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Craig, D. W.

Publications and source records attributed to Craig, D. W..

11 recordsLinked to original sources

Custom Probe-Based Spatial Transcriptomics Enables Microbiome Detection in FFPE Colorectal Cancer Tissue

Intratumoral bacteria have emerged as functionally relevant components of the tumor microenvironment, yet the spatial relationship between these bacterial communities and host gene expression remains poorly characterized, in part due to methodological constraints. Existing spatial transcriptomics approaches for microbial detection rely on fresh frozen tissue, excluding FFPE specimens which dominate clinical archives. Here, we describe a custom probe design pipeline targeting the variable regions of bacterial 16S rRNA, compatible with the probe-based chemistry of the 10x Genomics Visium CytAssist platform, enabling spatially resolved bacterial profiling in FFPE tissue. Applied to a pilot cohort of six FFPE colorectal cancer tumor and normal adjacent tissue specimens, we show that integration of custom microbial probes into the Visium workflow preserves host transcriptomic structure, with clustering analysis recapitulating expected colonic cell type architecture. Bacterial signal was detected across all samples in a spatially patterned and focal manner, with one tumor sample exhibiting markedly elevated signal intensity and a distinct invasive distribution pattern, driven by spatially structured Bacteroides-Phocaeicola and Porphyromonas signals with divergent intratumoral trajectories. These findings establish the feasibility of probe-based spatial metatranscriptomics in FFPE tissue and provide a generalizable framework for studying host-microbiome interactions in relevant clinical samples.

cancer biology↗

Extrachromosomal DNA as a Causal Instrument for Spatial Multi-Omics

Spatial transcriptomics, multiplex imaging, and computational pathology now map tissue organization at cellular resolution, but the analyses applied to these data remain correlational. Clustering and co-occurrence statistics describe which features appear together; they cannot say which feature drives the others. We propose a framework for causal inference in spatial multi-omics built on a specific feature of extrachromosomal DNA (ecDNA). ecDNA carries no centromere; it does not attach to the mitotic spindle and partitions randomly to daughter cells at division. Two neighboring cells in the same microenvironment can therefore inherit very different oncogene copy numbers for reasons unrelated to local signaling. Together, these cell-intrinsic randomization properties of ecDNA provide the framework for ecDNA copy number serving as an instrumental variable (IV) separating the effects of oncogene dosage from the downstream cellular effects. In this study, we formalize this within a structural causal model, implement two-stage least squares estimation with sibling-comparison and falsification diagnostics, and provide sensitivity analyses for residual confounding. To benchmark these methods against ground truth, we built CAUSANTA, an agent-based simulator that generates spatial tissue with a known causal graph and stochastic ecDNA inheritance. The framework recovers known oncogene-to-phenotype effects from simulated data, and we outline its application to real tumor sections, including multi-instrument settings where independent ecDNA species carrying different oncogenes enable factorial designs within a single tumor.

cancer biology↗

Mechanistic Link Between Glyoxalase 1 Expression and Methylglyoxal-Induced Oncogenic Stress in Prostate Cancer

Prostate cancer (PCa) is the second leading cause of cancer-related death in American men, with African American/Black (AA/B) men experiencing higher incidence and mortality than European American (EA) men. Obesity, which disproportionately affects AA/B men, is linked to increased PCa mortality, potentially through metabolic dysregulation. We hypothesize that methylglyoxal (MG), a reactive byproduct of glucose, lipid, and protein metabolism that is elevated in obesity, contributes to PCa progression. MG forms covalent adducts on DNA, RNA, and protein. We found that MG-adducts are elevated in AA/B men with PCa compared to EA men with PCa, as well as men without PCa. AA/B men with PCa had a higher frequency of SNP rs1049346 in glyoxalase 1 (GLO1), the primary MG detoxification enzyme. PCa cell lines from EA (C4-2) and AA/B (MDA-PCa-2b) men showed differential rs1049346 status, with C4-2 cells heterozygous and MDA-PCa-2b cells homozygous for the variant. This was associated with altered GLO1 expression and activity, with MDA-PCa-2b cells exhibiting reduced GLO1 function and increased MG-adducts compared to C4-2 cells. MG altered DNA repair and RNA processing pathways and induced distinct metabolic shifts in MDA-PCa-2b compared to C4-2 cells, including increased glycolysis and reduced oxidative phosphorylation. Transcriptomic analysis revealed unique MG-induced stress responses including a tenfold higher induction of TXNIP in MDA-PCa-2b vs. C4-2 cells, a gene inversely linked to GLO1 expression and activity. These findings suggest that MG stress may contribute to PCa progression in AA/B men through metabolic reprogramming and impaired detoxification, offering insight into potential precision medicine applications. Statement of SignificancePatients with obesity and diabetes have an elevated risk of cancer mortality. Defining how metabolic alterations contribute to this link is critical to understanding disease progression and identifying strategies to improve outcomes.

cancer biology↗

Integrated single-cell whole genome sequencing and spatial transcriptomics reveal latent intra-tumoral heterogeneity in ovarian cancer

The mortality rate of ovarian cancer remains disproportionately high compared to its incidence. This is partly due to a high level of intra-tumoral heterogeneity that promotes disease recurrence and treatment failure. In this study, we describe degrees of heterogeneity revealed by single-cell whole genome sequencing and spatial transcriptomics of five epithelial ovarian carcinomas. At the cellular level, we describe pseudo-diploid cells that match the malignant cell population in both somatic variant and copy number patterns. At the clonal and subclonal levels, we describe diversification associated with copy number gains and whole genome doubling. In multi-clonal samples, we infer evolutionary relationships from single cell copy number, loss of heterozygosity analysis, and somatic variant detection, and correlate these with tissue histology and gene expression programs. In one sample, we identify functionally consequential copy number alterations that contribute to molecular diversity, cell proliferation, and inflammation in a minor clone that persisted without major expansion alongside a more complex major clone. In another, we describe a complex evolutionary history including a spontaneous reversion of a driver mutation in a secondary clone, which correlated with a switch in oncogenic expression programs.

cancer biology↗

Decreased SNCA Expression in Whole-Blood RNA Analysis of Parkinson's Disease Adjusting for Lymphocytes

Blood-based RNA transcriptomics offers a promising avenue for identifying biomarkers of Parkinsons Disease (PD) progression and may provide mechanistic insights into the systemic biological processes underlying its pathogenesis beyond the well-defined neurodegenerative features. Previous studies have indicated an age-dependent increase in neutrophil-enriched gene expression, alongside a reduction in lymphocyte counts, in individuals with PD. These immune cell changes can obscure disease-relevant transcriptomic signals. In this study, we performed differential expression (DE) analysis of whole-blood RNA sequencing data from PD cohorts, incorporating a correction for immune cell-enriched gene expression, particularly neutrophil-related pathways, to improve the resolution of PD-associated molecular changes. Using 1,254 Parkinsons Progression Markers Initiative (PPMI) samples with complete blood count (CBC) data, we developed a predictive model to estimate neutrophil percentages in a 6,987 PPMI and Parkinsons Disease Biomarkers Program (PDBP) samples. We mitigated the confounding effects of immune cell-enriched gene expression by integrating predicted neutrophil percentages as a covariate in DE analysis. This approach revealed a consistent and significant downregulation of SNCA across all PD cohorts, a finding previously obscured by immune cell signatures. Lowered SNCA expression was found in individuals with known predisposition genes (e.g., SNCA, GBA, LRRK2) and in non-genetic PD cohorts lacking known pathogenic mutations, suggesting it may represent a key transcriptomic hallmark of the disease.

bioinformatics↗

The Foundational data initiative for Parkinsons disease (FOUNDIN-PD): enabling efficient translation from genetic maps to mechanism

The FOUNdational Data INitiative for Parkinsons Disease (FOUNDIN-PD) is an international collaboration producing fundamental resources for Parkinsons disease (PD). FOUNDIN-PD generated a multi-layered molecular dataset in a cohort of induced pluripotent stem cell (iPSC) lines differentiated to dopaminergic (DA) neurons, a major affected cell type in PD. The lines were derived from the Parkinsons Progression Markers Initiative study including participants with PD carrying monogenic PD (SNCA) variants, variants with intermediate effects and variants identified by genome-wide association studies and unaffected individuals. We generated genetic, epigenetic, regulatory, transcriptomic, and longitudinal cellular imaging data from iPSC-derived DA neurons to understand molecular relationships between disease associated genetic variation and proximate molecular events. These data reveal that iPSC-derived DA neurons provide a valuable cellular context and foundational atlas for modelling PD genetic risk. We have integrated these data into a FOUNDIN-PD data browser (https://www.foundinpd.org) as a resource for understanding the molecular pathogenesis of PD.

genomics↗

Multi-Modality Machine Learning Predicting Parkinsons Disease

BackgroundPersonalized medicine promises individualized disease prediction and treatment. The convergence of machine learning (ML) and available multi-modal data is key moving forward. We build upon previous work to deliver multi-modal predictions of Parkinsons Disease (PD). MethodsWe performed automated ML on multi-modal data from the Parkinsons Progression Marker Initiative (PPMI). After selecting the best performing algorithm, all PPMI data was used to tune the selected model. The model was validated in the Parkinsons Disease Biomarker Program (PDBP) dataset. Finally, networks were built to identify gene communities specific to PD. FindingsOur initial model showed an area under the curve (AUC) of 89.72% for the diagnosis of PD. The tuned model was then tested for validation on external data (PDBP, AUC 85.03%). Optimizing thresholds for classification, increased the diagnosis prediction accuracy (balanced accuracy) and other metrics. Combining data modalities outperforms the single biomarker paradigm. UPSIT was the largest contributing predictor for the classification of PD. The transcriptomic data was used to construct a network of disease-relevant transcripts. InterpretationWe have built a model using an automated ML pipeline to make improved multi-omic predictions of PD. The model developed improves disease risk prediction, a critical step for better assessment of PD risk. We constructed gene expression networks for the next generation of genomics-derived interventions. Our automated ML approach allows complex predictive models to be reproducible and accessible to the community. FundingNational Institute on Aging, National Institute of Neurological Disorders and Stroke, the Michael J. Fox Foundation, and the Global Parkinsons Genetics Program. RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSPrior research into predictors of Parkinsons disease (PD) has either used basic statistical methods to make predictions across data modalities, or they have focused on a single data type or biomarker model. We have done this using an open-source automated machine learning (ML) framework on extensive multi-modal data, which we believe yields robust and reproducible results. We consider this the first true multi-modality ML study of PD risk classification. Added value of this studyWe used a variety of linear, non-linear, kernel, neural networks, and ensemble ML algorithms to generate an accurate classification of both cases and controls in independent datasets using data that is not involved in PD diagnosis itself at study recruitment. The model built in this paper significantly improves upon our previous models that used the entire training dataset in previous work1. Building on this earlier work, we showed that the PD diagnosis can be refined using improved algorithmic classification tools that may yield potential biological insights. We have taken careful consideration to develop and validate this model using public controlled-access datasets and an open-source ML framework to allow for reproducible and transparent results. Implications of all available evidenceTraining, validating, and tuning a diagnostic algorithm for PD will allow us to augment clinical diagnoses or risk assessments with less need for complex and expensive exams. Going forward, these models can be built on remote or asynchronously collected data which may be important in a growing telemedicine paradigm. More refined diagnostics will also increase clinical trial efficiency by potentially refining phenotyping and predicting onset, allowing providers to identify potential cases earlier. Early detection could lead to improved treatment response and higher efficacy. Finally, as part of our workflow, we built new networks representing communities of genes correlated in PD cases in a hypothesis-free manner, showing how new and existing genes may be connected and highlighting therapeutic opportunities.

genomics↗

Integrated multi-omics analysis reveals common and distinct dysregulated pathways for genetic subtypes of Frontotemporal Dementia

Understanding the molecular mechanisms underlying frontotemporal dementia (FTD) is essential for the development of successful therapies. Systematic studies on human post-mortem brain tissue of patients with genetic subtypes of FTD are currently lacking. The Risk and Modyfing Factors of Frontotemporal Dementia (RiMod-FTD) consortium therefore has generated a multi-omics dataset for genetic subtypes of FTD to identify common and distinct molecular mechanisms disturbed in disease. Here, we present multi-omics datasets generated from the frontal lobe of post-mortem human brain tissue from patients with mutations in MAPT, GRN and C9orf72 and healthy controls. This data resource consists of four datasets generated with different technologies to capture the transcriptome by RNA-seq, and small RNA-seq, and supplemented this date with CAGE-seq, and methylation profiling. We show concrete examples on how to use the resulting data and confirm current knowledge about FTD and identify new processes for further investigation. This extensive multi-omics dataset holds great value to reveal new research avenues for this devastating disease.

neuroscience↗

Deep sncRNA-seq of the PPMI cohort to study Parkinson's disease progression

Coding and non-coding RNAs have diagnostic and prognostic importance in Parkinsons diseases (PD). We studied circulating small non-coding RNAs (sncRNAs) in 7, 003 samples from two longitudinal PD cohorts (Parkinsons Progression Marker Initiative (PPMI) and Luxembourg Parkinsons Study (NCER-PD)) and modelled their influence on the transcriptome. First, we sequenced sncRNAs in 5, 450 blood samples of 1, 614 individuals in PPMI. The majority of 323 billion reads (59 million reads per sample) mapped to miRNAs. Other covered RNA classes include piRNAs, rRNAs, snoRNAs, tRNAs, scaRNAs, and snRNAs. De-regulated miRNAs were associated with the disease and disease progression and occur in two distinct waves in the third and seventh decade of live. Originating mostly from a characteristic set of immune cells they resemble a systemic inflammation response and mitochondrial dysfunction, two hallmarks of PD. By profiling 1, 553 samples from 1, 024 individuals in the NCER-PD cohort using an independent technology, we validate relevant findings from the sequencing study. Finally, network analysis of sncRNAs and transcriptome sequencing of the original cohort identified regulatory modules emerging in progressing PD patients.

neuroscience↗

Complex genetic network underlying the convergent of Rett Syndrome like (RTT-L) phenotype in neurodevelopmental disorders

Mutations of the X-linked gene encoding methyl-CpG-binding protein 2 (MECP2) cause classical forms of Rett syndrome (RTT) in girls. Patients with features of classical Rett syndrome, but do not fulfill all the diagnostic criteria (e.g. absence of a MECP2 mutation), are defined as atypical Rett syndrome. Genes encoding for cyclin-dependent kinase-like 5 (CDKL5) and forkhead box G1 (FOXG1) are more commonly found in patients with atypical Rett syndrome. Nevertheless, a subset of patients who are recognized to have an overlapping phenotype with RTT but are lacking a mutation in a gene that causes typical or atypical RTT are described as having Rett syndrome like phenotype (RTT-L). Whole Exome Sequencing (WES) of 8 RTT-L patients from our cohort revealed mutations in the genes GABRG2, GRIN1, ATP1A2, KCNQ2, KCNB1, TCF4, SEMA6B, and GRIN2A, which are seemingly unrelated to Rett syndrome genes. We hypothesized that the phenotypic overlap in RTT and RTT-L is caused by mutations in genes that affect common cellular pathways critical for normal brain development and function. We annotated the list of genes identified causing RTT-L from peer-reviewed articles and performed a protein-protein interaction (PPI) network analysis. We also investigated their interaction with RTT (typical or atypical) genes such as MECP2, CDKL5, NTNG1, and FOXG1. We found that the RTT-L-causing genes were enriched in the biological pathways such as circadian entrainment, the CREB pathway, and RET signaling, and neuronal processes like ion transport, synaptic transmission, and transcription. We conclude that genes that significantly interact with the PPI network established by RTT genes cause RTT-L, explaining the considerable feature overlap between genes that are indicated for RTT-L and RTT.

genomics↗

Resolving sub-clonal heterogeneity within cell-line growths by single cell sequencing genomic DNA

We performed shallow single-cell sequencing of genomic DNA across 1,475 cells from a well-studied cell-line, COLO829, to resolve overall tumor complexity and clonality. This melanoma tumor-line has been previously characterized by multiple technologies and provides a benchmark for evaluating somatic alterations, though has exhibited conflicting and indeterminate copy number states. We identified at least four major sub-clones by discriminant analysis of principal components (DAPC) of single cell copy number data. Break-point and loss of heterozygosity (LOH) analysis of aggregated data from sub-clones revealed a complex rearrangement of chromosomes 1, 10 and 18 that was maintained in all but two sub-clones. Likewise, two of the sub-clones were distinguished by loss of 1 copy of chromosome 8. Re-analysis of previous spectral karyotyping data and bulk sequencing data recapitulated these sub-clone hallmark features and explains why the original bulk sequencing experiments generated conflicting copy number results. Overall, our results demonstrate how shallow copy number profiling together with clustering analysis of single cell sequencing can uncover significant hidden insights even in well studied cell-lines.

genomics↗