pathways known to play critical roles in essential cellular processes such as cell division, maintenance of genomic stability, apoptosis, autophagy, immune response, and regulation of tumor microenvironment (TME) . T. T126]. TLR) gene expression in T-lymphocytes and to a lesser extent in macrophages in a way that is dependent on genetic stress and the host genetic background [Wild-type p53 controls Toll-like receptor  induce illness. Targeting senescent cells through SASP regulation, or cellular reprogramming could be a new therapeutic path for cancer and age-related illnesses like neurodegeneration, pulmonary fibrosis, and renal failure. The A repair .TP53 and RB2/P130 [P53 and RB2/P130 were identified as the major regulators of senescence in human MSCs [ELK1 could be targeting the promoters of RB2/P130 . This fiman MSCs –146.In the context of senescence, p53 plays a critical role in deciding the fate of cells, and its activation can be DDR-dependent or DDR-independent . In the The role of DDR activation as a necessary and causal element in p53 activation and senescence induction has lately been questioned. Many recent investigations have shown that many OIS routes can really activate p53 without going through the DDR as in fact they might occur throughAKT via downregulation of MnSOD, through the onco-suppressor PTEN depletion, prompting mTORC1 and mTORC2 to bind to p53 instead of MDM2, and MAPK p38 by direct phosphorylation of p53 . These fBecause wild-type p53 is an efficient promoter of apoptosis and senescence  in tumorSeveral studies have shown that transfection of cancer cells with wild-type p53 expressing plasmids can induce apoptosis and/or growth arrest, implying that a gene therapy method for cancer treatment could be based on restoring normal p53 expression and function. Several clinical research investigations using viral and non-viral vectors delivering p53 genes, alone or combined with other therapeutic agents, have been completed to far .Some tumor-derived mutations that cause wild-type p53 loss-of-function can be restored by other point mutations that help stabilize the p53 protein, indicating that the structural change is reversible . PhiKan0When zinc is added to the structural mutants G245C and G245D, the wild-type structure is for the most part restored  As a conCompounds like APR-246, PK11007, and COTI-2 are promising treatments for patients with trible negative (TN) breast tumours because p53 is mutated in the great majority of them. However,mutant p53 can work as a biomarker in breast cancer, is not clearly defined .Critical Outcome Techonologies Inc.'s (COTI-2) third-generation thiosemicarbazone, as well as particular peptides, have recently been shown to convert reactive mutant p53 protein to a form with wild-type characteristics. These drugs have been demonstrated to have anticancer action in preclinical models expressing mutant p53, which is consistent with the reactivation of mutant p53. Two of these drugs, APR-246 and COTI-2, have made it to clinical trials so far. APR-246 had no major side effects in a phase I/IIa clinical trial. APR-246 is now being tested in patients with advanced serous ovarian cancer in a phase Ib/II trial, while COTI-2 is being tested in patients with advanced gynaecological tumours in a phase I trial. However, whether any mutant p53 reactivating chemical is effective in the therapy of human cancer remains to be seen .Shenzhen SiBiono GeneTech Co. Ltd. produced Gendicine (recombinant human p53 adenovirus), which was approved by the China Food and Drug Administration (CFDA) in 2003 as a first-in-class gene therapy treatment to treat head and neck cancer and went on the market in 2004. Gendicine is a biological therapy that can be administered via intratumoral injection, intracavity infusion, or intravascular infusion. Depending on the cellular stress circumstances, the wild-type (wt) p53 protein expressed by Gendicine-transduced cells is a tumour suppressor that is triggered by cellular stress and promotes cell-cycle arrest and DNA repair, or produces apoptosis, senescence, and/or autophagy. Based on more than 30 published clinical trials and 12 years of commercial use in over 30,000 patients, Gendicine has a proven track record of safety, and when combined with chemotherapy and radiotherapy, it has shown to produce much higher response rates than traditional therapies alone. In addition to head and neck cancer, Gendicine has been used to successfully treat a variety of other cancer kinds and stages. Thirteen published trials with long-term survival data found that Gendicine combination regimens result in considerably longer progression-free survival periods than standard treatments alone. Despite the fact that the p53 gene is mutated in more than half of all human malignancies, the presence of a p53 mutation had no effect on efficacy or long-term survival in Ad-p53-treated patients .The growing understanding of mutant p53 actions has contributed to the identification of a number of compounds with promising therapeutic potential. However, further experiments are required to fully characterize mutant p53 function in cancer. The fact that mutant p53 might play a role in promoting metastasis – the primary cause of cancer-related mortality – is particularly attractive in terms of possible therapeutic applications. Although many tumors express mutant p53, it is unclear if the many mutations present on this protein have similar activity, and we might have to personalize therapy depending on the presence of a particular mutation rather than only consider whether a cancer express a wild-type vs a mutant p53 mutant.


The transcription factor p73 is a structural and functional homolog of TP53, the most famous and frequently mutated tumor-suppressor gene. The TP73 gene can synthesize an overwhelming number of isoforms via splicing events in 5′ and 3′ ends and alternative promoter usage. Although it originally came into the spotlight due to the potential of several of these isoforms to mimic p53 functions, it is now clear that TP73 has its own unique identity as a master regulator of multifaceted processes in embryonic development, tissue homeostasis, and cancer. This remarkable functional pleiotropy is supported by a high degree of mechanistic heterogeneity, which extends far-beyond the typical mode of action by transactivation and largely relies on the ability of p73 isoforms to form protein–protein interactions (PPIs) with a variety of nuclear and cytoplasmic proteins. Importantly, each p73 isoform carries a unique combination of functional domains and residues that facilitates the establishment of PPIs in a highly selective manner. Herein, we summarize the expanding functional repertoire of TP73 in physiological and oncogenic processes. We emphasize how TP73’s ability to control neurodevelopment and neurodifferentiation is co-opted in cancer cells toward neoneurogenesis, an emerging cancer hallmark, whereby tumors promote their own innervation. By further exploring the canonical and non-canonical mechanistic patterns of p73, we apprehend its functional diversity as the result of a sophisticated and coordinated interplay of: (a) the type of p73 isoforms (b) the presence of p73 interaction partners in the cell milieu, and (c) the architecture of target gene promoters. We suppose that dysregulation of one or more of these parameters in tumors may lead to cancer initiation and progression by reactivating p73 isoforms and/or p73-regulated differentiation programs thereof in a spatiotemporally inappropriate manner. A thorough understanding of the mechanisms supporting p73 functional diversity is of paramount importance for the efficient and precise p73 targeting not only in cancer, but also in other pathological conditions where TP73 dysregulation is causally involved. In bony fishes, the p63/p73 and the p53 genes are separated into distinct entities and undergo positive selection to control different processes: p63 got specialized in the production of epithelial cells and p73 in neuronal development, whereas p53 is better adapted as a tumor suppressor , the core DNA-binding domain (DBD) and the oligomerization domain (OD), with DBD being the most conserved domain. They have an extra SAM  domain in their C-terminus, which confers protein stability . Althougppressor . These frganisms . These cN classes of isoforms, (b) alternative splicing in the 5′ end, resulting in amino-truncated ΔTA isoforms  that partially or entirely lack the transactivation domain and, together with ΔN, constitute the so called DN isoforms, (c) alternative splicing in the 3′ end, putting forth several C-terminal splice variants   use of an extrinsic (P1) and alternative intrinsic promoter (P2) in the 5′ end, generating TA and Δ, and θ)   . We also, and θ) . In the , and θ) , while D, and θ) . In cont, and θ) . Using k, and θ) . The ΔTA, and θ)  and, anaitiation  and cancitiation . For insitiation . Consistitiation . For a dper se. Furthermore, the p53/p63 cell content often influences p73 functions, because p73 can form stable hetero-oligomers with p63 or mutant p53  cells in the developing brain. Hippocampal dysgenesis appears to be a consequence of deprivation of CR cells that are physiologically deputed to direct brain architecture during embryonic development. These effects appear to be highly isoform-dependent , which are vital for respiration, neurogenesis and fertility. Moreover, TAp73 shows a significant ability to regulate cellular metabolism. The abovementioned physiological functions of p73 in these organ systems have been extensively reviewed elsewhere . BesidesLast but not least, several lines of evidence imply a key role of TP73 protein products in the differentiation and homeostasis in several types of muscle tissues. In skeletal muscles, TAp73α but not TAp73β isoform suppresses myogenic differentiation , while ΔOncogenic transformation occurs through progressive acquisition of key adaptations, the so-called cancer hallmarks, which include sustaining proliferative signaling, resisting cell death, evading growth suppressors, activating invasion, enabling replicative immortality, inducing angiogenesis, reprogramming energy metabolism and evading immune destruction. Transformation is further facilitated by tumor-promoting inflammation and genome instability. Strikingly, TAp73 isoforms inhibit all these hallmarks [reviewed in detail in It is noteworthy that in some cancer-related processes and cell-contents, TAp73 isoforms show an effect inconsistent with their traditional tumor suppressive function. For example, TAp73 activates anabolic pathways compatible with proliferation and cancer promotion by regulating glucose metabolism to control cellular biosynthetic pathways and antioxidant capacity [reviewed in Using our previous work on melanoma as a representative example of p73’s regulation of cancer outcomes via orchestrating molecular networks, we further made the striking observation that TAp73/DNp73-controlled pathways can co-ordinate extracellular changes in the TME and intracellular gene regulation by intercepting with major cellular signaling cascades which respond to growth factors in the TME. These intersections suggest that p73 isoforms internalize the information from extracellular signals that are received by cell surface receptors and convey them to the nucleus, leading to global changes in p73-transcriptome. In particular, we used the melanoma paradigm and constructed a comprehensive regulatory map by exploiting own high-throughput and experimental data from melanoma tissue culture, mouse metastasis models and patient tumor samples. These results were integrated with data from the literature, previous mathematical models describing sections of the map , and parOf particular interest is that neurodevelopmental defects and cancer-related phenotypes co-exist in TAp73 knockout and, to a much lesser extent, in ΔNp73 KO mice. The ability of p73 isoforms to modulate tumor initiation and progression may be relevant to their neurological functions. In particular, it is becoming increasingly evident that cancer and neuronal cells develop reciprocal interactions via mutual production and secretion of neuronal growth factors, neurothrophins and/or axon guidance molecules in the TME. Intriguingly, tumors can stimulate their own innervation during cancer progression, and this phenomenon is termed neoneurogenesis. Tumors produce and excrete neurogenic factors that modulate the TME and induce formation of new nerves that eventually infiltrate tumors . RecentlIt is possible that the ability of p73 isoforms to regulate several cancer hallmarks may not be independent from their neurological and/or immunomodulatory functions, but instead might imply co-options of relevant p73-governed pathways in a cancer cell context. Looking at the processes regulated by p73 isoforms in normal and cancerous tissues more closely, analogies can be found between some physiological processes and cancer hallmarks. It is well-accepted that ΔNp73 overexpression becomes a positive advantage for tumor progression due to co-option of its pro-angiogenic capacity in tumors that trigger neoangiogenesis . In an aIn view of these data, our results suggest that p73 isoforms co-regulate stemness and neurodifferentiation to control tumor progression. The tumor-specific p73ΔEx2/3 isoforms, which are established metastasis inducers and CSC regulators, have the ability to activate key neurodifferentiation players. Increased cancer stemness, together with the loss of original cell identity (de-differentiation), and the acquisition of characteristics of neuronal cell types upon p73 isoform expression, are overall indicative for their ability to switch from a melanocyte to a neuronal-like cell phenotype which would be theoretically able to foster a newly-emerged dangerous liaison between melanomas and the nervous system .In light of the overwhelming functional diversity of the products of TP73, it is reasonable to assume that the typical mode of direct transactivation/transrepression that has been described for TAp73 and DNp73’s, on its own, is not sufficient to support such high level of functional pleiotropy across several organ systems. Besides the TA/DN ratio, isoforms synthesized by other members of the p53 family, like p53 and p63, also influence p73 activity . HoweverIn this section we provide compelling evidence to support that p73-regulated functions are a result of a sophisticated combination of at least three parameters: (a) the type of p73 isoforms, (b) the presence of p73 interacting partners in the cellular milieu, and (c) the intrinsic properties of promoters of the target genes. Tissues are characterized by distinct proteomes, hence the expression of the same p73 isoform in one tissue content could lead to functional diversification, since this isoform is able to form protein–protein interactions (PPIs) with a particular set of factors that is contained in one tissue but not in another. In addition, the C-terminus of p73 determines whether PPI occurs, because even if the interactor is present in the cell, the expressed p73 splice variant may not interact with it. Furthermore, p73 isoforms form protein complexes with several other key transcriptional regulators on target gene promoters to fine-tune their transcriptional regulation. Besides, some p73 isoforms appear to have non-transcriptional functions by interacting with proteins other than transcriptional regulators outside the nucleus , suggestThe p73 protein interactome is a decisive parameter of the tissue- and/or cell context-specific p73 activity. Several proteins have been described to physically associate with p73 isoforms, by recognizing the TA domain, the DBD domain, the OD or the C-terminus. These interactions usually take place in the nucleus and regulate the transactivation activity of p73 either positively or negatively. However, in some cases the interactions occur in the cytoplasm . AnotherSeveral p73 protein binding partners induce post-translational modification, proteolytic degradation, phosphorylation-dependent activation or inhibition, acetylation or gene target co-regulation, often in a p73 C-terminus-dependent manner . Other pd13/d13 mice, in which a switch from the α to β form through knockout of exon 13 results in distinct phenotypic abnormalities that do not overlap with those of other p73 knockout mice  the yes-associated protein YAP, a phosphoprotein that interacts with a non-receptor Src tyrosine kinase encoded by the c-yes protooncogene, (b) NEDD4-like ubiquitin protein ligase 2 (NEDL2), (c) cytoplasmic tumor suppressor oxidoreductase (Wwox), and (d) E3 ubiquitin ligase Itch. YAP, NEDL2, and Wwox enhance the transcriptional activity of p73, while Itch is a YAP antagonist, that leads to p73 ubiquitination and degradation and impairs transcriptional activity  nalysis:  candidatnalysis:  candidatnalysis:  the likenalysis: .b) Tethering scenario: we checked ORTFBS in DNp73 genes that are highly increased  or decreased  and found non-overlapping groups of ORTFBS between both groups (A2 vs. B2) (∗) in a non-overlapping manner.( vs. B2) . In A2, In summary, the interplay between p73 isoforms with different C-termini, their interacting partners, and the architecture of the target gene promoter supports a high degree of heterogeneity in the mechanisms underlying tissue-specific p73-driven functions in a variety of organ systems including immunity, neurodevelopment and reproduction. Such sophisticated mechanistical patterns may explain, at least partially, the different and sometimes opposing results across different cell contents and experimental settings. Dysregulation of one or more of the above parameters in tumors could lead to tumor initiation and progression by, at least in some cases, reactivating p73-regulated differentiation programs in a spatiotemporally inappropriate manner.The main body of research on TP73 has been initially performed in the cancer setting, due to its functional and structural similarity with the tumor-suppressor TP53. In cancer, the networks controlled by the typically anti-oncogenic TAp73 isoforms offer functional redundancy to the intricate circuitries regulated by p53, through activating fully or partially overlapping pathways, which can circumvent blocks attributed to mutations in TP53 or its downstream effectors . Neverthde novo combinatorial activation of multiple distinct and developmentally distant transcriptional modules (In view of recent findings, the potential of p73 isoforms to modulate cancer initiation and progression might not be independent from the physiological functions of TP73. Instead, it could reflect the recapitulation of the same p73-regulated developmental/tissue homeostasis pathways within the cancer cell content. Specifically, a new idea proposes that metastatic transcriptional programs arise from  modules . Althoug modules . This pr modules .The functional pleiotropy of TP73 assumes existence of mechanistic heterogeneity that extends beyond its typical transactivation mode of action. The PPI-mode of action of TP73 can provide a basis for its multifunctionality and tissue specificity. We propose that multiple p73 isoforms can establish PPIs with many proteins via several motifs in the N-terminus, core domain, and C-terminus, with the C-terminus being particularly enriched in protein binding motifs and residues. In different tissues, p73 isoforms with different C-termini may have different binding partners that can be recognized by the appropriate interacting regions of 73, causing the formation of a variety of p73 coregulator complexes. The localization of these complexes can also lead to functional diversification. Thus, p73-containing complexes within the nucleus select direct and indirect p73 target genes and regulate them via composite and tethering mechanisms based on a specified promoter architecture. In addition, p73 isoforms may be able to bind to other proteins as transcriptional regulators, such as the miRNA processing complex and SAC. p73 isoforms also physically associate outside the nucleus with proteins localized in a number of subcellular organelles. The ER- and Golgi-related interactome reflects to some extent the well-established tendency of TP73 gene products to undergo post-translational modifications. However, their potential to interact directly with proteins in intracellular compartments, such as the cell membrane, lysosomes, endosomes, and mitochondria might indicate novel, non-transcription-mediated functions of p73 isoforms that are worth unveiling in the future. A comprehensive scheme of the heterogeneity of mechanisms supporting p73 functional pleiotropy and diversity is shown in From the therapeutic perspective, these new insights provide a roadmap for efficient and selective manipulation of p73 isoforms toward precision medicine. On the shoulders of structural systems pharmacology , these mSL and BP conceived the review and took the lead in writing. SM developed bioinformatics pipelines and analyzed high-throughput data. CR conducted CoIP-MS and data analysis. NM, CR, and SL performed literature search. AS, SM, and SL crafted the illustrations. All authors contributed to and approved the final manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


TP53) plays a central role in response to various cellular stresses. A variety of biological processes are regulated by p53 such as cell cycle arrest, apoptosis, senescence and metabolism. Besides these well-known roles of p53, accumulating evidence show that p53 also regulates innate immune and adaptive immune responses. p53 influences the innate immune system by secreted factors that modulate macrophage function to suppress tumourigenesis. Dysfunction of p53 in cancer affects the activity and recruitment of T and myeloid cells, resulting in immune evasion. p53 can also activate key regulators in immune signaling pathways which support or impede tumor development. Hence, it seems that the tumor suppressor p53 exerts its tumor suppressive effect to a considerable extent by modulating the immune response. In this review, we concisely discuss the emerging connections between p53 and immune responses, and their impact on tumor progression. Understanding the role of p53 in regulation of immunity will help to developing more effective anti-tumor immunotherapies for patients with TP53 mutation or depletion.As a key transcription factor, the evolutionarily conserved tumor suppressor p53 (encoded by  As an intensively studied protein, the fame of p53 mainly stemming from its role as a tumor suppressor which is activated when responding to stress signals such as genotoxic damage, or nutrient deprivation . MutatioBesides the capability of governing cellular homeostasis to curb tumourigenesis, accumulating observations suggest that p53 also plays the role in inflammatory reactions . ChronicChronic inflammation enhances the risk of cancer. As the crucial transcription factor, NF-κB is constitutively activated in most tumors. p53 and NF-κB pathways play crucial roles in response to various stresses and the NF-κB activity usually shows an antagonistic relationship with that of p53 . In contIntriguingly, the reciprocal activation of p53 and NF-κB has been also found in certain cases . It has As the most frequently genetic alterations in cancer, p53 mutations exist in over half of human cancers. However, many p53 mutants (mutp53) gain new activities to augment pro-inflammatory and survival properties, termed gain-of-function (GOF). Several studies have shown that GOF mutp53 can activate some of the NF-κB target genes . For exavia IKK, which is required for nuclear localization. At the transcriptional level, r-Roscovitine inhibits the transcription of NF-κB-regulated genes such as MCP-1, ICAM-1, COX2, FLIP, and IL-8  . The TMEIn the TME, cancer-associated fibroblasts (CAFs) play an important role in modulating tumor progression and metastasis . In CAFsOne of the most important components in TME is the extracellular matrix (ECM), which are comprised of various macromolecules that regulate cellular functions in tumors. Tumor cells manipulate the arrangement and orientation of ECM to enhance tumor progression and create a positive tumourigenic feedback loop . PreviouImmune cells are important cellular compartments in TME that are heterogeneous across tumor types and are associated with cancer progression and prognosis , b. Prod–/– mice show more susceptibility to inflammation and auto-immunity which favors tumor establishment and progression , including retinoic-acid- inducible gene I (RIG-I)-like receptors (RLRs), stimulator of IFN genes (STING) protein, and Toll-like receptors (TLRs) . The rolThe TLRs are membrane glycoproteins and previous studies reveal that p53 transcriptionally regulate several TLRs, constituting a crucial bridge between cellular stresses and TLR-induced innate immune response . Notablyin vivo via up-regulation of type I IFNs  that enhance NK cell-mediated cytotoxicity . However+ T cells, and the direct role of p53 in DC antigen presentation requires further exploration.The development of effective immunotherapies for oncology patients is now becoming a clinical reality. Notably, the interaction between T cells and DCs is developing as one of the key targets for immunotherapy. As an important sensor to activate adaptive immune responses, p53-mediated activation of innate immune cells, particularly DC, is expected to promote adaptive immunity. Although the direct effect of p53 on the function of DCs has not been clarified, many results suggest that p53 activation is necessary for DC function. Treatment with Nutlin 3, an MDM2 inhibitor that activates wild-type p53, has been reported to increase the ability of DCs to stimulate T-cell proliferation, suggesting that p53 is involved in the activation of DCs . It has via PD-L1  and cytotoxic T lymphocyte antigen 4 (CTLA4), which are important for T cell tolerance to evade immune attack . Links bia PD-L1 . Consistia PD-L1 .During the resolution of injury and infection, normal cell turnover and clearance is an important process in preventing autoimmunity and triggering immune recognition of antigens by dying cells . FailureAs mentioned above, the regulation of p53 in the tumor immune response exhibits a yin-yang balance. On the one hand, p53 counteracts pro-inflammatory factors, such as NF-κB and STAT3, to promote tissue homeostasis and avoid excessive immune responses. On the other hand, p53 contributes to the recognition of non-self antigens and thus activates anti-tumor immunity through multiple pathways. All these p53 features will allow us to develop more effective tumor therapies in combination with current immunotherapies.Cancer cells are always accompanied by unstable genetic changes and produce new antigens that distinguish cancer cells from normal cells. The accumulation of p53 hotspot mutations in cancer has been considered as immunologically active neoantigens for immunotherapy. However, progress in this field has been limited by the lack of efficiency of recognition of mutant p53 antigens in cells . A recenAlthough significant advances have been made in antitumour immune checkpoint inhibitor (ICI) therapy, only a minority of cancer patients respond well to immune checkpoint inhibitors . An effeAs a tumor suppressor, the cell-autonomous function of p53 in suppressing malignant tumors has been extensively studied. More recently, growing evidence suggest a potential link between p53 and immune function, and dysfunction of p53 is also associated with inflammatory diseases. Dysfunction of p53 in tumors is shown to regulate not only immune recognition but also affect the stromal compartment, which plays an important role in controlling tumor progression. Thus, as a “guardian of genomic integrity,” p53 also functions in response to homeostatic stress, including innate and adaptive immunity as described above. There are still many uncharacterized issues that presumably have a broad impact on immunity and inflammation, which may ultimately lead to tumor development. For instance, how exactly p53 dysregulation affects the immune response to various external or internal stimuli, and what is the role of p53 in immune cell development. Moreover, depletion or mutation of p53 is likely to reprogram the microenvironment, especially the extracellular components in tumors, but the molecular regulatory mechanisms involved remain still largely unknown. p53 mutations can promote tumor cell metastasis. How the immune regulation and response are changed during this process, and in particular which immune cells’ functions are altered. In addition, the role of p53 in the remote regulation and communication between different tissues or organs will also be a highly anticipated research direction. There is no doubt that, understanding these issues will significantly improve our knowledge of both biologic and pathologic functions of p53, allowing for the development of targeted therapeutic approaches in the future.DS and PJ conceived the manuscript. DS reviewed the literature, drafted the manuscript, and drew the figures. PJ revised the manuscript. Both authors read and approved the final manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Efficient proteostasis is crucial for somatic maintenance, and its decline during aging leads to cellular dysfunction and disease. Selective autophagy is a form of autophagy mediated by receptors that target specific cargoes for degradation and is an essential process to maintain proteostasis. The protein Sequestosome 1 (p62/SQSTM1) is a classical selective autophagy receptor, but it also has roles in the ubiquitin-proteasome system, cellular metabolism, signaling, and apoptosis. p62 is best known for its role in clearing protein aggregates via aggrephagy, but it has recently emerged as a receptor for other forms of selective autophagy such as mitophagy and lipophagy. Notably, p62 has context-dependent impacts on organismal aging and turnover of p62 usually reflects active proteostasis. In this review, we highlight recent advances in understanding the role of p62 in coordinating the ubiquitin-proteasome system and autophagy. We also discuss positive and negative effects of p62 on proteostatic status and their implications on aging and neurodegeneration. Finally, we relate the link between defective p62 and diseases of aging and examine the utility of targeting this multifaceted protein to achieve proteostatic benefits. The health and survival of an organism is reliant on efficient proteostasis, and breakdown of this process results in accumulation of toxic protein aggregates that contribute to aging and age-related diseases . The maivia the nuclear UPS machinery  are more apparent in p62 cluster formation than K48-linked cargoes, suggesting the K63-linked chains specifically triggered clustering, while K48-linked chains needed higher concentrations for clustering, which might occur during proteasomal inhibition  accounts for nearly 80% of protein degradation in the cell . p62 colThe UPS and autophagy are two major intracellular degradation routes and p62 is a key mediator of crosstalk between these pathways . ProteasLike the UPS, inhibiting autophagy also causes accumulation of p62, but this accumulation delays delivery of ubiquitinated proteins to the proteasome and thus reduces flux through the UPS . AutophaToxoplasma gondii by encapsulating them in vesicles that do not fuse with lysosomes  . p62 expression is age- and disease-dependent, characterized by a decline of expression with age and senescence in mice  and flie(ALS) ; however(ALS) .Drosophila melanogaster, but only in females that have the overexpression initiated at middle-age. This follows the endogenous expression pattern: transcript levels of p62 are increased in early adulthood, but the sharp decrease in expression after midlife can be rescued by p62 overexpression only at that stage  causes SQST-1/p62 to accumulate, leading to a decrease in lifespan  is a progressive neurodegenerative disease that destroys memory and cognitive functions and is characterized by accumulation of amyloid-β (Aβ) and hyperphosphorylated tau, causing amyloid plaques and tau tangles, respectively, in the brain. Low expression of p62 has been observed in the frontal cortex of AD patients as well as in transgenic AD mouse models; however, the remaining p62 is associated with tangles, and is believed to play an important role in tau degradation . In addiParkinson’s Disease (PD) is characterized by intracellular accumulation of Lewy bodies and Lewy neurites, which consist of p62-associated aggregated proteins, including α-synuclein, parkin, and ubiquitinated proteins . DisruptC. elegans model of ALS showed defective autophagy and increased levels of p62; however the removal of p62 alleviated the locomotion defect without restoring the autophagy defects  is characterized by ubiquitin-p62 positive intraneuronal inclusions, with increased levels of p62 in the spinal cord and motor neurons. Some cases of ALS are associated with p62 mutations in the UBA, LIR, or KIR domains. UBA domain mutations prevent interactions with ubiquitinated proteins tagged for degradation , and LIR defects , suggestFrontotemporal Lobar Degeneration (FTLD) is characterized by neuronal cytoplasmic inclusions of TDP-43, which are correlated with neurodegeneration. Overexpression of p62 leads to the mislocalization of TDP-43 to the cytoplasm, causing aggregates and neuronal death . p62 hasHuntington’s Disease (HD) is characterized by neuronal degeneration associated with a CAG repeat expansion (polyQ) in the huntingtin gene (mHTT). Early work investigating mTOR inhibition by rapamycin treatment in a HD mouse model demonstrated reduce HD pathology ; however2O2 leads to inhibition of proteasome activity and an increase in p62 expression in RPE cells. Silencing of p62 increases the accumulation of protein aggregates in RPE cells that showed decreased autophagy and Nrf2-mediated antioxidant response , leading to the accumulation of cellular waste such as lipofuscin. Oxidative stress induced by Hresponse . RPE provia autophagy induction.p62 is commonly upregulated in human tumors , and theMost degenerative diseases are characterized by protein aggregation, so naturally it would seem that targeting autophagy would be the best option to clear large aggregates. But what is the best way to induce this process when it requires coordination of multiple steps for autophagy to work? Since p62 has a multitude of roles beyond protein turnover and is also not essential for autophagy , specifiLoss of p62 leads to accelerated aging due to a decline in proteostasis, dysregulation of signaling pathways, and inability to sufficiently respond to oxidative stress. However, overexpression of p62 has potential detrimental effects, including accumulation of cellular p62-aggregates , tumorig


APOB gene. Haplotype analysis showed that these patients presented the same mutant haplotype. Moreover, there was a decrease in plasma levels of PCSK9 in affected individuals compared to the non-affected and a significant positive correlation between circulating PCSK9 and ApoB levels in all studied probands and their family members. Some of the p.(Arg490Trp) carriers suffered from diabetes, hepatic steatosis or neurological problems. In conclusion, the p.(Arg490Trp) pathogenic variant seems a cause of FHBL in patients from Lebanese origin, accounting for approximately 70% of the probands with FHBL presumably as a result of a founder mutation in Lebanon. This study is crucial to guide the early diagnosis, management and prevention of the associated complications of this disease.Familial hypobetalipoproteinemia (FHBL) is a codominant genetic disorder characterized by reduced plasma levels of low-density lipoprotein cholesterol and apolipoprotein B. To our knowledge, no study on FHBL in Lebanon and the Middle East region has been reported. Therefore, we conducted genetic studies in unrelated families and probands of Lebanese origin presenting with FHBL, in order to identify the causes of this disease. We found that 71% of the recruited probands and their affected relatives were heterozygous for the p.(Arg490Trp) variant in the  APOB gene which encodes the apolipoprotein B [PCSK9 gene [APOB gene is located on the short arm of chromosome 2 at position 2p24.1 [Familial hypobetalipoproteinemia  is the most common monogenic form of primary hypobetalipoproteinemia . It is arotein B ,7. Less SK9 gene ,6, a pron 2p24.1 . This gen 2p24.1 ,12,13. Mn 2p24.1 ,15. Thesn 2p24.1 . The firn 2p24.1 ,17.APOB gene are generally asymptomatic but present an increased risk of developing liver disease [MTP) gene [Patients who are heterozygous for mutations in the  disease . Truncat disease , and thu disease . PatientTP) gene ,11. ThesTP) gene ,11,12. LPL gene causing type I hyperlipoproteinemia [ABCA1 gene causing Tangier disease [APOB gene responsible for this phenotype, presumably as a result of a founder effect.To our knowledge, no genetic study on FHBL has been conducted in the Middle East (ME) region. In fact, the ME region is characterized by a high prevalence of consanguineous marriages, metabolic syndrome, dyslipidemia characterized by low high-density lipoprotein cholesterol (HDL-C) levels and high triglycerides levels, familial hypercholesterolemia (FH), diabetes , and nonteinemia , and mor disease . In thisIn the first family , the proIn the second family , the proIn the third family , the proIn addition to these families, four unrelated probands were also recruited. Proband 4 was a 49-year-old male who presented low LDL-C levels (0.45 mmol/L)  and was APOB gene, in which the pathogenic c.1468 C>T variation (rs771541567) has been previously reported by Burnett et al. in an extended Lebanese family living in Australia [We sequenced exon 11 of the ustralia ,17. It wustralia  considerIn the first family, sequencing of exon 11 revealed the presence of the p.(Arg490Trp) variant at the heterozygous state in the proband (II.1) and the affected relatives  and its absence in non-affected individuals (I.2 and II.3), highlighting a segregation with the FHBL phenotype in the family . In the −6 in the general population according to GnomAD.Interestingly, the amino acid arginine at position 490 is well conserved among species. The p.(Arg490Trp) variant was predicted to be disease causing on Mutation Taster (Grantham Matrix score of 101), deleterious on PROVEAN (with a score of −5.204), with probably damaging consequences on the functionality of the protein according to Polyphen-2 , and presented a CADD score of 34 suggesting that this variant is predicted to be among the top 0.1% of the most deleterious substitutions in the human genome. The allele frequency of this variant is estimated to be 7.96 × 10PCSK9 gene in which loss-of-function mutations have been associated with hypocholesterolemia [We also sequenced all the exons of the erolemia . The seqerolemia   while others presented the E3/E3 isoforms (I.1 and II.2)  . The seqnd II.2) .The relatively high frequency of the p.(Arg490Trp) variant in the present FHBL cohort and its occurrence in Lebanese families living abroad raised questions about its origin and the presence of a founder effect. Therefore, we conducted a haplotype analysis to determine whether the chromosomal background of the variation detected in some individuals is identical. The latter showed that in families 1 , 2 and 3For the HincII and PvuII markers, all heterozygous carriers presented at both sites a nucleotide substitution which is predominant compared to the wild type nucleotide, with a prevalence of 86% and 93% respectively. For the AluI marker, all heterozygous individuals did not present a polymorphism at this site, the wild type amino acid Ala was conserved, with a prevalence of 47%. For the XbaI marker, all heterozygous individuals had a nucleotide substitution, with a prevalence of almost 50%. For the MspI and EcoR1 markers, all heterozygotes had the wild type amino acid conserved. The prevalence of the wild type amino acid is respectively around 90% and 81%. These prevalences are reported in Non-Finnish European according to GnomAD and in an Austrian population reported in previous studies .p = 0.004) as well as in FHBL patients carrying the p.(Arg490Trp) variant .We measured plasma and serum PCSK9 levels in family 1 and in some of the other individuals of the present cohort, for whom plasma was available. The results are presented in APOB.This is the first study conducted to investigate the genetic causes of hypobetalipoproteinemia in Lebanon, and to measure PCSK9 levels in familial hypobetalipoproteinemia caused by a missense mutation. We searched for the p.(Arg490Trp) variant which was described for the first time in 2003 by Burnett et al. in an extended family with FHBL of Lebanese origin living in Australia ,17. ThisAPOE gene and two others for the p.Leu21dup in exon 1 of the PCSK9 gene. The presence of a single E2 allele is thought to be associated with a ≈ 10 mg/dL reduction in TC compared to people with other APOE allele combinations [PCSK9 gene was detected, we observed that there was a significant decrease in PCSK9 levels in affected individuals of family 1 compared to the unaffected. These results are consistent with the work of Fazio et al., who studied the association between plasma PCSK9 levels and low LDL-C levels in subjects with FHBL due to truncations in the ApoB protein and found that PCSK9 levels were decreased in subjects harboring a truncating mutation [Moreover, two carriers of the p.(Arg490Trp) were heterozygous for the E2 allele in the inations , and theinations . While nmutation .APOB gene are at increased risk to develop fatty liver because of a defective synthesis and export of VLDL from the liver, which causes an accumulation of TG in the hepatocytes [APOB gene variants does not seem to cause an increase in the risk of diabetes mellitus (DM) [The pathogenicity of this variant has been proven in both in vitro and in vivo functional studies ,26. It matocytes . In the atocytes ,33,34, watocytes . Relatiotus (DM) . Moreovetus (DM) ,39,40. Itus (DM) . Therefotus (DM) .Another interesting observation is that three individuals from the present cohort carrying the p.(Arg490Trp) suffered from a neurological manifestation ranging from neuropathies to Parkinson’s disease and multiple system atrophy. It is not clear whether the neurological disorders developed by these individuals are caused by FHBL or by other factors. In fact, Parkinson’s disease  and a caThree unrelated families and four unrelated probands from different regions in Lebanon were recruited in collaboration with endocrinologists and gastroenterologists at the Endocrinology and Gastroenterology Clinics of Hôtel Dieu de France hospital in Beirut upon perceiving permanent low LDL-C levels . Inclusi® analyzer .Blood samples were obtained after the subjects had fasted overnight, plasma and serum were prepared. Lipid measurements were determined on a COBAS INTEGRA® DNA Kit from Qiagen  according to the manufacturer’s instructions. The exon 11 of the APOB and all exons of the PCSK9 genes and their flanking exon-intron boundaries, as well as the region containing the two SNPs for genotyping the APOE gene [® Software.Genomic DNAs of all the participants were extracted from peripheral blood leukocytes using FlexigenePOE gene  were amphttp://gnomad.broadinstitute.org/, accessed on 7 April 2021), and for pathogenicity prediction we used Polymorphism Phenotyping version 2 , Protein Variation Effect Analyzer , Mutation Taster , and the Combined Annotation Dependent Depletion score .For frequency determination of the variant, we used the Genome Aggregation database  that modify a recognition site by a specific restriction enzyme: p.Ala618Val (AluI), p.Thr2515 (XbaI), p.Arg3638Gln (MspI), p.Glu4181Lys (EcoRI), and two occurring in intron 4 (HincII and PvuII) [These diallelic markers resulted from the following substitutions in d PvuII) . We measured plasma and serum PCSK9 levels using a commercial ELISA kit  and the Bio-Plex Pro assay technology  as previously described .APOB gene, in several Lebanese families and probands presenting with FHBL (in approximately 70% of the probands), presumably because of a founder mutation. Therefore, in the case of a patient presenting with primary hypobetalipoproteinemia or with a hepatic steatosis without any risk factors and low LDL-C levels, family investigation should be conducted in order to identify a familial aspect of the hypobetalipoproteinemia. Molecular diagnosis should be implemented by searching firstly for the p.(Arg490Trp) variant in exon 11 of the APOB gene. Moreover, these patients and their families should be advised on the risk of consanguineous marriage, especially that its prevalence in Lebanon was of 35.5% [In this article, we highlight the presence of a pathogenic variant, p.(Arg490Trp), in exon 11 of the of 35.5% , knowingof 35.5% . In conc


Importance: The protein p53 is an unequivocal tumor suppressor that is altered in half of all cancers. The immune system produces systemic p53 autoantibodies (p53 Abs) in many cancer patients. Objective: This systemic review and meta-analysis focuses on the prognostic value of p53 Abs expressed in the serum of patients with solid tumors. Data Sources: All the clinical investigations were searched on PubMed from the first study dated 1993 until May 2021 (date of submission of the manuscript). Study Selection: Studies were included that met the following criteria: (1) participants with cancer; (2) outcome results expressed in relation to the presence of a p53 antibody; (3) a primary outcome  expressed as hazard ratio (HR). The following exclusion criteria were used: (1) insufficient data available to evaluate outcomes; (2) animal studies; (3) studies with less than 10 participants. As a result, 12 studies were included in the analysis. Data Extraction and Synthesis: PRISMA guidelines were used for abstracting and assessing data quality and validity by three independent observers. The summary estimates were generated using a fixed-effect model (Mantel–Haenszel method) or a random-effect model (DerSimonian–Laird method), depending on the absence or presence of heterogeneity (I2). Main Outcome(s) and Measure(s): The primary study outcome was to determine the prognostic value of p53 Abs from a large population of patients with solid tumors, as determined before data collection. Results: In total, 12 clinical studies involving 2094 patients were included in the meta-analysis, and it was determined that p53 Abs expression in the serum significantly correlated with poorer survival outcomes of cancer patients . Conclusions and Relevance: This is the first meta-analysis proving the diagnostic utility of p53-Abs for cancer patients in predicting poorer outcomes. The serum-p53 value  may be useful for future theranostics. The P53 protein is an unequivocal tumor suppressor mutated in almost half of human cancers ,2,3,4. IMice lacking MDM2 show embryonic lethality, while the dual presence of p53 and MDM2 can rescue lethality . The p53Prognostic biomarkers have a crucial role in measuring the progression of diseases from samples of patients, such as metastasis in cancer, and they can aid clinicians in intervening with more precise medical interventions. In addition to the common theory that in humans the loss of p53 increases genomic instability, this loss has been linked to the proliferation of the stem-cell characteristic that ultimately leads to highly aggressive cancers with invasive and metastatic properties. p53 antibodies (s-p53-Abs) are stably expressed in the sera of cancer patients, and could have an important prognostic application. Many clinical studies have assessed in cancer patients the correlation between the expression of s-p53-Abs with tumor invasiveness grades, metastasis and prognosis .In our review, we performed a meta-analysis of the current literature, investigating the prognostic role of serum p53-Abs in cancer patients.p < 0.0001, 2 = 19%).After screening the article according to flow chart in The funnel plot  of the iThe meta-analysis showed that high levels of p53 antibodies significantly correlated with worse clinical outcomes. However, our study had some limitations. First, the retrospective nature of the study was intrinsically susceptible to biases. Second, different forms of solid tumors were included pre- or post-treatment with various types of therapies, as the typology requirements were at different stages. Consequently, in our analysis patients were observed independently of treatment and tumor type because of the relatively low number of randomized studies at our disposal. Third, there was a lack of follow-up with patients from different clinical trials. Thus, differences in survival probability may have been influenced by the durations of the studies. This may have given rise to different age populations, which could ultimately have affected the data. All these variables may ultimately have influenced the results.As medicine advances, studies involving greater numbers of patients could help to evaluate the impact of our findings and treatment response.In summary, p53 is a well-established tumor suppressor, and its absence is commonly found in patients diagnosed with cancer. The p53 protein has been demonstrated to be absent or mutated in approximately one out of two malignancies. It is known that p53-wt cancers have a better prognosis compared to p53-mut cancers. Our data are not in contradiction with this notion. Although both mutated and wild type p53 antibodies can be detected in cancer patients, their role is still controversial and a matter for debate. Recently, a few studies have reported that these antibodies are statistically associated with the survival of patients diagnosed with different malignancies. To the best of our knowledge, our meta-analysis is original and is the first study gathering p53 (wild type/mutated) antibody data generated from 1993 thus far. Overall, the investigation includes 12 studies and a total of 2094 patients.The studies were identified according to the following inclusion criteria: (1) participants with cancer; (2) outcome results expressed in relation to the presence of a p53 antibody; (3) a primary outcome  expressed as hazard ratio (HR). The following exclusion criteria were used: (1) insufficient data available to evaluate outcomes; (2) animal studies; (3) studies with less than 10 participants.Two independent researchers revised the included studies, and all potential disputes that could have arisen were evaluated with the corresponding author.2). A subgroup analysis was performed to highlight any differences between studies in terms of Overall Survival (OS), Disease-Free Survival (DFS), Progression-Free Survival (PFS), as summarized in The summary estimates were generated using a fixed-effect model (Mantel–Haenszel method)  or a ranWhen we used the keywords “p53 antibodies in early cancer“, p53 antibodies in metastatic cancer”, “p53 antibodies impact on cancer progression”, the PubMed search yielded 1375 potentially relevant articles. Studies as duplicates, animal studies, cellular studies, or letters to the editor or reviews were excluded. After viewing the titles and abstracts, the full texts of 34 studies were retrieved and 12 studies ,19,20,21We observed that serum antibodies generated in the blood of cancer patients against p53 (and mostly p53-wt) were deleterious. Given the straightforward detection in blood of p53 antibodies as a biomarker for cancer survival, as summarized in a simple workflow in The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in, or financial conflict with, the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.


LDLR, APOB, PCSK9, LDLRAP1 and STAP1) has been performed by next-generation sequencing (NGS). In cases where a copy number variation (CNV) has been detected by NGS, confirmation by multiplex ligation-dependent probe amplification (MLPA) has also been performed. We identified 47 causal or potentially causal (including variants of uncertain significance) LDLR and APOB variants in 44 index patients. The most common variant in the APOB gene was the c.10580G>A p.(Arg3527Gln) missense alteration, this being in accordance with literature data. Several missense variants in the LDLR gene were detected in more than one index patient. LDLR variants in the Hungarian population largely overlap with variants detected in neighboring countries.Familial hypercholesterolemia (FH) is one of the most common autosomal, dominantly inherited diseases affecting cholesterol metabolism, which, in the absence of treatment, leads to the development of cardiovascular complications. The disease is still underdiagnosed, even though an early diagnosis would be of great importance for the patient to receive proper treatment and to prevent further complications. No studies are available describing the genetic background of Hungarian FH patients. In this work, we present the clinical and molecular data of 44 unrelated individuals with suspected FH. Sequencing of five FH-causing genes ( LDLR and APOB genes are responsible for familial hypercholesterolemia, and sometimes alterations in the PCSK9 gene, but more FH-related genes have also been described, such as the LDLRAP1 and STAP1 genes. Variants in these genes have a negative effect on the proper elimination of low-density lipoproteins (LDL) from the blood, which results in an elevated LDL cholesterol (LDL-C) and total cholesterol (TC) level.Familial hypercholesterolemia FH, MIM#143890) is one of the most common autosomal, dominantly inherited diseases; it is a disorder of the cholesterol metabolism and one of the major risk factors for early arteriosclerosis and cardiovascular complications. Disease prevalence is 1:200–250. The estimated FH prevalence in Hungary was found to be 1:340 [43890 is In the case of a heterozygous alteration, the FH patient has an LDL-C level higher than 5 mmol/L, whereas homozygous or compound heterozygous alterations generally result in an LDL-C level higher than 13 mmol/L [LDLR and 250 APOB pathogenic variants have been described in the literature . No mutational hotspots have been identified in the LDLR gene; the genetic variants are spread from the promoter region to the very last exon of the gene. According to literature data, pathogenic variants occur more frequently in some regions of the gene, such as the ligand-binding domain and the epidermal growth factor-like domain.To date, over 2100 APOB gene is located on chromosome 2, consists of 29 exons and encodes 4563 amino acids. Apolipoproteins play an important role in binding the lipoprotein particle to lipoprotein receptors. One of the main roles of lipoproteins is supplying the cells with lipids and transporting the unnecessary lipids. ApoB has two forms, ApoB 100 and ApoB 48. Both forms are the product of the APOB gene, but the ApoB 48 is a truncated version of ApoB 100. ApoB 100 is produced in the liver, whereas ApoB 48 is produced in the proximal small intestines.The LDLR gene has five functional domains: an N-terminal ligand-binding region, an epidermal growth factor (EGF)-precursor homology region, a region containing O-linked sugars, a transmembrane domain, and a C-terminal cytosolic domain.The human LDLR is encoded by a gene of ~44.5 kb located on chromosome 19p13.3. The 18 exons of the gene are translated into an 860 amino acid-long type I transmembrane protein. The LDLR variants have been found in patients with familial hypercholesterolemia (FH) that can impair LDLR activity at different levels and therefore are classified according to their phenotypic behavior as follows: class 1 (no protein synthesis), class 2  and class 5 (diminished LDLR recycling capacity) [The LDLR mediates the clearance of cholesterol and cholesteryl ester-containing low-density lipoprotein (LDL) particles from blood . LDLR plOur goal was to examine the genetic background of patients with familial hypercholesterolemia in Hungary to map variants in the Hungarian population, as no such data have been available to date. Here, we describe the clinical and molecular data of 44 unrelated suspected FH patients.Patients clinically diagnosed with FH were referred to our laboratory for genetic testing from all around Hungary. Family members of index patients who tested positive were also examined when available. All participants or their legal representatives gave informed consent to genetic testing according to national regulations. Relevant clinical data were available in the case of most patients.Clinical diagnosis, whenever possible, was established by a clinical geneticist or internist based on the patient’s laboratory findings and possible phenotypic symptoms. Among the patients with a pathogenic alteration, there were 31 female and 13 male patients. The mean age of the patients was 48 years (14–78 years). The average LDL-cholesterol level was 7.2 (mmol/L) and the average total cholesterol level was 10.4 (mmol/L) in those who had at least one FH-causing variant.Genomic DNA was isolated from peripheral blood leukocytes using the QIAamp Blood Mini kit .APOB: NM_000384.3, LDLR: NM_000527.5, PCSK9: NM_174936.4, LDLRAP1: NM_015627.3, STAP1: NM_012108.4.Genes related to familial hypercholesterolemia were analyzed using the Devyser FH v2 kit . This amplicon-based library preparation kit can examine the coding region and exon/intron boundaries (minimum +/− 10 base pairs) of the following genes: Sequencing was performed on Illumina MiSeq with 300 cycles according to the manufacturer’s protocol. Raw data analysis was carried out with the NextGENe  software, with a minimum coverage requirement of 40x. Copy number variation detection was performed based on coverage data.https://primer3.ut.ee/) (accessed on 15 October 2021). Primers are listed in The detected pathogenic variants were confirmed by Sanger sequencing. Primers were designed using Primer3 software  according to the manufacturer’s protocol.All detected variants with an MAF > 0.01  in the gnomAD population database were filtered. The remaining variants were classified according to the ACMG standards and guidelines . A web-bLDLR gene surrounding the deletion to determine the suspected breakpoints  precursor homology domain, one in the O-linked sugar region, and a further 3 variants were found in the cytoplasmic domain  [We detected a pathogenic alteration in the In two index patients, we detected two missense variants , c.10438A>G, p.(Lys3480Glu)) where pathogenicity is unclear, but they are missing from population databases, therefore not present in the normal population. Furthermore, we did not detect any other alteration in the genes examined in either of the patients. According to the ACMG classification criteria, they are classified as variants of uncertain significance (VUS), so further testing is needed to decide their pathogenicity.APOB gene, the c.13242delG frameshift variant was detected. This deletion results in a premature stop codon at the 4415 amino acid position, leading to a truncated protein. As the variant is located in the last exon, it is unlikely that the consequence of it would be nonsense-mediated mRNA decay, rather, the truncated protein likely lacks 148 amino acids from its C-terminal. The variant is not present in the population databases, but ClinVar describes it as a variant of uncertain significance with a single submission of a clinical test result (ClinVar: SCV001357780). The prediction algorithms classify it as VUS/Likely Pathogenic. Downstream to this variant, other pathogenic truncating alterations have been described, which also suggests that this variant is a pathogenic one [APOB variants in details.In exon 29 of the enic one . Table 1LDLR gene in a total of 37, patients of which three patients carried variants in the compound heterozygous form .We detected 40 alterations in the LDLR gene were present in more than one index patient. The most common variant, c.1618G>A, p.(Ala540Thr), was present in four families, the c.1130G>A, p.(Cys377Tyr) change in three families, and the c.2054C>T, p.(Pro685Leu) was also detected in three families. The c.662A>G, p.(Asp221Gly), c.1775G>A, p.(Gly592Glu), c.2000G>A, p.(Cys667Tyr) and c.1865A>G, p.(Asp622Gly) alterations were detected in 2-2 families  and c.337G>T, p.(Glu113*) nonsense variants were detected in 22 families, whereas the c.1048C>T, p.(Arg350*) alteration was present in one family in heterozygous form. In patient 13, the c.337G>T, p.(Glu113*) variant was detected together with another pathogenic missense change .We detected variants resulting in a splicing defect in eight patients . Two of LDLR gene in one patient (patient 34)  ..LDLR gene in a homopolymer region consisting of five guanines. As a result of the insertion, there will be six guanines instead of five, which disrupts the reading frame, leading to a premature stop codon. The variant belongs to class three as it results in a defective receptor.The c.2416dupG, p. variant was detected in two families in a heterozygous form. This is a 1 bp insertion in exon 17 of the LDLR gene disrupts the reading frame and leads to a premature stop codon. In this patient, we also detected a second heterozygous missense change, the c.862G>A, p.(Glu288Lys).We detected the c.2167delG, p.(Glu723Argfs*7) alteration in patient 28 in a heterozygous form. This small deletion in exon 15 of the An in-frame deletion ), was detected in a single patient in our cohort .We had three patients in our cohort with a compound heterozygote genotype . The clinical symptoms were severe in all three patients, as can be seen in Patient 13 had a nonsense ) and a missense ) variant in a heterozygous form. Cascade screening revealed that the patient inherited the nonsense variant from the mother. The father had previously died because of a cardiovascular complication, so the paternal DNA sample was not available for testing .In Patient 28, we detected a missense ) and a frameshift ) heterozygous variant.The c.2167delG variant was described previously, where a patient had highly elevated TC level . Our patLDLR gene. Only one of the two short 32 bp-long homologue DNA sequences before and after exon 7 was detected in the PCR product. The copy loss of this sequence indicates that deletion of exon 7 occurred ) and deletion of the whole exon 7 (c.941-190_c.1061-270del). We performed targeted testing of the available family members as well, which proved that the two variants were inherited in trans . We haveoccurred .LDLR gene in three patients, in one case in a compound heterozygous form in the above-described patient 12.We detected a copy number variation in the In patient 38, we detected the duplication of exons 4–8 and in patient 33 we detected the deletion of exons 3–8 in a heterozygous form based on coverage from NGS data .Patients clinically diagnosed with FH were examined with a next-generation sequencing method that detects not only the small-scale variants but large-scale deletions and insertions as well.LDLR alterations (c.1706-2A>G) is described here the first time, whereas the other 39 LDLR variants have already been reported in the literature. Although the deletion of exon 7 of the LDLR gene has been described previously [LDLR variants, most of them are in the ligand-binding domain and the epidermal growth factor-like domain.One of the eviously , we haveLDLR gene in the Polish population can be found in our cohort as well [To our knowledge, no study describing the variants related to familial hypercholesterolemia in the Hungarian population has been published before. Regarding the mutation spectrum, there is a large overlap between Hungary and neighboring countries, such as Poland or the Czech Republic. The most common variants in the  as well ,26.The 1618G>A, p.(Ala540Thr) missense change was detected in four index patients , and in a further two family members during cascade testing. This pathogenic variant has been described in several European and Latin American populations, and it is a commonly occurring alteration among our patients as well. The phenotypes of our patients are similar to the ones described in the literature. This variant is located in the epidermal growth factor (EGF)-precursor homology domain. Its pathogenicity is demonstrated by the fact that, as described in the literature before, patients homozygous for this variant present with severe clinical symptoms at a young age such as premature coronary heart disease, tendon xanthomas and high total cholesterol level ,30.The c.1130G>A, p.(Cys377Tyr) missense change was detected in three families. First, it was described in a study examining Swedish FH patients . Its patA missense change, c.2054C>T, p.(Pro685Leu) was detected in three families in our cohort. According to a comprehensive study describing more than 2000 FH-causing variants, this alteration is pathogenic. It should be noted that seven alterations were found in this study which have been described in several countries on every continent, and one of these is the c.2054C>T, p.(Pro685Leu) missense change . This suThe c.1775G>A, p.(Gly592Glu) missense change was detected in two patients in a heterozygous form. This variant has been previously described in several populations and was associated with a strong founder effect in a Greek region according to a previous study, because it was present in almost one-third of the patients examined and detected in a homozygous form in several cases. According to literature data, this class 5 variant has no drastic cholesterol-elevating effect associated with its homozygous form, suggesting that it results only in a small decrease in LDLR activity .The nonsense alterations have been previously described as FH-causing pathogenic variants. These alterations result in a truncated form of the LDLR protein which is nonfunctional. The uptake of LDL particles is reduced in the liver and other tissues.LDLR gene. A luciferase-based functional assay has suggested that this variant may significantly reduce LDLR promoter activity [The c.-153C>T promoter variant has been previously detected in Czech FH patients. This variant is located in the conserved sterol-dependent regulatory element 2 (SRE2) in the promoter region of the activity .The c.940_940+14del variant has been previously described in a study examining a Scandinavian population, where it segregated with the disease in the family tested . In thatInterestingly, the c.694+2 T>C variant was previously described as a founder variant in the Icelandic population that could be traced back to the 18th century. It affects a conservative splicing site and its pathogenicity has been proved by segregation studies as well .The pathogenicity of the novel c.1706-2A>G variant is suggested by the fact that variants with other nucleotide changes have been previously identified in the same position . The c.1706-2A>T variant has been detected in two Arabic families, and further testing showed that the A>T exchange at this position is pathogenic because of the large reduction in LDLR mRNA levels .The c.2416dupG, p. variant was first described in a Swedish population study, then it appeared in Pakistani and other population studies as well. Elevated TC levels of the index patient and further eight family members were typical for heterozygous FH patients (7.76–12.93 mmol/L), whereas the 5-year-old child of the index patient had a TC level typical for homozygous FH patients (23.25 mmol/L) and had typical xanthoma [The c.654_656delTGG, p.(Gly219del) deletion has been previously described in the Ashkenazi Jewish population as a founder variant, and identified as the FH-causing variant in more than 21% of the cases . The varLDLR gene and it belongs to class 1 as it results in a null allele. It is a known pathogenic variant and was first described by Hobbs et al. [The c.337G>T, p.Glu113*) variant is in exon 4 of the 3* varians et al. .The c.862G>A, p.(Glu288Lys) missense change was first described in German patients . In ItalAPOB testing was characteristic because of the size of the APOB gene, examining the most common FH-causing variants as well [APOB gene is available, therefore more FH and other lipid metabolism disorder-related alterations have been described [Earlier, targeted  as well . Nowadayescribed .APOB gene, the most common variant in the Hungarian population is the c.10580G>A p.(Arg3527Gln) missense change, which is in accordance with literature data. By testing the whole APOB coding region, we could detect a further three variants, of which two missense alterations have not been described before, and the third frameshift variant ) has been previously reported in the ClinVar database, and is also related to FH. This result reflects on the fact that the sequencing of the whole APOB gene instead of targeted APOB testing can greatly improve the diagnostic sensitivity.In the LDLR gene, while most of the variants that are present in the Hungarian population have already been described.Our study revealed one novel variant in the The proportion of variant types and their localization within the gene are also highly similar in population studies.Indicating the importance of establishing a genetic diagnosis, the timing of initiation of lipid-lowering therapies may greatly influence the onset and severity of expected complications. According to a large study that followed therapy in children for 20 years, early treatment greatly reduced or delayed the risk of cardiovascular complications in adulthood . To init


APRT sequence variants in a diverse set of large genomic databases. A thorough search was carried out for all APRT variants that have been confirmed as pathogenic under recessive mode of inheritance, and the frequency of the identified variants examined in six population genomic databases: the deCODE genetics database, the UK Biobank, the 100,000 Genomes Project, the Genome Aggregation Database, the Human Genetic Variation Database and the Korean Variant Archive. The estimated frequency of homozygous genotypes was calculated using the Hardy-Weinberg equation. Sixty-two pathogenic APRT variants were identified, including six novel variants. Most common were the missense variants c.407T>C (p.(Met136Thr)) in Japan and c.194A>T ) in Iceland, as well as the splice-site variant c.400 + 2dup (p.(Ala108Glufs*3)) in the European population. Twenty-nine variants were detected in at least one of the six genomic databases. The highest cumulative minor allele frequency (cMAF) of pathogenic variants outside of Japan and Iceland was observed in the Irish population (0.2%), though no APRT deficiency cases have been reported in Ireland. The large number of cases in Japan and Iceland is consistent with a founder effect in these populations. There is no evidence for widespread underdiagnosis based on the current analysis.Adenine phosphoribosyltransferase deficiency is a rare, autosomal recessive disorder of purine metabolism that causes nephrolithiasis and progressive chronic kidney disease. The small number of reported cases indicates an extremely low prevalence, although it has been suggested that missed diagnoses may play a role. We assessed the prevalence of APRT deficiency based on the frequency of causally-related  Adenine phosphoribosyltransferase (APRT) deficiency  is a rare disorder of purine metabolism that is inherited in an autosomal recessive manner [APRT gene, located on chromosome 16q24, is 2466 base pairs long and contains five exons encoding a 180 amino acid protein (NP_000476.1) [The human 00476.1) . The Hum00476.1) , a T ins00476.1) . Individ00476.1) .Although the small number of reported cases worldwide indicates that APRT deficiency is an extremely rare condition, the large number of patients identified in Japan, France and Iceland has raised concerns that the prevalence may be underestimated in many populations. Furthermore, the commonly reported occurrence of delayed or missed diagnosis suggests that lack of awareness among clinicians might contribute to the low number of reported cases , 10, 11.APRT variants confirmed as disease-causing under recessive mode of inheritance that have been reported in the literature and registered in online disease databases to date, and assess the frequency of these variants in a set of large population genomic databases containing information on individuals from diverse geographic locations and ethnic groups.As whole-genome and whole-exome sequencing have become more widely available, studies on the molecular basis of rare diseases using open-access population-based data have provided further insight into their pathogenesis and allele frequency –16. The The study was approved by the National Bioethics Committee of Iceland (NBC 09-072) and the Icelandic Data Protection Authority.APRT variants in reported cases of APRT deficiency using PubMed, HGMD [Medical literature and databases. We performed a web-based search and assessement of ed, HGMD , OMIM [1ed, HGMD , and Clied, HGMD .APRT variants, we also assessed the full text of published articles, conference abstracts and book chapters identified using PubMed® (https://pubmed.ncbi.nlm.nih.gov/) and Google® (http://www.google.com), with the terms “APRT deficiency”, “adenine phosphoribosyltransferase deficiency”, “2,8-dihydroxyadenine”, “2,8-dihydroxyadeninuria” and “2,8-DHA”.Expanded web sources. To search for http://www.rarekidneystones.org). Information on APRT variants was obtained from the APRT Deficiency Registry and through personal communication to the research group. The RKSC was established in 2009 to study hereditary kidney stone disorders and has since maintained a private database with clinical information on participating individuals. As of September 2019, 63 patients from eight countries were enrolled in the APRT Deficiency Registry, 56 of whom had undergone genetic testing with the identification of biallelic disease-causing APRT variants.APRT Deficiency Registry of the Rare Kidney Stone Consortium  and transcript number (ENST00000426324.6).https://varseak.bio/).As consequence at the RNA level has not been reported for the vast majority of the variants, a prediction analysis of the variant effect at the splice junction on the splicing was carried out using consensus sequence frequencies, maximum entropy modeling and the varSEAK Online splice prediction tool   browser The Human Genetic Variation Database of Japan , 25 compThe Korean Variant Archive (KOVA)  includesWe used six databases in all, containing information on sequence variation for over 300,000 individuals from various geographic locations and ethnic groups:The minor allele frequency (MAF) of each causally-related sequence variant among different ancestries was exctracted.APRT variants, we used the sum of the allele frequency of individual variants in databases of genome and exome sequences available for a given geographic area or ethnic group. We note that each of the individual variants is rare and none are reported to be present on the same haplotype. As APRT deficiency is an autosomal recessive disease, each affected individual is expected to carry two copies of a disease-causing variant, either the same (homozygous) or different variants (compound heterozygous). The Hardy-Weinberg equation (p2 + 2pq + q2 = 1) was used to calculate the expected genotype frequencies for heterozygous (2pq) and homozygous (p2) genotypes, using the minor and total allele counts, where p is equal to the cMAF of causally-related variants and q is (1 − p).To determine the cumulative minor allele frequency (cMAF) of the reported APRT gene were identified in homozygous or compound heterozygous patients with APRT deficiency ), from Australia and the c.346G>A (p.(Ala116Thr)) missense variant from China. These variants were quite common in the databases searched; in gnomAD, the p.(Ala116Thr) variant had an allele frequency of 0.23%  in the East Asian population and p.(Arg89Gln) an allele frequency of 0.41%  in the South Asian population. However, neither of the two variants have been observed in confirmed cases of APRT deficiency and, thus, were not included in our set of 62 causally-related variants.APRT variants, 56 had already been reported in the literature. The missense variants c.407T>C (p.(Met136Thr)) and c.194A>T ) and the splice-site variant c.400 + 2dup (p.(Ala108Glufs*3)) were most commonly reported. Additionally, six novel pathogenic variants were discovered through our APRT Deficiency Research Program. A C-to-G substitution in intron 1 (c.81-3C>G) was identified in the homozygous state in two affected siblings in the US and one patient in Italy, and a C-to-T substitution in exon 1 ) was detected in a homozygous patient in the UK. Two compound heterozygous patients from the US had novel sequence variants; one of these patients had a frameshift variant in exon 1, c.23dup ), and a deletion in exon 5, c.522_524del (p.(Ser175del)), while the other had a missense variant in exon 3, c.264G>T (p.(Lys88Asn)), in addition to a variant that had already been reported. The sixth variant was identified in a patient from India who was referred to our program but had already been found to be a compound heterozygote when evaluated in his home institution, carrying a missense variant in exon 3, c.227C>T ), in addition to one previously reported variant.Of the 62 pathogenic All cases harboring the three most common variants causing APRT deficiency in a homozygous or compound heterozygous state are presented in Table The T-to-C missense variant at codon 136 in exon 5, p.(Met136Thr), has been described in 134 patients of Japanese descent, all but one of whom were previously reported in the literature. The additional case, identified in the APRT Deficiency Registry, was a Japanese patient living in the US. This pathogenic variant was also found in the Human Genetic Variation Database in Japanese cohorts from Tohoku University  and the University of Tokyo . This geographic distribution is consistent with previous reports [APRT variant in 1299 alleles, yielding a minor allele frequency (MAF) of 1.2% . Icelanders have ancestries originating from Scandinavia and the British Islands. Out of 14,904 whole-genome sequenced Scandinavian individuals at deCODE, only two heterozygotes carrying the p. variant were detected, and one single heterozygote was identified in the UK  variant have been reported. Of these, 37 individuals are from Iceland, all of whom are homozygous. One patient from Spain and one from the UK were homozygous for the same variant and two other patients were compound heterozygotes, one from France and one from Australia. On whole-genome sequencing of 53,964 Icelanders in the deCODE database, we observed this n = 29), Germany, Austria, Belgium, Italy and Poland. The variant has also been found in patients in the US (n = 6) and Australia (n = 1). The variant was consistently present in European populations in the genomic databases  and p., are clearly indicative of a founder effect in Japan and Iceland, respectively, with very low frequency in the public databases. The third variant, c.400 + 2dup, shows a wide distribution among cases from multiple European countries and the US, and is the disease-causing variant with the highest occurence in public databases. Besides these three variants, the most commonly reported ones were c.294G>A (p.(Trp98*)) in 26 Japanese cases and the c.521_523del (p.(Phe174del)) variant which was observed in six cases from European countries and three in the US.APRT variants in the deCODE database (Table Iceland is the country where the highest proportion of the population has been sequenced (around one in six Icelanders). In addition to the p. variant, we discovered five individuals heterozygous for one of the two other known pathogenic se Table . Of thesBased on the Hardy-Weinberg principle, the cMAF of p. in the Icelandic population was 1.2%, with a predicted frequency of homozygous individuals of 1 in 6840 Table . In the The overall frequency of the reported pathogenic variants in gnomAD was similar among the European (non-Finnish) population, with a calculated allele frequency of 0.05% . Frequencies of causally-related variants in Latinos and East Asians was similar to that observed in gnomAD for Europeans, whereas other groups had lower frequencies. Thus, there could be as many as 300 cases in the East Asian population (1.7 billion) and up to 170 cases in the Latin American population (642 million).APRT variants in a total of 311 patients undergoing molecular diagnosis. Six novel variants were discovered through the APRT Deficiency Registry. Three variants are by far most common among the APRT deficiency cases, namely the missense variants p.(Met136Thr) and p. and the splice variant c.400 + 2dup, collectively accounting for 64% of the disease-associated alleles.A comprehensive search for APRT deficiency cases, using multiple web-based resources and the APRT Deficiency Registry, identified 62 pathogenic Twenty-nine of the 62 pathogenic variants were detected in large population genomic databases and were assessed for the individual and cumulative frequencies. Evaluation of the frequency of the Icelandic missense variant p. in large datasets from ancestral populations in Scandinavia and UK revealed that the variant is approximately 100 times less common in these geographic locations. Thus, the high carrier rate of the variant of 1.2% in the Icelandic population clearly represents a founder effect. Not surprisingly, analysis of the large Icelandic genomic data disclosed a number of additional undiagnosed homozygous individuals.The high frequency of the missense variant p.Met136Thr) in the Japanese population is also consistent with a founder effect. A previous study assessing the frequency of p.(Met136Thr) observed an allele frequency of 0.13% (3/2326) among Japanese and 0.05% (1/1898) among Koreans, while this variant was absent in samples from Taiwanese individuals. In the same study, the geographic distribution in Japan was determined to be rather uniform [6Thr in tThe c.400 + 2dup variant was observed in cases from many countries in Europe, the US and Australia, as well as in most of the public databases used in the current study. In a report of a French APRT deficiency cohort, this variant was found in 54% of the patient population, all of whom originated from metropolitan France except for one Italian family . The allWhen calculating the cMAF of reported causally-related variants to assess the expected genotypic frequency, we found that there may be as many as 200 individuals in Japan homozygous for p.(Met136Thr) and 50 in Iceland homozygous for p.. As expected, the two described founder variants in Japan and Iceland are very rare outside of their respective countries. In the European (non-Finnish) population, the cMAF of any pathogenic variant was 0.05%. The cMAF in the Irish population was higher and is consistent with approximately 30 homozygous individuals. Interestingly, no cases have been diagnosed in Ireland to the best of our knowledge, indicating that the disease may be underreported or underdiagnosed.APRT missense variants, c.266G>A (p.(Arg89Gln)) [APRT variants. It is unclear if and to what extent individuals homozygous for these variants will develop clinical manifestations of APRT deficiency. Multiple copies of both variants were present in the genomic databases and the allele frequencies were quite high in both the East Asian and South Asian populations. If proven to be disease-causing, there could be roughly 20,000 homozygotes in South Asia and 5000 in East Asia, assuming the Hardy-Weinberg equilibrium. However, as no cases of APRT deficiency caused by these two variants have been reported, a classical phenotype appears unlikely as the disease would then be seriously underdiagnosed. Hence, homozygosity for these variants would either be expected to cause a very mild clinical phenotype or no disease at all.Two g89Gln))  and c. 3g89Gln)) , have beOf the 62 pathogenic variants, 33 were not observed in any of the public genomic databases. Most were detected in one or two cases except for c.188G>A (p.(Gly63Asp)) which was found in six patients, most of whom were of Lebanese decent. The reason for this may be the high proportion of consanguineous marriages in Lebanon, previously reported to be ~35%, and/or that Middle Eastern populations might be underrepresented in the genomic databases used in this study .We also examined sequence variants that have not been detected in patients with APRT deficiency in gnomAD, which is the largest population database with the highest allele count  in the Icelandic population clearly represents a founder effect and the same appears to hold true for the missense variant p.(Met136Thr) in Japan. While there is no definite indication of extensive underdiagnosis, this may nevertheless be the case in certain areas. It is important to recognize that genomic data are still lacking for many countries. Hence, future work should assess the frequency of pathogenic APRT variants in countries and populations where the number of expected cases is higher than currently reported. An updated list of all variants causally related to APRT deficiency will be maintained and made publicly available on the RKSC website.In conclusion, we identified 62 confirmed pathogenic Supplementary_Tables_1_To_7_Clean


The coronavirus disease 2019 (COVID-19) pandemic has had a significant impact on the mental health and wellbeing of Ontario's youth. Our study investigated the psychological impacts of COVID-19 on the pediatric population of Ontario, using a survey derived from the Revised Children's Anxiety and Depression Scale (RCADS) system to identify children who may benefit from seeking professional help. Our cross-sectional study examined the potential risk factors that contributed to worsening mental health and wellbeing in children, including changes in sleep patterns, appetite, and physical activity levels, as well as the diagnosis of a family member with COVID-19. Our study found that 24%, 9.4%, and 15.5% of participants exhibited symptoms of depression, anxiety, and obsessive-compulsive disorder (OCD), respectively, according to the RCADS system. Furthermore, there were significant associations between the presence of symptoms and the diagnosis of a family member with COVID-19 or a frontline worker in the family. This suggests a need to create interventions to support the families of frontline workers and those directly affected by a COVID-19 diagnosis. The coronavirus disease 2019 (COVID-19) pandemic has had a significant impact on the mental health of people across the whole world, including Ontario's youth . For chiThis is highlighted in a recent study by McArthur et al., where 846 mothers and children participated in a COVID-19 mental health questionnaire in Calgary, Canada [Another longitudinal study by Bignardi et al. that assessed childhood depression symptoms during the pandemic found that there was a significant increase in depression symptoms as a result of the lockdown in the UK . Mental Our study aimed to investigate the psychological impacts of COVID-19 on the pediatric population of Ontario, using a survey completed by parents. Although a survey is not an appropriate diagnostic tool for mental health conditions, our survey uses the Revised Children's Anxiety and Depression Scale (RCADS) system to identify children who may benefit from seeking professional help. Our cross-sectional study examined the potential risk factors that contribute to worsening mental health and wellbeing in children, including changes in sleep patterns, appetite, and physical activity levels, as well as the diagnosis of a family member with COVID-19.Our study is a cross-sectional study in which we asked the parents of children in grades 3 to 12 of all races and genders to fill out an anonymous online survey investigating any possible development of symptoms of anxiety, depression, and obsessive-compulsive disorder during the COVID-19 pandemic. Furthermore, our survey asked questions pertaining to noticeable behavioral changes in the child concerning aspects such as physical activity, sleep, and appetite. Children were excluded from the study if they had psychological or physical comorbidities prior to the beginning of the COVID-19 pandemic. Parents were asked to provide informed consent on behalf of the child prior to filling out the survey. Anonymity was preserved through a lack of personal identifying information. Our survey was a standardized survey that was derived from the Revised Children’s Anxiety and Depression Scale (RCADS) that is available for use at no cost, given in a Likert scale, and designed using Cognito Forms  (Appendix 1). A poster with an easily accessible link and QR code that took parents to the survey form was used to recruit participants (Appendix 2). The poster was shared on various social media sites including Facebook and Instagram. Research Ethical Board approval was obtained through the Scarborough Health Network . The data collection period was July 8 to October 8, 2021, and we were able to recruit 246 children in total.To contextualize this time period, by early July in 2021, the province had seen continued improvement in key public health and health care indicators such as hospitalizations, ICU occupancy, and the weekly cases incidence rates . On JulyThe demographic characteristics of the participants are presented in Table A total of 246 children participated in this study. The majority of participants were male (52.9%), in Grade 3 (14.23%), had no personal or family COVID-19 diagnosis (83.74%), and had no frontline workers in the family (59.35%).Depression symptoms2 (n = 59) = 49.358, p = 0.014) or a frontline worker in the family (X2 (n = 59) = 45.004, p = 0.039). The difference in depressive symptoms between genders was nonsignificant (X2 (n = 59) = 36.549, p = 0.191).Twenty-four percent  of participants were found to be positive for symptoms of depression using the RCADS clinical cut-off of T-scores over 65. As shown in Table Anxiety symptoms2 (n = 23) = 52.733, p = 0.000) or a frontline worker in the family (X2 (n = 23) = 35.600, p = 0.017; see Table 2 (n = 23) = 26.403, p = 0.153)  developed symptoms of anxiety using the RCADS clinical cut-off of T-scores over 65. The presence of a personal or family diagnosis of COVID-19 (X developeOCD symptoms2 (n = 38) = 55.596, p = 0.000) and a frontline worker in the family (X2 (n = 38) = 48.453, p = 0.000; see Table 2 (n = 38) = 20.919, p = 0.191)  of participants had symptoms of anxiety. Significant associations were found between anxiety symptoms and the presence of a personal or family diagnosis of COVID-19 (XThis cross-sectional study aimed to assess the impact of the COVID-19 pandemic on Ontario’s pediatric population by examining potential risk factors. We discovered that there were significant differences in the development of children’s symptoms of depression, anxiety, and OCD when associated with the child having family members working on the frontline or if there was a positive COVID-19 diagnosis in the family. Gender was not a significant risk factor for the development of anxiety, depression, or OCD symptoms in children during COVID-19.Our findings are consistent with other studies that also suggest the rise of youth mental health difficulties during the COVID-19 pandemic . This maCost et al. examined COVID-19 exposure as a potential risk factor for pediatric mental health concerns . HoweverThe association found between the development of pediatric mental health disorders and the chronic and acute stressors associated with frontline work is alarming, suggesting a heightened need to create effective interventions to help frontline workers cope with and adequately support their children and families. Perhaps this is indicative of a weakness in the Canadian response to the pandemic. Efforts must be made by workplaces when implementing occupational health measures to look beyond people’s function as frontline responders and to adopt a more holistic approach that takes into account their societal roles as parents, spouses, and offspring. A study among Moroccan healthcare workers explores the effects of how 40% of healthcare workers marry each other, causing a “childcare crisis” that causes disruption to traditional solidarity systems, potentially exposing children of healthcare workers to additional emotional challenges caused by the absence of both parents .This prompts one to consider the state of childcare for frontline workers in Canada. As of March 17, 2020, licensed child care centers were closed, aside from select locations to support health care and other frontline workers. Then, from June 12, 2020, child care centers were allowed to reopen throughout the province . It was On the other hand, the findings of our study may hint at something greater than an infrastructure issue such as parents being ill-equipped to face the novel challenges posed by parenting due to the pandemic. In a few studies, it was shown that a rise in parental stress levels is associated with harsher parenting while perceived control over stressful environments while supportive family environments act as a buffer to decrease stress -22. COVIThis study has several limitations. The study design was cross-sectional, preventing us from making causal inferences on the relationship between frontline worker families, families with a positive COVID-19 diagnosis, and symptoms of pediatric mental health disorders. Longitudinal research is required to evaluate these potential risk factors and capture their effect in light of changing COVID-19 restrictions. The RCADS scale used in this study while a validated screening tool for symptoms of depression, anxiety, and OCD cannot ascertain cases with certainty. A clinician needs to be present to review each individual case in order to make a diagnosis. Another limitation of the study was that the survey collected limited data regarding participant characteristics, preventing a more holistic examination of sociodemographic risk and protective factors for the development of pediatric mental health concerns. Additionally, while advertisements were shared in networks serving Ontarians and participants were required to self-identify as Ontario residents, it cannot be said with certainty that all respondents were from Ontario exclusively. While the results were consistent with the general literature, our findings cannot be broadly generalized. Finally, our survey was only administered in English, which excludes the experiences of non-English-speaking parents in Ontario. However, as of 2016, only 2.5% of Ontarians were found to be non-English speaking so perhaps these results may still be generalizable to the Ontario population .Despite these limitations, our findings illustrate the important associations of COVID-19 diagnosis exposure, frontline workers within families, and pediatric mental health disorders. Our findings have widespread implications for prevention and intervention programming such as developing the infrastructure to provide frontline workers with more flexibility and access to childcare supports. This may include increasing flexibility for workers with families and reducing barriers to accessing care for these vulnerable populations such as subsidizing transportation costs and expanding no-cost emergency childcare programs. In addition, the findings imply that parents may benefit from public health messaging and improved family-centered healthcare that better equips them with the difficulties of raising children in the context of a global pandemic. Finally, our study suggests that there is a gap in the literature regarding the effects of occupational stress on frontline workers' and their families' well-being, warranting further research.In conclusion, this study examined the psychological impact of COVID-19 diagnosis exposure and family members who are frontline workers on a pediatric sample in Ontario. Institutions with frontline workers and parents will benefit from knowing about the potential downstream impacts of occupational stress on children and adolescents. In addition, public health and education agencies may benefit from a greater understanding of stressors affecting children, allowing integration into intervention programs. The current research provides preliminary insight into the myriad of factors affecting pediatric individuals.