OBJECTIVE: The management of indeterminate thyroid nodules remains a topic of ongoing debate, particularly regarding the differentiation of malignancy. Somatic mutation analysis offers crucial insights into tumor characteristics. This study aimed to assist the clinical management of indeterminate nodules with somatic mutation analysis.
METHODS: Aspiration samples from 20 indeterminate thyroid nodules were included in the study. A next-generation sequencing panel containing 67 genes was used for molecular profiling. The results were compared with pathology data from surgical material, which is considered the gold standard. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated.
RESULTS: Variants in six genes (NRAS, BRAF, TP53, TERT, PTEN, PIK3CA) were detected in 10 out of 20 samples. We identified nine Tier 1 or 2 variants in 10 (67 %) out of 15 malignant nodules (NRAS, BRAF, TP53, TERT, PTEN, PIK3CA) and one Tier 2 (PIK3CA) variant in one out of five benign nodules. The study demonstrated an NPV of 40 %, a PPV of 90 %, a specificity of 80 %, and a sensitivity of 60 %.
CONCLUSIONS: Based on the detected molecular markers, at least nine patients (45 %) could be managed correctly without needing a repeat FNAB attempt. This study underscores the clinical practicality of molecular tests in managing nodules with indeterminate cytology. Additionally, this study emphasizes the importance of considering the patient\'s age when determining the DNA- or RNA-based genetic testing method. Finally, we discussed the significance of the somatic mutation profile and its impact on the current pathological classification.

The production of transgenic animals using non-viral methods has raised questions regarding their long-term health and genomic stability. In this study, we evaluated these aspects in transgenic cattle over ten years, using transposon-mediated gene transfer. Our longitudinal analysis included a comprehensive health assessment and whole-genome DNA resequencing. We found no significant alterations in physiological parameters or health complications in transposon-mediated transgenic cattle that exceeded 10 years of age. Genomic analysis revealed that the rates of somatic mutations and copy number variations in transgenic cattle were comparable to those in non-transgenic cattle. Furthermore, structural variants were infrequent, suggesting that transposon-mediated gene insertion did not compromise genomic integrity. These findings highlight the viability of transposon systems for generating transgenic livestock, potentially expanding their applications in agriculture and biotechnology. This study contributes significantly to our understanding of the long-term implications of transgenesis in large animals and supports the safety and stability of this method.

BACKGROUND: The von Hippel-Lindau (VHL) mutation is an important alteration in clear cell renal cell carcinoma (ccRCC); however, its imaging phenotype remains unclear.
OBJECTIVE: To investigate whether MRI features can reflect the VHL mutation status.
METHODS: Retrospective.
UNASSIGNED: 3 T/fast spin echo T2-weighted, spin-echo echo planar diffusion-weighted, gradient recalled echo T1-weighted, gradient recalled echo chemical-shift T1-weighted, and contrast-enhanced gradient recalled echo T1-weighted sequences.
METHODS: One hundred five patients with ccRCC who underwent preoperative contrast-enhanced MRI and subsequent genomic sequencing: 59 consecutive patients from our institution (38 [64.41%] with VHL mutations) formed a training cohort, and 46 from The Cancer Genome Atlas (TCGA) database (24 [52.17%] with VHL mutations) formed an independent test cohort.
RESULTS: Two radiologists, with 23 and 33 years of experience respectively, jointly evaluated the semantic MRI features of the primary lesion in ccRCCs to propose potential features related to VHL mutations in both cohorts. Three additional readers, with 5, 7, and 10 years of experience respectively, independently reviewed all lesions to assess the interobserver agreement of MRI features. A VHL mutational likelihood score (VHL-MULIS) system was constructed using the training cohort and validated using the independent test cohort.
METHODS: Fisher\'s test or chi-square test, t-test or Mann-Whitney U test, logistic regression, Cohen\'s kappa (κ), area under the receiver operating characteristic curve (AUC). A two-sided P value <0.05 was considered statistically significant.
RESULTS: In both the local and public cohorts, T2-weighted signal intensity and presence of microscopic fat from primary lesions were significantly associated with VHL mutation status. The VHL-MULIS incorporated maximum diameter, T2-weighted signal intensity, and presence of microscopic fat in the training cohort and demonstrated promising diagnostic ability (AUC, 0.82; sensitivity, 0.79; specificity, 0.82) and substantial interobserver agreement (κ, 0.787) in the test cohort.
CONCLUSIONS: The VHL mutation exhibited a distinct MRI phenotype. Integrating multiple semantic MRI features has potential to reflect the mutation status in patients with ccRCC.
METHODS: 3 TECHNICAL EFFICACY: Stage 2.

Understanding somatic mutations and structural variations in domestic pigs (Sus scrofa domestica) is critical due to their increasing importance as model organisms in biomedical research. In this study, we conducted a comprehensive analysis through whole-genome sequencing of skin, organs, and blood samples. By examining two pig pedigrees, we investigated the inheritance and sharedness of structural variants among fathers, mothers, and offsprings. Utilizing single-cell clonal expansion techniques, we observed significant variations in the number of somatic mutations across different tissues. An in-house developed pipeline enabled precise filtering and analysis of these mutations, resulting in the construction of individual phylogenetic trees for two pigs. These trees explored the developmental relationships between different tissues, revealing insights into clonal expansions from various anatomical locations. This study enhances the understanding of pig genomes, affirming their increasing value in clinical and genomic research, and provides a foundation for future studies in other animals, paralleling previous studies in mice and humans. This approach not only deepens our understanding of mammalian genomic variations but also strengthens the role of pigs as a crucial model in human health and disease research.

Immune checkpoint inhibitors (ICIs) exert clinical efficacy against various types of cancers by reinvigorating exhausted CD8+ T cells that can expand and directly attack cancer cells (cancer-specific T cells) among tumor-infiltrating lymphocytes (TILs). Although some reports have identified somatic mutations in TILs, their effect on antitumor immunity remains unclear. In this study, we successfully established 18 cancer-specific T cell clones, which have an exhaustion phenotype, from the TILs of four patients with melanoma. We conducted whole-genome sequencing for these T cell clones and identified various somatic mutations in them with high clonality. Among the somatic mutations, an SH2D2A loss-of-function frameshift mutation and TNFAIP3 deletion could activate T cell effector functions in vitro. Furthermore, we generated CD8+ T cell-specific Tnfaip3 knockout mice and showed that Tnfaip3 function loss in CD8+ T cell increased antitumor immunity, leading to remarkable response to PD-1 blockade in vivo. In addition, we analyzed bulk CD3+ T cells from TILs in additional 12 patients and identified an SH2D2A mutation in one patient through amplicon sequencing. These findings suggest that somatic mutations in TILs can affect antitumor immunity and suggest unique biomarkers and therapeutic targets.

ABO blood group discrepancies in healthy individuals were caused by body-wide chimerism and mosaicism. They can be evaluated with new diagnostic options for disease-related cell clones that are typically associated with mosaicism. The observations raise the attention for sporadic mixed-field observations of any blood group antigen. Commentary on: Dauber et al. Body-wide chimerism and mosaicism are predominant causes of naturally occurring ABO discrepancies. Br J Haematol 2024 (Online ahead of print). doi:10.1111/bjh.19618.

Evaluating the mutagenic properties of chemicals is crucial for understanding their potential cancer risks. Recent Illumina-based error-corrected sequencing techniques have enabled the direct detection of mutations induced de novo by mutagens. However, as the Illumina platform lacks intrinsic error-correction capabilities, complex library preparations and bioinformatic processes are necessary to identify these rare mutations. In this study, we evaluated whether long-read PacBio-based HiFi sequencing (HiFi seq), which has integrated error-correction, can detect de novo mutations induced by mutagens in C57BL/6 mouse tissues. Using HiFi seq, dose-dependent increases in mutation frequencies were found in tissues from mice exposed to 7,12-dimethylbenz[a]anthracene, procarbazine, and N-propyl-N-nitrosourea. Furthermore, the mutational signatures derived from these exposures were consistent with those previously reported for these mutagens. This study demonstrates that HiFi seq can complement established mutation detection assays to facilitate the identification of hazardous compounds.

In the long history of human relations with flowering cherry trees in Japan, \'Somei-Yoshino\' occupies an exceptional position among a variety of flowering trees: it is a self-incompatible interspecific hybrid but has been enthusiastically planted by grafting throughout Japan, due most likely to its flamboyant appearance upon full bloom. Thus, \'Somei-Yoshino\' gives us a rare opportunity to trace and investigate the occurrence and distribution of somatic mutations within a single plant species through analysis of the genomes of the clonally propagated trees grown under a variety of geographical and artificial environments. In the studies presented here, a total of 46 samples of \'Somei-Yoshino\' trees were collected and their genomes were analysed. We identified 684 single nucleotide mutations, of which 71 were present in more than two samples. Clustering analysis of the mutations indicated that the 46 samples were classified into eight groups, four of which included 36 of the 46 samples analysed. Interestingly, all the four tree samples collected in Ueno Park of Tokyo were members of the four groups mentioned above. Based on comparative analysis of their mutations, one of the four trees growing in Ueno Park was concluded to be the closest to the original ancestor. We propose that somatic mutations may be used as tracers to establish the ancestral relationship amongst clonally propagated individuals.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases, < 1% were transmitted by misfolded PrP, ~ 15% are inherited, and ~ 85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate localized initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of > 5000× across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a localized presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

UNASSIGNED: Age is a major risk factor for differentiated thyroid cancer (DTC); however, the mechanisms underlying aging-regulated progression of DTC remains unclear.
UNASSIGNED: Based on multi-omics data (transcriptional files, somatic mutation files, methylation files) derived from the TCGA database, we comprehensively investigated the genomic and biological features associated with aging in patients with DTC.
UNASSIGNED: We confirmed that age was an independent risk factor for overall survival and progression-free survival of patients with DTC, and confirmed that 55 years of age (adopted in the 8th AJCC staging system) is an appropriate cutoff for patients with DTC rather than 45 years (adopted in the 7th AJCC staging system). Using 55 years as the cutoff, we demonstrated DNA methylation-driven transcriptional regulation during aging, and identified the landscape of somatic mutations in young and old patients with DTC along with two aging-related mutations: TTN and EIF1AX. Subsequently, we investigated the infiltration of immune cells in DTC, and found that old patients exhibited decreased CD8+ T cells infiltration with lower cytotoxicity. Finally, we constructed a prognosis prediction model based on three age-related genes (PTK2B, E2F1, and GHR) that showed satisfactory performance in predicting patients prognosis.
UNASSIGNED: We comprehensively investigated the complex interplay between age and biological features of DTC, which may provide new insights into the role of aging in DTC.