Clinical and Genetic Analysis of Li-Fraumeni Syndrome with Novel TP53 Mutations

Clinical and Genetic Analysis of Li-Fraumeni Syndrome with Novel TP53 Mutations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clinical and Genetic Analysis of Li-Fraumeni Syndrome with Novel TP53 Mutations Yiping Tian, Zhengxiao Ma, Conghui Wu, Xu Chen, Zhuo Yue, Lisha Ying, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3811434/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective Mutations in the TP53 gene can cause Li-Fraumeni syndrome (LFS), an autosomal dominant genetic syndrome that increases susceptibility to various tumors. This study aims to explore the clinical and pathological features as well as the genetic characteristics of LFS to provide a theoretical basis for genetic counseling in affected families. Methods We conducted a retrospective analysis of clinical data and family history in three LFS cases with TP53 germline mutations. High-throughput sequencing technology was used to screen for hereditary tumor-related genes in the probands, and Sanger sequencing was used to confirm and analyze candidate pathogenic variant sites in their family members. Results Three different types of TP53 mutation variants were found in our study. The first family, spanning four generations and consisting of 30 individuals, included 9 adults diagnosed with 8 different types of cancer. Genetic testing revealed the TP53 c.642_643delTA p.H214Qfs*7 mutation in this family, showing that the age of onset tended to become younger in successive generations. The second family, with two patients having four different malignant tumors, carried the TP53 c.742C > T p.R248W mutation. This family had an average diagnosis age younger than the first family. The third proband, a 13-year-old boy, carried the TP53 c.844C > T p.R282W mutation and had no family history, indicating that this may be a new TP53 germline mutation in his family. Conclusion Our study identified and reported the pathogenic variant TP53 p.H214Qfs*7 frameshift mutation for the first time, expanding the mutation spectrum of the TP53 gene. We recommend timely genetic counseling and TP53 germline mutation testing for patients with childhood tumors or multiple familial tumors. Systematic monitoring of individuals carrying these mutations is crucial for early intervention to prevent primary and secondary tumors. TP53 mutations Li-Fraumeni syndrome Genetic Counseling Hereditary Tumors Figures Figure 1 Figure 2 Figure 3 1. Introduction LFS is a rare autosomal dominant hereditary cancer susceptibility syndrome characterized by a heightened risk of tumor development and an early onset of malignancies. Individuals with LFS have a 24-fold increased probability of developing cancer compared to the general population, with the highest incidence occurring before the age of 30. The median age of initial cancer diagnosis for females is approximately 31 years, while for males, it is around 46 years. Many LFS patients experience multiple primary tumors throughout their lives [ 1 ] . Common tumor types associated with LFS include soft tissue sarcomas, osteosarcomas, brain and central nervous system (CNS) tumors, adrenal cortical carcinomas, acute leukemias, and breast cancer. Various diagnostic criteria exist for LFS, with the Chompret criteria [ 2 , 3 , 4 ] being the most widely utilized. Given the elevated cancer risk and heterogeneity associated with this condition, current recommendations for cancer screening in LFS patients revolve around multimodal, high-frequency evaluations centered on whole-body MRI. Research has demonstrated that cancer screening in individuals with this syndrome can improve survival rates, but excessive screening may lead to patient anxiety, uncertainty, and increased treatment burden [ 5 ] . TP53 germline mutations have been identified in approximately 70% of LFS patients, making it the sole known genetic cause of LFS. The gene of TP53 is located on chromosome 17p13.1 and serves as a crucial tumor suppressor gene involved in various biological processes, including DNA repair, cell cycle regulation, and apoptosis. When the TP53 gene undergoes mutation, cells lose their ability to effectively repair DNA damage, consequently increasing the risk of cancer. TP53 variants impact different functions of the p53 protein, thereby affecting its tumor-suppressing activity. TP53 variants can be categorized based on their functional consequences: those associated with loss of function (LOF) resulting in haploinsufficiency, those conferring gain of function (GOF) to the p53 protein, and those linked to dominant negative effects (DNE) and impaired transactivation activity [ 6 ] . Carriers of pathogenic or potentially pathogenic germline TP53 variants exhibit considerable variability in cancer phenotype, penetrance, and expressivity. Few genotype-phenotype studies have assessed how the functional properties of TP53 variants influence cancer risk. TP53 gene mutations represent the primary pathogenic factor in LFS, with their diverse mutation forms contributing to phenotypic variations among LFS patients. In our study, we delving into the clinical and genetic characteristics of three LFS patients and their families. Combined with high-throughput sequencing and Sanger sequencing technology, we conducted genotype-phenotype correlations as well as tumor analyses, providing further genetic evidence for the diagnosis of LFS. The results of this study not only enrich the spectrum of TP53 gene mutations but also offer a more comprehensive genetic counseling, monitoring, and treatment strategy for LFS patients and their families, better addressing the complexity and challenges posed by this syndrome. In the following sections, we will provide a detailed overview of our research methods, results, and discussions to gain deeper insights into the relationship between LFS and TP53 gene mutations. 2. Object and Methods 2.1 Object In family 1, there were a total of 30 individuals across 4 generations, among which 9 individuals had cancer. One individual had multiple primary cancers including gastric cancer, renal cancer, colorectal cancer, and lung cancer. Two individuals had breast cancer, 2 had lung cancer, 1 had liver cancer, 1 had lymphoma, 1 had thyroid cancer, and 1 had an unknown type of tumor. As of December 2022, 3 individuals had passed away: 1 due to breast cancer, 1 due to lung cancer, and 1 due to an unknown type of tumor. Clinical data on family 1 can be found in Fig. 1 A. Family 2 consisted of 7 individuals across 3 generations, of which 2 individuals had cancer. One individual had breast and ovarian cancer, while another individual had breast cancer, liver cancer, malignant fibrous histiocytoma, and ovarian malignant melanoma. As of December 2022, both afflicted individuals had passed away due to tumor recurrence or progression. Clinical data on family 2 can be found in Fig. 1 B. In family 3, only the index case had mediastinal neuroendocrine carcinoma and died at November 2022. 2.2 Methods 2.2.1 Genetic Testing of Index Cases Using medical and family history of the index cases, we performed whole-exome sequencing of 71 genes related to genetic risk in the three families' index cases. 2.2.2 Bioinformatics Analysis The Gene + seq-2000 gene sequencing instrument was used to perform sequencing and controlled the quality of the raw data using in-house scripts. The commercial software Sentieon was applied to align the genome (GRC37/hg19). Variant data were further annotated and filtered by NCbamlnfo software, RealDcaller software and self-built software. Samples with an effective depth of 500X or more were considered qualified. ACMG guidelines were used to grade the pathogenicity of the variant locus, combined with population data, specific variant type judgment, disease database, peer-reviewed literature, and computer prediction. 2.2.3 Mutation Site Detection of Family Members Based on the pathogenic variant loci detected in the index cases of the three families, and with the principle of voluntary participation, first-generation sequencing technology was performed corresponding genetic testing of the variant loci on other family members (with informed consent obtained). This study was approved by the Medical Ethics Committee of Zhejiang Cancer Hospital (IRB-2020-421). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). 3. Results 3.1 Pedigree Analysis The first p roband (III3) is a 34-year-old female with a family history of mutiple tumors. In 2020, the proband underwent surgery for excision of the right and isthmus of thyroid gland with diagnosis of poorly differentiated / undifferentiated thyroid cancer (poorly differentiated turned to undifferentiated thyroid cancer). In addition, immunohistochemical (IHC) analysis showed positive staining for TTF1 and PAX8, and local positive staining for CK7. Considering that the combined positive score (CPS) for PD-L1 expression was 70%, the patient received chemotherapy and immune checkpoint inhibitor treatment which resulted in significant improvement in her condition. In this family, female member I1 had a history of malignant tumors (details unknown); male member II1 had multiple tumors, including gastric, renal, lung, and colon cancer since 2008 at the age of 46; female member II3 was diagnosed with breast cancer in 2006 at the age of 42; female member II7 was diagnosed with breast cancer in 2006 at the age of 36 and passed away at 2015; male member II9 was diagnosed with liver cancer in 2011 at the age of 36; male member III1 was diagnosed with lymphoma in 2009 at the age of 35 and male member III5 was diagnosed with invasive pulmonary adenocarcinoma in 2018 at the age of 35. Male member III11 was diagnosed with lung cancer in 2018 at the age of 22 and passed away in 2019. Other members of the family (II5, III8, III10, IV1, IV2, IV3, IV4, IV5, IV6, and IV7) had no history of tumors. The second proband (II2) diagnosed with malignant melanoma of the ovary in 2021 at the age of 31 (considered a malignant transformation of cystic teratoma) with positive staining for SOX10, HMB45, Melan-A by IHC. She had a history of multiple malignancies, including malignant fibrous histiocytoma in 2006, left breast cancer in 2017 and liver cancer precursor lesions in 2020. After undergoing palliative ovary resection surgery, chemotherapy, targeted therapy and immunotherapy, her condition progressed, and she passed away one year later. Her mother (I2), at the age of 21 (the year of 1989) and 23 (the year of 1991), was diagnosed with breast cancer and ovarian teratoma respectively. I2 underwent cystic teratoma resection, and she suffered malignant transformation of mature cystic teratoma in 2003, leading to her death the same year. Other members of the family (I1, I3, II3, and III1) had no history of tumors. The third proband, a 11-year-old boy, was diagnosed with right mediastinal neuroendocrine carcinoma in 2021. The IHC showed positive for CD56 and Syn and the Ki67 index showed 95%. Due to the ineffectiveness of chemotherapy combined with targeted therapy and immunotherapy, he underwent palliative radiotherapy and died one year later due to disease progression. No tumor history was identified in this family. 3.2 Genetic Testing Results of the probands The first proband III3 of Family 1 was found to carry a frameshift mutation TP53 c.642_643delTA (p.H214Qfs*7) through NGS tumor multi-gene testing (Fig. 2 A). This mutation results in a shift in the reading frame starting at amino acid 214 and terminating 7 residues downstream causing a premature truncation of the 393 amino acid p53 protein, which leads to a dysfunctional or inactivated protein (PVS1). This variant is not found in the Thousand Genomes Project, gnomADe, genomADg or the HUABIAO Project (PM2). The ClinVar database reported this variant as a pathogenic mutation with a reliability rating of two stars (PP5). Based on the ACMG guidelines, considering the above evidence collectively, this variant is classified as a pathogenic mutation. In pedigree 2, the proband III2, after NGS tumor multi-gene testing, was found to carry a TP53 c.742C > T (p.R248W) missense mutation (Fig. 2 B). This mutation results in the substitution of arginine with tryptophan at position 248 of the encoded protein. This specific mutation is not documented in the Thousand Genomes Project database or the Hua's Gene Variant Database, with frequencies in gnomAD and ExAC databases being < 0.00001 and 0.000008, respectively (PM2). This variant resides within the DNA-binding domain of the p53 protein and is recognized as one of the hotspot mutations in the TP53 gene (PM1) [ 7 ] . Multiple prediction algorithms suggest the deleterious nature of this mutation, with a BayesDel score > 0.16 and an Align GVGD score of 65, supporting its pathogenicity (PP3). The ClinVar database has reported it as a pathogenic variant with a three-star reliability rating (PP5). Studies have indicated that this variant exhibits reduced or even absent transcriptional activation function (PS3) and demonstrates a dominant negative effect [ 8 ] . Furthermore, this variant has been reported in multiple LFS pedigrees [ 9 – 11 ] (PS4), and evidence of familial co-segregation has been observed in several pedigree patients. According to the ACMG guidelines, considering the cumulative evidence, this variant is classified as pathogenic. In pedigree 3, the proband, due to testing requirements, underwent NGS tumor multi-gene testing and was found to carry a TP53 c.844C > T (p.R282W) missense mutation (Fig. 2 C). This mutation results in the substitution of arginine with tryptophan at position 282 of the encoded protein. This particular mutation is not documented in the Thousand Genomes Project database or the Hua's Gene Variant Database, with frequencies in gnomAD and ExAC databases being < 0.00001 and 0.000017, respectively (PM2). This mutation is also located within the DNA-binding domain of the p53 protein and is recognized as one of the hotspot mutations in the TP53 gene [ 7 ] (PM1). It is expected to lead to the loss of transactivation function of TP53 gene, thereby inhibiting apoptosis. The ClinVar database has reported it as a pathogenic variant with a two-star reliability rating (PP5). Computational software predicts that this mutation is deleterious to protein structure/function (PP3), and it also exhibits a dominant negative effect. According to the ACMG guidelines, considering the cumulative evidence, this variant is classified as a suspected pathogenic mutation. 3.3 Identifying mutation sites among family members. In family 1, direct relatives of the first proband underwent genetic testing on blood or oral swab samples, with the exception of III7 who refused testing. Nine individuals had TP53 c.642_643delTA (p.H214Qfs*7) mutations, and five adults (II1, II3, II9, III1, III5) had a history of malignant tumors, while three children (IV1, IV4, IV5) and one adult (III10, age 28) had no tumors (Fig. 2 A, 3 A). No related mutations were found in phenotypically normal adult family members. In family 2, the TP53 c.742C > T (p.R248W) genetic testing of the second proband's son (IV1) and her younger brother (III3) revealed that the son (aged 8 years old) carries the mutation (Fig. 2 B, 3 B). Family 3 showed no history of malignant tumors. The proband’s mother and younger brother were tested for TP53 c.844C > T (p.R282W) on blood samples, but no relevant mutations found. Therefore, proband 3 may result in a new TP53 germline mutation in this family (Fig. 2 C, 3 C). 4. Discussion LFS is a rare autosomal dominant hereditary cancer susceptibility syndrome. In 1969, Li and Fraumeni conducted a retrospective study on four pediatric rhabdomyosarcoma pedigrees, providing the first report of this disease [ 12 – 14 ] . In 1990, germline pathogenic variants in the TP53 tumor suppressor gene were discovered, representing the sole known cause of LFS [ 15 ] . To date, approximately 1,000 families from 172 different countries worldwide have been affected by this syndrome [ 16 ] . Clinically, individuals carrying germline TP53 mutations exhibit an 80% penetrance of tumors by the age of 70. However, the penetrance of TP53 germline variants varies due to age, gender, and mutation type. Adrenal cortical carcinoma, choroid plexus carcinoma, rhabdomyosarcoma, and medulloblastoma are common tumors that occur between infancy and adolescence (between the ages of 0–15), accounting for 22% of all ages. Breast cancer, osteosarcoma, leukemia, gliomas, gastrointestinal cancer, lung cancer, and various sarcomas are common in young adults between the ages of 16 and 50, accounting for 51% of all ages. Lung and colorectal cancers occur frequently in middle-aged and elderly individuals (51–80 years old, 27%). Females are most likely to develop breast cancer, while males are more likely to develop brain tumors [ 17 – 20 ] . TP53 is a tumor suppressor gene, and the most common type of variant is missense mutations. The encoded p53 protein is a homotetrameric protein consisting of 393 amino acids, encompassing five major functional domains: two N-terminal transactivation domains (TADI, 1–42, and TADII, 43–62), a proline-rich domain (PRD, 64–92), a core DNA-binding domain (DBD, 102–292), an oligomerization domain (OD, 323–356), and a C-terminal regulatory domain (RD, 363–393). Mutations in different regions have varying effects on transcriptional function, leading to phenotypic differences among TP53 variant carriers. Boettcher et al. [ 8 ] used CRISPR-Cas9 technology to generate human leukemia cell lines with TP53 missense mutations in the DBD region. The loss of p53 protein function (LOF) was revealed through functional, DNA-binding, and transcriptional analyses. Moreover, mutational scanning of p53 single amino acid variants showed that missense mutations in the DBD region exhibit dominant negative effects (DNE), where the mutated protein not only lacks function but also hinders or interferes with the normal protein's physiological function. The mutations in families 2 and 3 described in this article are both missense mutations located in the DBD region and exhibit DNE. Research has shown that such mutations are associated with an earlier median age of onset compared to carriers of loss-of-function and rearrangement mutations (21.3 years vs. 28.5 years vs. 35.8 years; P < 0.05). In the pediatric population, they are the most prevalent mutation type and may have a poorer prognosis in specific cases [ 8 , 21 , 22 ] . Our research shows a statistically significant difference in the average age of tumor onset between families 1 and 2 (38.75 years vs. 18.5 years, P = 0.012), which consistent with previous research findings. According to missense mutation allelic genes described by the International Agency for Research on Cancer (IARC) dataset and their ability to activate a set of human target sequences, the p53 missense variant forms can be classified into partially defective (PD) allelic genes, severely defective (SD) allelic genes and specific severe defects (O-SD) allelic genes [ 23 ] . In this article, both p53 p.R282W and p53 p.R248W belong to SD-type variants, while the p53 p.H214Qfs*7 frameshift mutation belongs to O-SD-type variants. Studies have indicated that p53 proteins with SD genotypes are more likely to exhibit DNE, while those with PD genotypes are less likely to show DNE [ 24 ] . Analysis of TP53 genotype and phenotype has revealed that patients with SD genotypes have an earlier median age of onset compared to those with O-SD genotypes (15 years vs. 25 years, P = 0.07) and a higher degree of cancer risk [ 24 , 25 ] , emphasizing the need for early attention and inclusion in clinical monitoring and management. Currently, treatment options specifically targeting LFS are limited, with treatment mainly focused on symptomatic management of different cancers. During treatment, efforts should be made to avoid radiation exposure, radiotherapy, and alkylating agent therapy to prevent the development of second malignancies [26]. Studies have shown that radiotherapy and genotoxic chemotherapy increase the risk of tumor progression in LFS mouse models [ 26 ] . Yoon IN et al. [ 27 ] suggest minimizing radiotherapy whenever possible when alternative treatment options are available, and if radiation therapy is necessary, it can be adapted through proton therapy, image guidance, and minimizing the irradiated volume. Furthermore, immunotherapy has emerged as a new treatment for malignant genetic heterogeneity. Hassin et al. [ 28 ] have proposed p53-related immunotherapy strategies involving the recognition and targeting of cancer cells carrying TP53 mutations by the immune system, enhancing the sensitivity of cancer cells to immune checkpoint inhibitors through the restoration of p53 function, among other treatment approaches. Yang et al. [ 29 ] demonstrated increased sensitivity of triple-negative breast cancer to PD-1 immunotherapy by restoring the activity of p53 protein carrying TP53 mutations. Megyesfalvi Z et al. [ 30 ] found widespread inactivation of the TP53 gene in small cell lung cancer, suggesting potential efficacy of immunotherapy in this context. Chen et al. [ 31 ] reported the first case of CAR-T cell therapy in an LFS patient with hematological malignancy, suggesting that CAR-T cell therapy may be an alternative option compared to traditional chemotherapy and allogeneic hematopoietic stem cell transplantation. In our research, the CPS of PD-L1 in the proband 1 was 70%, and the patient received radical resection, postoperative chemotherapy combination with immunotherapy. The progression-free survival (PFS) of the proband 1 is more than 3 years according to the recent follow-up. Due to the characteristic development of multiple tumors in LFS, early surgical intervention maybe result in better survival. Thus, screening, early diagnosis, and personalized treatment for TP53 variant carriers are crucial. LFS patients and their relatives are advised to undergo regular cancer surveillance [ 32 ] and special screening for different cancer types and mutation types. To explore the importance of cancer surveillance, Villani et al [ 33 , 34 ] conducted an 11-year follow-up study of LFS patients in the United States and Canada and found that the 5-year overall survival rate was higher in the surveillance group (88.8% vs 59.6%, P < 0.05). Studies have shown that exposure to radioactive substances should be avoided as much as possible during cancer screening [ 32 ] . Whole-body MRI should be performed instead of CT and X-ray examinations [ 32 ] . Adrenal cortical carcinoma should undergo abdominal ultrasound every 6 months, and if ultrasound does not provide sufficient imaging, cortisol levels can be measured. For patients who have received abdominal radiotherapy or have a family history of colorectal cancer, colonoscopy should be performed every 5 years from the age of 18. For female patients, annual breast MRI is recommended from the age of 20 to 65. For adults, annual brain MRI is recommended until the age of 50 [ 35 ] . The frequency of new TP53 mutations is approximately 7%-20%, and the average age of first cancer in these patients is 5–6 years, with 80% having multiple primary cancers. The accurate identification of new TP53 germline mutations is also of crucial clinical significance for the identification and screening of LFS patients. Even if there is no family history of tumors, patients with a history of early-onset multiple primary cancers should receive genetic counseling, cancer screening, and prevention as early as possible [ 36 , 37 ] . In this study, the genetic characteristics of three families and the gene testing results of TP53 germline mutations were analyzed. TP53 p.H214Qfs*7 frameshift mutation was reported as the first case of a family inheritance mutation, providing more genetic pathogenic causes for LFS diagnosis and enriching the mutation spectrum of the TP53 gene. Differences in tumor occurrence time between individuals may be related to mutation types and the interaction of genetic and environmental factors. DNE missense mutations in the DBD region of the TP53 gene maybe associated with early-onset childhood tumors and poor prognosis; SD-type TP53 mutations may have an earlier age of onset, higher tumor risk and the p53 protein carrying SD mutations perhaps prone to DNE. These functionally impaired TP53 mutations can serve as potential biomarkers for LFS and may need more active monitoring and treatment. Declarations Authors claim there are no financial interests that are directly or indirectly related to the work submitted for publication. 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J Med Genet 46:689–693. 10.1136/jmg.2008.058958 Renaux-Petel M, Charbonnier F, Théry J-C, Fermey P, Lienard G, Bou J et al (2018) Contribution of de novo and mosaic TP53 mutations to Li-Fraumeni syndrome. J Med Genet 55:173–180. 10.1136/jmedgenet-2017-104976 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3811434","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":263917942,"identity":"0ec98b21-ad97-4fe3-a83e-af6151367ea2","order_by":0,"name":"Yiping Tian","email":"","orcid":"","institution":"Zhejiang Cancer Hospital, Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yiping","middleName":"","lastName":"Tian","suffix":""},{"id":263917944,"identity":"feb26f98-4089-422f-8d08-34d82253eca8","order_by":1,"name":"Zhengxiao Ma","email":"","orcid":"","institution":"Postgraduate training base Alliance of Wenzhou Medical University (Zhejiang Cancer Hospital)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhengxiao","middleName":"","lastName":"Ma","suffix":""},{"id":263917945,"identity":"18033e01-a9bf-4120-97bd-9cdd7fc9a07f","order_by":2,"name":"Conghui Wu","email":"","orcid":"","institution":"Postgraduate training base Alliance of Wenzhou Medical University (Zhejiang Cancer Hospital)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Conghui","middleName":"","lastName":"Wu","suffix":""},{"id":263917947,"identity":"7411e2f1-bf9f-4379-a206-a4745b3f1e03","order_by":3,"name":"Xu Chen","email":"","orcid":"","institution":"Postgraduate training base Alliance of Wenzhou Medical University (Zhejiang Cancer Hospital)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Chen","suffix":""},{"id":263917952,"identity":"5a714870-283b-4bf4-bb1c-020e1e99c982","order_by":4,"name":"Zhuo Yue","email":"","orcid":"","institution":"Postgraduate training base Alliance of Wenzhou Medical University (Zhejiang Cancer Hospital)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhuo","middleName":"","lastName":"Yue","suffix":""},{"id":263917954,"identity":"ed61bcc7-5344-4353-abee-869b6d42bb3e","order_by":5,"name":"Lisha Ying","email":"","orcid":"","institution":"Postgraduate training base Alliance of Wenzhou Medical University (Zhejiang Cancer Hospital)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lisha","middleName":"","lastName":"Ying","suffix":""},{"id":263917956,"identity":"fc5e943d-2f06-4770-97cd-82635cc3f15a","order_by":6,"name":"Dan Su","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYHACAwaGCgZmUrWcIVkLYxsp6uVnJG/8XDivjp2//QDj44pfDPLmBK24kVYsPXMbG7PEmQRmw7N9DIY7GwhpkcgxkObdxsNsIMHAJtnYw5BgcICgw3KMf/POkSBBC8ONHDNp3gYDiJaGH0RoMTjzrMya51gC0C+JzYaNDRKGGwg6rD15822emrpk/vbDBx82/LGRJ+wwKEhmYGBsAEaQBJHqgcAOQv0hXscoGAWjYBSMHAAAB4I3MwWbOi8AAAAASUVORK5CYII=","orcid":"","institution":"Postgraduate training base Alliance of Wenzhou Medical University (Zhejiang Cancer Hospital)","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Su","suffix":""}],"badges":[],"createdAt":"2023-12-27 08:59:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3811434/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3811434/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49125329,"identity":"7d7d4b63-e058-4501-aeb0-07c33853a7a7","added_by":"auto","created_at":"2024-01-03 14:48:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75083,"visible":true,"origin":"","legend":"\u003cp\u003ePedigree Charts. \u003cstrong\u003eFigure 1A\u003c/strong\u003ePedigree chart of family 1. \u003cstrong\u003eFigure 1B\u003c/strong\u003e Pedigree chart of family 2.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3811434/v1/30bda584b982e85097c0cbc6.png"},{"id":49125328,"identity":"f4084d96-74e9-46c6-8a5d-b43af13f0ba7","added_by":"auto","created_at":"2024-01-03 14:48:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1220001,"visible":true,"origin":"","legend":"\u003cp\u003eshows the results of high-throughput tumor gene sequencing (visualization of BAM files). Figure 2A shows a white area within a red box, which indicates a TA deletion in the \u003cem\u003eTP53\u003c/em\u003egene (\u003cem\u003eTP53\u003c/em\u003e c.642_643delTA). \u003cstrong\u003eFigure 2B\u003c/strong\u003e the red box indicates a G\u0026gt;A mutation in the \u003cem\u003eTP53\u003c/em\u003e gene (\u003cem\u003eTP53\u003c/em\u003ec.742C\u0026gt;T). \u003cstrong\u003eFigure 2C\u003c/strong\u003e the red box indicates a G\u0026gt;A mutation in the \u003cem\u003eTP53\u003c/em\u003e gene (\u003cem\u003eTP53\u003c/em\u003e c.844C\u0026gt;T).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3811434/v1/54a4d9f38af3ba007b088d4f.png"},{"id":49125330,"identity":"e82dede3-2561-4b83-b01e-e511d16ef666","added_by":"auto","created_at":"2024-01-03 14:48:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":306473,"visible":true,"origin":"","legend":"\u003cp\u003eshows the results of Sanger sequencing validation of family members. \u003cstrong\u003eFigure A\u003c/strong\u003e is for family 1, \u003cstrong\u003eFigure B\u003c/strong\u003e is for family 2, \u003cstrong\u003eFigure C\u003c/strong\u003e is for family 3.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3811434/v1/0ff9bf74bae27fd36d8e35b5.png"},{"id":50313680,"identity":"e6825c57-750c-4a49-bba2-d240d5ea9784","added_by":"auto","created_at":"2024-01-29 15:23:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1006289,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3811434/v1/23966028-a5d7-45b4-8d61-8e6e11f46d3e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Clinical and Genetic Analysis of Li-Fraumeni Syndrome with Novel TP53 Mutations","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLFS is a rare autosomal dominant hereditary cancer susceptibility syndrome characterized by a heightened risk of tumor development and an early onset of malignancies. Individuals with LFS have a 24-fold increased probability of developing cancer compared to the general population, with the highest incidence occurring before the age of 30. The median age of initial cancer diagnosis for females is approximately 31 years, while for males, it is around 46 years. Many LFS patients experience multiple primary tumors throughout their lives \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Common tumor types associated with LFS include soft tissue sarcomas, osteosarcomas, brain and central nervous system (CNS) tumors, adrenal cortical carcinomas, acute leukemias, and breast cancer. Various diagnostic criteria exist for LFS, with the Chompret criteria \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e being the most widely utilized. Given the elevated cancer risk and heterogeneity associated with this condition, current recommendations for cancer screening in LFS patients revolve around multimodal, high-frequency evaluations centered on whole-body MRI. Research has demonstrated that cancer screening in individuals with this syndrome can improve survival rates, but excessive screening may lead to patient anxiety, uncertainty, and increased treatment burden \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTP53\u003c/em\u003e germline mutations have been identified in approximately 70% of LFS patients, making it the sole known genetic cause of LFS. The gene of \u003cem\u003eTP53\u003c/em\u003e is located on chromosome 17p13.1 and serves as a crucial tumor suppressor gene involved in various biological processes, including DNA repair, cell cycle regulation, and apoptosis. When the \u003cem\u003eTP53\u003c/em\u003e gene undergoes mutation, cells lose their ability to effectively repair DNA damage, consequently increasing the risk of cancer. \u003cem\u003eTP53\u003c/em\u003e variants impact different functions of the p53 protein, thereby affecting its tumor-suppressing activity. \u003cem\u003eTP53\u003c/em\u003e variants can be categorized based on their functional consequences: those associated with loss of function (LOF) resulting in haploinsufficiency, those conferring gain of function (GOF) to the p53 protein, and those linked to dominant negative effects (DNE) and impaired transactivation activity \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Carriers of pathogenic or potentially pathogenic germline \u003cem\u003eTP53\u003c/em\u003e variants exhibit considerable variability in cancer phenotype, penetrance, and expressivity. Few genotype-phenotype studies have assessed how the functional properties of \u003cem\u003eTP53\u003c/em\u003e variants influence cancer risk.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTP53\u003c/em\u003e gene mutations represent the primary pathogenic factor in LFS, with their diverse mutation forms contributing to phenotypic variations among LFS patients. In our study, we delving into the clinical and genetic characteristics of three LFS patients and their families. Combined with high-throughput sequencing and Sanger sequencing technology, we conducted genotype-phenotype correlations as well as tumor analyses, providing further genetic evidence for the diagnosis of LFS. The results of this study not only enrich the spectrum of \u003cem\u003eTP53\u003c/em\u003e gene mutations but also offer a more comprehensive genetic counseling, monitoring, and treatment strategy for LFS patients and their families, better addressing the complexity and challenges posed by this syndrome. In the following sections, we will provide a detailed overview of our research methods, results, and discussions to gain deeper insights into the relationship between LFS and \u003cem\u003eTP53\u003c/em\u003e gene mutations.\u003c/p\u003e"},{"header":"2. Object and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Object\u003c/h2\u003e \u003cp\u003eIn family 1, there were a total of 30 individuals across 4 generations, among which 9 individuals had cancer. One individual had multiple primary cancers including gastric cancer, renal cancer, colorectal cancer, and lung cancer. Two individuals had breast cancer, 2 had lung cancer, 1 had liver cancer, 1 had lymphoma, 1 had thyroid cancer, and 1 had an unknown type of tumor. As of December 2022, 3 individuals had passed away: 1 due to breast cancer, 1 due to lung cancer, and 1 due to an unknown type of tumor. Clinical data on family 1 can be found in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Family 2 consisted of 7 individuals across 3 generations, of which 2 individuals had cancer. One individual had breast and ovarian cancer, while another individual had breast cancer, liver cancer, malignant fibrous histiocytoma, and ovarian malignant melanoma. As of December 2022, both afflicted individuals had passed away due to tumor recurrence or progression. Clinical data on family 2 can be found in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. In family 3, only the index case had mediastinal neuroendocrine carcinoma and died at November 2022.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Methods\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Genetic Testing of Index Cases\u003c/h2\u003e \u003cp\u003eUsing medical and family history of the index cases, we performed whole-exome sequencing of 71 genes related to genetic risk in the three families' index cases.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Bioinformatics Analysis\u003c/h2\u003e \u003cp\u003eThe Gene\u0026thinsp;+\u0026thinsp;seq-2000 gene sequencing instrument was used to perform sequencing and controlled the quality of the raw data using in-house scripts. The commercial software Sentieon was applied to align the genome (GRC37/hg19). Variant data were further annotated and filtered by NCbamlnfo software, RealDcaller software and self-built software. Samples with an effective depth of 500X or more were considered qualified. ACMG guidelines were used to grade the pathogenicity of the variant locus, combined with population data, specific variant type judgment, disease database, peer-reviewed literature, and computer prediction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Mutation Site Detection of Family Members\u003c/h2\u003e \u003cp\u003eBased on the pathogenic variant loci detected in the index cases of the three families, and with the principle of voluntary participation, first-generation sequencing technology was performed corresponding genetic testing of the variant loci on other family members (with informed consent obtained). This study was approved by the Medical Ethics Committee of Zhejiang Cancer Hospital (IRB-2020-421). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Pedigree Analysis\u003c/h2\u003e \u003cp\u003eThe first \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ep\u003c/span\u003eroband (III3) is a 34-year-old female with a family history of mutiple tumors. In 2020, the proband underwent surgery for excision of the right and isthmus of thyroid gland with diagnosis of poorly differentiated / undifferentiated thyroid cancer (poorly differentiated turned to undifferentiated thyroid cancer). In addition, immunohistochemical (IHC) analysis showed positive staining for TTF1 and PAX8, and local positive staining for CK7. Considering that the combined positive score (CPS) for PD-L1 expression was 70%, the patient received chemotherapy and immune checkpoint inhibitor treatment which resulted in significant improvement in her condition. In this family, female member I1 had a history of malignant tumors (details unknown); male member II1 had multiple tumors, including gastric, renal, lung, and colon cancer since 2008 at the age of 46; female member II3 was diagnosed with breast cancer in 2006 at the age of 42; female member II7 was diagnosed with breast cancer in 2006 at the age of 36 and passed away at 2015; male member II9 was diagnosed with liver cancer in 2011 at the age of 36; male member III1 was diagnosed with lymphoma in 2009 at the age of 35 and male member III5 was diagnosed with invasive pulmonary adenocarcinoma in 2018 at the age of 35. Male member III11 was diagnosed with lung cancer in 2018 at the age of 22 and passed away in 2019. Other members of the family (II5, III8, III10, IV1, IV2, IV3, IV4, IV5, IV6, and IV7) had no history of tumors.\u003c/p\u003e \u003cp\u003eThe second proband (II2) diagnosed with malignant melanoma of the ovary in 2021 at the age of 31 (considered a malignant transformation of cystic teratoma) with positive staining for SOX10, HMB45, Melan-A by IHC. She had a history of multiple malignancies, including malignant fibrous histiocytoma in 2006, left breast cancer in 2017 and liver cancer precursor lesions in 2020. After undergoing palliative ovary resection surgery, chemotherapy, targeted therapy and immunotherapy, her condition progressed, and she passed away one year later. Her mother (I2), at the age of 21 (the year of 1989) and 23 (the year of 1991), was diagnosed with breast cancer and ovarian teratoma respectively. I2 underwent cystic teratoma resection, and she suffered malignant transformation of mature cystic teratoma in 2003, leading to her death the same year. Other members of the family (I1, I3, II3, and III1) had no history of tumors.\u003c/p\u003e \u003cp\u003eThe third proband, a 11-year-old boy, was diagnosed with right mediastinal neuroendocrine carcinoma in 2021. The IHC showed positive for CD56 and Syn and the Ki67 index showed 95%. Due to the ineffectiveness of chemotherapy combined with targeted therapy and immunotherapy, he underwent palliative radiotherapy and died one year later due to disease progression. No tumor history was identified in this family.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Genetic Testing Results of the probands\u003c/h2\u003e \u003cp\u003eThe first proband III3 of Family 1 was found to carry a frameshift mutation \u003cem\u003eTP53\u003c/em\u003e c.642_643delTA (p.H214Qfs*7) through NGS tumor multi-gene testing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This mutation results in a shift in the reading frame starting at amino acid 214 and terminating 7 residues downstream causing a premature truncation of the 393 amino acid p53 protein, which leads to a dysfunctional or inactivated protein (PVS1). This variant is not found in the Thousand Genomes Project, gnomADe, genomADg or the HUABIAO Project (PM2). The ClinVar database reported this variant as a pathogenic mutation with a reliability rating of two stars (PP5). Based on the ACMG guidelines, considering the above evidence collectively, this variant is classified as a pathogenic mutation.\u003c/p\u003e \u003cp\u003eIn pedigree 2, the proband III2, after NGS tumor multi-gene testing, was found to carry a \u003cem\u003eTP53\u003c/em\u003e c.742C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.R248W) missense mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This mutation results in the substitution of arginine with tryptophan at position 248 of the encoded protein. This specific mutation is not documented in the Thousand Genomes Project database or the Hua's Gene Variant Database, with frequencies in gnomAD and ExAC databases being \u0026lt;\u0026thinsp;0.00001 and 0.000008, respectively (PM2). This variant resides within the DNA-binding domain of the p53 protein and is recognized as one of the hotspot mutations in the \u003cem\u003eTP53\u003c/em\u003e gene (PM1) \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Multiple prediction algorithms suggest the deleterious nature of this mutation, with a BayesDel score\u0026thinsp;\u0026gt;\u0026thinsp;0.16 and an Align GVGD score of 65, supporting its pathogenicity (PP3). The ClinVar database has reported it as a pathogenic variant with a three-star reliability rating (PP5). Studies have indicated that this variant exhibits reduced or even absent transcriptional activation function (PS3) and demonstrates a dominant negative effect \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Furthermore, this variant has been reported in multiple LFS pedigrees \u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e (PS4), and evidence of familial co-segregation has been observed in several pedigree patients. According to the ACMG guidelines, considering the cumulative evidence, this variant is classified as pathogenic.\u003c/p\u003e \u003cp\u003eIn pedigree 3, the proband, due to testing requirements, underwent NGS tumor multi-gene testing and was found to carry a \u003cem\u003eTP53\u003c/em\u003e c.844C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.R282W) missense mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This mutation results in the substitution of arginine with tryptophan at position 282 of the encoded protein. This particular mutation is not documented in the Thousand Genomes Project database or the Hua's Gene Variant Database, with frequencies in gnomAD and ExAC databases being \u0026lt;\u0026thinsp;0.00001 and 0.000017, respectively (PM2). This mutation is also located within the DNA-binding domain of the p53 protein and is recognized as one of the hotspot mutations in the \u003cem\u003eTP53\u003c/em\u003e gene \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e (PM1). It is expected to lead to the loss of transactivation function of \u003cem\u003eTP53\u003c/em\u003e gene, thereby inhibiting apoptosis. The ClinVar database has reported it as a pathogenic variant with a two-star reliability rating (PP5). Computational software predicts that this mutation is deleterious to protein structure/function (PP3), and it also exhibits a dominant negative effect. According to the ACMG guidelines, considering the cumulative evidence, this variant is classified as a suspected pathogenic mutation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Identifying mutation sites among family members.\u003c/h2\u003e \u003cp\u003eIn family 1, direct relatives of the first proband underwent genetic testing on blood or oral swab samples, with the exception of III7 who refused testing. Nine individuals had \u003cem\u003eTP53\u003c/em\u003e c.642_643delTA (p.H214Qfs*7) mutations, and five adults (II1, II3, II9, III1, III5) had a history of malignant tumors, while three children (IV1, IV4, IV5) and one adult (III10, age 28) had no tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). No related mutations were found in phenotypically normal adult family members. In family 2, the \u003cem\u003eTP53\u003c/em\u003e c.742C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.R248W) genetic testing of the second proband's son (IV1) and her younger brother (III3) revealed that the son (aged 8 years old) carries the mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Family 3 showed no history of malignant tumors. The proband\u0026rsquo;s mother and younger brother were tested for \u003cem\u003eTP53\u003c/em\u003e c.844C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.R282W) on blood samples, but no relevant mutations found. Therefore, proband 3 may result in a new \u003cem\u003eTP53\u003c/em\u003e germline mutation in this family (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eLFS is a rare autosomal dominant hereditary cancer susceptibility syndrome. In 1969, Li and Fraumeni conducted a retrospective study on four pediatric rhabdomyosarcoma pedigrees, providing the first report of this disease \u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. In 1990, germline pathogenic variants in the \u003cem\u003eTP53\u003c/em\u003e tumor suppressor gene were discovered, representing the sole known cause of LFS \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. To date, approximately 1,000 families from 172 different countries worldwide have been affected by this syndrome \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Clinically, individuals carrying germline \u003cem\u003eTP53\u003c/em\u003e mutations exhibit an 80% penetrance of tumors by the age of 70. However, the penetrance of \u003cem\u003eTP53\u003c/em\u003e germline variants varies due to age, gender, and mutation type. Adrenal cortical carcinoma, choroid plexus carcinoma, rhabdomyosarcoma, and medulloblastoma are common tumors that occur between infancy and adolescence (between the ages of 0\u0026ndash;15), accounting for 22% of all ages. Breast cancer, osteosarcoma, leukemia, gliomas, gastrointestinal cancer, lung cancer, and various sarcomas are common in young adults between the ages of 16 and 50, accounting for 51% of all ages. Lung and colorectal cancers occur frequently in middle-aged and elderly individuals (51\u0026ndash;80 years old, 27%). Females are most likely to develop breast cancer, while males are more likely to develop brain tumors \u003csup\u003e[\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTP53\u003c/em\u003e is a tumor suppressor gene, and the most common type of variant is missense mutations. The encoded p53 protein is a homotetrameric protein consisting of 393 amino acids, encompassing five major functional domains: two N-terminal transactivation domains (TADI, 1\u0026ndash;42, and TADII, 43\u0026ndash;62), a proline-rich domain (PRD, 64\u0026ndash;92), a core DNA-binding domain (DBD, 102\u0026ndash;292), an oligomerization domain (OD, 323\u0026ndash;356), and a C-terminal regulatory domain (RD, 363\u0026ndash;393). Mutations in different regions have varying effects on transcriptional function, leading to phenotypic differences among \u003cem\u003eTP53\u003c/em\u003e variant carriers. Boettcher et al. \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e used CRISPR-Cas9 technology to generate human leukemia cell lines with \u003cem\u003eTP53\u003c/em\u003e missense mutations in the DBD region. The loss of p53 protein function (LOF) was revealed through functional, DNA-binding, and transcriptional analyses. Moreover, mutational scanning of p53 single amino acid variants showed that missense mutations in the DBD region exhibit dominant negative effects (DNE), where the mutated protein not only lacks function but also hinders or interferes with the normal protein's physiological function. The mutations in families 2 and 3 described in this article are both missense mutations located in the DBD region and exhibit DNE. Research has shown that such mutations are associated with an earlier median age of onset compared to carriers of loss-of-function and rearrangement mutations (21.3 years vs. 28.5 years vs. 35.8 years; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the pediatric population, they are the most prevalent mutation type and may have a poorer prognosis in specific cases \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Our research shows a statistically significant difference in the average age of tumor onset between families 1 and 2 (38.75 years vs. 18.5 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012), which consistent with previous research findings.\u003c/p\u003e \u003cp\u003eAccording to missense mutation allelic genes described by the International Agency for Research on Cancer (IARC) dataset and their ability to activate a set of human target sequences, the p53 missense variant forms can be classified into partially defective (PD) allelic genes, severely defective (SD) allelic genes and specific severe defects (O-SD) allelic genes \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In this article, both p53 p.R282W and p53 p.R248W belong to SD-type variants, while the p53 p.H214Qfs*7 frameshift mutation belongs to O-SD-type variants. Studies have indicated that p53 proteins with SD genotypes are more likely to exhibit DNE, while those with PD genotypes are less likely to show DNE \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Analysis of \u003cem\u003eTP53\u003c/em\u003e genotype and phenotype has revealed that patients with SD genotypes have an earlier median age of onset compared to those with O-SD genotypes (15 years vs. 25 years, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.07) and a higher degree of cancer risk \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, emphasizing the need for early attention and inclusion in clinical monitoring and management.\u003c/p\u003e \u003cp\u003eCurrently, treatment options specifically targeting LFS are limited, with treatment mainly focused on symptomatic management of different cancers. During treatment, efforts should be made to avoid radiation exposure, radiotherapy, and alkylating agent therapy to prevent the development of second malignancies [26]. Studies have shown that radiotherapy and genotoxic chemotherapy increase the risk of tumor progression in LFS mouse models \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Yoon IN et al. \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e suggest minimizing radiotherapy whenever possible when alternative treatment options are available, and if radiation therapy is necessary, it can be adapted through proton therapy, image guidance, and minimizing the irradiated volume.\u003c/p\u003e \u003cp\u003eFurthermore, immunotherapy has emerged as a new treatment for malignant genetic heterogeneity. Hassin et al. \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e have proposed p53-related immunotherapy strategies involving the recognition and targeting of cancer cells carrying \u003cem\u003eTP53\u003c/em\u003e mutations by the immune system, enhancing the sensitivity of cancer cells to immune checkpoint inhibitors through the restoration of p53 function, among other treatment approaches. Yang et al. \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e demonstrated increased sensitivity of triple-negative breast cancer to PD-1 immunotherapy by restoring the activity of p53 protein carrying \u003cem\u003eTP53\u003c/em\u003e mutations. Megyesfalvi Z et al. \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e found widespread inactivation of the \u003cem\u003eTP53\u003c/em\u003e gene in small cell lung cancer, suggesting potential efficacy of immunotherapy in this context. Chen et al. \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e reported the first case of CAR-T cell therapy in an LFS patient with hematological malignancy, suggesting that CAR-T cell therapy may be an alternative option compared to traditional chemotherapy and allogeneic hematopoietic stem cell transplantation. In our research, the CPS of PD-L1 in the proband 1 was 70%, and the patient received radical resection, postoperative chemotherapy combination with immunotherapy. The progression-free survival (PFS) of the proband 1 is more than 3 years according to the recent follow-up. Due to the characteristic development of multiple tumors in LFS, early surgical intervention maybe result in better survival. Thus, screening, early diagnosis, and personalized treatment for \u003cem\u003eTP53\u003c/em\u003e variant carriers are crucial.\u003c/p\u003e \u003cp\u003eLFS patients and their relatives are advised to undergo regular cancer surveillance \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e and special screening for different cancer types and mutation types. To explore the importance of cancer surveillance, Villani et al \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e conducted an 11-year follow-up study of LFS patients in the United States and Canada and found that the 5-year overall survival rate was higher in the surveillance group (88.8% vs 59.6%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Studies have shown that exposure to radioactive substances should be avoided as much as possible during cancer screening \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Whole-body MRI should be performed instead of CT and X-ray examinations \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Adrenal cortical carcinoma should undergo abdominal ultrasound every 6 months, and if ultrasound does not provide sufficient imaging, cortisol levels can be measured. For patients who have received abdominal radiotherapy or have a family history of colorectal cancer, colonoscopy should be performed every 5 years from the age of 18. For female patients, annual breast MRI is recommended from the age of 20 to 65. For adults, annual brain MRI is recommended until the age of 50 \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. The frequency of new \u003cem\u003eTP53\u003c/em\u003e mutations is approximately 7%-20%, and the average age of first cancer in these patients is 5\u0026ndash;6 years, with 80% having multiple primary cancers. The accurate identification of new \u003cem\u003eTP53\u003c/em\u003e germline mutations is also of crucial clinical significance for the identification and screening of LFS patients. Even if there is no family history of tumors, patients with a history of early-onset multiple primary cancers should receive genetic counseling, cancer screening, and prevention as early as possible \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, the genetic characteristics of three families and the gene testing results of \u003cem\u003eTP53\u003c/em\u003e germline mutations were analyzed. \u003cem\u003eTP53\u003c/em\u003e p.H214Qfs*7 frameshift mutation was reported as the first case of a family inheritance mutation, providing more genetic pathogenic causes for LFS diagnosis and enriching the mutation spectrum of the \u003cem\u003eTP53\u003c/em\u003e gene. Differences in tumor occurrence time between individuals may be related to mutation types and the interaction of genetic and environmental factors. DNE missense mutations in the DBD region of the \u003cem\u003eTP53\u003c/em\u003e gene maybe associated with early-onset childhood tumors and poor prognosis; SD-type \u003cem\u003eTP53\u003c/em\u003e mutations may have an earlier age of onset, higher tumor risk and the p53 protein carrying SD mutations perhaps prone to DNE. These functionally impaired \u003cem\u003eTP53\u003c/em\u003e mutations can serve as potential biomarkers for LFS and may need more active monitoring and treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthors claim there are no financial interests that are directly or indirectly related to the work submitted for publication.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYiping Tian and Zhengxiao Ma wrote the main manuscript text and Conghui Wu and Chen Xu prepared figures 1-3. 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J Med Genet 55:173\u0026ndash;180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1136/jmedgenet-2017-104976\u003c/span\u003e\u003cspan address=\"10.1136/jmedgenet-2017-104976\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"TP53 mutations, Li-Fraumeni syndrome, Genetic Counseling, Hereditary Tumors","lastPublishedDoi":"10.21203/rs.3.rs-3811434/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3811434/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eMutations in the \u003cem\u003eTP53\u003c/em\u003e gene can cause Li-Fraumeni syndrome (LFS), an autosomal dominant genetic syndrome that increases susceptibility to various tumors. This study aims to explore the clinical and pathological features as well as the genetic characteristics of LFS to provide a theoretical basis for genetic counseling in affected families.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe conducted a retrospective analysis of clinical data and family history in three LFS cases with \u003cem\u003eTP53\u003c/em\u003e germline mutations. High-throughput sequencing technology was used to screen for hereditary tumor-related genes in the probands, and Sanger sequencing was used to confirm and analyze candidate pathogenic variant sites in their family members.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThree different types of \u003cem\u003eTP53\u003c/em\u003e mutation variants were found in our study. The first family, spanning four generations and consisting of 30 individuals, included 9 adults diagnosed with 8 different types of cancer. Genetic testing revealed the \u003cem\u003eTP53\u003c/em\u003e c.642_643delTA p.H214Qfs*7 mutation in this family, showing that the age of onset tended to become younger in successive generations. The second family, with two patients having four different malignant tumors, carried the \u003cem\u003eTP53\u003c/em\u003e c.742C\u0026thinsp;\u0026gt;\u0026thinsp;T p.R248W mutation. This family had an average diagnosis age younger than the first family. The third proband, a 13-year-old boy, carried the \u003cem\u003eTP53\u003c/em\u003e c.844C\u0026thinsp;\u0026gt;\u0026thinsp;T p.R282W mutation and had no family history, indicating that this may be a new \u003cem\u003eTP53\u003c/em\u003e germline mutation in his family.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study identified and reported the pathogenic variant \u003cem\u003eTP53\u003c/em\u003e p.H214Qfs*7 frameshift mutation for the first time, expanding the mutation spectrum of the \u003cem\u003eTP53\u003c/em\u003e gene. We recommend timely genetic counseling and \u003cem\u003eTP53\u003c/em\u003e germline mutation testing for patients with childhood tumors or multiple familial tumors. Systematic monitoring of individuals carrying these mutations is crucial for early intervention to prevent primary and secondary tumors.\u003c/p\u003e","manuscriptTitle":"Clinical and Genetic Analysis of Li-Fraumeni Syndrome with Novel TP53 Mutations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-03 14:48:33","doi":"10.21203/rs.3.rs-3811434/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7f4522bd-5ec5-4ae6-ba9f-c6c9988fc1e7","owner":[],"postedDate":"January 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-29T15:14:53+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-03 14:48:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3811434","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3811434","identity":"rs-3811434","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}