Citations

Abstract Somatic mutations that arise post-zygotically create genetic diversity among normal human cells and provide key insights into human development and aging. Fibroblast-derived induced pluripotent stem cells (iPSCs) have proved to be a useful system for disease modelling; however, due to their clonal nature, iPSC lines carry somatic mutations inherited from the founder cells, raising concerns about their genomic integrity. At the same time, this clonality enables single-cell–level discovery of somatic mutations and the reconstruction of developmental lineages. In living individuals, though, this approach requires invasive biopsies and is limited to skin-derived lineages. Here, we generated 33 urine-derived iPSC lines from four males representing two father– son relationships, performed shallow whole-genome sequencing of the lines and analyzed somatic mutations. Derived iPSCs representing single cells from urine carried a few hundred of somatic single-nucleotide variants per genome, dominated by endogenous, clock-like mutational signatures and lacking environmental imprints such as UV-associated mutations. Copy-number analysis identified somatic CNVs in most of the lines and revealed higher CNV burdens in fathers than in sons, consistent with age-related structural mosaicism. Shared mutations across lines enabled reconstruction of cell lineage phylogenetic trees. In summary, urine-derived iPSCs showed genomic alterations comparable to those in fibroblast-derived iPSC lines and represent a valuable non-invasive alternative for disease modeling. Overall, this study provides the first genome-wide characterization of somatic mutations in urine-derived iPSCs and establishes them as a practical and non-invasive platform for charting somatic mutation landscapes and tracing developmental lineages in living humans.

Abstract Background: Most colorectal cancer (CRC) arises from polyps and is mainly prevented by polypectomy. The most important polyps to manage with colonoscopy are those with highest CRC risk- namely, advanced polyps (> 1cm, villous histology or high-grade dysplasia (HGD). Yet, 48% of advanced polyps recur within 1 to 3 years of removal, and up to 5% of advanced polyps under surveillance still progress to CRC. We performed this pilot study to investigate molecular and microenvironment heterogeneities of polyps and how they might impact their clinical behavior. Methods: GeoMx spatial transcriptomics was performed on FFPE tissues from three different polyp outcome phenotypes (POPs) including the polyp that does not recur (POP-NR), that recurs following polypectomy but cured by colonoscopy (POP-R) or the polyp despite polypectomy develops CRC at the polypectomy(ies) site (POP-CRC). Normal colon and polyp with low or HGD from the index polyp were assessed from 6 patients with POP-NR, 9 with POP-R and 12 with POP-CRC with a minimum of 2 follow up colonoscopies at 3-year intervals. Epithelium was identified as PanCK positive, and stroma identified as PanCK negative and positive nuclear staining. Cell type deconvolution was implemented using single-cell adult human intestinal tract catalogue (Elmentaite et al., https://www.gutcellatlas.org/). Differential gene expression (DEG), cell type abundance and functional enrichment analyses was performed using linear mixed effects model, and observations with p-value < 0.05 and log2FC >= |1| are reported. Results: The greatest number of DEGs were identified in stroma of the index POP-CRC compared to POP-NR polyps (n = 17 down and 11 upregulated genes), followed by epithelium of the index POP-CRC vs POP-NR polyps (n = 10 down and one upregulated gene(s)). Between the index POP-CRC and POP-R, epithelium showed downregulation of 7 genes, and upregulation of no genes. In stroma two genes were down and none upregulated. Four genes were downregulated in stroma of the index POP-R vs POP-NR polyp and 3 in epithelium, and one gene was upregulated in stroma and another in epithelium. MZT2B was upregulated in stroma of both the index POP-R (log2FC = 1.09, p-value = 0.0014) and POP-CRC (log2FC = 1.29, p-value = 0.0001) and has been implicated as a prognostic marker associated with worse prognosis in certain cancers. IgM and IgA plasma cells, proximal progenitors, MMP9+ inflammatory macrophages and myofibroblasts were found to be downregulated in epithelium of index POP-CRC and POP-R compared to POP-NR polyps; while microfold cells were downregulated in stroma of index POP-CRC and POP-R compared to POP-NR polyps. Conclusions: The stromal microenvironment and less so, epithelial features present in the index polyp differ based on a polyp’s future clinical behavior. The polyp-immune interaction warrants further study as a potential prevention target against polyp progression. Citation Format: Mrunal Dehankar, Lisa A. Boardman, Daniel O'Brien, E. Aubrey Thompson, Jennifer M. Kachergus, Ji Shi, Alexej Abyzov, Milovan Suvakov, Rondell P. Graham, Chen Wang. Stromal immune composition significantly contributes to adenomatous polyp recurrence or progression to cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4018.

Abstract Background Current literature suggestsisocitrate dehydrogenase (IDH)-mutant astrocytoma contains several molecular subgroups. In this study, we are interested in determining the connection between different molecular subgroups with grade and/or survival. Methods A cohort of 470 Mayo Clinic adult patients (≥18 years, 56.2% male) with primary IDH-mutant astrocytoma diagnosed by World Health Organization (WHO) 2021 criteria were examined. Results were validated in an independent cohort of 614 Mayo Clinic Neuropathology consult patients and 235 The Cancer Genome Atlas (TCGA) patients. Results The Mayo Clinic Practice cohort confirmed the association of CDKN2A/B deletion with overall survival (OS, homozygous vs hemizygous vs intact, 2.7 vs 9.6 vs 17.2 years, P < .001). Phosphatase and tensin homolog (PTEN) deletion was also associated with poor OS (7.3 vs 17.4 years, P < .001). Increased number of copy number alterations was associated with OS (continuous variable, HR = 1.027, P < .001). Carrying one or more copies of the germline risk allele at rs55705857 was associated with earlier age of onset (median age 33 vs 35 years, P = .01), and a shorter OS after adjusting for age, grade, sex and treatment (HR = 1.81, P = .007). The Mayo Clinic Neuropathology Consult cohort and TCGA were utilized to validate age of onset and survival, respectively. Unsupervised clustering of the copy number alterations identified several clinically significant groups that may define pathways to disease progression. Losses of chromosomes 11p, 13q, 1p, and 10q were all associated with reduced overall survival in the Mayo Clinic cohort. Conclusions Patients with hemizygous loss of CDKN2A/B, loss of PTEN, increased number of copy number alterations, specific chromosomal arm losses or rs55705857 germline risk allele have reduced overall survival.

Xeroderma Pigmentosum group C (XP-C) is an autosomal recessive disorder caused by mutations in the XPC gene, leading to defective nucleotide excision repair. This defect leads to genomic instability and a profound cancer predisposition. To model this disease, we generated induced pluripotent stem cells (iPSCs) from an XP-C patient carrying a novel homozygous nonsense mutation in the XPC gene (c.1830C>A). The resulting iPSCs demonstrated typical pluripotent characteristics, including expression of key markers and trilineage differentiation capability. However, genomic assessment revealed progressive karyotypic instability during extended culture. While initial whole-genome sequencing detected no major chromosomal abnormalities, subsequent G-banding analysis identified acquired trisomy 12 in two lines (CL12 and CL27) and a derivative X chromosome in a third line (CL30). These abnormalities were absent in early-passage analyses, indicating that they were acquired and selected for during extended culture. The acquisition of a derivative X chromosome in CL30, alongside recurrent trisomy 12, represents a unique cytogenetic signature likely attributable to the underlying XPC defect. We hypothesize that the loss of GG-NER creates a permissive genomic environment, accelerating the accumulation of DNA damage and chromosomal missegregation under replicative stress. This temporal divergence in genomic integrity highlights how culture pressures drive chromosomal evolution in XP-C iPSCs independently of initial reprogramming. Our findings emphasize that XP-C iPSCs require continuous genomic surveillance and provide a model for investigating how DNA repair deficiencies interact with in vitro culture stress.

Abstract The WHO 2021 classification of central nervous system tumors incorporated molecular markers to better define diffuse glioma subgroups. We analyzed array-based copy number, targeted next-generation sequencing, germline genotyping, and Illumina EPIC array methylation data from 404 primary IDH-mutant astrocytomas to further understand the relationship between copy number and outcomes. Loss of chromosome arm 13q was associated with poor overall survival after adjusting for age, sex, tumor grade, and treatment status (hazard ratio [HR]=2.04 (95% confidence interval: 1.25-3.33), p=0.004). Chromosome 13q loss was also significantly associated with the total number of copy number alterations (p<0.001). Loss of arm 11p was associated with poor survival in the adjusted analysis (HR=2.29 (1.38-3.79), p=0.001). Unsupervised clustering of the raw copy number data identified 12 clusters, where each cluster was defined by distinct copy number alteration(s). Clusters that contained 13q loss exhibited worse overall survival compared to other clusters. The clusters were significantly associated with age at diagnosis (p=0.015), tumor grade (p<0.001) and copy number complexity (p<0.001). The clusters with a high proportion of grade 4 tumors and with the highest complexity had the poorest overall survival. These findings highlight the prognostic relevance of chromosome 13q and 11p losses in IDH-mutant astrocytomas and support the integration of additional copy number alterations into molecular classification frameworks of IDH-mutant astrocytomas to improve risk stratification and patient management.

Somatic mosaicism is increasingly recognized as a fundamental feature of human biology, yet the detection of somatic mutations remains challenging. The SMaHT Network conducted four large-scale benchmarking experiments involving cell-lines and donor tissues, to evaluate sequencing technologies, experimental approaches, and computational methods for detecting different types of somatic mutations, generating community resource with >1,000X short-read and 100-400X long-read data for each of the nine analyzed samples. We determined effective strategies for utilizing short- and long-reads sequencing for mutation detection and demonstrated that using donor-specific assemblies and human pangenome improved calling, extending mutation catalogs to challenging genomic regions. We benchmarked six duplex technologies and showed that single-cell sequencing resolves cell type-specific mutational patterns and heterogeneity. Our results indicate that bulk, single-cell, and duplex analyses are complementary - and leveraging all three provides comprehensive characterization of mosaicism within tissues. Together, these findings provide a roadmap for accurate, genome-wide somatic mutation discovery and analysis.

Germline mutations modulate the risk of developing schizophrenia (SCZ). Much less is known about the role of mosaic somatic mutations in the context of SCZ. Deep (239×) whole-genome sequencing (WGS) of brain neurons from 61 SCZ cases and 25 controls postmortem identified mutations occurring during prenatal neurogenesis. SCZ cases showed increased somatic variants in open chromatin, with increased mosaic CpG transversions (CpG>GpG) and T>G mutations at transcription factor binding sites (TFBSs) overlapping open chromatin, a result not seen in controls. Some of these variants alter gene expression, including SCZ risk genes and genes involved in neurodevelopment. Although these mutational processes can reflect a difference in factors indirectly involved in disease, increased somatic mutations at developmental TFBSs could also potentially contribute to SCZ.

We developed a generally applicable method, CRISPR/Cas9-targeted long-read sequencing (CTLR-Seq), to resolve, haplotype-specifically, the large and complex regions in the human genome that had been previously impenetrable to sequencing analysis, such as large segmental duplications (SegDups) and their associated genome rearrangements. CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to isolate intact large (i.e., up to 2,000 kb) genomic regions that encompass previously unresolvable genomic sequences. These targets are then sequenced (amplification-free) at high on-target coverage using long-read sequencing, allowing for their complete sequence assembly. We applied CTLR-Seq to the SegDup-mediated rearrangements that constitute the boundaries of, and give rise to, the 22q11.2 Deletion Syndrome (22q11DS), the most common human microdeletion disorder. We then performed de novo assembly to resolve, at base-pair resolution, the full sequence rearrangements and exact chromosomal breakpoints of 22q11.2DS (including all common subtypes). Across multiple patients, we found a high degree of variability for both the rearranged SegDup sequences and the exact chromosomal breakpoint locations, which coincide with various transposons within the 22q11.2 SegDups, suggesting that 22q11DS can be driven by transposon-mediated genome recombination. Guided by CTLR-Seq results from two 22q11DS patients, we performed three-dimensional chromosomal folding analysis for the 22q11.2 SegDups from patient-derived neurons and astrocytes and found chromosome interactions anchored within the SegDups to be both cell type-specific and patient-specific. Lastly, we demonstrated that CTLR-Seq enables cell-type specific analysis of DNA methylation patterns within the deletion haplotype of 22q11DS.

Abstract Summary Copy number variation (CNV) and alteration (CNA) analysis is a crucial component in many genomic studies and its applications span from basic research to clinic diagnostics and personalized medicine. CNVpytor is a tool featuring a read depth-based caller and combined read depth and B-allele frequency (BAF) based 2D caller to find CNVs and CNAs. The tool stores processed intermediate data and CNV/CNA calls in a compact HDF5 file—pytor file. Here, we describe a new track in igv.js that utilizes pytor and whole genome variant files as input for on-the-fly read depth and BAF visualization, CNV/CNA calling and analysis. Embedding into HTML pages and Jupiter Notebooks enables convenient remote data access and visualization simplifying interpretation and analysis of omics data. Availability and implementation The CNVpytor track is integrated with igv.js and available at https://github.com/igvteam/igv.js. The documentation is available at https://github.com/igvteam/igv.js/wiki/cnvpytor. Usage can be tested in the IGV-Web app at https://igv.org/app and also on https://github.com/abyzovlab/CNVpytor.

The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (1) editing cultured cells; (2) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, whereas cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, whereas detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10–20 passages. Notably, clones also differed in copy number alterations (CNAs) that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and CNAs acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.