Do not overlook the possibility of genome-edited somatic cells ending up in the human germline.

Most debates on human germline genome editing have limited discussions to just genetic modifications of sperm and eggs (gametes), their precursors within testicular or ovarian tissues, and preimplantation human embryos. What has largely been overlooked is that genome editing of somatic (non-reproductive) cells can also become heritable and can potentially be transmitted to future generations of human offspring under specific experimental conditions, due to the emergence of various new technology platforms. Most notably, the reprogramming of human somatic cells to a pluripotent "embryonic stem cell-like" state (i.e. induced pluripotent stem cells), has opened up the possibility that genome editing performed on human somatic cells can also be transmitted to future generations of human offspring when combined with other new technology platforms, such as in vitro gametogenesis, chimeric and synthetic embryos. Additionally, due to high levels of plasticity and extensive tissue remodeling within the human fetus during gestation, it is speculated that genome editing performed on fetal somatic cells intended for fetal gene therapy in utero may be unintentionally transmitted to the human germline. Hence, there should be strict regulatory oversight to ensure that any genome-edited somatic cell that ends up in the human germline via such aforementioned technology platforms does so in strict compliance with relevant legislation and ethical guidelines, especially that pertaining to safety issues with genome editing technology and its potential misuse in human enhancement and eugenics.

Nodule-specific NRF2-targeted upregulation in patients with KEAP1 mutations and familial nontoxic multinodular goiter.

CONTEXT: Kelch-like ECH-associated protein 1 (KEAP1) is associated with nuclear factor erythroid-2 related factor 2 (NRF2) and promotes NRF2 degradation in normal conditions. Genetic abnormality in KEAP1 is a rare disease and presents with familial multinodular goiter. OBJECTIVE: This study assessed the clinical and molecular findings concerning nodular formation in the thyroid gland of patients harboring KEAP1 germline mutations. METHODS: Next-generation sequencing analysis targeting goiter-associated genes was performed on 39 patients with familial multinodular goiter. The expression of NRF2-targeted genes from surgical thyroid specimens of patients with KEAP1 mutations were analyzed using a whole transcript expression array and immunohistochemistry. RESULTS: We found five probands with pathogenic heterozygous mutations in KEAP1 (p.Q86*, p.L136P, p.V411fs, p.R415C, and p.R483H), which had no meaningful concomitance with mutations of other goiter-associated genes in germline and somatic levels. Their common histopathological features showed multinodular goiters in the entire thyroid gland with few degenerative lesions or complications of malignancy and slow proliferation indicating < 1% at the Ki-67 labeling index. Among 42 NRF2-targeted genes, antioxidant genes were most frequently upregulated (11/12) in the nodule, followed by detoxification genes (6/11). Immunohistochemical analysis showed relatively high expression of glutathione peroxidase 2 and NAD(P)H quinone oxidoreductase 1 (representative NRF2-targeted genes) in the nodules of various patients harboring KEAP1 mutations. CONCLUSION: KEAP1 germline heterozygous mutations exert excessive NRF2 activity in the thyroid gland and may confer cytoprotective effects even under abundant reactive oxygen species associated with thyroid hormone production, resulting in thyroid hyperplasia with scarce degradation.

Double Hit in Clear-Cell Renal Cell Carcinoma With Germline Pathogenic ATM Mutation and Somatic VHL Mutation.

While renal cell carcinoma (RCC) is often linked to smoking, obesity, and hypertension, hereditary forms also account for about 3% of RCC cases. Notably, NCCN guidelines identify 7 major hereditary syndromes associated with an increased RCC risk. Inherited mutations in DNA repair genes, such as ATM, BRCA, and TP53, significantly increase the risk of various cancers. Biallelic pathogenic mutations in ATM cause Ataxia-Telangiectasia (A-T) syndrome, while heterozygous germline pathogenic ATM mutations, present in about 1% of the population, also elevate cancer risk. RCC has not traditionally been associated with germline pathogenic ATM mutations, only limited retrospective analyses have identified such mutations. This case report presents a 68-year-old woman with a germline pathogenic ATM mutation (c.8786+1 G>A) who developed high-risk clear cell RCC followed by an acquired somatic VHL mutation in RCC and a 3-cm serous cystadenoma, illustrating the double-hit phenomenon. Her brother, who shares the same germline pathogenic mutation, was diagnosed with pancreatic cancer and prostate cancer. This case highlights the potential use for enhanced screening protocols for RCC in patients who have germline pathogenic ATM mutations and the importance of research in targeted treatments for tumors driven by dual genetic mechanisms. Increased awareness and vigilant screening for RCC are crucial in managing hereditary cancer syndromes effectively.

The TOP-2/condensin II axis silences transcription during germline specification in C. elegans.

In C. elegans, the germline is specified via a preformation mechanism that relies on the PIE-1 protein's ability to globally silence mRNA transcription in germline precursor cells, also known as the P lineage. Recent work from our group has identified additional genome silencing events in C. elegans during oogenesis and in starved L1 larvae, and these require the condensin II complex, topoisomerase II (TOP-2), and components of the H3K9me/heterochromatin pathway. Interestingly, silencing in oocytes also requires PIE-1, but this is not the case in starved L1s. Here, we ask if additional genome silencing components besides PIE-1 are required to repress gene expression in the P lineage of early embryos, and we find that condensin II and TOP-2 are required and the H3K9me/heterochromatin pathway is not. We show that depletion of TOP-2/condensin II activates the normally suppressed RNA polymerase II to inappropriately transcribe somatic genes in the P lineage. We also present evidence that while both PIE-1 and TOP-2/condensin II are required for genome silencing in the P lineage, PIE-1 can silence transcription independently of TOP-2/condensin II when misexpressed in somatic cells. Thus, in oocytes, all three genome silencing systems (TOP-2/condensin II, H3K9me, and PIE-1) are operational while in both early embryos and starved L1s two of the three are active. Our data show that multiple, redundantly acting genome silencing mechanisms act in a mix and match manner to repress transcription at different developmental stages in the C. elegans germline.

Somatic and germline mutations in endometrial cancer.

Endometrial cancer is a complex disease influenced by both somatic and germline mutations. While individual mutations in genes such as PTEN, PIK3CA, and members of the DNA mismatch repair (MMR) system have been extensively studied, comprehensive analyses comparing somatic and germline mutations within the same cohort are limited. This study compares these mutations using whole exome sequencing (WES) data from tumor and blood samples in patients with endometrial cancer. Thirteen female patients with histologically confirmed endometrial cancer were selected. Tumor tissues and matched blood samples were collected and subjected to WES at the CeGaT laboratory, followed by bioinformatics analysis and annotation using the Geneyx platform. WES revealed significant somatic and germline DNA mutations, with key pathogenic variants identified in genes such as PTEN, PIK3CA, TP53, MLH1, and MSH2. Comparative analysis showed distinct and overlapping mutation profiles, highlighting the importance of integrating somatic and germline data in endometrial cancer research.

A Simple and Efficient Procedure for Developing Mouse Germline Stem Cell Lines with Gene Knock-in via CRISPR/Cas9 Technology.

Cultured mammalian spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), hold great promise for applications such as fertility preservation, gene therapy, and animal breeding, particularly in conjunction with accurate gene editing. Although the in vitro development of mouse GSC (mGSC) lines, and gene-targeting procedures for such lines, were initially established about two decades ago, it remains challenging for beginners to efficiently accomplish these tasks, partly because mGSCs proliferate more slowly and are more resistant to lipid-mediated gene transfection than pluripotent stem cells (PSCs). Meanwhile, methods for mGSC culture and gene editing have been evolving constantly to become simpler and more efficient. Here, we describe how to develop mGSC lines from small mouse testis samples and how to carry out gene knock-in in these cells using CRISPR/Cas9 technology, detailing three basic protocols that constitute a streamlined procedure. Using these simple and efficient procedures, site-specific knock-in mGSC lines can be obtained in 3 months. We hope that these protocols will help researchers use genetically modified GSCs to explore scientific questions of interest and to accumulate experience for application to GSC research in other mammalian species. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Establishment of mouse GSCs lines from small testicular samples Basic Protocol 2: Preparation of plasmids for gene knock-in using the CRISPR/Cas9 system Basic Protocol 3: Establishment of gene knock-in mGSC lines by electroporation gene delivery.

Nuclear actin is a critical regulator of Drosophila female germline stem cell maintenance.

Nuclear actin has been implicated in regulating cell fate, differentiation, and cellular reprogramming. However, its roles in development and tissue homeostasis remain largely unknown. Here we uncover the role of nuclear actin in regulating stemness using Drosophila ovarian germline stem cells (GSCs) as a model. We find that the localization and structure of nuclear actin is dynamic in the early germ cells. Nuclear actin recognized by anti-actin C4 is found in both the nucleoplasm and nucleolus of GSCs. The polymeric nucleoplasmic C4 pool is lost after the 2-cell stage, whereas the monomeric nucleolar pool persists to the 8-cell stage, suggesting that polymeric nuclear actin may contribute to stemness. To test this idea, we overexpressed nuclear targeted actin constructs to alter nuclear actin polymerization states in the GSCs and early germ cells. Increasing monomeric nuclear actin, but not polymerizable nuclear actin, causes GSC loss that ultimately results in germline loss. This GSC loss is rescued by simultaneous overexpression of monomeric and polymerizable nuclear actin. Together these data reveal that GSC maintenance requires polymeric nuclear actin. This polymeric nuclear actin likely plays numerous roles in the GSCs, as increasing monomeric nuclear actin disrupts nuclear architecture causing nucleolar hypertrophy, distortion of the nuclear lamina, and heterochromatin reorganization; all factors critical for GSC maintenance and function. These data provide the first evidence that nuclear actin, and in particular, its ability to polymerize, are critical for stem cell function and tissue homeostasis in vivo.

BRCA1-associated-protein-1 inactivated melanocytic tumours: characterisation of the clinicopathological spectrum and immunohistochemical expression pattern of preferentially expressed antigen in melanoma.

AIMS: BRCA1-associaed protein-1 (BAP1) inactivated tumours (BIMT) are rare melanocytic tumours that may be mistaken for Spitz tumours or melanoma. They occur sporadically or in association with the BAP1 tumour predisposition syndrome (BAP1-TPDS), which may be complicated by uveal or cutaneous melanoma, mesothelioma, basal cell carcinoma and renal cell carcinoma. The aim of this study was to characterise the clinicopathological features and the immunohistochemical expression pattern of preferentially expressed antigen in melanoma (PRAME) of BIMT in a large patient cohort. METHODS AND RESULTS: Ethical approval was obtained, haematoxylin and eosin-stained slides were reviewed, PRAME immunohistochemistry was performed and clinical follow-up was obtained from patient records. Sixty-five BIMT from 38 patients (F:M = 4.4:1) were identified. BIMT were typically located on the trunk and head and neck (median size = 0.5 cm). Seven patients with BAP1-TPDS (range = 16-66 years, median = 25) had multiple BIMT (median = 5), while sporadic BIMT were solitary (median patient age = 39 years). One of seven patients with BAP1-TPDS developed additional malignancies (mesothelioma and cutaneous spindle cell melanoma) and died of complications of mesothelioma. All other patients are alive without recurrence of BIMT (median follow-up = 42 months). BIMT presented as intradermal, nodular aggregates of epithelioid melanocytes with low mitotic activity and moderate to severe cytological atypia in 63% of cases. A background conventional naevus was present in 64%. PRAME immunohistochemistry showed negative or weakly patchy positive staining in all BIMT. CONCLUSIONS: BIMT are more common in a sporadic setting and behave indolently, despite worrying cytological atypia. PRAME immunohistochemistry is a reassuring tool in distinguishing BIMT from melanoma.

Whole exome sequencing in relapsed or refractory childhood cancer: case series.

Background: The prognosis for relapsed or refractory childhood cancer is approximately 20%. Genetic alterations are one of the significant contributing factors to the prognosis of patients. Objective: To investigate the molecular profile of relapsed or refractory childhood cancers in Thai cases. Methods: The study design is a descriptive study of patients <18 years old, suspected or diagnosed of relapsed or refractory childhood cancer who underwent whole exome sequencing (WES). Results: WES was successfully performed in both the tumor and the blood or saliva samples obtained from 4 unrelated patients. Six different variants were identified in the NCOR2, COL6A3, TP53, and SMAD4 genes. These alterations were found to be associated with tumor aggressiveness. Conclusion: This study is the first one to demonstrate genetic alterations by using WES in relapsed or refractory childhood cancer in Thai cases.

Molecular characterization of gliomas and glioneuronal tumors amid Noonan syndrome: cancer predisposition examined.

Introduction: In the setting of pediatric and adolescent young adult cancer, increased access to genomic profiling has enhanced the detection of genetic variation associated with cancer predisposition, including germline syndromic conditions. Noonan syndrome (NS) is associated with the germline RAS pathway activating alterations and increased risk of cancer. Herein, we describe our comprehensive molecular profiling approach, the association of NS with glioma and glioneuronal tumors, and the clinical and histopathologic characteristics associated with the disease. Methods: Within an institutional pediatric cancer cohort (n = 314), molecular profiling comprised of paired somatic disease-germline comparator exome analysis, RNA sequencing, and tumor classification by DNA methylation analysis was performed. Results: Through the implementation of paired analysis, this study identified 4 of 314 (1.3%) individuals who harbored a germline PTPN11 variant associated with NS, of which 3 individuals were diagnosed with a glioma or glioneuronal tumor. Furthermore, we extend this study through collaboration with a peer institution to identify two additional individuals with NS and a glioma or glioneuronal tumor. Notably, in three of five (60%) individuals, paired genomic profiling led to a previously unrecognized diagnosis of Noonan syndrome despite an average age of cancer diagnosis of 16.8 years. The study of the disease-involved tissue identified signaling pathway dysregulation through somatic alteration of genes involved in cellular proliferation, survival, and differentiation. Discussion: Comparative pathologic findings are presented to enable an in-depth examination of disease characteristics. This comprehensive analysis highlights the association of gliomas and glioneuronal tumors with RASopathies and the potential therapeutic challenges and importantly demonstrates the utility of genomic profiling for the identification of germline cancer predisposition.

Germline Variants in Patients Affected by Both Uveal and Cutaneous Melanoma.

Uveal melanoma (UM) and nonacral cutaneous melanoma (CM) are distinct entities with varied genetic landscapes despite both arising from melanocytes. There are, however, similarities in that they most frequently affect people of European ancestry, and high penetrance germline variants in BAP1, POT1 and CDKN2A have been shown to predispose to both UM and CM. This study aims to further explore germline variants in patients affected by both UM and CM, shedding light on the underlying genetic mechanism causing these diseases. Using exome sequencing we analysed germline DNA samples from a cohort of 83 Australian patients diagnosed with both UM and CM. Eight (10%) patients were identified that carried pathogenic mutations in known melanoma predisposition genes POT1, MITF, OCA2, SLC45A2 and TYR. Three (4%) patients carried pathogenic variants in genes previously linked with other cancer syndromes (ATR, BRIP1 and MSH6) and another three cases carried monoallelic pathogenic variants in recessive cancer genes (xeroderma pigmentosum and Fanconi anaemia), indicating that reduced penetrance of phenotype in these individuals may contribute to the development of both UM and CM. These findings highlight the need for further studies characterising the role of these genes in melanoma susceptibility.

Hereditary and Familial Traits in Urological Cancers and Their Underlying Genes.

Early recognition of hereditary urological cancers may influence diagnostic and therapeutic decision-making, and potentially alter the fate of patients and family members. Here, we introduce readers to the current knowledge on germline genetic testing and clinical practice in prostate, bladder, renal, and testicular carcinoma. Considering all urological cancer patients, routine inquiries about familial cancer history should become a standard practice in clinical settings. If suspicion arises, patients can opt for two avenues: referral to genetic counseling or undergoing genetic tests after consultation with the treating urologist. Patient summary: Tumors of the urogenital tract (prostate, kidney, bladder, and testes) can sometimes be related to genetic mutations that are present in all the cells of the body. Such mutations can be inherited and run in families. Therefore, it is relevant to obtain information on the incidence of all cancers in the family history. The information obtained may initiate genetic testing, leading to the identification of mutations that are related to cancer in the current or next generation. In addition, these mutations may offer alternative treatment options for patients.

Germline genetic variants that predispose to myeloproliferative neoplasms and hereditary myeloproliferative phenotypes.

Epidemiological evidence of familial predispositions to myeloid malignancies and myeloproliferative neoplasms (MPN) has long been recognised, but recent studies have added to knowledge of specific germline variants in multiple genes that contribute to the familial risk. These variants may be common risk alleles in the general population but have low penetrance and cause sporadic MPN, such as the JAK2 46/1 haplotype, the variant most strongly associated with MPN. Association studies are increasingly identifying other MPN susceptibility genes such as TERT, MECOM, and SH2B3, while some common variants in DDX41 and RUNX1 appear to lead to a spectrum of myeloid malignancies. RBBP6 and ATM variants have been identified in familial MPN clusters and very rare germline variants such as chromosome 14q duplication cause hereditary MPN with high penetrance. Rarely, there are hereditary non-malignant diseases with an MPN-like phenotype. Knowledge of those genes and germline genetic changes which lead to MPN or diseases that mimic MPN helps to improve accuracy of diagnosis, aids with counselling regarding familial risk, and may contribute to clinical decision-making. Large scale population exome and genome sequencing studies will improve our knowledge of both common and rare germline genetic contributions to MPN.

Familial Acute Promyelocytic Leukemia: A Case Report and Review of the Literature.

Acute promyelocytic leukemia (APL) is characterized by a reciprocal translocation t (15;17) (q24;q21), which leads to the fusion of PML and RARα genes known as PML-RARα fusion. A few cases of potentially hereditary leukemia-related genes in APL have been reported, but no instances of familial aggregation of APL have been documented. Here, we describe a family in whom two members successively affected by APL。The potential familial association observed in these two cases of APL highlights the need for further investigation and more definitive genetic lineage tracing in order to understand the genetic basis of this disease.

Identification and functional validation of a novel pathogenic POT1 germline variant p.G95V in familial melanoma.

POT1 variants have been identified in familial melanoma (FM) as well as a number of other germline and somatic malignancies. The functional validation of variants identified from the screening of patients with melanoma gene susceptibility panels is key to understanding the clinical significance of identified variants. Here we report a novel, likely pathogenic POT1 missense variant (p.G95V) in FM and investigate its functional impact. We demonstrate loss of function owing to the inability of the mutant POT1 protein to bind telomeric DNA compared to its wild-type counterpart. This study provides important functional validation of a novel POT1 variant in FM.

Colorectal cancer with a germline BRCA1 variant inherited paternally: a case report.

BRCA genes have well-known associations with breast and ovarian cancers. However, variations in the BRCA gene, especially germline variations, have also been reported in colorectal cancer (CRC). We present the case of a rectal cancer with a germline BRCA1 variation inherited from the paternal side. A 39-year-old male was admitted with rectal cancer. The patient underwent surgical resection and the pathologic diagnosis was adenocarcinoma. Next-generation sequencing was performed and a BRCA1 variant was detected. Reviewing the public database and considering the young age of the patient, the variant was suggested to be germline. The patient's father had had prostate cancer and next-generation sequencing testing revealed an identical BRCA1 variant. In the BRCA cancer group, there is relatively little attention paid to male cancers. The accumulation of male CRC cases linked to BRCA variations may help clarify the potential pathological relationship between the two.

Clinical Impact of Constitutional Genomic Testing on Current Breast Cancer Care.

The most commonly diagnosed cancer in women worldwide is cancer of the breast. Up to 20% of familial cases are attributable to pathogenic mutations in high-penetrance (BReast CAncer gene 1 [BRCA1], BRCA2, tumor protein p53 [TP53], partner and localizer of breast cancer 2 [PALB2]) or moderate-penetrance (checkpoint kinase 2 [CHEK2], Ataxia-telangiectasia mutated [ATM], RAD51C, RAD51D) breast-cancer-predisposing genes. Most of the breast-cancer-predisposing genes are involved in DNA damage repair via homologous recombination pathways. Understanding these pathways can facilitate the development of risk-reducing and therapeutic strategies. The number of breast cancer patients undergoing testing for pathogenic mutations in these genes is rapidly increasing due to various factors. Advances in multigene panel testing have led to increased detection of pathogenic mutation carriers at high risk for developing breast cancer and contralateral breast cancer. However, the lack of long-term clinical outcome data and incomplete understanding of variants, particularly for moderate-risk genes limits clinical application. In this review, we have summarized the key functions, risks, and prognosis of breast-cancer-predisposing genes listed in the National Health Service (NHS) England National Genomic Test Directory for inherited breast cancer and provide an update on current management implications including surgery, radiotherapy, systemic treatments, and post-treatment surveillance.

A dual biomarker in non-small cell lung cancer that predicts Li Fraumeni syndrome : Lung cancer and Li Fraumeni.

Non-small cell lung cancer is the most common cause of cancer death globally. When apparent incidental pathogenic germline variants (PGVs) are uncovered with routine next generation sequencing (NGS) of NSCLCs, germline testing (GT) is recommended to confirm that PGV. Because it is far more common that an uncovered tumor TP53 variant is related to a somatic event than an incidental PGV, however, GT for Li Fraumeni syndrome (LFS) is not recommended based solely on uncovering a NSCLC TP53 variant. Because nearly all tumor EGFR variants are also somatic in origin, GT is not recommended based solely on uncovering a tumor EGFR variant.However, there is evidence that patients with coexisting NSCLC variants in both EGFR and TP53 have significant likelihoods of having LFS. For patients with LFS, there are recommended measures for prevention and early detection of LFS-associated cancers and cascade GT of relatives for LFS. Although co-existing genetic variants in NSCLC are not currently used as a biomarker for GT to identify patients with PGVs, given the evidence reviewed here, select patients with NSCLCs that harbor this dual biomarker (i.e., co-existing TP53/EGFR variants) might reasonably be considered for GT for LFS.

Multi-Gene Panel Testing for Hereditary Cancer Predisposition Among Patients Sixty-Five Years and Above Diagnosed With Breast Cancer.

Background: The availability and affordability of germline genetic testing (GGT) has resulted in a broader utilization in daily clinical practice. However, adherence to testing guidelines is low, especially among older patients, where testing is often not offered. Methods: In this study, consecutive, newly diagnosed patients with breast cancer (BC) aged ≥ 65 years and eligible for GGT, as per the National Comprehensive Cancer Network (NCCN) guidelines (version 1, 2021), were invited to participate, from March 2021 to December 2022. Patients were offered a restricted (two- or 20-gene panel), or an expanded 84-gene panel. Results: During the study period, 204 patients were enrolled. The mean (standard deviation (SD)) age at BC diagnosis was 70.5 (5.13) years, ranging 65 - 81 years. All patients were Arab and the majority were Jordanian. The majority (n = 188, 92.2%) had early-stage (stages I and II) disease. One hundred three (50.5%) patients were tested with a restricted two-gene (n = 13) or 20-gene (n = 90) panel, while the remaining 101 (49.5%) patients had an expanded 84-gene panel. Family history of close blood relative(s) with BC was the most common indication for testing (n = 110, 53.9%). Among the entire study cohort, 22 (10.8%) had pathogenic/likely pathogenic germline variants (PGVs) and another 97 (47.5%) had ≥ 1 variants of uncertain significance (VUS). PGV rates were significantly higher with the expanded panel (14.9%) compared to restricted testing (6.8%) (P = 0.032). Similarly, VUS rates were significantly higher with the expanded panel (64.4%) compared to the restricted panel (31.1%) (P < 0.001). The most prevalent genes with PGVs were BRCA1/2 (31.3% of all PGV-positive patients), CHEK2 (23.1%) and ATM (19.2%). Conclusion: GGT should not be overlooked in older BC patients, as this study demonstrates that > 10% of patients have PGVs, largely in potentially actionable genes.