Biology subjects

Huang, A. Y.

Publications and source records attributed to Huang, A. Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Genomic, Clinical, and Spatial Predictors of Durable Response to BRAF/MEK Inhibition in BRAF-Mutant Melanoma

BRAF-targeted therapy (BRAFi/MEKi) and immune checkpoint blockade (anti-PD-1/anti-CTLA-4) have transformed the treatment of BRAF-mutant metastatic melanoma. While most patients who respond to targeted therapy eventually progress, a subset derives durable benefit, and biomarkers to identify this subset would inform optimal treatment selection. In this study, we analyzed pre-treatment tumor samples from a clinically annotated cohort of 155 patients with BRAF-mutant metastatic melanoma treated with first-line BRAFi/MEKi and followed for up to five years. We stratified patients into durable responders (PFS [≥] 24 months) and rapid progressors (PFS < 6 months with progression) and found that a global metric of tumor genomic heterogeneity, rather than individual gene alterations, distinguished these groups. Combining genomic heterogeneity with baseline tumor burden (e.g., lactate dehydrogenase (LDH) or radiographic lesion dimensions), we developed a parsimonious model that predicted durable responders with high precision and specificity. Notably, the analogous population of patients treated instead with immunotherapy were not durable responders, suggesting that the selected predictors of durable responders are targeted therapy specific. Spatial profiling of a subset of pre-treatment biopsies (n = 47) demonstrated that high intratumoral, but not peritumoral, CD8+ T-cell infiltration correlated with prolonged survival on BRAF-targeted therapy and served as an independent predictive factor when considered with genomic heterogeneity and features of clinical tumor burden. Together, these findings highlight the distinct baseline intrinsic and extrinsic features underlying durable response to BRAF-targeted therapy and support their potential implication in guiding treatment selection for patients with BRAF-mutant metastatic melanoma. One-Sentence SummaryIntegrated clinical, tumor genomic, and immune microenvironmental features predict durable responses to BRAF-targeted therapy.

cancer biology↗

bioRxiv2026

Somatic mutation in human cerebellum illustrates neuron type-specific patterns of age-related mutation

Human neurodegenerative disorders are characterized by exquisite specificity for neuronal types, but the basis of this is unknown. Here, we show that cerebellar granule neurons (GN)--the most abundant neuronal type in the human brain--accumulate somatic mutations in patterns highly distinct from cerebral cortical neurons, and more closely resembling oligodendroglia and other dividing cells. We find shared mutational signatures between normal aging GNs and medulloblastoma subtypes, suggesting the GN lineage as a tumor cell of origin. Whole-genome sequence of multiple single GNs from the same donor allowed analysis of specific times of neurogenesis, revealing a rich lineage tree that includes GNs that become postmitotic 2 years or more after birth, yet migrating postnatally to populate both the cerebellar vermis and the distant cerebellar hemisphere. Our results show that neuronal type-specific somatic mutation patterns enlighten normal development, cancer origins and potentially the cell type-specificity of neurodegeneration.

neuroscience↗

bioRxiv2025

Cell-type-specific patterns and consequences of somatic mutation in development and aging brain

Elucidating the role of somatic mutations in cancer, healthy tissues, and aging depends on methods that can accurately characterize somatic mosaicism across different cell types, as well as assay their impact on cellular function. Current technologies to study cell-type-specific somatic mutations within tissues are low-throughput. We developed Duplex-Multiome, incorporating duplex consensus sequencing to accurately identify somatic single-nucleotide variants (sSNV) from the same nucleus simultaneously analyzed for single-nucleus ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq). By introducing strand-tagging into the construction of snATAC-seq libraries, duplex sequencing reduces sequencing error by >10,000-fold while eliminating artifactual mutational signatures. When applied to 98%/2% mixed cell lines, Duplex-Multiome identified sSNVs present in 2% of cells with 92% precision and accurately captured known sSNV mutational spectra, while revealing unexpected subclonal lineages. Duplex-Multiome of > 51,400 nuclei from postmortem brain tissue captured sSNV burdens and spectra across all major brain cell types and subtypes, including those difficult to assay by single-cell whole-genome sequencing (scWGS). This revealed for the first time that diverse neuronal and glial cell types show distinct rates and patterns of age-related mutation, while also directly discovering developmental cell lineage relationships. Duplex-Multiome identified clonal sSNVs occurring at increased rates in glia of certain aged brains, as well as clonal sSNVs that correlated with changes in expression of nearby genes, in both neurotypical and autism spectrum disorder (ASD) individuals, directly demonstrating that somatic mutagenesis can contribute to gene expression phenotypes. Duplex-Multiome can be easily adopted into the 10X Multiome protocol and will bridge somatic mosaicism to a wide range of phenotypic readouts across cell types and tissues.

genomics↗

bioRxiv2025

Neurons accumulate disease-specific somatic genomic changes across taupathologic states in Alzheimer's disease

Tau deposition within neurons marks Alzheimers disease (AD) neuropathology, suggesting that tau may contribute to cellular dysfunction and death. In AD, somatic mutations accumulate in neurons, with features that suggest deleterious effects on cellular function. To examine the relationship between tau and somatic mutation, we isolated neurons according to tau pathology and performed single-cell whole-genome sequencing on tau+, tau-, and tau-agnostic neurons from 13 individuals with AD, as well as neurons from 15 control individuals. We found that AD neurons, regardless of tau status, exhibited an increased burden of somatic single-nucleotide variants (sSNVs) and insertions and deletions (sIndels). Mutational signature analyses revealed disease-specific patterns, including sIndels characterized by two-basepair (2bp) deletions, indicating shared mutagenic mechanisms in AD neurons across tau pathologic cell states. Somatic mutations are associated with tissue-wide tau pathology, suggesting that tangles do not confer cell-autonomous genotoxicity to neurons and that non-tangle components drive somatic mutation in AD.

genomics↗

bioRxiv2025

Clonal Hematopoiesis Mutations Increase Risk of Alzheimer's Disease with APOE ϵ3/ϵ3 Genotype

Clonal hematopoiesis of indeterminate potential (CHIP) represents clonal expansion of blood cells, and increases the risk of hematological malignancies and cardiovascular disorders. Recent studies have studied CHIP mutations in individuals with Alzheimers disease (AD), but it is unclear whether their role in AD pathogenesis is protective, detrimental, or neutral. In this study, we used molecular-barcoded deep gene panel sequencing ([~]400X) to examine CHIP mutations in 298 blood samples from AD and neurotypical individuals 60 years and older. The AD patients exhibited a significantly higher burden of CHIP mutations compared to the age-matched controls (p < 2e-7, odds ratio (OR) = 2.89), particularly in low-frequency variants often not captured by standard whole exome or whole genome sequencing (WGS). This increase was driven by individuals with the APOE {varepsilon}3/{varepsilon}3 genotype and absent in {varepsilon}4 carriers. Analysis of an independent dataset from the Alzheimers Disease Sequencing Project (ADSP), comprised of WGS data from [~]30,000 individuals, confirmed increased CHIP mutations in AD versus control (p < 0.02, OR = 1.32), again driven by individuals with APOE {varepsilon}3/{varepsilon}3 genotype. CHIP mutations in AD patients also showed stronger positive selection than in controls. Our results indicate that AD patients show significantly more CHIP mutations in their blood than controls, involving more than one third of AD patients, and contributing to AD risk through a mechanism independent of APOE {varepsilon}4.

genetics↗

bioRxiv2025

Recurrent patterns of widespread neuronal genomic damage shared by major neurodegenerative disorders

Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimers disease (AD) represent two major categories of neurodegenerative disorders--TDP-43 and tau proteinopathies--for which the mechanisms driving neuronal death remain unclear. Single-cell whole-genome sequencing of 469 neurons from C9ORF72 ALS, C9ORF72 FTD, AD, and control brains revealed increased somatic single nucleotide variants (sSNVs) and insertions/deletions (sIndels) in all three diseases. Mutational signature analysis identified a disease-associated sSNV signature consistent with oxidative damage and an sIndel process affecting 22% of ALS, 76% of FTD, and 61% of AD neurons--but only 2% of control neurons--resembling signature ID4, previously linked to topoisomerase 1 (TOP1)-mediated mutagenesis. Rapid Approach to DNA Adduct Recovery (RADAR) assays confirmed increased TOP1-DNA covalent complexes and duplex sequencing confirmed the increased sIndels and identified single-strand events as likely precursor lesions. TOP1-associated sIndel mutagenesis and genome instability thus represents a mechanism shared by both TDP-43 and tau neurodegeneration.

genomics↗

bioRxiv2025

Diverse somatic genomic alterations in single neurons in chronic traumatic encephalopathy

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease that is linked to exposure to repetitive head impacts (RHI), yet little is known about its pathogenesis. Applying two single-cell whole-genome sequencing methods to hundreds of neurons from prefrontal cortex of 15 individuals with CTE, and 4 with RHI without CTE, revealed increased somatic single-nucleotide variants in CTE, resembling a pattern previously reported in Alzheimers disease (AD). Furthermore, we discovered remarkably high burdens of somatic small insertions and deletions in a subset of CTE individuals, resembling a known pattern, ID4, also found in AD. Our results suggest that neurons in CTE experience stereotyped mutational processes shared with AD; the absence of similar changes in RHI neurons without CTE suggests that CTE involves mechanisms beyond RHI alone.

genomics↗

bioRxiv2024

Stratified analysis identifies HIF-2α as a therapeutic target for highly immune-infiltrated melanomas

While immune-checkpoint blockade (ICB) has revolutionized treatment of metastatic melanoma over the last decade, the identification of broadly applicable robust biomarkers has been challenging, driven in large part by the heterogeneity of ICB regimens and patient and tumor characteristics. To disentangle these features, we performed a standardized meta-analysis of eight cohorts of patients treated with anti-PD-1 (n=290), anti-CTLA-4 (n=175), and combination anti-PD-1/anti-CTLA-4 (n=51) with RNA sequencing of pre-treatment tumor and clinical annotations. Stratifying by immune-high vs -low tumors, we found that surprisingly, high immune infiltrate was a biomarker for response to combination ICB, but not anti-PD-1 alone. Additionally, hypoxia-related signatures were associated with non-response to anti-PD-1, but only amongst immune infiltrate-high melanomas. In a cohort of scRNA-seq of patients with metastatic melanoma, hypoxia also correlated with immunosuppression and changes in tumor-stromal communication in the tumor microenvironment (TME). Clinically actionable targets of hypoxia signaling were also uniquely expressed across different cell types. We focused on one such target, HIF-2, which was specifically upregulated in endothelial cells and fibroblasts but not in immune cells or tumor cells. HIF-2 inhibition, in combination with anti-PD-1, enhanced tumor growth control in pre-clinical models, but only in a more immune-infiltrated melanoma model. Our work demonstrates how careful stratification by clinical and molecular characteristics can be leveraged to derive meaningful biological insights and lead to the rational discovery of novel clinical targets for combination therapy.

cancer biology↗

bioRxiv2024

Cell-type-informed genotyping of mosaic focal epilepsies reveals cell-autonomous and non-cell-autonomous disease-associated transcriptional programs

Recent studies demonstrate growing roles for genetic mosaicism in neurodevelopmental and neuropsychiatric disorders, with the paradigm being drug-resistant pediatric focal epilepsy related to activating somatic variants in the PI3K-mTOR pathway. While identifying the genotype-associated changes at the single-cell level is fundamental to understanding disease pathophysiology, this remains technically challenging in human tissue samples with existing methods. Here, we performed single-nucleus RNA-sequencing (snRNA-seq) of 20 focal cortical dysplasia (FCD) samples removed surgically for treatment of drug-resistant epilepsy, and 10 non-FCD controls, and we developed a new approach, Genotyping Of Transcriptomes Enhanced with Nanopore sequencing (GO-TEN), that combines targeted complementary (c)DNA sequencing with snRNA-seq to perform concurrent single-nucleus genotyping and transcriptional analysis. We find that mosaic pathogenic variants in FCD do not produce a detectable novel cell identity, but instead we observe conserved cell types present both in FCD cases and non-FCD control specimens. Similarly, GO-TEN analysis shows that most pathogenic variant-carrying cells have well-differentiated neuronal or glial identities and are enriched for layer II-III excitatory neurons. We identify cell-intrinsic disruption of glutamate and GABA-A signaling pathways in variant-carrying neurons and altered intercellular signaling, making potential mechanisms for epileptogenesis in FCD. In summary, by addressing genotype-specific changes in mosaic epilepsy-associated lesions, our study highlights new potential disease mechanisms and therapeutic targets.

genetics↗

bioRxiv2024

Neuropathologically-directed profiling of PRNP somatic and germline variants in sporadic human prion disease

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 focal 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 >5,000X 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 focal presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

genetics↗

bioRxiv2024

Mature and migratory dendritic cells promote immune infiltration and response to anti-PD-1 checkpoint blockade in metastatic melanoma

Immune checkpoint inhibitors (ICIs) have heralded a remarkable shift in cancer care, significantly extending survival for advanced cancer patients. However, despite their remarkable clinical successes, a substantial majority of patients fail to achieve a lasting response to ICIs. To address this challenge and gain insights into the complex landscape of the tumor microenvironment (TME), we conducted an extensive analysis using single-cell RNA sequencing (scRNA; [~]216K cells across 39 samples) and single-nucleus transposase-accessible chromatin sequencing (snATAC; [~]43K cells from 15 samples) in a metastatic melanoma cohort. This systematic approach delineates 14 distinct cell types and 55 cell subtypes, including the identification of 15 transcriptional hallmarks of malignant cells. Through correlation analysis of cell subtype proportions, we unveiled six distinct clusters associated with varying tumor responses. Particularly intriguing was the identification of the mature DC enriched in immunoregulatory molecules (mregDC) subtype exhibiting correlations with naive T and B cells, forming an anti-tumor program that underscores the importance of multiple immune cell types in mediating anti-tumor immunity. Notably, we found that mregDC abundance represents a good prognostic predictor of progression-free survival (PFS) in the context of ICI treatment, and when combined with the TCF7+/- CD8 T cell ratio, it reliably predicts patient PFS across treatments beyond ICI. We validated our findings using an independent cohort of 274 ICI-treated melanoma samples analyzed using tissue-level expression. We next compared mregDCs and conventional dendritic cell types 1 and 2 (cDC1 and cDC2) using transcriptome signature, differentiation trajectory, interactome, cytokine milieu, and epigenome landscape analyses. This comparative analysis shed light on the unique attributes of mregDCs within the TME. Finally, we investigated cell type/subtype-specific genes, pathways, immune response enrichment, and ligand-receptor interactions closely associated with the proportions of mregDCs within the TME. These molecular and cellular insights, with their critical roles in enhancing the immune response against cancer, offer valuable prospects for predicting the efficacy of ICI regimens, and potentially guiding the selection of rational combinatorial therapies.

cancer biology↗

bioRxiv2024

Epstein-Barr Virus Orchestrates Spatial Reorganization and Immunomodulation within the Classic Hodgkin Lymphoma Tumor Microenvironment

Classic Hodgkin Lymphoma (cHL) is a tumor composed of rare malignant Hodgkin and Reed-Sternberg (HRS) cells nested within a T-cell rich inflammatory immune infiltrate. cHL is associated with Epstein-Barr Virus (EBV) in 25% of cases. The specific contributions of EBV to the pathogenesis of cHL remain largely unknown, in part due to technical barriers in dissecting the tumor microenvironment (TME) in high detail. Herein, we applied multiplexed ion beam imaging (MIBI) spatial pro-teomics on 6 EBV-positive and 14 EBV-negative cHL samples. We identify key TME features that distinguish between EBV-positive and EBV-negative cHL, including the relative predominance of memory CD8 T cells and increased T-cell dysfunction as a function of spatial proximity to HRS cells. Building upon a larger multi-institutional cohort of 22 EBV-positive and 24 EBV-negative cHL samples, we orthogonally validated our findings through a spatial multi-omics approach, coupling whole transcriptome capture with antibody-defined cell types for tu-mor and T-cell populations within the cHL TME. We delineate contrasting transcriptomic immunological signatures between EBV-positive and EBV-negative cases that differently impact HRS cell proliferation, tumor-immune interactions, and mecha-nisms of T-cell dysregulation and dysfunction. Our multi-modal framework enabled a comprehensive dissection of EBV-linked reorganization and immune evasion within the cHL TME, and highlighted the need to elucidate the cellular and molecular fac-tors of virus-associated tumors, with potential for targeted therapeutic strategies.

cancer biology↗

bioRxiv2024

Somatic cancer driver mutations are enriched and associated with inflammatory states in Alzheimer's disease microglia

Alzheimers disease (AD) is an age-associated neurodegenerative disorder characterized by progressive neuronal loss and pathological accumulation of the misfolded proteins amyloid-{beta} and tau1,2. Neuroinflammation mediated by microglia and brain-resident macrophages plays a crucial role in AD pathogenesis1-5, though the mechanisms by which age, genes, and other risk factors interact remain largely unknown. Somatic mutations accumulate with age and lead to clonal expansion of many cell types, contributing to cancer and many non-cancer diseases6,7. Here we studied somatic mutation in normal aged and AD brains by three orthogonal methods and in three independent AD cohorts. Analysis of bulk RNA sequencing data from 866 samples from different brain regions revealed significantly higher ([~]two-fold) overall burdens of somatic single-nucleotide variants (sSNVs) in AD brains compared to age-matched controls. Molecular-barcoded deep (>1000X) gene panel sequencing of 311 prefrontal cortex samples showed enrichment of sSNVs and somatic insertions and deletions (sIndels) in cancer driver genes in AD brain compared to control, with recurrent, and often multiple, mutations in genes implicated in clonal hematopoiesis (CH)8,9. Pathogenic sSNVs were enriched in CSF1R+ microglia of AD brains, and the high proportion of microglia (up to 40%) carrying some sSNVs in cancer driver genes suggests mutation-driven microglial clonal expansion (MiCE). Analysis of single-nucleus RNA sequencing (snRNAseq) from temporal neocortex of 62 additional AD cases and controls exhibited nominally increased mosaic chromosomal alterations (mCAs) associated with CH10,11. Microglia carrying mCA showed upregulated pro-inflammatory genes, resembling the transcriptomic features of disease-associated microglia (DAM) in AD. Our results suggest that somatic driver mutations in microglia are common with normal aging but further enriched in AD brain, driving MiCE with inflammatory and DAM signatures. Our findings provide the first insights into microglial clonal dynamics in AD and identify potential new approaches to AD diagnosis and therapy.

genomics↗

bioRxiv2023

Somatic Mosaicism in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Reveals Widespread Degeneration from Focal Mutations

Although mutations in many genes cause familial amyotrophic lateral sclerosis and frontotemporal dementia, most cases are sporadic (sALS and sFTD) with unclear etiology. We tested whether somatic mutations contribute to sALS and sFTD by deep targeted sequencing of 88 neurodegeneration-related genes in postmortem brain and spinal cord samples from 399 sporadic cases and 144 controls. Predicted deleterious somatic variants in ALS/FTD genes were observed in 2.1% of sporadic cases lacking deleterious germline variants. These variants occurred at very low allele fractions (typically <2%) and were often focal and enriched in disease-affected regions. Analysis of bulk RNA-seq data from an additional cohort identified deleterious somatic variants in DYNC1H1 and LMNA, genes associated with pediatric motor neuron degeneration. Targeted long-read sequencing further identified one sFTD case with de novo somatic C9orf72 repeat expansions. Together, these findings suggest that rare, focal somatic variants can contribute to sALS and sFTD and drive widespread neurodegeneration.

genetics↗

bioRxiv2023

Multivariate links between the developmental timing of adversity exposure and white matter tract integrity in adulthood

Early-life adversity is pervasive worldwide and represents a potent risk factor for increased mental health burden across the lifespan. However, there is substantial individual heterogeneity in associations between adversity exposure, neurobiological changes, and mental health problems. Accounting for key features of adversity such as the developmental timing of exposure may clarify associations between adversity, neurodevelopment, and mental health. The present study leverages sparse canonical correlation analysis to characterize modes of covariation between age of adversity exposure and the integrity of white matter tracts throughout the brain in a sample of 107 adults. We find that adversity exposure during middle childhood (ages 5-6 and 8-9 in particular) is consistently linked with alterations in white matter tract integrity, such that tracts supporting sensorimotor functions display higher integrity in relation to adversity exposure while tracts supporting cortico-cortical communication display lower integrity. Further, latent patterns of tract integrity linked with adversity experienced across preschool age and middle childhood (ages 4-9) were associated with trauma-related symptoms in adulthood. Our findings underscore that adversity exposure may differentially affect white matter in a function- and developmental-timing specific manner and suggest that adversity experienced between ages 4-9 may shape the development of global white matter tracts in ways that are relevant for adult mental health.

neuroscience↗

bioRxiv2023

Cell lineage analysis with somatic mutations reveals late divergence of neuronal cell types and cortical areas in human cerebral cortex

The mammalian cerebral cortex shows functional specialization into regions with distinct neuronal compositions, most strikingly in the human brain, but little is known in about how cellular lineages shape cortical regional variation and neuronal cell types during development. Here, we use somatic single nucleotide variants (sSNVs) to map lineages of neuronal sub-types and cortical regions. Early-occurring sSNVs rarely respect Brodmann area (BA) borders, while late-occurring sSNVs mark neuron-generating clones with modest regional restriction, though descendants often dispersed into neighboring BAs. Nevertheless, in visual cortex, BA17 contains 30-70% more sSNVs compared to the neighboring BA18, with clones across the BA17/18 border distributed asymmetrically and thus displaying different cortex-wide dispersion patterns. Moreover, we find that excitatory neuron-generating clones with modest regional restriction consistently share low-mosaic sSNVs with some inhibitory neurons, suggesting significant co-generation of excitatory and some inhibitory neurons in the dorsal cortex. Our analysis reveals human-specific cortical cell lineage patterns, with both regional inhomogeneities in progenitor proliferation and late divergence of excitatory/inhibitory lineages.

neuroscience↗

bioRxiv2023

Full-length isoform sequencing for resolving the molecular basis of Charcot-Marie-Tooth 2A

ObjectivesTranscript sequencing of patient derived samples has been shown to improve the diagnostic yield for solving cases of likely Mendelian disorders, yet the added benefit of full-length long-read transcript sequencing is largely unexplored. MethodsWe applied short-read and full-length isoform cDNA sequencing and mitochondrial functional studies to a patient-derived fibroblast cell line from an individual with neuropathy that previously lacked a molecular diagnosis. ResultsWe identified an intronic homozygous MFN2 c.600-31T>G variant that disrupts a branch point critical for intron 6 spicing. Full-length long-read isoform cDNA sequencing after treatment with a nonsense-mediated mRNA decay (NMD) inhibitor revealed that this variant creates five distinct altered splicing transcripts. All five altered splicing transcripts have disrupted open reading frames and are subject to NMD. Furthermore, a patient-derived fibroblast line demonstrated abnormal lipid droplet formation, consistent with MFN2 dysfunction. Although correctly spliced full-length MFN2 transcripts are still produced, this branch point variant results in deficient MFN2 protein levels and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A (CMT2A). DiscussionThis case highlights the utility of full-length isoform sequencing for characterizing the molecular mechanism of undiagnosed rare diseases and expands our understanding of the genetic basis for CMT2A.

genetics↗

bioRxiv2022

Genomic heterogeneity and ploidy identify patients with intrinsic resistance to PD-1 blockade in metastatic melanoma

While the introduction of immune checkpoint blockade (ICB) has dramatically improved clinical outcomes for patients with advanced melanoma, a significant proportion of patients develop resistance to therapy, and mechanisms of resistance are poorly elucidated in most cases. Further, while combination ICB has higher response rates and improved progression free survival compared to single agent therapy in the front line setting, there is significantly increased toxicity with combination ICB, and biomarkers to identify patients who would disproportionately benefit from combination therapy vs aPD-1 ICB are poorly characterized. To understand resistance mechanisms to single vs combination ICB therapy, we analyze whole-exome-sequencing (WES) of pre-treatment tumor and matched normals of 4 cohorts (n=140) of previously ICB-naive aPD-1 ICB treated patients. We find that high intratumoral genomic heterogeneity and low ploidy identify patients with intrinsic resistance to aPD-1 ICB. Comparing to a melanoma cohort from a pre-targeted therapy and ICB time period ("untreated" cohort), we find that genomic heterogeneity specifically predicts response and survival in the ICB treated cohorts, but not in the untreated cohort, while ploidy is also prognostic of overall survival in the "untreated" (by targeted therapy or ICB) group. To establish clinically actionable predictions, we optimize a simple decision tree using genomic ploidy and heterogeneity to identify with high confidence (90% PPV) a subset of patients with intrinsic resistance to and significantly worse survival on aPD1 ICB treatment. We then validate this model in independent cohorts, and further show that a significant proportion of patients predicted to have intrinsic resistance to single agent aPD-1 ICB respond to combination ICB, which suggests that nominated patients may benefit disproportionately from combination ICB. We further show that the features and predictions of the model are independent of known clinical features and previously nominated molecular biomarkers. These findings highlight the clinical and biological importance of genomic heterogeneity and ploidy, and sets a concrete framework towards clinical actionability, broadly advancing precision medicine in oncology.

cancer biology↗